VIRULENCE

2023, VOL. 14, NO. 1, 2265015

https://doi.org/10.1080/21505594.2023.2265015

REVIEW ARTICLE

Pathogenicity and virulence of Borrelia burgdorferi

a,b a a,b
Martin Strnad , Natalie Rudenko , and Ryan O.M. Rego
a
Biology Centre CAS, Institute of Parasitology, České Budějovice, Czech Republic; bFaculty of Science, University of South Bohemia,
Branišovská, Czech Republic

ABSTRACT ARTICLE HISTORY

Infection with Borrelia burgdorferi often triggers pathophysiologic perturbations that are further Received 9 November 2022
augmented by the inflammatory responses of the host, resulting in the severe clinical conditions of Revised 23 August 2023
Lyme disease. While our apprehension of the spatial and temporal integration of the virulence Accepted 25 September
determinants during the enzootic cycle of B. burgdorferi is constantly being improved, there is still 2023
much to be discovered. Many of the novel virulence strategies discussed in this review are undeter­ KEYWORDS
mined. Lyme disease spirochaetes must surmount numerous molecular and mechanical obstacles in Borrelia burgdorferi; Lyme
order to establish a disseminated infection in a vertebrate host. These barriers include borrelial disease; virulence
relocation from the midgut of the feeding tick to its body cavity and further to the salivary glands, determinants; pathogenicity;
deposition to the skin, haematogenous dissemination, extravasation from blood circulation system, clinical manifestations; tick-
evasion of the host immune responses, localization to protective niches, and establishment of local as borne disease
well as distal infection in multiple tissues and organs. Here, the various well-defined but also possible
novel strategies and virulence mechanisms used by B. burgdorferi to evade obstacles laid out by the
tick vector and usually the mammalian host during colonization and infection are reviewed.

Introduction
The bacterial pathogen Borrelia burgdorferi, also known
as Borreliella burgdorferi, is the causative agent of Lyme
disease (LD) and is transmitted to humans through
Ixodes ticks. LD is the most common tick infection,
with approximately 476 000 cases in the United States
[1] and 650 000–850 000 cases in Europe annually [2].
LD occurs in a variety of clinical manifestations, ran­
ging from skin lesions, through musculoskeletal, neu­
rological, to cardiovascular symptoms [3]. The
organotrophy associated with B. burgdorferi species
has an impact on the development of the disease.

CONTACT Martin Strnad martin.strnad.cze@gmail.com

© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the
Accepted Manuscript in a repository by the author(s) or with their consent.
2 M. STRNAD ET AL.
Although there is already an abundance of knowledge
on the causative agent of the disease, an efficacious
strategy to reliably overcome LD has not been found.
There is currently no vaccine for human use available,
although new ways how to cope with the malicious
pathogen are being explored [4]. Multiple genes com­
mon for B. burgdorferi sensu lato species (referred to as
B. burgdorferi in this Review) display high sequence
variability between the species of LD spirochaetes.
Genes required for optimal infectivity often differ
markedly at both the nucleotide and amino acid level
[5,6]. Such variability significantly complicates the
design of efficacious immunization strategies including
vaccines since the immune responses do not protect
against all infectious species to the same extent. This
highlights the current strategy which involves clinical
development of a multivalent protein vaccine that tar­
gets all common serotypes of B. burgdorferi [7].
Virulence determinants of B. burgdorferi are found
both on the linear chromosome and on linear and
circular plasmids. Chromosomal virulence genes
include p66, bgp, plzA, rpoN, rpoS, or bosR whereas
virulence-associated genes that are present on plasmids
encode proteins such as DbpA/B, BBK32, OspC, PncA,
VlsE [8]. B. burgdorferi possesses a unique, segmented
genome comprised of the largest plasmid complement
of any characterized bacterium. The plasmids are sub­
stantially variable between species and strains, both in
number and in sequence [9]. In contrast to a majority
of other bacteria, circular plasmids represent a minority
of the genome in B. burgdorferi. With the exception of
the cp-26 plasmid, all circular plasmids are related to
each other and are derived from the cp-32 plasmid
family. A unique characteristic of linear replicons is
that they are terminated by covalently closed hairpin
ends [10].
B. burgdorferi exists in an enzootic cycle alternating
between a tick vector and a vertebrate host (Figure 1).
Acquisition of B. burgdorferi by a tick occurs through
haematophagous bites of the tick. In order to feed, tick
chelicerae cut the host epidermis and the hypostome
penetrates through the layers of the skin to secret saliva
into the tick bite site on the host and draw blood,
possibly, together with Borrelia [11]. The spirochaete
enters the tick when the larval/nymphal tick takes
a bloodmeal on an infected host and colonizes the
tick midgut. During subsequent feeding, a handful of
spirochaetes colonizing the tick exit the midgut by
traversing a peritrophic membrane, a layer of epithelial
cells and a basal membrane [12]. The spirochaetes then
migrate into the haemocoel as they navigate toward the
tick salivary glands, from which they are transmitted to
the host dermis via saliva. The salivary route of trans­
mission is highly favourable to the pathogen as it delays
the contact with borreliacidal factors present in the host
blood. Borrelia then migrate through the extracellular
matrix (ECM) constituents for haematogenous dissemi­
nation into their colonization niches where they cause
the damage that can result in development of disease
and complete the enzootic cycle [13,14].
To be able to establish an infection and stay inside
the host, B. burgdorferi must evade the immune pres­
sure exerted by the host’s defence system. The genome
sequences of B. burgdorferi species revealed that spir­
ochaetes lack the classically defined virulence determi­
nants common to a large number of microbial and,
specifically, bacterial pathogens, such as lipopolysac­
charides, specialized secretion systems, and toxins
[15,16]. Borrelia has developed several well-defined
mechanisms such as utilization of tick and host pro­
teins, complement evasion, localization into specific
niches, dynamic genetic regulation and antigenic varia­
tion, in order to survive in the host [8]. There are also
several alleged virulence mechanisms not yet uncondi­
tionally accepted by the expert community. These
include transformations of spiral-shaped replicating
Borrelia into alternative structural forms such as
round bodies or biofilm-like structures [17]. These
pleomorphic variants are hypothesized to be able to
survive the host immune response or prolonged courses
of antibiotic treatment in cases when the subpopulation
of Borrelia cells switch the phenotype from susceptible
to persistent under hostile pressure [18]. Intracellular
localization of the spirochaetes, shedding of borrelial
outer membrane vesicles, nutritional virulence, modu­
lation of tick microbiota and tick behaviour, and
Borrelia-induced structural and mechanical transfor­
mations of host cells are other potential virulence
mechanisms of LD Borrelia [19–25].
There are a number of outstanding reviews focus­
ing either on a narrow range of topics in borrelial
virulence mechanisms [26–29] or which provide
a detailed overview of multiple widely accepted viru­
lence mechanisms [8]. Herein, we guide the reader
not only through the complexities of classical but
importantly also through the trending virulence
mechanisms employed by LD Borrelia. In the context
of this review, similarly as in the other review of
borrelial pathogenic mechanisms [8], the term “viru­
lence determinants” encompasses all genes that
impact the infection process, eventhough these fac­
tors were often studied and defined in the host
 VIRULENCE 3

Figure 1. Enzootic cycle responsible for maintaining B. burgdorferi in tick populations. Ixodes ticks undergo a three-stage life
cycle (larva, nymph and adult), with one bloodmeal per stage. Uninfected larval ticks hatch from eggs and feed on a variety of small
mammals that can host B. burgdorferi. The larva takes an infected bloodmeal and moults into a next tick stage, which is a nymph.
Nymphs are responsible for transmitting the majority of infections to humans, leading to variety of clinical manifestations. Deer are
important hosts for adult ticks but they are not effective reservoirs for the spirochaetes. Engorged females lay thousands of eggs,
however, the spirochaetes are not transmitted through the eggs to the larval progeny.

animals which are not very susceptible to the disease
or show few signs of LD, e.g. mice. This definition
includes also genes involved in fundamental meta­
bolic processes necessary for borrelial survival in the
host. When the genes associated with a suspected
virulence trait are specifically deactivated, it results
in a noticeable reduction in infection potential. To
sum up, in addition to a better understanding of the
bacterial pathogenesis process, identification of viru­
lence factors in the spirochaetal genome may allow
the design of new, more efficient and, importantly,
more specific intervention strategies than those that
are currently employed in combating of LD.
Diagnosis of Lyme disease
LD has a broad spectrum of symptoms and clinical
manifestations. The initial step in distinguishing LD
from other illnesses involves the review of all signs
and symptoms and gathering subjective and objective
data from the patient. If there is potential tick exposure
in an endemic area and the individual exhibits an
expanding skin lesion called erythema migrans (EM),
immediate suspicion for LD should arise. Recognizing
EM can be challenging, since EM can be present in
various morphological forms, different from the classic
bull’s eye [30].
4 M. STRNAD ET AL.
The current reference method utilized for confirma­
tion of physician-diagnosed LD is serological testing.
Standard two-tiered testing (STTT) involves an initial
enzyme-linked immunoassay (ELISA) as the primary
test, followed by supplemental IgM and/or IgG immu­
noblots. The STTT is recognized for its limited sensi­
tivity in detecting early localized infection, as opposed
to very high sensitivity in detecting late infection [31].
Modified two-tier testing (MTTT) is an updated two-
tiered testing algorithm, in which confirmation of the
infection is done with an ELISA instead of an immu­
noblot. Multiple studies, both in North America and
Europe, have shown that MTTT has improved sensitiv­
ity compared with STTT, without losing specificity
[32,33]. Some MTTT algorithms have already received
FDA clearance for use in serological diagnostics of LD.
The interpretation of serology results can be intri­
cate. It usually takes several days to a few weeks for the
immune system to generate a Borrelia-specific IgM
response, while the development of a Borrelia-specific
IgG response can take up to two months [34]. Methods
to detect IgM antibodies are mainly useful during early
disease, since later during infection they often generate
false positive signal due to cross-reactions with other
infections [35,36]. Another regular weakness of current
commercial diagnostic tests is the low sensitivity for
less prevalent or rare Borrelia species, especially those
which exhibit high polymorphism in the commonly
detected immunogenic proteins [37,38]. In addition,
establishing a clear connection between antibody status
and actual infection can be challenging. Non-infected
individuals may exhibit immunity long after active LD
and test positive, while infected individuals might
experience a delay in their antibody response and test
negative [39]. Furthermore, specifically in Lyme neuro­
borreliosis, the antibody production does not always
follow the typical immune response of initial IgM
secretion followed by secretion of IgG [40].
Treatment & clinical manifestations of Lyme
disease
The course of LD can be divided into three distinct
stages – early localized LD, early disseminated LD and
late LD. Early localized infection, typically presented as
a painless skin rash (EM), is treated with antibiotics.
The therapeutic success of LD is dependent on how
early is the treatment with antibiotics initiated. A short
course of oral antibiotics in most cases efficiently elim­
inates the pathogen and cures the majority of LD cases.
It is recommended that patients with EM are treated
with a 10-day course of doxycycline or a 14-day course
of amoxycillin or, possibly, cefuroxime rather than
longer treatment periods [41]. When LD is diagnosed
later, after the spirochaetes have moved from the tick
bite site to the secondary sites of infection, the stage is
called early disseminated infection. This stage can occur
days, weeks, or even months after an infected tick
transmits the spirochaetes. Either oral or intravenous
antibiotic therapy is recommended for treatment.
Clinical manifestations of late LD include a variety of
symptoms that are often arthritic and neurologic, and
occur months after the transmission of the pathogen
[42,43]. Even though the majority of LD cases are cured
by antibiotic therapy, around 10–20% of patients
experience persistent disease that does not resolve
within an extended period of time even after repeated
courses of antibiotic therapy [44]. Symptoms such as
headache, fatigue, and joint pain are commonly asso­
ciated with this stage of the infection, which has been
defined as post-treatment Lyme disease syndrome
(PTLDS) [44]. PTLDS is considered a subset of the
term chronic LD, which is an umbrella term with
a wide and imprecise definition, often applied to indi­
viduals experiencing non-specific symptoms that are
believed to stem from an assumed ongoing
B. burgdorferi infection. These patients may or may
not exhibit evidence of past or present LD [45].
Considerable confusion and controversy still exist and
questions about the possible existence of this fourth
stage of LD, its frequency or the cause of PTLDS is
actively discussed by the scientific and medical com­
munity. The exact mechanisms and molecular triggers
leading to the long-term sequelae of the infection are
still undetermined. It is hypothesized that the reduced
efficacy of antibiotic therapy may be caused by: a) host-
adapted spirochaetes that persist in the tissues or pos­
sibly intracellularly inside certain cell types such as
fibroblasts and thus are inaccessible to antibiotics
[19]; b) excessive inflammatory responses to residual
antigenic molecules such as peptidoglycan from killed
microbes or autoimmune responses [46]; c) tissue
damage after clearance of Borrelia [42].
The genetic heterogeneity of B. burgdorferi species
seems to be the main factor of the diverse clinical
manifestations with serious multi-system disorders of
human LD. It is not surprising, as out of 22 currently
recognized LD spirochaete species, 12 were already
detected or isolated from human patients, diagnosed
with LD or expected to have a spirochaete infection
[47,48]. Within the species that are detected in, or
isolated from humans, the frequency of recovery varies
greatly. While B. afzelii, B. bavariensis, B. garinii,
B. burgdorferi sensu stricto (ss) or B. mayonii are
responsible for the majority of LD cases worldwide,
the number of patients in which B. bissettii,

B. kurtenbachii, B. spielmanii or B. valaisiana have been
detected is low, and detection of B. americana,
B. lusitaniae or B. andersonii in humans is rare [49,
50]. Diverse clinical manifestations of the disease are
connected to different organ tropism that individual
LD spirochaete species possess. For example, infection
with B. burgdorferi ss, the major case of LD in the
United States, results in the most common for North
America musculoskeletal disorder, Lyme arthritis, that
was diagnosed in at least 60% of untreated human
patients with EM [51]. In Europe, where B. afzelii and
B. garinii represent the most pathogenic spirochaete
species and are recovered from environmental and
samples of human origin much more often than
B. burgdorferi ss, the number of diagnosed cases of
arthritis did not exceed 15% [52]. Widely distributed
B. garinii was confirmed to be related to increased
number of neuroborreliosis cases in Europe [53,54].
Occasionally, infections with B. burgdorferi ss and
B. afzelii result in neuroborelliosis as well, however,
the number of diagnosed cases is comparably lower.
B. afzelii predominantly causes skin infections such as
EM, acrodermatitis chronica atrophicans (ACA) or
borrelial lymphocytoma [55]. Rare cases of ACA were
described in patients infected with B. garinii as well
[56–58]. In addition to the major recognized LD caus­
ing species, some other species are occasionally
detected in humans as well. While connection of
B. bissettii [59,60] with cardiovascular disorders was
described in patients from Europe [61,62], the status
of B. lusitaniae [63], and B. valaisiana [64] as a human
pathogen still needs a valid confirmation [65]. Tick co-
infection with other pathogens such as Rickettsia and A.
phagocytophilum does not significantly increase in clin­
ical symptoms or disease duration [66].
Vaccine development
Although there are currently no approved vaccines
against LD for human use, multiple studies have built
a solid foundation for further vaccine development.
B. burgdorferi produces multiple antigenic and immu­
nologically accessible molecules which were shown to
be promising vaccine candidates but which have never
reached the market. These molecules include mostly the
surface-exposed borrelial proteins such as BBA52,
BB0405, BBI39, DbpA, BBK32, OspC, with data show­
ing that combination vaccines composed of DbpA,
BBK32, and OspC are more powerful than single for­
mulation or double component cocktails [67–73].
The only licenced vaccine to prevent LD was
LYMERix, with efficacy of nearly 80% [74]. After
being available on the market few years, the vaccine
VIRULENCE 5
was pulled from the US market in 2002 due to numer­
ous reports of severe side effects. There was
a hypothesis suggesting that the vaccine antigen, outer
surface protein A (OspA), acted as an autoantigen,
potentially causing arthritis. However, the adverse
effects were never definitively substantiated [75]. The
Valneva-Pfizer “VLA15” is currently the only LD vac­
cine candidate in advanced clinical development
(phase 3). This multivalent protein subunit vaccine
targets OspA and provides protection against the six
prevalent OspA serotypes expressed by the
B. burgdorferi species commonly found in North
America and Europe [7,76].
In order to address the considerable variability
observed in proteins across different strains and species
of B. burgdorferi, effort has been put on creating
a multivalent chimeric vaccines that offer protective
effects against various species of LD, aiming to tackle
the heterogeneity inherent in the pathogen [70,77,78].
Multivalent OspA-based formulations are currently
common targets in vaccine development. To create self-
assembling nanoparticles, OspA was combined with
bacterial ferritin. These OspA-ferritin nanoparticles
induced robust and long-lasting antibody responses
against the major serotypes in both mice and non-
human primates [79]. To achieve broad protection
with a single recombinant antigen, a strategy called
grafting, or epitope reshaping, has been successfully
tested for production of neutralizing antibodies against
the six clinically most relevant OspA serotypes [80].
Besides vaccines based on recombinant protein tech­
nology, DNA tattoo and lipid nanoparticle-
encapsulated DNA vaccination represents a promising
strategy for prevention of LD [81–83]. DNA vaccines
present several potential benefits compared to conven­
tional strategies, such as the activation of both B- and
T-cell responses, enhanced vaccine stability, the
absence of any infectious agents, and the straightfor­
ward large-scale production process [84].
Methods used to identify virulence mechanisms
and determinants
Genetic modifications
Virulence is defined by the ability of a microorganism
to cause disease in the host. Modern strategies that are
being used to uncover the fine details of bacterial viru­
lence mechanisms encompass a combination of bio­
chemical, genetic, imaging, and computational
techniques. The currently used genetic tools for manip­
ulating B. burgdorferi are sufficiently mastered for pre­
cise and effective genetic investigations and were
6 M. STRNAD ET AL.
recently reviewed in great detail by Rosa and Jewett
[85]. Targeted genes can be selectively rendered inac­
tive and the resulting mutant phenotype can be exam­
ined for infectivity in the experimental mouse-tick
transmission cycle, thus elucidating the actual functions
of borrelial virulence factors during infection of the
host. Genetic manipulation of B. burgdorferi is typically
achieved via allelic exchange. The transformation pro­
tocol often makes use of electroporation for transfer of
foreign DNA [86]. The selection of a mutant with
either gene disruption or gene complementation is
achieved by using a selectable antibiotic marker.
Suicide vectors are commonly utilized for targeted
gene inactivation, as the mutant is generated by allelic
replacement of the wild-type gene with a disrupted
copy [85,87]. The functional complementation of the
mutant is typically achieved through shuttle and suicide
vectors for trans and cis complementation, respec­
tively [85].
Transposon mutagenesis and signature tagged muta­
genesis (STM) are more recent genetic approaches that
were developed to identify novel bacterial virulence
factors and were already exploited in borrelial research
[88–90]. Both methods represent a technical advance
over other random mutagenesis approaches such as
radiation and chemical mutangenesis [91] as they uti­
lize transposable genetic elements to randomly inacti­
vate multiple genes, with each specific mutation
occurring in an individual clone. A Himar1-based
transposon suicide vector is often used for the delivery
of the gene insertions [88,92]. The principle of STM is
based on molecular barcoding with unique DNA
sequences (signature tags), which allow mutants to be
differentiated from each other. This allows high-
throughput screens of large number of different
mutants within a single animal. By comparing the
signature tags of the input pool of mutants, i.e. those
which were inoculated into an animal, and the recov­
ered pool of mutants, i.e. those which survived in the
animal, the important virulence factors can be
identified.
Gene expression analysis
DNA microarrays have been used to study changes in
the global expression profile of B. burgdorferi grown
under various in vitro and in vivo conditions [93,94].
For mimicking the growth conditions in the mamma­
lian host, a dialysis membrane chamber implanted
into the rat peritonea is considered a top-notch
model as it allows to obtain high number of host-
adapted spirochaetes for subsequent analysis [95,96].
By comparison of expression profiles of spirochaetes
grown under fed-tick, unfed-tick, and mammalian
growth conditions, it was shown that mostly the extra­
cellular lipoproteins are dynamically regulated during
borrelial transitions between the tick vector and the
host, hinting to their important role in borrelial
pathogenesis [93]. The common drawback of the
DNA microarray technology is that only the annotated
coding regions of the genome can be profiled. To meet
the limitations of probe performance in DNA micro­
arrays, RNA sequencing (RNA-Seq) has been used to
explore for instance the antisense transcripts and non­
coding/small RNAs [97,98].
The in vivo expression technology (IVET) is
a genetic tool that allows selection of genes specifically
expressed in vivo in complex environments. In contrast
to DNA microarrays technology and RNA-Seq, IVET
does not require borrelial RNA isolation for the tran­
scriptomic investigation and provides purely qualitative
data with no information about the quantity of studied
gene product. This promoter-trapping technique pro­
vides means for genome-wide identification of the bor­
relial genes that are expressed for instance during
murine infection. Using IVET for the first time in
B. burgdorferi, an RpoS-independent gene bbk46 was
identified and shown to be important for evasion of the
host adaptive immunity [99]. Using an IVET reporter
cassette containing antibiotic resistance aacC1 gene and
borrelial pncA, Casselli and Bankhead [100] identified
a number of constitutively expressed promoters,
in vitro-induced promoters, and in vivo-induced pro­
moters in B. burgdorferi.
Imaging techniques
The rapid development of imaging technologies has
revolutionized our ability to visualize the delicate intri­
cacies of Borrelia-host interactions at very different
scales. Imaging methods and applications encompass
a palette of options from which one might select, span­
ning from whole animal imaging techniques to mod­
alities that are able to reach subatomic resolution
[101,102]. Bioluminescent imaging has enabled one to
follow the infection of Borrelia in an entire model
animal over time by non-invasive means in natural
environment [101]. Another sophisticated technique
that has allowed the investigation of Borrelia pathogen­
esis, including dissemination and invasion of the host,
is intravital microscopy [14]. Intravital imaging allows
real-time characterization of a pathogen in a living host
at specific locations. For intravital imaging, spinning
disk confocal microscopy (SDCM) and two-photon
microscopy (2PM) are often employed [14,103–105].
The spinning disk in the SDCM setup allows for

imaging of very fast dynamic processes in live speci­
mens and reduces phototoxicity. With improved laser
penetration by low energy photons and significantly
reduced phototoxicity, 2 PM is very convenient for
real-time intravital imaging of Borrelia in the mouse
dermis [104,105]. Using SDCM in an intravital setting,
the details of vascular extravasation of B. burgdorferi
were visualized, showing that the bacterium engages in
multi-stage interactions with the endothelial cells,
including tethering and dragging interactions, and sta­
tionary adhesion to the vasculature [14]. Intravital ima­
ging is typically accomplished by using fluorescent
labelling of the pathogen, preferably by expression of
a fluorescent fusion protein such as GFP. To avoid the
potential issues with bulkiness of the GFP, new small
fluorescent probes for investigation of spirochaete mor­
phology and motility are being explored [106].
Light and electron microscopy are usually used to
provide a cellular-level understanding of mechanisms
of Borrelia infection (spirochaete-host cell interac­
tions) [22,107]. To address the structure-functional
contributions of borrelial proteins to bacterial viru­
lence and disease pathogenesis, modern high-
resolution imaging modalities can be utilized. Atomic
force microscopy-based techniques enable the study of
protein–protein interactions at the single-molecule
level, for instance, in uncovering the important inter­
actions that facilitate the spirochaete to efficiently
translocate in the extracellular matrix of a host [13].
Nuclear magnetic resonance-based techniques can go
even deeper with resolution power and determine the
location of the binding site and the dynamic structural
rearrangements that occur upon binding, with amino
acid precision [102,108].
Borrelia-specific versus common virulence
factors
Virulence factors can be classified as pathogen-
specific (no orthologous protein in other bacteria)
and common (one or more orthologous proteins in
different pathogenic bacteria) based on whether the
virulence determinant is present in a single species or
it is found in multiple different organisms.
B. burgdorferi contains a high number of borrelia-
specific virulence factors such as OspC, OspA, VlsE
but also contains several virulence determinants that
can be found in other microorganisms. For instance,
B. burgdorferi utilizes non-toxin adenylate cyclase
cyaB, an enzyme that produces secondary messenger
cyclic adenosine monophosphate (cAMP), to modify
borrelial gene expression and protein production in
order to enhance its virulence in the vertebrate host
VIRULENCE 7
[109]. CyaB ortholog can be found in other patho­
genic bacteria such as Pseudomonas aeruginosa or
Vibrio vulnificus [110,111]. Cyclic dimeric guanosine
monophosphate (c-di-GMP) is an ubiquitous second
messenger found in many prokaryotes such as
Mycobacterium tuberculosis, Vibrio cholerae, or
Salmonella enterica that regulates a wide span of
physiological processes including bacterial protein
secretion, multicellular behaviour, virulence, motility,
and cell development and differentiation [112,113].
Although c-di-GMP has various downstream effec­
tors in bacteria [114], PlzA protein is the only uni­
versal c-di-GMP binding protein in B. burgdorferi.
The regulatory processes of c-di-GMP are mediated
through binding with the PilZ domain, the
C-terminal part of PlzA [115]. In B. burgdorferi, c-di-
GMP regulates the adaptation processes to the tick
and mammalian environment via the interaction with
PlzA [116]. PilZ domains are found in multiple bac­
terial taxa such as actinobacteria, proteobacteria, or
spirochaetes [112].
LuxS is another virulence factor found in Borrelia
[117] as well as many other pathogens such as
Bacillus anthracis [118], Staphylococcus epidermidis
[119], or Haemophilus parasuis [120]. In addition to
the mostly referred genes, such as ospA, ospB, ospC,
crasp(s), erp(s), vls, dbpA/B, flaB, p66, rpo(s), the
group of genes involved in sensing and transduction
of environmental signals deserves more attention. To
differentially synthesize proteins during the infectious
cycle, Borrelia is equipped with regulatory networks
to sense its environmental signals. Quorum sensing is
a mechanism by which many prokaryotes monitor
their surroundings by producing and responding to
signalling molecules known as autoinducers [121].
The genome of B. burgdorferi encodes for a number
of enzymes involved in quorum sensing, such as
MetK, LuxS, and Pfs [15] that enables the spirochaete
to synthesize an autoinducer type 2 (AI-2) mediating
in this way quorum sensing that can function in both
the mammalian host and the tick vector. Therefore, it
is possible to regulate the differential expression of
Borrelia genes in different environments [117,122].
The bacterium has to interact with different tissues
during its life cycle, derive nutrition mostly from
warm-blooded mammalian hosts as well as from the
tick vector, and avoid clearance by host and vector
immune responses. By exploiting quorum sensing,
a whole borrelial population can synchronize produc­
tion of molecules required for infection and survival.
There are additional advantages in having the whole
population of Borrelia to coordinate particular func­
tions. For example, simultaneous transmission of
8 M. STRNAD ET AL.
large numbers of spirochaetes from a tick to a warm-
blooded host might improve the odds that at least
some spirochaetes survive the host’s immune
responses and establish disseminated infection [122].
The quorum sensing mediated by LuxS is considered
as the main system used for cell-to-cell communica­
tion. Although an earlier study suggested that luxS is
redundant for B. burgdorferi tick colonization, trans­
mission to a mammal, or establishment of infection
[123], a more recent study demonstrated that the
luxS mutant was markedly less infectious than the
wild type and that luxS gives a fundamental advan­
tage to the spirochaete during vertebrate infec­
tion [124].
Tick transmission factors of B. burgdorferi
B. burgdorferi naturally persists in an enzootic cycle
that primarily involves Ixodes ticks and mammals.
Adult and nymphal ticks are the most important stages
in the transmission of the bacterium. B. burgdorferi is
not propagated from the adult female tick to their off­
spring, or it happens only rarely [125,126]. The transo­
varial transfer of B. burgdorferi has been occasionally
documented in the literature, but these records may
probably stem from confusion between LD species
and relapsing fever (RF) Borrelia [44]. Nymphs are
the most harmful tick stage for humans, because the
risk of an infection in the majority of cases comes from
the bite of this developmental stage of the tick vector.
Tick larvae are very rarely infected and the adult ticks
are large enough to be noticed early and removed
appropriately. Ixodes ticks take only one bloodmeal
per life stage (larva, nymph, adult) after hatching
from eggs. At least seven tick-borne zoonoses are trans­
mitted by members of the Ixodes complex in the north­
ern hemisphere, and include diseases such as LD,
ehrlichiosis, babesiosis, anaplasmosis, tick-borne ence­
phalitis, Rocky Mountain spotted fever, Powassan ence­
phalitis disease, etc.
B. burgdorferi species are host-propagated pathogens
that shuttle between tick vectors and vertebrate hosts.
B. burgdorferi in the unfed ticks is predominantly
located in the midgut lumen, where the spirochaetes
have to avoid the tick innate immune responses, man­
ifested especially in the form of antimicrobial peptides
(AMPs) [127,128]. Further down the road primarily in
the tick haemocoel, the bacteria have to avoid the
haemocyte-mediated processes (phagocytosis, encapsu­
lation and nodulation) as well as damage mediated by
AMPs, lysozyme, complement-like factors, and reactive
oxygen and nitrogen species [129–132]. Borrelia multi­
ply in the midgut to many thousands of cells [133].
Spirochaetes reside in the midgut during tick starvation
phases as well as tick moults. Upon blood feeding,
Borrelia escape from the gut to navigate toward the
salivary glands and onwards into a new host [134].
An alternative transmission pathway, in which the
tick salivary glands are considered unessential for suc­
cessful transmission, might be exploited by some of the
major European species. In the study by Pospisilova
et al. [135], the authors show that B. afzelii did not
reach the tick salivary glands at any stage of feeding and
that tick saliva is redundant for infectivity of this parti­
cular species. The possible differences in the transmis­
sion mechanisms in B. burgdorferi genospecies were
further strengthened by a study demonstrating no pre­
sence of B. afzelii in the salivary glands of unfed ticks
[136]. On the contrary, in the same study, other
European species B. lusitaniae, B. spielmanii and
B. garinii were detected in both midgut and salivary
glands suggesting that the migration of these spiro­
chaetes between midgut and salivary glands might not
be activated by the bloodmeal [136].
Protein interactions between LD Borrelia and ticks
Borrelia adapts to the transition between the tick vector
and vertebrate host by preferential gene expression. To
optimize colonization and persistence in the tick mid­
gut, Borrelia has evolved complex strategies, how to
exploit the tick midgut milieu (Figure 2). The successful
tick colonization depends on and is governed by com­
plex molecular interplay of spirochaetes with the gut
epithelium receptors. The temporal pattern of ospA
(outer surface protein A) gene expression together
with a number of functional studies [137,138] indicate
that the abundantly expressed OspA plays a vital role in
spirochaete persistence in the vector. OspA-deficient
spirochaetes are unable to colonize the tick since they
fail to bind to the tick gut [137]. The impaired attach­
ment of B. burgdorferi to the tick gut effectively
obstructs all subsequent downstream transmission
events. Tick receptor for OspA (TROSPA), located on
the luminal surface of gut epithelium, interacts with the
spirochaete OspA lipoprotein and facilitates the tick
colonization. TROSPA interacts specifically with OspA
and was shown to be upregulated when spirochaetes
are ingested [139]. RNA interference of TROSPA pre­
vents efficient colonization of the tick and reduces
borrelia transmission to the host [139]. Similarly, bor­
relial outer surface lipoprotein BBE31 binds to the
vector molecule TRE31 located in the tick gut [140].
Borrelia infection induces TRE31 expression and silen­
cing of TRE31 results in a significant decrease of the
 VIRULENCE 9

Figure 2. Molecular interactions at the tick midgut luminal interface involved in transmission and acquisition/persistence
of B. burgdorferi in ticks. When an infected tick starts feeding on the host, spirochaetes present in the tick midgut multiply,
traverse the gut barrier to reach the haemocoel (1), navigate toward the tick salivary glands (2), and ultimately infect the host (3). (a)
upon blood intake, the spirochete changes its protein coat to produce molecules such as OspC and BBE31, which enable the
pathogen to escape from gut and invade the tick salivary glands. Tick epithelial cells express molecules Ixofin3D and TRE31, which
facilitate the process of gut penetration. (b) Borrelia cells ingested in the bloodmeal bind to the tick gut and stay there until a next
tick feeding. B. burgdorferi expresses several-exposed lipoproteins such as OspA, OspB and BptA, which protect spirochaetes from
bactericidal components present in the host blood and enable them to colonize the tick gut. OspA interacts specifically with the tick
receptor TROSPA, thereby enabling efficient colonization of the vector.

borrelial loads in the tick haemolymph and salivary
glands [140].
Outer surface protein B (OspB) is another surface-
exposed lipoprotein essential for adherence, survival, and
persistent infection of the tick midgut [137]. OspB-
deficient microbes are capable of migrating to the feeding
ticks together with the bloodmeal but their ability to
adhere to the tick gut and survive within the arthropod
vector is significantly diminished [141]. While the binding
partners for BBE31 and OspA are known, the mechanism
how OspB exerts its function is still unclear. Studies have
also identified borrelial proteins required for persistence
inside the tick, which are critical for survival during long
off-host periods between bloodmeals. BptA (bbe16),
a lp25-encoded surface-exposed lipoprotein, was found
to be essential for this persistence of Borrelia in ticks but
the molecular mechanisms by which BptA promotes the
persistence remains to be elucidated [142]. DksA is
involved in the transcriptional response to nutrient lim­
itation and supports the transcription and translation of
genes contributing to B. burgdorferi infectivity such as of
virulence-associated lipoprotein OspC [143,144]. Another
potentially important factor in the tick vector stage of
B. burgdorferi is an outer membrane protein BBA52.
Tick acquisition of mutants lacking functional bba52
was shown to be reduced [145]. Using a yeast surface
display library, Ixofin3D and I. scapularis dystroglycan-
like protein (ISDLP) were identified as other candidate
tick gut binding partners of B. burgdorferi [146,147]. The
expression of both Ixofin3D and ISDLP is increased in
Borrelia-infected tick gut during bloodmeal ingestion.
Ixofin3D was suggested to facilitate borrelial congregation
to the tick gut that ultimately helps Borrelia to navigate
and escape from the gut [146].
The migration of the spirochaetes toward the tick
salivary glands is accompanied by downregulation of
tick gut specific borrelial proteins such as OspA and
concomitant upregulation of proteins such as OspC
that helps Borrelia to reach the tick salivary glands
[148] and infect and colonize the host [149]. The
I. scapularis salivary protein Salp15 binds specifically
to OspC, thereby enabling the bacterium to avoid clear­
ance mediated by host anitbodies [150]. The same
function was demonstrated for the homologue of
Salp15 from I. ricinus Iric1. In addition, Iric2 and
Iric3 from I. ricinus are capable of OspC binding with
high affinity [151]. Although Borrelia evolved its own
mechanisms to combat immune defence of the host,
the transmission via tick saliva gives spirochaetes cer­
tain adaptive benefits. Salivary gland products also
10 M. STRNAD ET AL.

provide the first line of defence for Borrelia against the
host innate immune system. Tick salivary proteins play
a role in the inhibition of the host immune and homo­
eostatic responses that are raised against them. This
includes the suppression of activation of phagocytic
dendritic cells and macrophages, including the inhibi­
tion of natural killer cells that are responsible for che­
mokine and cytokine production, and disabling of
granulocyte recruitment to the tick bite site [152].
When an uninfected tick feeds on an infected host,
the spirochaetes are directed toward the tick bite site by
specific environmental and chemotactic stimuli. Tick
protein Salp12 is released into the skin tissue as
a component of the tick saliva, where it is sensed by
resident Borrelia [153]. This recognition contributes to
attraction and relocation of the spirochaetes into the
tick feeding pit. Salp12 is expressed and present in the
salivary glands as well as midgut. It was shown that
during tick feeding, the expression of Salp12 in midgut
of the tick was significantly higher than Salp12 expres­
sion in the salivary glands at various investigated time
points, possibly creating a concentration gradient
which directs the bacteria toward the tick midgut.
Passive immunization and disruption of Salp12 in the
tick salivary glands were shown to decrease acquisition
of the spirochaetes by the vector [153]. Using similar
experimental procedure, it was shown that the tick
antioxidant protein Salp25D also play a fundamental
role in the acquisition of B. burgdorferi [154] (Figure 3).
Pathogenic mechanisms at the host–pathogen
interface
Genome sequencing revealed that B. burgdorferi lacks
virulence factors common to many microbial and bac­
terial pathogens, such as specialized secretion systems,
toxins, or extracellular proteases with the exception of
HtrA [15,155]. As a notorious extracellular pathogen,
the spirochaete most likely does not damage the host
cells by penetrating them or multiplying in them.
Neither does Borrelia produce toxins that would
impose direct damage to the host cells membranes,
kill the host immune cells, or inhibit protein synthesis.
The outer membrane of B. burgdorferi is structurally
distinctive among bacterial cell envelopes and it is
a common target of host immunity [156]. Unlike typi­
cal diderms, B. burgdorferi does not produce the endo­
toxin lipopolysaccharides but secrete extensive surface
lipoproteins at the host–pathogen interface [15]. These
outer membrane lipoproteins play an essential in the
borrelial virulence by suppressing immune responses of
the host and by mediating interactions with the targets
tissues [157]. Despite the activation of both the innate

Figure 3. Molecular factors involved in transmission and acquisition of B. burgdorferi by ticks. numerous molecules produced
by both a tick vector and Borrelia are required for successful early-stage infection of the host and tick acquisition of B. burgdorferi.
Many tick proteins (Salp15, evasins, TSLPI, tHRF, etc.) and borrelial proteins (e.g. OspC, DbpA, DbpB, BBK32) are known to be
necessary for transmission. Chemotactic tick protein Salp12 and antioxidant Salp25D, together with borrelial BBA52, were shown to
be critical for host-to-tick acquisition of Borrelia.

and adaptive immune systems, Borrelia is often able to
survive in the hostile environment to cause infection,
which then can become persistent. LD has not been
associated with severe inflammatory over-reaction such
as cytokine storms known from cytomegalovirus,
Epstein-Barr virus, streptococcus, and many other
microbes. Borrelia is better known for its immune-
evasion mechanisms, expression of immunomodulatory
surface proteins, downregulation of immunogenic pro­
teins, and antigenic variation, which help to elude
immune responses of the host [158].
Evasion of host innate immunity
The mammalian host immune response to infection with
LD spirochaetes begins with effector components of the
innate immune system. Nitric oxide (NO), generated in
multiple biologic tissues and cells by specific nitric oxide
synthases, is a known player in innate immunity
responses against bacterial pathogens [159]. Although
NO is a powerful antimicrobial agent and its production
is elevated during borrelial infection [160], B. burgdorferi
is not significantly affected by NO during mouse infection
[161]. In contrary to elevated levels of NO during infec­
tion, the spirochaete is able to suppress the production of
additional common toxic molecules such as reactive oxy­
gen species (ROS) via the mTOR pathway in order to
avoid killing [162,163]. B. burgdorferi produces multiple
proteins which were implicated to ameliorate the effects
of ROS exposure, including BmtA, SodA [164,165].
Recently, a gene cluster bb_0554-bb_0556 (xdhACB)
encoding a xanthine dehygrogenase and transmembrane
proteins BB0017, BB0164, and BB0202 were shown to
play a significant role in ROS resistance [166,167]. In
addition, the capacity of lysozyme, an antimicrobial
enzyme and a major constituent of innate immunity, to
kill B. burgdorferi is quite limited [168].
Complement evasion by LD Borrelia
The complement system is considered a functional
bridge between innate and adaptive immune responses
and is known to be activated via three pathways – the
classical pathway, lectin pathway, and alternative path­
way. Upon activation by the pathogen, the comple­
ment system opsonizes bacterial surfaces and induce
a series of proinflammatory responses that ultimately
lead to bacteriolysis [169]. Vertebrate host range for
Borrelia is determined by spirochaetes sensitivity to
complement of particular animal species; the ability
to bind factor H (FH) and factor-H-like protein 1
VIRULENCE 11
(FHL-1) appears to depend on the Borrelia genotype
[170, 171]. B. burgdorferi species vary in their suscept­
ibility to complement. B. afzelii and B. burgdorferi ss
are strongly serum (complement) resistant while
B. garinii is more sensitive to most mammalian
serum [172,173]. The spirochaete has evolved mechan­
isms to evade destruction from the complement sys­
tem by producing a set of outer surface lipoproteins
that interact with host complement molecules and
manipulate the native activities of the defence system
[174]. Borrelia can either bind directly to complement
components or the spirochaete can interact with the
host-derived regulators of the complement system in
order to attenuate complement activation.
Direct interference is mediated by a spectrum of
outer surface lipoproteins such as BBK32, OspC, and
BBA70. BBK32 specifically inhibits the classical path­
way through high-affinity binding of the C1r subcom­
ponent of C1, entrapping C1 in its zymogen form
[175]. OspC was shown to bind C4b in both
B. garinii and B. burgdorferi ss, blocking formation
of C3 convertase and thereby restricting killing via
the classical and lectin pathways [176]. OspC, together
with other surface molecules such as OspA, CspA,
Erp-family proteins, and BBA70, are capable of bind­
ing plasminogen, which is a known complement inhi­
bitor [177–182]. Indirect strategies include the
production of complement-regulator acquiring surface
proteins (CRASPs) which encompass CspA, CspZ, and
OspE paralogs to inhibit the complement responses.
These molecules attenuate complement activation on
the borrelial surface by binding to complement regu­
lator FH or FH-related protein 1 (FHR-1) [183,184].
CspA (CRASP-1) attenuates complement on two cen­
tral activation levels, C3b generation and assembly of
the terminal complement membrane attack complex
[178,185]. European species B. bavariensis produces
surface proteins BGA66 and BGA71 similar to CspA.
Both molecules bind complement components C7, C8
and C9, and prevent assembly of the terminal comple­
ment complex [186]. CspZ (or CRASP-2), a second
FH/FHL-1-binding protein, also downregulates the
formation of C3 and C5 convertases on the spiro­
chaete surface. A third type of FH-binding proteins
are OspE paralogs ErpP/ErpC/ErpA (also known as
CRASP-3, CRASP-4 and CRASP-5). The outer surface
protein OspE binds to Factor H as well as various
members of complement factor H-related (CFHR)
proteins [187]. ErpA and ErpP, the OspE-related pro­
teins, bind the complement factor H-related proteins
CFHR1, CFHR2, and CFHR5, while ErpC binds only
CFHR1 and CFHR2 [174,188].
12 M. STRNAD ET AL.
Antigenic diversity and variation in LD Borrelia
After activation of adaptive immunity, antigen present­
ing cells utilize bacterial peptides and present them to
B and T cells. T cells release cytokines and stimulate
macrophage activation while B cells produce specific
antibodies that bind borrelial outer membrane epitopes.
The escape from adaptive immune responses is
mediated primarly by the VlsE antigenic variation sys­
tem [189]. Antigenic diversity can fundamentally
extend the time a pathogen maintains an infection
within a host and avoids eradication by the host
immune system. The initial set of surface-presented
antigenic molecules stimulate an immune response
against the dominant antigens. If the pathogen changes
the composition of its antigenic coat to new variants,
the microbe escapes the immune response and con­
tinues infection until the host generates a new response
against the latest variants [190]. LD Borrelia species are
well known for differential gene expression and altera­
tions in antigenic structure during their life cycle.
A prototypical example is downregulation of OspA
lipoprotein during transmission while the expression
of OspC is upregulated. Subsequently, OspC, which is
critical for early stage of mammalian infection is down­
regulated and Borrelia produces genetic variants at the
vls (variable membrane protein-like sequences) locus to
enable long-term infection of the host [149,191].
OspC is a dominant highly polymorphic antigen of
LD bacteria, that is heavily targeted by the host
immune system. Borrelia ospC is more diverse than
any other studied gene to date; it is known to be able
to establish the secondary immune response or
immune memory in hosts [192], association between
ospC genotypes and invasiveness in human patients and
infected animals have been reported in multiple studies
[193–195]. The gene encoding for OspC is mapped to
cp26, a 26-kb circular plasmid that is a stable compo­
nent of the segmented B. burgdorferi genome [196].
OspC is transiently but absolutely required during the
early stage of infection and neither vlsE nor ospA can
compensate for the absence of OspC and restore infec­
tivity to an ospC mutant [191]. OspC is a protective
antigen [197], due to its high sequence variability, pro­
tection is generally strain specific [198]. Multiple alleles
are circulating among reservoir hosts and tick vectors
[192]. OspC alleles A, B, and L were detected in Europe
and North America in vectors and hosts including
humans. Six ospC alleles are prevalent in Europe and
four of them were detected in human samples. Ten
ospC alleles were identified in the western United
States. Four ospC alleles were abundant in the south­
eastern United States [199]. OspC has also been
suggested to play a role in host selectivity [192], plas­
minogen binding in hosts [200], defining Borrelia inva­
siveness in rodents [200], dissemination during
mammalian infection [201], salivary gland migration
in the tick [148], evasion of innate immunity [202],
binding a tick salivary protein that inhibits complement
[150,203], conferring bloodstream survival [204], and
contributing to Borrelia strain-specific differences in
tissue tropism [205]. OspC suppresses the classical
and lectin complement pathways and competes with
complement protein C2 for C4b binding [176]. OspC
is important for Borrelia in macrophage phagocytosis,
reducing the uptake of the bacterium by human and
murine macrophages [206].
Decorin binding proteins A and B (DbpA/B), that
are important for motility, bind primarily to the con­
nective tissue proteoglycan decorin and facilitate host
colonization by the spirochaete [13]. Although not
absolutely essential for infection, the important role of
these adhesins for the overall virulence of B. burgdorferi
was demonstrated in a number of studies [67,207,208].
DbpA and DbpB deletion mutants display marked
attenuation in mammals, but particularly early during
the course of infection [209,210]. Recovery of mutant
bacteria from tissues distant to the inoculation site is
diminished as well [208,210]. Besides binding to speci­
fic glycosaminoglycan (GAG) ligands in order to
anchor the spirochaete in the destination niches,
DbpA and DbpB help in host colonization by enhan­
cing borrelial translational motility in the low shear
stress environments [13]. DbpA sequence variability
among different species of B. burgdorferi is high, result­
ing in pronounced differences in their GAG affinities
[6,211,212]. It was shown that allelic variations of
B. burgdorferi DbpA affect tissue tropism and disease
manifestation of different LD genospecies [213].
Besides temporal changes in the antigenic coat as
a result of tick vector-host circulation and high
sequence variability between borrelial genospecies,
Borrelia are capable of in situ antigenic variation to
avoid detection from host adaptive immune response.
Recombinational switching of a gene locus, or the pro­
cess of alteration of the pathogen’s surface antigens in
order to avoid detection from host adaptive immune
response, is one of the most effective attributes of
immune evasion by human pathogens. Concomitant
with the development of the host acquired immune
response and OspC downregulation, robust synthesis
of VlsE is initiated. In B. burgdorferi strain B31, the vlsE
gene is encoded on the 28-kb linear plasmid, lp28–1,
less than 100 bp from the right telomere. VlsE is dis­
similar to OspC, OspA, or Dbps, as it is abundantly
present on the spirochaetes surface during persistent

infection [214, 215]. The silent cassettes at the vls locus
vary during the course of infection as well as between
species and within species [216–218]. The VlsE anti­
genic variation system contains the vlsE expression site
and 15 silent cassettes, thus providing multiple possible
recombination events for producing a variety of VlsE
epitopes through unidirectional, segmental gene con­
version [189,219]. The vlsE cassette region can have
over 90% nucleotide sequence identity with the vls
cassettes. European species B. garinii and B. afzelii
were shown to have less vls silent cassettes than
B. burgdorferi B31. B. garinii strain Ip90 carries 11 vls
silent cassettes and B. afzelii strain ACAI encodes 14 vls
silent cassettes [220]. The cassettes can recombine in
seemingly random manner (although there is some
preference for certain silent cassettes), ranging from
single nucleotide substitutions to almost full replace­
ment of the vlsE site, with switch rate of approximately
0.033 per cell generation [221]. The RuvAB helicase
complex apparently facilitates the vlsE recombination
[222]. There are two types of sequence changes; tem­
plated and non-templated. The templated changes are
those which correspond to sequences in the vls silent
cassettes, whereas non-templated changes are often
represented by point mutations. The degree of recom­
bination events and switch length of nucleotides are
influenced by the immune pressure. The spirochaetes
in wild-type immunocompetent mice display longer
stretches of recombination and average switching rate
than in immunodeficient mice [221]. The antigenic
variability of VlsE facilitates another novel escape
mechanism from innate immunity described as epitope
shielding [223]. The VlsE protein can effectively block
the binding of antibodies which target immunogenic
borrelial proteins such as arthritis-related protein Arp.
Motility-driven pathogenesis
B. burgdorferi possesses surface adhesins that are well
adapted to aid in its dissemination and colonization
strategies (Figure 4). During its infectious cycle,
B. burgdorferi needs to cope with high blood flow-
mediated shear stress in the host vasculature. BBK32
and P66 borrelial proteins are key players mediating
the stabilizing interactions and the adhesion to the
cells lining the vascular lumen [103,224,225].
Recently, it was suggested that VlsE also promotes
transient binding to the vasculature under flow via
binding to dermatan sulphate and that a complex
temporal choreography of P66, DbpA/B and OspC
is required for the escape process from postcapillary
venules [226,227]. Blood circulation is not the only
VIRULENCE 13
mechanical stress that B. burgdorferi needs to over­
come in order to infect the target tissues. During the
vascular extravasation and after reaching the extra­
cellular space, the bacterium faces a milieu of much
lower shear stress than is experienced in the vascu­
lature. Here, however, the pathogen needs to able to
penetrate through dense and highly viscous struc­
tures with pore sizes much smaller than the diameter
of borrelial cell. This is allowed by forming transi­
ently stable interactions that enable pushing against
the surrounding structures [104]. It was shown that
DbpA and DbpB are critical borrelial surface mole­
cules that allow for efficient dissemination of Borrelia
in low shear stress and dense environments such as
the extracellular matrix [13].
In contrast to pathogens that circulate only pas­
sively in the vasculature of a host, B. burgdorferi can
actively influence its dissemination in the host in
order to infect various organs and tissues. Multiple
studies have shown that motility of B. burgdorferi is
a critical virulence determinant and absolutely vital
for pathogenesis throughout the enzootic cycle and
the loss of motility leads to a non-infectious or
attenuated phenotype [228–230]. The bacterium is
able to adopt distinct motility states and transition
between them as the pathogen colonizes the host
[104]. The unique flat wave rotational movement of
Borrelia is generated by 7–11 flagella located beneath
the outer membrane in the periplasmic space. Since
borrelial flagella do not extend outwards from the
bacterial surface, B. burgdorferi is able to migrate
with ease in highly viscous media, in which most
other bacteria are significantly decelerated [231].
Therefore, the spirochaetes like to hide in the extra­
cellular structures, making them less subject to cir­
culating leukocytes. Borrelia with a mutation in the
major flagellin gene flaB that did not synthesize
flagella, was shown to be non-motile and rod-
shaped [232]. As such, the flagella are arguably one
of, if not the most critical virulence factors in the LD
infection. Additionally, B. burgdorferi possesses an
unusual peptidoglycan cell wall which significantly
contributes to structural and morphological integrity
of the bacterium and allows the spirochaete to with­
stand the high torque generated by the periplasmic
flagella during borrelial translocation [233]. BpiP
protein aids in sustaining the cellular integrity cell
by binding the peptidoglycan wall and it influences
the borrelial virulence in the host [234]. Notably,
peptidoglycan was shown to be a persistent immuno­
gen contributing to inflammation during acute infec­
tion and post-Lyme arthritis [46].
14 M. STRNAD ET AL.

Figure 4. Molecular factors directly involved in active migration and dissemination of Borrelia in humans. (a) B. burgdorferi
has to overcome high shear forces generated by blood flow in order to adhere to vascular surfaces for extravasation. The interactions
of borrelial protein BBK32 with vascular fibronectin allow active migration in high shear stress environment and borrelial proteins
P66 and VlsE aid in vascular transmigration. (b) in low shear stress niches such as extracellular matrix of target skin tissue, borrelial
surface-exposed proteins DbpA and DbpB facilitate the translational motion by interactions with matrix molecules such as decorin.

Localization into specific niches in extracellular
matrix
In contrast to RF Borrelia, LD Borrelia are rapidly
cleared from the blood circulation and cannot multiply
there [235]. When the adaptive immune responses of
the host are fully activated, B. burgdorferi disappears
from the bloodstream and disseminates to the organs,
collagenous tissues, joints, and synovial fluid of the host
[236]. The spirochaetes need to survive until they are
transmitted back to competent ticks. At this time the
successful persistence of the spirochaete within the host
depends on evading the host’s immune system, rather
than exploiting the host tissues for reproduction or
growth [237]. The extracellular matrix provides an
immune-privileged milieu for the pathogen [238]. The
generally accepted theory is, after vascular
extravasation, the spirochaetes colonize the ECM of
multiple tissues and organs, which contain molecules
that can be bound by Borrelia. These are commonly
proteoglycans, biomolecules composed of a core pro­
tein together with long and negatively charged polysac­
charide chains called glycosaminoglycans (GAGs). For
example in joints, ECM of the articular cartilage con­
tains a variety of GAGs such as chondroitin sulphates,
keratan sulphate, dermatan sulphate as well as multiple
collagen types [239,240]. LD Borrelia bind to different
proteoglycans, which promotes tissue colonization and
bacterial attachment to the cells [211,213,241,242]. The
binding interactions with the host ECM components
are mediated by outer surface molecules of Borrelia
(Table 1). B. burgdorferi displays a dizzying diversity
of abundant surface lipoproteins. At least 120 proteins
 VIRULENCE 15

Table 1. Surface proteins of B. burgdorferi which are known to mediate attachment to the
extracellular matrix (ECM) components of the vertebrate host.
ECM component Protein Reference
Collagen CspA, BBA33 [243, 244]
Decorin DbpA, DbpB [245, 246]
Fibronectin BBK32, RevA, RevB, BB0347, OspC [205, 242, 247–249]
Integrins BBK32, P66, BB0172, BBB07 [250–253]
Laminin BmpA, ErpX, BB0406, CspA [,256]
Glycosaminoglycans DbpA, DbpB, BBK32, Bgp, OspC, Lmp1, VlsE [205, 226, 257–260]

that possess a lipid moiety are encoded in the borrelial
genome [261], which represent nearly 8% of all borre­
lial open reading frames [262]. Out of 125 lipoproteins,
86 of these were shown to be secreted to the outer
surface [261].
DbpA and DbpB exhibit binding activity toward
various components of the ECM, including the proteins
decorin, biglycan and various GAG molecules such as
dermatan sulphate or heparin [108,211,241,245,263].
Fibronectin-binding activity was demonstrated for at
least four outer membrane proteins, BBK32, RevA,
RevB, and BB0347 [242,247–249]. BBK32 mutants dis­
play a significant defect in infectivity in the mouse
infection studies and highlight the fundamental role
of the protein in B. burgdorferi pathogenesis
[248,264,265]. Similarly to BBK32, the lack of RevA
significantly affects the pathogenesis in the mouse
model of LD, although revA is not absolutely essential
for infection [266]. The role of RevB and BB0347 in
pathogenesis remains to be elucidated. BmpA, ErpX
and BB0406 were shown to interact with laminin
[254–256]. B. burgdorferi produces also collagen-
binding proteins such as CspA and BBA33 [243,244]
and it is able to bind several types of host integrins such
as α3β1 integrin using P66, BB0172, and BBB07 pro­
teins [250–253].
Alternative pathogenic mechanisms
With the rise of modern research technologies and
inflow of new studies, novel virulence mechanisms
that might be exploited by the LD pathogen have
come to light. These novel virulence activities are still
either not fully accepted by the expert borrelial com­
munity or have not been thoroughly investigated yet.
Confirmation of any of the purported virulence
mechanisms (Figure 5) would significantly expedite
our comprehension of B. burgdorferi infection, provid­
ing valuable insights to leverage in the creation of novel
vaccines and other countermeasures. The pattern of the
disease can be resolved into several discrete stages –
host invasion, dissemination, colonization, and
persistence. Interestingly, the discussed novel virulence
mechanisms bear almost exclusively on the persistent
stage of the disease.
Structural variants of B. burgdorferi
Whereas no significant debate surrounds the issue of
B. burgdorferi dissemination pathways within the host,
the strategy for persistence inside the host is a different
story. Under optimal conditions, B. burgdorferi has the
typical flat wave morphology. The bacterium adopts the
spiral form during multiple phases of its enzootic
cycle – migration from the feeding tick midgut to the
haemocoel and salivary glands, deposition in the skin,
haematogenous dissemination, adhesion to and trans­
migration through the endothelium, and establishment
of infection in distal niches, as it was evidenced by live
and intravital imaging [12,14,105,267].
In the face of the hostile microenvironment conditions,
the situation around the morphology of Borrelia becomes
less clear. It was shown in vitro that B. burgdorferi can alter
its cell shape into a different pleomorphic form under
physiological stress, immune pressure, or antibiotic treat­
ment [17,19,268–271], even inside tick midgut [272]. The
morphologic variants, usually spherical and non-motile
forms, of the B. burgdorferi are known under various
terms in the literature. The terms such as L-forms, cystic
forms, spheroplasts, or round bodies are commonly used. It
was shown that the round bodies retain their flagella in the
periplasmic space, have an intact and flexible cell envelope,
and are able to dynamically transition back into the motile
spirochaete under favourable conditions in vitro [17,271].
In addition, it was determined that the round bodies display
lower metabolic activity and present different protein pro­
files and antigenicity than the spiral form [17,268].
The morphologic variants are often suggested to be
associated with chronic or persistent LD and PTLDS, as
they may enhance survival in hostile environmental
conditions [18]. Chronic LD describes the cluster of
symptoms that start after getting LD, persist despite
antibiotic therapy and that result into long-term seque­
lae of infection. Although a systematic review of
B. burgdorferi pleomorphs does not reveal a clear role
16 M. STRNAD ET AL.

Figure 5. Potential virulence mechanisms of B. burgdorferi. (a) B. burgdorferi can change its morphology as a response to
environmental stress. Pleomorphic forms of B. burgdorferi such as biofilm-like structures (or aggregates) and round bodies could
possibly help the spirochaetes to overcome prolonged stress conditions exerted for instance by antibiotics. (b) Although
B. burgdorferi is considered extracellular parasite, the spirochete is occasionally found inside the nonphagocytic cells. The
intracellular niche might help them to hide from immune responses. (c) outer membrane vesicles produced by the bacterium
may modulate host immune responses. (d) structural transformation in cell shape and actin cytoskeleton of human cells were shown
upon contact with Borrelia. (e) B. burgdorferi relies on uptake of essential nutrients such as amino acids, fatty acids and nucleosides
from its host environments for survival and infection. Nutritional virulence might constitute important virulence factors for B.
burgdorferi.

in chronic LD [273], it was demonstrated that LD
patients might have more intense responses to borrelial
circular forms in comparison to spiral forms of
Borrelia. These results suggest that round bodies
might play a certain role in LD pathogenesis [268].
Using an ex-vivo murine skin model, it was observed
that Borrelia can also form biofilm-like colonies made
by spiral-shaped bacteria [274]. Biofilm represents an
alternative lifestyle in which the microbes grow as
structrured aggregates and adopt a multicellular beha­
viour that facilitates their survival under unfavourable
conditions. The bacteria are typically held together by
self-produced polymer matrixes. The presence of algi­
nate-like polysaccharide was revealed in a study that
explored the Borrelia aggregates in vitro [275].
Taken together, it is evident that the spiral, highly
motile form is absolutely required for borrelial dis­
semination and colonization of the tick vector and
the vertebrate host. We know that the motility states
are dynamic as the spirochaetes were observed to
transition between them in the dermis of the host
[104] and the midgut of the tick [12]. It is also
apparent that B. burgdorferi is exposed to many hos­
tile environments and antimicrobial molecules dur­
ing the pathogenic cycle and that the importance of
morphologic variants for survival in hostile condi­
tions should be considered in future research.
Further studies need to be done to assess the poten­
tial contribution of morphologic and motility var­
iants to virulence of B. burgdorferi.
 VIRULENCE 17

Intracellular niche metabolic pathways needed to produce its own nutri­

ents. Recently, it has been suggested that Borrelia is
The ability of B. burgdorferi to maintain themselves for
able to hijack the molecular machinery of host cells
extended periods of time in the vertebrate hosts is
to gain survival benefits [162,282], in a similar man­
critical for continuation of their enzootic cycle. The
ner as was evidenced in many other human patho­
spirochaete can persist in multiple tissue sites despite
gens [283–285]. Metabolism generates energy as well
strong immune pressure. Since B. burgdorferi is known
as fundamental building blocks for every vital aspect
to be an extracellular pathogen, the ECM is considered
of cell biology, including the formation of the cytos­
to be the ultimate protective niche for the spirochaete,
keleton and extracellular matrix. Since many pro­
where the organism is sequestered from the immune
ducts of the cellular machinery are structural
responses [238]. Nonetheless, one of the questions that
components of the cell and at the same time LD is
is still a matter of debate, is whether B. burgdorferi is
characterized by tissue transformations of the host, it
truly an obligate extracellular pathogen, or if the spir­
might be tempting to hypothesize that Borrelia
ochaete is capable of living, and possibly reproducing,
directly affects the structural and mechanical proper­
inside the host cells, when the conditions outside the
ties of the host cells. Significant upregulation of
cells are not favourable (for instance due to presence of
genes coding for decorin or collagen type I was
strong immunity or antibiotics). A number of studies
demonstrated in human fibroblasts upon cultivation
reported the intracellular localization of the bacteria
with B. burdorferi species [278]. Induced alterations
inside non-phagocytic cells, especially fibroblasts
in cell shape and actin cytoskeleton of human cells
[276–278], but also in synovial cells [279], endothelial
were also confirmed upon contact with Borrelia
cells [280], or glial and neuronal cells [281]. On the
[286]. The structural rearrangements of the host
other hand, a recent study was not able to confirm the
cells will likely be highly beneficial for the pathogen.
intracellular niche in the non-phagocytic mammalian
For instance, the skeletal changes can provide a way
cells using advanced correlative imaging approach, but
for Borrelia to escape phagocytosis. Specifically, it
they proved the propensity of the spirochaete for extra­
was shown that uptake of Borrelia by macrophages
cellular surface structures [107]. The stimuli that drives
is not a simple, one-directional process but rather
the pathogen to internalize into the non-phagocytic
a lengthy battle, where the back-and-forth movement
cells has yet to be determined.
of the spirochaete might create “tunnels” inside the
Studies on B. burgdorferi infected murine fibroblasts
macrophage [22]. To what extent is the pathogenesis
has also suggested cyst-like Borrelia located inside these
caused by structural changes in the host organism is
cells to possibly aid in host immune evasion by har­
now a question worth pursuing as mechanical prop­
bouring the bacterium in an inactive state. When the
erties of the host cells might also be the critical
friendly environment is restored, the spirochaetes can
determinant of the pathogen virulence [287].
revert back to a motile spirochaetal form [270,271,276].
Based on the intimate interactions that Borrelia
Notably, coiling phagocytosis, the preferential phago­
form with the host cells, one can assume that the
cytic mechanism for B. burgdoferi, has been demon­
physical contact of Borrelia with the cell (probably
strated to occur less often with non-motile forms than
with some of the outer surface virulent molecules of
motile spirochaetes [268], suggesting that different
Borrelia) triggers the modulatory effects on cellular
receptors are present in the bacterial membrane of
metabolism, which ultimately results in changes of
spiral and non-spiral Borrelia. Moreover, intracellular
cellular architecture and nanomechanical properties
spirochaetes were observed in a variety of shapes, while
of the cell. In the literature, there are numerous studies
simultaneously avoiding lysosomal colocalization dur­
showing that cells affected by a disease exhibit differ­
ing the coculture [19]. The differences in antigenic
ent nanomechanical properties [288] and that cell
expression between borrelial pleomorphs plausibly
elasticity is a critical determinant of proper cell func­
explain the ability of the bacterium to adapt to different
tion [289]. Specifically in LD Borrelia, exposure of
milieus and survive in highly adverse environ­
endothelial cells to spirochaetes led to decreased moti­
ments [19].
lity and physical forces in host cell monolayers and
affected the mechanotransduction of the endothelial
cells [25]. An understanding of the structural rearran­
Borrelia-induced structural transformations of host
gements and mechanical responses of human cells to
cells
pathogen infection may shed light on the origin of
Borrelia is a pathogen that depends distinctly on its structure-related clinical manifestations such as skin
host to survive, since the spirochaete lacks many fibrosis, morphea, or hyperkeratosis.
18 M. STRNAD ET AL.
Secretion to extracellular space
The capacity of bacteria to secrete proteins, polysac­
charides, and various macromolecular membrane-
derived complexes beyond the bacterial cell surface
is often essential in the understanding of microbial
pathogenesis. Frequently, bacterial pathogenicity
depends fundamentally on the ability to secrete viru­
lence-associated molecules which are located on the
bacterial outer surface, released into the extracellular
space or introduced directly into the host cell.
Secretion systems have been thoroughly studied in
a broad spectrum of Gram-negative species [290].
Contrary to the other bacterial systems, secretion of
soluble proteins into the surrounding environment
has been demonstrated rather sporadically in
Borrelia due to limited secretory capabilities of spir­
ochaetes [291].
Bacterial outer membrane vesicles (OMVs) have
multifaceted roles as they can function as signal deliv­
ery vehicles for proteins, toxins, and other virulence
factors, as well as genetic material and have the ability
to modulate host immune responses [20,292]. OMVs
are usually formed by two different mechanisms: by
membrane blebbing or explosive cell lysis, with size
between 10 and 300 nm and with a single membrane
bilayer, which consist of almost the same outer mem­
brane proteins as the membranes of the bacteria that
the OMVs originated from [293,294]. Although the
secretion of proteins to extracellular space is rare and
largely uncharacterized in Borrelia, several studies have
shown that LD spirochaetes are able to produce OMVs.
Borrelia OMVs, also known as blebs in the literature,
have been revealed to be released near the sites of cell
division [295], and shedding can be observed both in
culture [20] or in vivo inside tick midguts mostly at the
outset of tick feeding [12]. The blebs were shown to be
likely a transitional stage between the spiral and the
round bodies form, with an expanded outer envelope
and with a folded protoplasmic cylinder inside [17].
OMVs from B. burgdorferi were demonstrated to be
on average 33 nm in diameter and contained antigenic
proteins OspA, OspC, p39, peptidoglycan, and neutro­
phil attracting protein NapA [296,297]. Another
B. burgdorferi protein termed Oms28 was reported to
be secreted from borrelial cells into extracellular envir­
onment [298] and associated with borrelial OMVs
[299]. GAG-binding outer surface membrane protein
Bgp has been shown to be secreted from the borrelial
cell as well [298,300]. The spirochaetes produce OMVs
that contain double stranded DNA [296,301], which
could presumably be a mechanism of horizontal gene
transfer. Through the release of OMVs, B. burgdorferi
can also transfer lipids and glycolipids to host epithelial
cells. This lipid exchange could be an important process
leading to antigen presentation and immune recogni­
tion that contributes to the pathogenesis of LD [302].
Nonetheless, the precise roles of OMVs in the patho­
genesis of Borrelia infections remain to be defined.
Nutritional virulence of LD Borrelia
Although LD spirochaetes are not known to significantly
affect the host by directly competing for scarce nutrients
with the host cells, the term “nutritional virulence” has
been recently mentioned in connection with borrelial
virulence [21,303]. Like many other obligate parasites,
LD Borrelia species have significantly reduced their
metabolic and biosynthetic capabilities. As
a consequence, B. burgdorferi lacks genes encoding
enzymes necessary for nucleic acid, amino acid, and
fatty acid biosynthesis. The survival of the spirochaete
in nature is thus fully dependent on salvage of essential
nutrients from their vertebrate hosts and tick vectors. B.
burgdorferi requires uptake of numerous components,
such as cholesterol and long chain saturated and unsa­
turated fatty acids [302,304], purines [305], transition
metals [164], and various carbon sources [306,307].
Several lines of evidence indicate that factors involved
in nutrient acquisition might constitute key virulence
determinants for B. burgdorferi [8]. Borrelia utilizes var­
ious scavenging strategies, obtaining macromolecules
from the microenvironment and breaking them down
to produce substrates for their own anabolic processes.
Typically, the bacterium utilizes highly selective/unspe­
cific porins (P66, P13, Oms28, BB0406) that open and
close in response to specific stimuli and allow passive
diffusion, or, active transport, which exploit the free
energy from ATP hydrolysis to pump the solutes against
concentration gradients [256,308,309].
Owing to its parasitic lifestyle, B. burgdorferi is
restricted to utilizing only the nutrients that are avail­
able in its microenvironment. It is probable that the
ability to exploit a variety of available carbohydrate
sources during the enzootic cycle is essential for the
survival of B. burgdorferi [307]. It has been reported
that B. burgdorferi can utilize at least seven carbohy­
drates as the carbon source – glucose, mannose,
N-acetylglucosamine, chitobiose, maltose, trehalose,
and glycerol [310,311]. When multiple nutrient sources
are available, the spirochaete likely takes advantage of
the rapidly absorbable and high energy carbon sources,
typically glucose. During the off-host starvation periods
in the tick, the spirochaetes were shown to use glycerol
as a carbohydrate source for glycolysis to maintain

maximal Borrelia fitness [306]. A common regulatory
mechanism in bacteria that represses genes associated
with the utilization of secondary carbon sources has
been coined carbon catabolite repression (CCR) [312].
There is no evidence of simple CCR in B. burgdorferi
[15], rather the switch in carbon usage is orchestrated
by mechanisms including various regulatory ele­
ments [313].
B. burgdorferi is unable to synthesize fatty acids de
novo, elongate fatty acid side chains or process exogenous
fatty acids via beta-oxidation [13]. Another noteworthy
aspect is that B. burgdorferi differs from other bacteria and
spirochaetes in that it mostly contains only two types of
phospholipids in the outer and cytoplasmic membranes,
phosphatidylglycerol and phosphatidylcholine [14]. In
order to obtain exogenous choline and fatty acids, the
spirochaete utilizes lipases to break down the lipids of
the host [314]. Using the phosphatidylcholine synthase
proteins, the spirochaete then synthesizes new lipids using
the building blocks acquired from the host [315]. The
lipolytic activity of multiple borrelial enzymes was
shown to contribute to optimal infectivity of the bacter­
ium [21,314], supporting fatty acid salvage as a virulence
mechanism of B. burgdorferi. In addition, fatty acids are
not only fundamental components of borrelial membrane
bilayers, but these moieties are part of many virulence-
associated, surface-exposed lipoproteins, needed for opti­
mal infectivity during tick and host colonization. Osp-,
Csp-, and Erp- lipoprotein families, DbpA/B, RevA/B,
and VlsE belong among the well-known, surface lipopro­
teins tightly associated with borrelial pathogenic potential
[261]. Palmitic acid is the most common fatty acid found
in the lipoproteins of B. burgdorferi [316]. The intercon­
nection of glycerol metabolism and glycolysis with lipo­
protein biosynthesis have been found to depend on
a function of two dehydrogenases, glycerol-3-phosphate
dehydrogenase (GlpD) and glycerol-3-phosphate dehy­
drogenase (GpsA) [317]. GpsA is required for survival
during nutrient stress and for mammalian infection [317].
The spirochaete is unable of de novo synthesis of NAD+
and therefore the molecule has to be recycled through the
salvage pathways of nicotinamide. The enzyme that cata­
lyzes the reaction (PncA) is a key element for mammalian
infection [318].
Conclusion
An understanding of the key aspects in the pathogen­
esis of LD is decisive to the effective care of patients
affected with the disease. The virulence strategies of the
infectious microbes are constantly evolving to maintain
their ability to infect, disseminate, and proliferate in the
host, which, in turn, forces the researchers to evolve
VIRULENCE 19
new ways to detect and combat the pathogen. In this
review, we have summarized the critical findings
regarding the well-defined virulence factors of
B. burgdorferi and potential novel virulence mechan­
isms that are being investigated under the scope of
current research. Although much has been explored
about the genes and proteins that play roles in disease
development [8], the need for further extensive
research is obvious to completely characterize the dif­
ferent factors underlying the infection caused by
B. burgdorferi. The recent improvements in high-end
technologies, which allow for instance whole-genome
sequencing and enable much better resolution in
microscopy and other structural biology techniques
such as NMR, has directly benefitted the understanding
of various aspects of LD, including borrelial phylogeny,
virulence mechanisms, subversion of immune system,
or host association of different Borrelia species. In this
regard, especially targeted mutagenesis and comple­
mentation are key methods for studying genes of
unknown function among bacterial pathogens includ­
ing the LD spirochaete. In addition, genome sequen­
cing of multiple B. burdorferi genospecies should
significantly accelerate the knowledge on this bacterial
complex. Until now, most of the data about borrelial
virulence determinants come from genetic experiments
performed on North American strains of a single spe­
cies, B. burgdorferi ss [9]. Recent whole-genome
sequences of several European and US originated spe­
cies/strains makes adaptation and use of genetic tech­
niques feasible in studying inherent differences between
them [16,319–321].
The complexity of the clinical picture of LD requires
a reliable diagnosis and a wide variety of pathologies. In
case of untreated infection or absence of antibiotic ther­
apy, disseminated spirochaetes can persist in the patient
for many months while evading immune responses.
Antibiotic treatment at the early stage of the disease is
the only effective measure to clear the infection as no
vaccine for human use is currently available. There are
a few novel promising vaccine candidates in the develop­
ment or exploratory stage [4,7]. In addition, the direct
anti-Borrelia measures can be coupled with anti-tick stra­
tegies to deliver a whole new level of human protection
[322]. Full characterization of the drivers involved in the
pathogenic processes, understanding of the regulatory
mechanisms, and further molecular and genetic analysis,
will enable us to design better control and diagnostic
approaches.
We aimed at highlighting a lot of the advances
made in understanding the various virulence determi­
nants of B. burgdorferi. It has also brought into shar­
per focus how much data we still need, to obtain
20 M. STRNAD ET AL.
a more in depth understanding of the virulence
mechanisms that have been identified and those yet
to be identified. The STM analysis was a major step
toward this goal but needs to be followed through at
the individual level for observing the virulence pheno­
type associated with each of the STM mutants. It is
abundantly clear that to be able to obtain better drug-
related interventions for patients, who suffer because
of misdiagnosis or belated treatment, use of faster
genetic methods/’omics’ studies is important. The
uniqueness/plasticity of the genomes of the various
Borrelia genospecies is the biggest challenge we face
in obtaining a broader spectrum of medical/diagnostic
options to counter this disease-causing bacterial
pathogen.
Acknowledgements
This work was supported by the Czech Science Foundation
grants No. 22-18647K and 23-06525J. MS is supported by the
Program for Prospective Human Resources by Czech
Academy of Sciences. NR is supported by grant from the
Ministry of Health of the Czech Republic (NV19-05-00191).
Figures were created with BioRender.com.
Disclosure statement
No potential conflict of interest was reported by the
authors.
Data Availability statement
Data sharing is not applicable to this article as no datasets
were generated or analysed in the current study.
ORCID
Martin Strnad http://orcid.org/0000-0002-1671-6252
Natalie Rudenko http://orcid.org/0000-0002-0506-2249
Ryan O.M. Rego http://orcid.org/0000-0001-6932-0940
References
[1] Kugeler KJ, Schwartz AM, Delorey MJ, et al.
Estimating the frequency of Lyme disease Diagnoses,
United States, 2010–2018. Emerg Infect Dis. 2021;27
(2):616–619. doi: 10.3201/eid2702.202731
[2] Sylvie Goddyn WF, Peterle A, Octavia Sârbu D, et al.
MOTION for a RESOLUTION on Lyme disease (bor­
reliosis) | B8-0514/2018 | European Parliament
[Internet]. [cited 2022 Oct 27]. Available from:
https://www.europarl.europa.eu/doceo/document/
B-8-2018-0514_EN.html
[3] Stanek G, Strle F. Lyme borreliosis–from tick bite to
diagnosis and treatment. FEMS Microbiol Rev. 2018;42
(3):233–258. doi: 10.1093/femsre/fux047
[4] Strnad M, Grubhoffer L, Rego ROM. Novel targets and
strategies to combat borreliosis. Appl Microbiol
Biotechnol. 2020;104(5):1915–1925. doi: 10.1007/
s00253-020-10375-8
[5] Jauris-Heipke S, Fuchs R, Motz M, et al. Genetic het­
erogenity of the genes coding for the outer surface
protein C (OspC) and the flagellin of Borrelia
burgdorferi. Med Microbiol Immunol. 1993;182
(1):37–50. doi: 10.1007/BF00195949
[6] Roberts WC, Mullikin BA, Lathigra R, et al. Molecular
analysis of sequence heterogeneity among genes encod­
ing decorin binding proteins a and B of Borrelia burg­
dorferi Sensu Lato. Infect Immun. 1998;66
(11):5275–5285. doi: 10.1128/IAI.66.11.5275-5285.1998
[7] Comstedt P, Schüler W, Meinke A, et al. The novel
Lyme borreliosis vaccine VLA15 shows broad protec­
tion against Borrelia species expressing six different
OspA serotypes. PLoS One. 2017;12(9):e0184357. doi:
10.1371/journal.pone.0184357
[8] Coburn J, Garcia B, Hu LT, et al. Lyme disease
pathogenesis. Curr Issues Mol Biol. 2021;42:473–518.
doi: 10.21775/cimb.042.473
[9] Strnad M, Rego ROM. The need to unravel the twisted
nature of the Borrelia burgdorferi sensu lato complex
across Europe. Microbiol (Reading). 2020;166
(5):428–435. doi: 10.1099/mic.0.000899
[10] Stewart PE, Byram R, Grimm D, et al. The plasmids of
Borrelia burgdorferi: essential genetic elements of a
pathogen. Plasmid. 2005;53(1):1–13. doi: 10.1016/j.plas
mid.2004.10.006
[11] Vancová M, Bílý T, Šimo L, et al. Three-dimensional
reconstruction of the feeding apparatus of the tick
Ixodes ricinus (Acari: ixodidae): a new insight into
the mechanism of blood-feeding. Sci Rep. 2020;10
(1):165. doi: 10.1038/s41598-019-56811-2
[12] Dunham-Ems SM, Caimano MJ, Pal U, et al. Live
imaging reveals a biphasic mode of dissemination of
Borrelia burgdorferi within ticks. J Clin Invest.
2009;119(12):3652–3665. doi: 10.1172/JCI39401
[13] Strnad M, Oh YJ, Vancová M, et al. Nanomechanical
mechanisms of Lyme disease spirochete motility
enhancement in extracellular matrix. Commun Biol.
2021;4(1):1–9. doi: 10.1038/s42003-021-01783-1
[14] Moriarty TJ, Norman MU, Colarusso P, et al. Real-
time high resolution 3D imaging of the lyme disease
spirochete adhering to and escaping from the vascula­
ture of a living host. PLOS Pathog. 2008;4(6):e1000090.
doi: 10.1371/journal.ppat.1000090
[15] Fraser CM, Casjens S, Huang WM, et al. Genomic
sequence of a Lyme disease spirochaete, Borrelia
burgdorferi. Nature. 1997;390(6660):580–586. doi: 10.
1038/37551
[16] Casjens SR, Mongodin EF, Qiu W-G, et al. Whole-
genome sequences of two Borrelia afzelii and two
Borrelia garinii Lyme disease agent isolates.
J Bacteriol. 2011;193(24):6995–6996. doi: 10.1128/JB.
05951-11
[17] Meriläinen L, Herranen A, Schwarzbach A, et al.
Morphological and biochemical features of Borrelia
burgdorferi pleomorphic forms. Microbiology
(Reading). 2015;161(3):516–527. doi: 10.1099/mic.0.
000027

[18] Rudenko N, Golovchenko M, Kybicova K, et al.
Metamorphoses of Lyme disease spirochetes: phenom­
enon of Borrelia persisters. Parasites Vectors. 2019;12
(1):237. doi: 10.1186/s13071-019-3495-7
[19] Karvonen K, Nykky J, Marjomäki V, et al. Distinctive
evasion mechanisms to allow persistence of Borrelia burg­
dorferi in different human cell lines. Front Microbiol.
2021;12:711291. doi: 10.3389/fmicb.2021.711291
[20] Karvonen K, Tammisto H, Nykky J, et al. Borrelia
burgdorferi Outer Membrane Vesicles Contain
Antigenic Proteins, but Do Not Induce Cell Death in
Human Cells. Microorganisms. 2022;10(2):212. doi: 10.
3390/microorganisms10020212
[21] Kuhn HW, Lasseter AG, Adams PP, et al. BB0562 is
a nutritional virulence determinant with lipase activity
important for Borrelia burgdorferi infection and survi­
val in fatty acid deficient environments. PLOS
Pathogens. 2021;17(8):e1009869. doi: 10.1371/journal.
ppat.1009869
[22] Klose M, Scheungrab M, Luckner M, et al. FIB-SEM-
based analysis of Borrelia intracellular processing by
human macrophages. J Cell Sci. 2021;134:jcs252320.
doi: 10.1242/jcs.252320
[23] Narasimhan S, Rajeevan N, Liu L, et al. Gut microbiota
of the tick vector Ixodes scapularis modulate coloniza­
tion of the Lyme disease spirochete. Cell Host Microbe.
2014;15(1):58–71. doi: 10.1016/j.chom.2013.12.001
[24] Herrmann C, Gern L. Search for blood or water is
influenced by Borrelia burgdorferi in Ixodes ricinus.
Parasites Vectors. 2015;8(1):6. doi: 10.1186/s13071-
014-0526-2
[25] Yuste RA, Muenkel M, Axarlis K, et al. Borrelia burg­
dorferi modulates the physical forces and immunity
signaling in endothelial cells. iScience. 2022;25
(8):104793. doi: 10.1016/j.isci.2022.104793
[26] Lin Y-P, Diuk-Wasser MA, Stevenson B, et al.
Complement evasion contributes to Lyme borreliae–
host associations. Trends Parasitol. 2020;36
(7):634–645. doi: 10.1016/j.pt.2020.04.011
[27] Coburn J, Leong J, Chaconas G. Illuminating the roles
of the Borrelia burgdorferi adhesins. Trends Microbiol.
2013;21(8):372–379. doi: 10.1016/j.tim.2013.06.005
[28] Hyde JA. Borrelia burgdorferi keeps moving and car­
ries on: a review of borrelial dissemination and
invasion. Front Immunol. 2017;8:114. doi: 10.3389/
fimmu.2017.00114
[29] Kurokawa C, Lynn GE, Pedra JHF, et al. Interactions
between Borrelia burgdorferi and ticks. Nat Rev
Microbiol. 2020;18(10):587–600. doi: 10.1038/s41579-
020-0400-5
[30] Schutzer SE, Berger BW, Krueger JG, et al. Atypical
erythema migrans in patients with PCR-Positive Lyme
disease. Emerg Infect Dis. 2013;19(5):815–817. doi: 10.
3201/eid1905.120796
[31] Waddell LA, Greig J, Mascarenhas M, et al. The accu­
racy of diagnostic tests for Lyme disease in humans,
a Systematic review and meta-analysis of North
American research. PLoS One. 2016;11(12):e0168613.
doi: 10.1371/journal.pone.0168613
[32] Baarsma ME, Schellekens J, Meijer BC, et al. Diagnostic
parameters of modified two-tier testing in European
patients with early Lyme disease. Eur J Clin Microbiol
VIRULENCE 21
Infect Dis. 2020;39(11):2143–2152. doi: 10.1007/
s10096-020-03946-0
[33] Davis IRC, McNeil SA, Allen W, et al. Performance of
a modified two-tiered testing enzyme immunoassay
algorithm for serologic diagnosis of Lyme disease in
Nova Scotia. J Clin Microbiol. 2020;58(7):e01841–19.
doi: 10.1128/JCM.01841-19
[34] Branda JA, Steere AC. Laboratory Diagnosis of Lyme
Borreliosis. Clin Microbiol Rev. 2021;34(2):e00018–19.
doi: 10.1128/CMR.00018-19
[35] Ang CW, Brandenburg AH, van Burgel ND, et al.
A Dutch nationwide evaluation of serological assays for
detection of Borrelia antibodies in clinically well-defined
patients. Diagn Microbiol Infect Dis. 2015;83
(3):222–228. doi: 10.1016/j.diagmicrobio.2015.07.007
[36] Joyner G, Mavin S, Milner R, et al. Introduction of IgM
testing for the diagnosis of acute Lyme borreliosis:
a study of the benefits, limitations and costs. Eur
J Clin Microbiol Infect Dis. 2022;41(4):671–675. doi:
10.1007/s10096-021-04366-4
[37] Sabin AP, Scholze BP, Lovrich SD, et al. Clinical eva­
luation of a Borrelia modified two-tiered testing
(MTTT) shows increased early sensitivity for Borrelia
burgdorferi but not other endemic Borrelia species in
a high incidence region for Lyme disease in Wisconsin.
Diagn Microbiol Infect Dis. 2023;105(1):115837. doi:
10.1016/j.diagmicrobio.2022.115837
[38] Wilske B, Fingerle V, Schulte-Spechtel U.
Microbiological and serological diagnosis of Lyme
borreliosis. FEMS Immunol Med Microbiol.
2007;49(1):13–21. doi: 10.1111/j.1574-695X.2006.
00139.x
[39] Kalish RA, McHugh G, Granquist J, et al. Persistence
of Immunoglobulin M or Immunoglobulin G Antibody
Responses to Borrelia burgdorferi 10–20 Years after
Active Lyme Disease. Clin Infect Dis. 2001;33
(6):780–785. doi: 10.1086/322669
[40] Krogen I, Skarphédinsson S, Jensen TG, et al. No
correlation between symptom duration and intrathecal
production of IgM and/or IgG antibodies in Lyme
neuroborreliosis – a retrospective cohort study in
Denmark. J Infect. 2022;85(5):507–512. doi: 10.1016/j.
jinf.2022.08.045
[41] Lantos PM, Rumbaugh J, Bockenstedt LK, et al. Clinical
practice guidelines by the infectious Diseases Society of
America (IDSA), American Academy of Neurology
(AAN), and American College of Rheumatology (ACR):
2020 guidelines for the Prevention, diagnosis, and treat­
ment of Lyme disease. Arthritis Care Res (Hoboken).
2021;73(1):1–9. doi: 10.1002/acr.24495
[42] Crossland NA, Alvarez X, Embers ME. Late dissemi­
nated Lyme disease. Am J Pathol. 2018;188(3):672–682.
doi: 10.1016/j.ajpath.2017.11.005
[43] Stanek G, Fingerle V, Hunfeld K-P, et al. Lyme borre­
liosis: Clinical case definitions for diagnosis and man­
agement in Europe. Clin Microbiol Infect. 2011;17
(1):69–79. doi: 10.1111/j.1469-0691.2010.03175.x
[44] Bobe JR, Jutras BL, Horn EJ, et al. Recent Progress in
Lyme Disease and Remaining Challenges. Front Med.
2021;8:666554. doi: 10.3389/fmed.2021.666554
[45] Wong KH, Shapiro ED, Soffer GK. A review of
post-treatment Lyme disease syndrome and chronic
22 M. STRNAD ET AL.
Lyme disease for the practicing immunologist. Clin Rev
Allergy Immunol. 2022;62(1):264–271. doi: 10.1007/
s12016-021-08906-w
[46] Jutras BL, Lochhead RB, Kloos ZA, et al. Borrelia
burgdorferi peptidoglycan is a persistent antigen in
patients with Lyme arthritis. Proc Natl Acad Sci
U S A. 2019;116(27):13498–13507. doi: 10.1073/pnas.
1904170116
[47] Rudenko N, Golovchenko M. Sexual transmission of
Lyme Borreliosis? The question that calls for an
answer. Trop Med Infect Dis. 2021;6(2):87. doi: 10.
3390/tropicalmed6020087
[48] Majerová K, Hönig V, Houda M, et al. Hedgehogs,
squirrels, and blackbirds as sentinel hosts for active
surveillance of Borrelia miyamotoi and Borrelia burg­
dorferi complex in urban and rural environments.
Microorganisms. 2020;8(12):E1908. doi: 10.3390/
microorganisms8121908
[49] Rudenko N, Golovchenko M, Grubhoffer L, et al.
Updates on Borrelia burgdorferi sensu lato complex
with respect to public health. Ticks Tick Borne Dis.
2011;2(3):123–128. doi: 10.1016/j.ttbdis.2011.04.002
[50] Clark KL, Leydet B, Hartman S. Lyme borreliosis in
human patients in Florida and Georgia, USA.
Int J Med Sci. 2013;10(7):915–931. doi: 10.7150/ijms.
6273
[51] Steere AC. Lyme disease. N Engl J Med. 1989;321
(9):586–596. doi: 10.1056/NEJM198908313210906
[52] Oschmann P, Dorndorf W, Hornig C, et al. Stages and
syndromes of neuroborreliosis. J Neurol. 1998;245
(5):262–272. doi: 10.1007/s004150050216
[53] Ornstein K, Berglund J, Nilsson I, et al.
Characterization of Lyme borreliosis isolates from
patients with erythema migrans and neuroborreliosis
in southern Sweden. J Clin Microbiol. 2001;39
(4):1294–1298. doi: 10.1128/JCM.39.4.1294-1298.2001
[54] Ruzić-Sabljić E, Maraspin V, Lotric-Furlan S, et al.
Characterization of Borrelia burgdorferi sensu lato
strains isolated from human material in Slovenia.
Wien Klin Wochenschr. 2002;114:544–550.
[55] Steere AC, Strle F, Wormser GP, et al. Lyme
borreliosis. Nat Rev Dis Primers. 2016;2(1):16090. doi:
10.1038/nrdp.2016.90
[56] Grange F, Wechsler J, Guillaume J-C, et al. Borrelia
burgdorferi-associated lymphocytoma cutis simulating
a primary cutaneous large B-cell lymphoma. J Am
Acad Dermatol. 2002;47(4):530–534. doi: 10.1067/
mjd.2002.120475
[57] Smetanick MT, Zellis SL, Ermolovich T.
Acrodermatitis chronica atrophicans: a case report
and review of the literature. Cutis. 2010;85:247–252.
[58] Picken RN, Strle F, Picken MM, et al. Identification of
three species of Borrelia burgdorferi sensu lato
(B. burgdorferi sensu stricto, B. garinii, and B. afzelii)
among isolates from acrodermatitis chronica atrophi­
cans lesions. J Invest Dermatol. 1998;110(3):211–214.
doi: 10.1046/j.1523-1747.1998.00130.x
[59] Strle F, Picken RN, Cheng Y, et al. Clinical findings for
patients with Lyme borreliosis caused by Borrelia burg­
dorferi sensu lato with genotypic and phenotypic simi­
larities to strain 25015. Clin Infect Dis. 1997;25
(2):273–280. doi: 10.1086/514551
[60] Margos G, Lane RS, Fedorova N, et al. Borrelia bisset­
tiae sp. nov. and Borrelia californiensis sp. nov. prevail
in diverse enzootic transmission cycles. Int J Syst Evol
Microbiol. 2016;66(3):1447–1452. doi: 10.1099/ijsem.0.
000897
[61] Rudenko N, Golovchenko M, Růzek D, et al. Molecular
detection of Borrelia bissettii DNA in serum samples
from patients in the Czech Republic with suspected
borreliosis. FEMS Microbiol Lett. 2009;292
(2):274–281. doi: 10.1111/j.1574-6968.2009.01498.x
[62] Rudenko N, Golovchenko M, Mokrácek A, et al.
Detection of Borrelia bissettii in cardiac valve tissue
of a patient with endocarditis and aortic valve stenosis
in the Czech Republic. J Clin Microbiol. 2008;46
(10):3540–3543. doi: 10.1128/JCM.01032-08
[63] Collares-Pereira M, Couceiro S, Franca I, et al. First
isolation of Borrelia lusitaniae from a human patient.
J Clin Microbiol. 2004;42(3):1316–1318. doi: 10.1128/
JCM.42.3.1316-1318.2004
[64] Diza E, Papa A, Vezyri E, et al. Borrelia valaisiana in
cerebrospinal fluid. Emerging Infect Dis. 2004;10
(9):1692–1693. doi: 10.3201/eid1009.030439
[65] Margos G, Sing A, Fingerle V. Published data do not
support the notion that Borrelia valaisiana is human
pathogenic. Infection. 2017;45(4):567–569. doi: 10.
1007/s15010-017-1032-1
[66] Eliassen KE, Ocias LF, Krogfelt KA, et al. Tick-
transmitted co-infections among erythema migrans
patients in a general practice setting in Norway:
a clinical and laboratory follow-up study. BMC Infect
Dis. 2021;21(1):1044. doi: 10.1186/s12879-021-06755-8
[67] Cassatt DR, Patel NK, Ulbrandt ND, et al. DbpA, but
not OspA, is expressed by Borrelia burgdorferi during
spirochetemia and is a target for protective antibodies.
Infect Immun. 1998;66(11):5379–5387. doi: 10.1128/
IAI.66.11.5379-5387.1998
[68] Hanson MS, Cassatt DR, Guo BP, et al. Active and
passive immunity against Borrelia burgdorferi decorin
binding protein a (DbpA) protects against infection.
Infect Immun. 1998;66(5):2143–2153. doi: 10.1128/IAI.
66.5.2143-2153.1998
[69] Brown EL, Kim JH, Reisenbichler ES, et al.
Multicomponent Lyme vaccine: three is not a crowd.
Vaccine. 2005;23(28):3687–3696. doi: 10.1016/j.vac
cine.2005.02.006
[70] Earnhart CG, Marconi RT. An octavalent lyme disease
vaccine induces antibodies that recognize all incorpo­
rated OspC type-specific sequences. Hum Vaccin.
2007;3(6):281–289. doi: 10.4161/hv.4661
[71] Kumar M, Kaur S, Kariu T, et al. Borrelia burgdorferi
BBA52 is a potential target for transmission blocking
Lyme disease vaccine. Vaccine. 2011;29(48):9012–9019.
doi: 10.1016/j.vaccine.2011.09.035
[72] Klouwens MJ, Trentelman JJ, Ersoz JI, et al.
Investigating BB0405 as a novel Borrelia afzelii vacci­
nation candidate in Lyme borreliosis. Sci Rep. 2021;11
(1):4775. doi: 10.1038/s41598-021-84130-y
[73] Singh P, Verma D, Backstedt BT, et al. Borrelia burg­
dorferi BBI39 paralogs, targets of protective immunity,
reduce pathogen persistence either in hosts or in the
vector. J Infect Dis. 2017;215(6):1000–1009. doi: 10.
1093/infdis/jix036

[74] Nigrovic LE, Thompson KM. The Lyme vaccine:
a cautionary tale. Epidemiol Infect. 2007;135(1):1–8.
doi: 10.1017/S0950268806007096
[75] Shaffer L. Inner workings: lyme disease vaccines face
familiar challenges, both societal and scientific. Proc
Natl Acad Sci U S A. 2019;116(39):19214–19217. doi:
10.1073/pnas.1913923116
[76] Bézay N, Hochreiter R, Kadlecek V, et al. Safety and
immunogenicity of a novel multivalent OspA-based
vaccine candidate against Lyme borreliosis:
a randomised, phase 1 study in healthy adults. Lancet
Infect Dis. 2023;S1473-3099(23):210–214. doi: 10.1016/
S1473-3099(23)00210-4
[77] Earnhart CG, Buckles EL, Marconi RT. Development
of an OspC-based tetravalent, recombinant, chimeric
vaccinogen that elicits bactericidal antibody against
diverse Lyme disease spirochete strains. Vaccine.
2007;25(3):466–480. doi: 10.1016/j.vaccine.2006.07.052
[78] Wressnigg N, Pöllabauer E-M, Aichinger G, et al.
Safety and immunogenicity of a novel multivalent
OspA vaccine against Lyme borreliosis in healthy
adults: a double-blind, randomised, dose-escalation
phase 1/2 trial. Lancet Infect Dis. 2013;13(8):680–689.
doi: 10.1016/S1473-3099(13)70110-5
[79] Kamp HD, Swanson KA, Wei RR, et al. Design of
a broadly reactive Lyme disease vaccine. NPJ Vaccines.
2020;5(1):33. doi: 10.1038/s41541-020-0183-8
[80] Nayak A, Schüler W, Seidel S, et al. Broadly protective
multivalent OspA vaccine against Lyme borreliosis,
developed based on surface shaping of the
C-Terminal fragment. Infect Immun. 2020;88(4):
e00917–19. doi: 10.1128/IAI.00917-19
[81] Klouwens MJ, Trentelman JJA, Wagemakers A, et al.
Tick-Tattoo: DNA vaccination against B. burgdorferi
or Ixodes scapularis Tick proteins. Front Immunol.
2021;12:615011. doi: 10.3389/fimmu.2021.615011
[82] Wagemakers A, Mason LMK, Oei A, et al. Rapid
outer-surface protein C DNA tattoo vaccination pro­
tects against Borrelia afzelii infection. Gene Ther.
2014;21(12):1051–1057. doi: 10.1038/gt.2014.87
[83] Pfeifle A, Thulasi Raman SN, Lansdell C, et al. DNA
lipid nanoparticle vaccine targeting outer surface pro­
tein C affords protection against homologous Borrelia
burgdorferi needle challenge in mice. Front Immunol.
2023;14:1020134. doi: 10.3389/fimmu.2023.1020134
[84] Saade F, Petrovsky N. Technologies for enhanced effi­
cacy of DNA vaccines. Expert Rev Vaccines. 2012;11
(2):189–209. doi: 10.1586/erv.11.188
[85] Rosa PA, Jewett MW. Genetic manipulation of
Borrelia. Curr Issues Mol Biol. 2021;42:307–332. doi:
10.21775/cimb.042.307
[86] Samuels DS. Electrotransformation of the spirochete
Borrelia burgdorferi. Methods Mol Biol.
1995;47:253–259.
[87] Rosa PA, Tilly K, Stewart PE. The burgeoning mole­
cular genetics of the Lyme disease spirochaete. Nat Rev
Microbiol. 2005;3(2):129–143. doi: 10.1038/
nrmicro1086
[88] Stewart PE, Hoff J, Fischer E, et al. Genome-wide
transposon mutagenesis of Borrelia burgdorferi for
identification of phenotypic mutants. Appl Environ
VIRULENCE 23
Microbiol. 2004;70(10):5973–5979. doi: 10.1128/AEM.
70.10.5973-5979.2004
[89] Botkin DJ, Abbott AN, Stewart PE, et al. Identification
of potential virulence determinants by Himar1 trans­
position of infectious Borrelia burgdorferi B31. Infect
Immun. 2006;74(12):6690–6699. doi: 10.1128/IAI.
00993-06
[90] Lin T, Gao L, Zhang C, et al. Analysis of an ordered,
Comprehensive STM mutant Library in infectious
Borrelia burgdorferi: insights into the genes required
for mouse infectivity. PLoS One. 2012;7(10):e47532.
doi: 10.1371/journal.pone.0047532
[91] Bose JL. Chemical and UV mutagenesis. Methods Mol
Biol. 2016;1373:111–115.
[92] Lin T, Gao L. Genome-Wide Mutagenesis in Borrelia
burgdorferi. Methods Mol Biol. 2018;1690:201–223.
[93] Revel AT, Talaat AM, Norgard MV. DNA microarray
analysis of differential gene expression in Borrelia
burgdorferi, the Lyme disease spirochete. Proc Natl
Acad Sci U S A. 2002;99(3):1562–1567. doi: 10.1073/
pnas.032667699
[94] Liang FT, Nelson FK, Fikrig E. DNA microarray
assessment of putative Borrelia burgdorferi lipoprotein
genes. Infect Immun. 2002;70(6):3300–3303. doi: 10.
1128/IAI.70.6.3300-3303.2002
[95] Akins DR, Bourell KW, Caimano MJ, et al. A new
animal model for studying Lyme disease spirochetes
in a mammalian host-adapted state. J Clin Invest.
1998;101(10):2240–2250. doi: 10.1172/JCI2325
[96] Caimano MJ. Cultivation of Borrelia burgdorferi in
dialysis membrane chambers in rat peritonea. Curr
Protoc Microbiol. 2005;(1). doi: 10.1002/
9780471729259.mc12c03s00
[97] Arnold WK, Savage CR, Brissette CA, et al. RNA-Seq
of Borrelia burgdorferi in multiple phases of growth
reveals insights into the dynamics of gene expression,
transcriptome architecture, and noncoding RNAs.
PLoS One. 2016;11(10):e0164165. doi: 10.1371/jour
nal.pone.0164165
[98] Lybecker MC, Samuels DS. Small RNAs of Borrelia
burgdorferi: characterizing functional regulators in
a sea of sRnas. Yale J Biol Med. 2017;90(2):317–323.
[99] Ellis TC, Jain S, Linowski AK, et al. In vivo expression
technology identifies a novel virulence factor critical for
Borrelia burgdorferi persistence in mice. PLOS Pathog.
2013;9(8):e1003567. doi: 10.1371/journal.ppat.1003567
[100] Casselli T, Bankhead T. Use of in vivo expression
technology for the identification of putative host adap­
tation factors of the Lyme disease spirochete. J Mol
Microbiol Biotechnol. 2015;25(5):349–361. doi: 10.
1159/000439305
[101] Hyde JA, Weening EH, Chang M, et al. Bioluminescent
imaging of Borrelia burgdorferi in vivo demonstrates
that the fibronectin-binding protein BBK32 is required
for optimal infectivity. Mol Microbiol. 2011;82
(1):99–113. doi: 10.1111/j.1365-2958.2011.07801.x
[102] Hejduk L, Rathner P, Strnad M, et al. Resonance
assignment and secondary structure of DbpA protein
from the European species, Borrelia afzelii. Biomol
NMR Assign. 2021;15(2):415–420. doi: 10.1007/
s12104-021-10039-2
24 M. STRNAD ET AL.
[103] Niddam AF, Ebady R, Bansal A, et al. Plasma fibro­
nectin stabilizes Borrelia burgdorferi–endothelial inter­
actions under vascular shear stress by a catch-bond
mechanism. PNAS. 2017;114(17):E3490–8. doi: 10.
1073/pnas.1615007114
[104] Harman MW, Dunham-Ems SM, Caimano MJ, et al.
The heterogeneous motility of the Lyme disease spir­
ochete in gelatin mimics dissemination through tissue.
Proc Natl Acad Sci, USA. 2012;109(8):3059–3064. doi:
10.1073/pnas.1114362109
[105] Bockenstedt LK, Gonzalez D, Mao J, et al. What ticks
do under your skin: Two-Photon intravital imaging of
Ixodes scapularis feeding in the presence of the Lyme
disease spirochete. Yale J Biol Med. 2014;87:3–13.
[106] Hillman C, Stewart PE, Strnad M, et al. Visualization of
spirochetes by labeling membrane proteins with fluor­
escent biarsenical dyes. Front Cell Infect Microbiol.
2019;9:287. doi: 10.3389/fcimb.2019.00287
[107] Strnad M, Elsterová J, Schrenková J, et al. Correlative
cryo-fluorescence and cryo-scanning electron micro­
scopy as a straightforward tool to study
host-pathogen interactions. Sci Rep. 2015;5(1):18029.
doi: 10.1038/srep18029
[108] Wang X. Solution structure of decorin-binding protein
a from Borrelia burgdorferi. Biochemistry. 2012;51
(42):8353–8362. doi: 10.1021/bi3007093
[109] Ante VM, Farris LC, Saputra EP, et al. The Borrelia
burgdorferi adenylate cyclase, CyaB, is important for
virulence factor production and mammalian infection.
Front Microbiol. 2021;12:676192. doi: 10.3389/fmicb.
2021.676192
[110] Topal H, Fulcher NB, Bitterman J, et al. Crystal struc­
ture and Regulation mechanisms of the CyaB Adenylyl
cyclase from the human pathogen Pseudomonas
aeruginosa. J Mol Biol. 2012;416(2):271–286. doi: 10.
1016/j.jmb.2011.12.045
[111] Kim YR, Kim SY, Kim CM, et al. Essential role of an
adenylate cyclase in regulating Vibrio vulnificus
virulence. FEMS Microbiol Lett. 2005;243(2):497–503.
doi: 10.1016/j.femsle.2005.01.016
[112] Galperin MY, Chou S-H, Stock AM. Structural
Conservation and diversity of PilZ-Related Domains.
J Bacteriol. 2020;202(4):10.1128/jb.00664–19. doi: 10.
1128/JB.00664-19
[113] Hong Y, Zhou X, Fang H, et al. Cyclic di-GMP med­
iates Mycobacterium tuberculosis dormancy and
pathogenecity. Tuberculosis (Edinb). 2013;93
(6):625–634. doi: 10.1016/j.tube.2013.09.002
[114] Amikam D, Galperin MY. PilZ domain is part of the
bacterial c-di-GMP binding protein. Bioinformatics.
2006;22(1):3–6. doi: 10.1093/bioinformatics/bti739
[115] Jusufovic N, Savage CR, Saylor TC, et al. Borrelia
burgdorferi PlzA is a c-di-GMP dependent DNA and
RNA binding protein. bioRxiv. 2023.
[116] Groshong AM, Grassmann AA, Luthra A, et al. PlzA is
a bifunctional c-di-GMP biosensor that promotes tick
and mammalian host-adaptation of Borrelia
burgdorferi. PLOS Pathog. 2021;17(7):e1009725. doi:
10.1371/journal.ppat.1009725
[117] Stevenson B, Babb K. LuxS-Mediated quorum sensing
in Borrelia burgdorferi, the Lyme disease spirochete.
Infect Immun. 2002;70(8):4099–4105. doi: 10.1128/IAI.
70.8.4099-4105.2002
[118] Jones MB, Peterson SN, Benn R, et al. Role of luxS in
bacillus anthracis growth and virulence factor
expression. Virulence. 2010;1(2):72–83. doi: 10.4161/
viru.1.2.10752
[119] Xu L, Li H, Vuong C, et al. Role of the luxS
Quorum-Sensing System in Biofilm Formation and
Virulence of Staphylococcus epidermidis. Infect
Immun. 2006;74(1):488–496. doi: 10.1128/IAI.74.1.
488-496.2006
[120] Zhang B, Ku X, Zhang X, et al. The AI-2/luxS quorum
sensing system affects the growth characteristics, bio­
film formation, and virulence of haemophilus parasuis.
Front Cell Infect Microbiol. 2019;9:62. doi: 10.3389/
fcimb.2019.00062
[121] Taga ME, Bassler BL. Chemical communication among
bacteria. Proc Natl Acad Sci U S A. 2003;100
(2):14549–14554. doi: 10.1073/pnas.1934514100
[122] Stevenson B, von Lackum K, Wattier RL, et al. Quorum
sensing by the Lyme disease spirochete. Microbes
Infect. 2003;5(11):991–997. doi: 10.1016/S1286-
4579(03)00184-9
[123] Blevins JS, Revel AT, Caimano MJ, et al. The luxS gene
is not required for Borrelia burgdorferi tick coloniza­
tion, transmission to a mammalian host, or induction
of disease. Infect Immun. 2004;72(8):4864–4867. doi:
10.1128/IAI.72.8.4864-4867.2004
[124] Arnold WK, Savage CR, Antonicello AD, et al.
Apparent Role for Borrelia burgdorferi LuxS during
Mammalian Infection. Infect Immun. 2015;83
(4):1347–1353. doi: 10.1128/IAI.00032-15
[125] Strnad M, Hönig V, Růžek D, et al. Europe-wide
meta-analysis of Borrelia burgdorferi Sensu Lato pre­
valence in questing Ixodes ricinus ticks. Appl
Environ Microbiol. 2017;83(15). doi: 10.1128/AEM.
00609-17
[126] van Duijvendijk G, Coipan C, Wagemakers A, et al.
Larvae of Ixodes ricinus transmit Borrelia afzelii and
B. miyamotoi to vertebrate hosts. Parasites Vectors.
2016;9(1):97. doi: 10.1186/s13071-016-1389-5
[127] Tonk M, Cabezas-Cruz A, Valdés JJ, et al. Defensins
from the tick Ixodes scapularis are effective against
phytopathogenic fungi and the human bacterial patho­
gen Listeria grayi. Parasites Vectors. 2014;7(1):554. doi:
10.1186/s13071-014-0554-y
[128] Cruz CE, Fogaça AC, Nakayasu ES, et al.
Characterization of proteinases from the midgut of
rhipicephalus (boophilus) microplus involved in the
generation of antimicrobial peptides. Parasites
Vectors. 2010;3(1):63. doi: 10.1186/1756-3305-3-63
[129] Pereira LS, Oliveira PL, Barja-Fidalgo C, et al. Production
of reactive oxygen species by hemocytes from the cattle
tick boophilus microplus. Exp Parasitol. 2001;99
(2):66–72. doi: 10.1006/expr.2001.4657
[130] Eggenberger LR, Lamoreaux WJ, Coons LB. Hemocytic
encapsulation of implants in the tick dermacentor
variabilis. Exp Appl Acarol. 1990;9(3–4):279–287. doi:
10.1007/BF01193434
[131] Fogaça AC, Sousa G, Pavanelo DB, et al. Tick immune
system: what is known, the interconnections, the gaps,

and the challenges. Front Immunol. 2021;12:628054.
doi: 10.3389/fimmu.2021.628054
[132] Tanaka T, Kawano S, Nakao S, et al. The identification
and characterization of lysozyme from the hard tick
haemaphysalis longicornis. Ticks Tick Borne Dis.
2010;1(4):178–185. doi: 10.1016/j.ttbdis.2010.09.001
[133] De Silva AM, Fikrig E. Growth and migration of
Borrelia burgdorferi in Ixodes ticks during blood
feeding. Am J Trop Med Hyg. 1995;53(4):397–404.
doi: 10.4269/ajtmh.1995.53.397
[134] Ribeiro JM, Mather TN, Piesman J, et al.
Dissemination and salivary delivery of Lyme disease
spirochetes in vector ticks (Acari: ixodidae). J Med
Entomol. 1987;24(2):201–205. doi: 10.1093/jmedent/
24.2.201
[135] Pospisilova T, Urbanova V, Hes O, et al. Tracking of
Borrelia afzelii transmission from infected Ixodes rici­
nus Nymphs to mice. Infect Immun. 2019;87(6):
e00896–18. doi: 10.1128/IAI.00896-18
[136] Lejal E, Moutailler S, Šimo L, et al. Tick-borne patho­
gen detection in midgut and salivary glands of adult
Ixodes ricinus. Parasites Vectors. 2019;12(1):152. doi:
10.1186/s13071-019-3418-7
[137] Yang XF, Pal U, Alani SM, et al. Essential role for
OspA/B in the life cycle of the Lyme disease
spirochete. J Exp Med. 2004;199(5):641–648. doi: 10.
1084/jem.20031960
[138] Pal U, de Silva AM, Montgomery RR, et al. Attachment
of Borrelia burgdorferi within Ixodes scapularis
mediated by outer surface protein a. J Clin Invest.
2000;106(4):561–569. doi: 10.1172/JCI9427
[139] Pal U, Li X, Wang T, et al. TROSPA, an Ixodes scapu­
laris receptor for Borrelia burgdorferi. Cell. 2004;119
(4):457–468. doi: 10.1016/j.cell.2004.10.027
[140] Zhang L, Zhang Y, Adusumilli S, et al. Molecular inter­
actions that enable movement of the Lyme disease agent
from the tick gut into the hemolymph. PLOS Pathog.
2011;7(6):e1002079. doi: 10.1371/journal.ppat.1002079
[141] Neelakanta G, Li X, Pal U, et al. Outer surface protein
B is critical for Borrelia burgdorferi adherence and
survival within Ixodes ticks. PLOS Pathogens. 2007;3
(3):e33. doi: 10.1371/journal.ppat.0030033
[142] Revel AT, Blevins JS, Almazán C, et al. bptA (bbe16) is
essential for the persistence of the Lyme disease spir­
ochete, Borrelia burgdorferi, in its natural tick vector.
Proc Natl Acad Sci U S A. 2005;102(19):6972–6977.
doi: 10.1073/pnas.0502565102
[143] Mason C, Thompson C, Ouyang Z. DksA plays an
essential role in regulating the virulence of Borrelia
burgdorferi. Mol Microbiol. 2020;114(1):172–183. doi:
10.1111/mmi.14504
[144] Boyle WK, Groshong AM, Drecktrah D, et al. DksA
controls the response of the Lyme disease spirochete
Borrelia burgdorferi to starvation. J Bacteriol. 2019;201
(4):e00582–18. doi: 10.1128/JB.00582-18
[145] Kumar M, Yang X, Coleman AS, et al. BBA52 facil­
itates Borrelia burgdorferi transmission from feeding
ticks to murine hosts. J Infect Dis. 2010;201
(7):1084–1095. doi: 10.1086/651172
[146] Narasimhan S, Coumou J, Schuijt TJ, et al. A tick gut
protein with fibronectin III domains aids Borrelia
burgdorferi congregation to the gut during
VIRULENCE 25
transmission. PLOS Pathogens. 2014;10(8):e1004278.
doi: 10.1371/journal.ppat.1004278
[147] Coumou J, Narasimhan S, Trentelman JJ, et al.
Ixodes scapularis dystroglycan-like protein pro­
motes Borrelia burgdorferi migration from the
gut. J Mol Med (Berl). 2016;94(3):361–370. doi:
10.1007/s00109-015-1365-0
[148] Pal U, Yang X, Chen M, et al. OspC facilitates Borrelia
burgdorferi invasion of Ixodes scapularis salivary
glands. J Clin Invest. 2004;113(2):220–230. doi: 10.
1172/JCI200419894
[149] Tilly K, Krum JG, Bestor A, et al. Borrelia burgdorferi
OspC protein required exclusively in a crucial early
stage of mammalian infection. Infect Immun. 2006;74
(6):3554–3564. doi: 10.1128/IAI.01950-05
[150] Ramamoorthi N, Narasimhan S, Pal U, et al. The Lyme
disease agent exploits a tick protein to infect the mam­
malian host. Nature. 2005;436(7050):573–577. doi: 10.
1038/nature03812
[151] Bierwagen P, Sliwiak J, Jaskolski M, et al. Strong inter­
actions between Salp15 homologues from the tick
I. ricinus and distinct types of the outer surface OspC
protein from Borrelia. Ticks Tick Borne Dis. 2021;12
(2):101630. doi: 10.1016/j.ttbdis.2020.101630
[152] Kotál J, Langhansová H, Lieskovská J, et al. Modulation
of host immunity by tick saliva. J Proteomics.
2015;128:58–68. doi: 10.1016/j.jprot.2015.07.005
[153] Murfin KE, Kleinbard R, Aydin M, et al. Borrelia
burgdorferi chemotaxis toward tick protein Salp12
contributes to acquisition. Ticks Tick Borne Dis.
2019;10(5):1124–1134. doi: 10.1016/j.ttbdis.2019.06.002
[154] Narasimhan S, Sukumaran B, Bozdogan U, et al. A tick
antioxidant facilitates the Lyme disease agent’s success­
ful migration from the mammalian host to the arthro­
pod vector. Cell Host Microbe. 2007;2(1):7–18. doi: 10.
1016/j.chom.2007.06.001
[155] Coleman JL, Crowley JT, Toledo AM, et al. The HtrA
protease of Borrelia burgdorferi degrades outer mem­
brane protein BmpD and chemotaxis phosphatase
CheX. Mol Microbiol. 2013;88(3):619–633. doi: 10.
1111/mmi.12213
[156] Boylan JA, Lawrence KA, Downey JS, et al. Borrelia
burgdorferi membranes are the primary targets of reac­
tive oxygen species. Mol Microbiol. 2008;68
(3):786–799. doi: 10.1111/j.1365-2958.2008.06204.x
[157] Radolf JD, Caimano MJ, Stevenson B, et al. Of ticks,
mice and men: understanding the dual-host lifestyle of
Lyme disease spirochaetes. Nat Rev Microbiol. 2012;10
(2):87–99. doi: 10.1038/nrmicro2714
[158] Tracy KE, Baumgarth N. Borrelia burgdorferi manip­
ulates innate and adaptive immunity to establish per­
sistence in rodent reservoir hosts. Front Immunol.
2017;8:116. doi: 10.3389/fimmu.2017.00116
[159] Bruch-Gerharz D, Ruzicka T, Kolb-Bachofen V. Nitric
oxide in human skin: current status and future
prospects. J Invest Dermatol. 1998;110(1):1–7. doi: 10.
1046/j.1523-1747.1998.00084.x
[160] Ma Y, Seiler KP, Tai KF, et al. Outer surface lipopro­
teins of Borrelia burgdorferi stimulate nitric oxide pro­
duction by the cytokine-inducible pathway. Infect
Immun. 1994;62(9):3663–3671. doi: 10.1128/iai.62.9.
3663-3671.1994
26 M. STRNAD ET AL.
[161] Seiler KP, Vavrin Z, Eichwald E, et al. Nitric oxide
production during murine Lyme disease: lack of invol­
vement in host resistance or pathology. Infect Immun.
1995;63(10):3886–3895. doi: 10.1128/iai.63.10.3886-
3895.1995
[162] Kerstholt M, Vrijmoeth H, Lachmandas E, et al. Role
of glutathione metabolism in host defense against
Borrelia burgdorferi infection. Proc Natl Acad Sci
U S A. 2018;115(10):E2320–8. doi: 10.1073/pnas.
1720833115
[163] Kerstholt M, Brouwer M, Te Vrugt M, et al. Borrelia
burgdorferi inhibits NADPH-mediated reactive oxygen
species production through the mTOR pathway. Ticks
Tick Borne Dis. 2022;13(4):101943. doi: 10.1016/j.
ttbdis.2022.101943
[164] Ouyang Z, He M, Oman T, et al. A manganese trans­
porter, BB0219 (BmtA), is required for virulence by the
Lyme disease spirochete, Borrelia burgdorferi. Proc
Natl Acad Sci U S A. 2009;106(9):3449–3454. doi: 10.
1073/pnas.0812999106
[165] Esteve-Gassent MD, Elliott NL, Seshu J. sodA is essen­
tial for virulence of Borrelia burgdorferi in the murine
model of Lyme disease. Mol Microbiol. 2009;71
(3):594–612. doi: 10.1111/j.1365-2958.2008.06549.x
[166] Phelan JP, Bourgeois JS, McCarthy JE, et al. A putative
xanthine dehydrogenase is critical for Borrelia burg­
dorferi survival in ticks and mice. Microbiology
(Reading). 2023;169(1):001286. doi: 10.1099/mic.0.
001286
[167] Ramsey ME, Hyde JA, Medina-Perez DN, et al. A
high-throughput genetic screen identifies previously
uncharacterized Borrelia burgdorferi genes important
for resistance against reactive oxygen and nitrogen
species. PLOS Pathog. 2017;13(2):e1006225. doi: 10.
1371/journal.ppat.1006225
[168] Lusitani D, Malawista SE, Montgomery RR. Borrelia
burgdorferi are susceptible to killing by a variety of
human polymorphonuclear leukocyte components.
J Infect Dis. 2002;185(6):797–804. doi: 10.1086/339341
[169] Dunkelberger JR, Song W-C. Complement and its role
in innate and adaptive immune responses. Cell Res.
2010;20(1):34–50. doi: 10.1038/cr.2009.139
[170] Kurtenbach K, De Michelis S, Etti S, et al. Host asso­
ciation of Borrelia burgdorferi sensu lato–the key role
of host complement. Trends Microbiol. 2002;10
(2):74–79. doi: 10.1016/S0966-842X(01)02298-3
[171] Stevenson B, El-Hage N, Hines MA, et al. Differential
binding of host complement inhibitor factor H by
Borrelia burgdorferi Erp surface proteins: a possible
mechanism underlying the expansive host range of
Lyme disease spirochetes. Infect Immun. 2002;70
(2):491–497. doi: 10.1128/IAI.70.2.491-497.2002
[172] van Dam AP, Oei A, Jaspars R, et al. Complement-
mediated serum sensitivity among spirochetes that
cause Lyme disease. Infect Immun. 1997;65:1228–1236.
doi: 10.1128/iai.65.4.1228-1236.1997
[173] Kurtenbach K, Peacey M, Rijpkema SG, et al.
Differential transmission of the genospecies of
Borrelia burgdorferi sensu lato by game birds and
small rodents in England. Appl Environ Microbiol.
1998;64(4):1169–1174. doi: 10.1128/AEM.64.4.1169-
1174.1998
[174] Skare JT, Garcia BL. Complement evasion by Lyme
disease spirochetes. Trends Microbiol. 2020;28
(11):889–899. doi: 10.1016/j.tim.2020.05.004
[175] Garcia BL, Zhi H, Wager B, et al. Borrelia burgdorferi
BBK32 inhibits the classical pathway by blocking acti­
vation of the C1 complement complex. PLOS Pathog.
2016;12(1):e1005404. doi: 10.1371/journal.ppat.
1005404
[176] Caine JA, Lin Y-P, Kessler JR, et al. Borrelia burgdor­
feri outer surface protein C (OspC) binds complement
component C4b and confers bloodstream survival. Cell
Microbiol. 2017;19(12):e12786. doi: 10.1111/cmi.12786
[177] Barthel D, Schindler S, Zipfel PF. Plasminogen Is
a Complement Inhibitor. J Biol Chem. 2012;287
(22):18831–18842. doi: 10.1074/jbc.M111.323287
[178] Hammerschmidt C, Koenigs A, Siegel C, et al. Versatile
roles of CspA orthologs in complement Inactivation of
serum-resistant Lyme disease spirochetes. Infect
Immun. 2014;82(1):380–392. doi: 10.1128/IAI.01094-13
[179] Koenigs A, Hammerschmidt C, Jutras BL, et al. BBA70
of Borrelia burgdorferi is a novel plasminogen-binding
protein. J Biol Chem. 2013;288(35):25229–25243. doi:
10.1074/jbc.M112.413872
[180] Önder Ö, Humphrey PT, McOmber B, et al. OspC Is
Potent Plasminogen Receptor on Surface of Borrelia
burgdorferi. J Biol Chem. 2012;287(20):16860–16868.
doi: 10.1074/jbc.M111.290775
[181] Fuchs H, Wallich R, Simon MM, et al. The outer sur­
face protein a of the spirochete Borrelia burgdorferi is
a plasmin(ogen) receptor. Proc Natil Acad Sci.
1994;91:12594–12598.
[182] Brissette CA, Haupt K, Barthel D, et al. Borrelia burg­
dorferi Infection-Associated Surface Proteins ErpP,
ErpA, and ErpC Bind Human Plasminogen. Infect
Immun. 2009;77(1):300–306. doi: 10.1128/IAI.01133-08
[183] Haupt K, Kraiczy P, Wallich R, et al. FHR-1, an addi­
tional human plasma protein, binds to complement
regulator-acquiring surface proteins of Borrelia
burgdorferi. Int J Med Microbiol. 2008;298:287–291.
doi: 10.1016/j.ijmm.2007.11.010
[184] Kraiczy P, Rossmann E, Brade V, et al. Binding of
human complement regulators FHL-1 and factor H to
CRASP-1 orthologs of Borrelia burgdorferi. Wien Klin
Wochenschr. 2006;118(21–22):669–676. doi: 10.1007/
s00508-006-0691-1
[185] Hallström T, Siegel C, Mörgelin M, et al. CspA from
Borrelia burgdorferi inhibits the terminal complement
pathway. MBio. 2013;4(4):4. doi: 10.1128/mBio.00481-13
[186] Hammerschmidt C, Klevenhaus Y, Koenigs A, et al.
BGA66 and BGA71 facilitate complement resistance of
Borrelia bavariensis by inhibiting assembly of the
membrane attack complex. Mol Microbiol. 2016;99
(2):407–424. doi: 10.1111/mmi.13239
[187] Hellwage J, Meri T, Heikkilä T, et al. The complement
regulator factor H binds to the surface protein OspE of
Borrelia burgdorferi. J Biol Chem. 2001;276
(11):8427–8435. doi: 10.1074/jbc.M007994200
[188] Kraiczy P, Hellwage J, Skerka C, et al. Immune evasion of
Borrelia burgdorferi: mapping of a complement-inhibitor
factor H-binding site of BbCRASP-3, a novel member of
the Erp protein family. Eur J Immunol. 2003;33
(3):697–707. doi: 10.1002/eji.200323571

[189] Zhang J-R, Norris SJ. Genetic variation of the Borrelia
burgdorferi gene vlsE involves cassette-specific, seg­
mental gene conversion. Infect Immun. 1998;66
(8):3698–3704. doi: 10.1128/IAI.66.8.3698-3704.1998
[190] Frank SA Benefits of antigenic variation [Internet].
Immunology and Evolution of infectious disease.
Princeton University Press; 2002 [cited 2022 Oct 12].
Available from: https://www.ncbi.nlm.nih.gov/books/
NBK2405/
[191] Tilly K, Bestor A, Rosa PA. Lipoprotein succession in
Borrelia burgdorferi: similar but distinct roles for OspC
and VlsE at different stages of mammalian infection.
Mol Microbiol. 2013;89(2):216–227. doi: 10.1111/mmi.
12271
[192] Brisson D, DE D. ospC diversity in Borrelia burgdor­
feri: different hosts are different niches. Genetics.
2004;168(2):713–722. doi: 10.1534/genetics.104.028738
[193] Brisson D, Baxamusa N, Schwartz I, et al. Biodiversity
of Borrelia burgdorferi strains in tissues of Lyme dis­
ease patients. PLoS One. 2011;6(8):e22926. doi: 10.
1371/journal.pone.0022926
[194] Ivanova L, Christova I, Neves V, et al. Comprehensive
seroprofiling of sixteen B. burgdorferi OspC: implica­
tions for Lyme disease diagnostics design. Clin
Immunol. 2009;132(3):393–400. doi: 10.1016/j.clim.
2009.05.017
[195] Dykhuizen DE, Brisson D, Sandigursky S, et al. The
propensity of different Borrelia burgdorferi sensu
stricto genotypes to cause disseminated infections in
humans. Am J Trop Med Hyg. 2008;78(5):806–810.
doi: 10.4269/ajtmh.2008.78.806
[196] Sadziene A, Wilske B, Ferdows MS, et al. The cryptic
ospC gene of Borrelia burgdorferi B31 is located on
a circular plasmid. Infect Immun. 1993;61
(5):2192–2195. doi: 10.1128/iai.61.5.2192-2195.1993
[197] Gilmore RD, Kappel KJ, Dolan MC, et al. Outer surface
protein C (OspC), but not P39, is a protective immu­
nogen against a tick-transmitted Borrelia burgdorferi
challenge: evidence for a conformational protective
epitope in OspC. Infect Immun. 1996;64
(6):2234–2239. doi: 10.1128/iai.64.6.2234-2239.1996
[198] Bockenstedt LK, Hodzic E, Feng S, et al. Borrelia burg­
dorferi strain-specific Osp C-mediated immunity in
mice. Infect Immun. 1997;65(11):4661–4667. doi: 10.
1128/iai.65.11.4661-4667.1997
[199] Rudenko N, Golovchenko M, Hönig V, et al. Detection
of Borrelia burgdorferi sensu stricto ospC alleles asso­
ciated with human lyme borreliosis worldwide in
non-human-biting tick Ixodes affinis and rodent
hosts in Southeastern United States. Appl Environ
Microbiol. 2013;79(5):1444–1453. doi: 10.1128/AEM.
02749-12
[200] Lagal V, Portnoï D, Faure G, et al. Borrelia burgdorferi
sensu stricto invasiveness is correlated with OspC–
plasminogen affinity. Microbes Infect. 2006;8
(3):645–652. doi: 10.1016/j.micinf.2005.08.017
[201] Seemanapalli SV, Xu Q, McShan K, et al. Outer surface
protein C is a dissemination-facilitating factor of
Borrelia burgdorferi during mammalian infection.
PLoS One. 2010;5(12):e15830. doi: 10.1371/journal.
pone.0015830
VIRULENCE 27
[202] Xu Q, McShan K, Liang FT. Essential protective role
attributed to the surface lipoproteins of Borrelia burg­
dorferi against innate defences. Mol Microbiol. 2008;69
(1):15–29. doi: 10.1111/j.1365-2958.2008.06264.x
[203] Hovius JW, Schuijt TJ, de Groot KA, et al. Preferential
protection of Borrelia burgdorferi Sensu Stricto by
a Salp15 homologue in Ixodes ricinus Saliva. J Infect
Dis. 2008;198(8):1189–1197. doi: 10.1086/591917
[204] Caine JA, Coburn J, Morrison RP. A short-term
Borrelia burgdorferi infection model identifies tissue
tropisms and bloodstream survival conferred by adhe­
sion proteins. Infect Immun. 2015;83(8):3184–3194.
doi: 10.1128/IAI.00349-15
[205] Lin Y-P, Tan X, Caine JA, et al. Strain-specific joint
invasion and colonization by Lyme disease spirochetes
is promoted by outer surface protein C. PLOS
Pathogens. 2020;16(5):e1008516. doi: 10.1371/journal.
ppat.1008516
[206] Carrasco SE, Troxell B, Yang Y, et al. Outer surface
protein OspC is an antiphagocytic factor that protects
Borrelia burgdorferi from phagocytosis by
macrophages. Infect Immun. 2015;83(12):4848–4860.
doi: 10.1128/IAI.01215-15
[207] Salo J, Jaatinen A, Söderström M, et al. Decorin bind­
ing proteins of Borrelia burgdorferi promote arthritis
development and joint specific post-treatment DNA
persistence in mice. PLoS One. 2015;10(3):e0121512.
doi: 10.1371/journal.pone.0121512
[208] Shi Y, Xu Q, McShan K, et al. Both decorin-binding
proteins a and B are critical for the overall virulence of
Borrelia burgdorferi. Infect Immun. 2008;76
(3):1239–1246. doi: 10.1128/IAI.00897-07
[209] Imai DM, Samuels DS, Feng S, et al. The early disse­
mination defect attributed to disruption of
decorin-binding proteins is abolished in chronic mur­
ine Lyme borreliosis. Infect Immun. 2013;81
(5):1663–1673. doi: 10.1128/IAI.01359-12
[210] Weening EH, Parveen N, Trzeciakowski JP, et al. Borrelia
burgdorferi lacking DbpBA exhibits an early survival
defect during experimental infection. Infect Immun.
2008;76(12):5694–5705. doi: 10.1128/IAI.00690-08
[211] Benoit VM, Fischer JR, Lin Y-P, et al. Allelic variation
of the Lyme disease spirochete adhesin DbpA influ­
ences spirochetal binding to Decorin, dermatan sulfate,
and mammalian cells. Infect Immun. 2011;79
(9):3501–3509. doi: 10.1128/IAI.00163-11
[212] Salo J, Loimaranta V, Lahdenne P, et al. Decorin bind­
ing by DbpA and B of Borrelia garinii, Borrelia afzelii,
and Borrelia burgdorferi sensu stricto. J Infect Dis.
2011;204(1):65–73. doi: 10.1093/infdis/jir207
[213] Lin Y-P, Benoit V, Yang X, et al. Strain-specific varia­
tion of the decorin-binding adhesin DbpA influences
the tissue tropism of the lyme disease spirochete. PLOS
Pathog. 2014;10(7):e1004238. doi: 10.1371/journal.
ppat.1004238
[214] Crother TR, Champion CI, Wu X-Y, et al. Antigenic
composition of Borrelia burgdorferi during infection of
SCID mice. Infect Immun. 2003;71(6):3419–3428. doi:
10.1128/IAI.71.6.3419-3428.2003
[215] Crother TR, Champion CI, Whitelegge JP, et al.
Temporal analysis of the antigenic composition of
28 M. STRNAD ET AL.
Borrelia burgdorferi during infection in rabbit skin.
Infect Immun. 2004;72(9):5063–5072. doi: 10.1128/
IAI.72.9.5063-5072.2004
[216] Coutte L, Botkin DJ, Gao L, et al. Detailed analysis of
sequence changes occurring during vlsE antigenic var­
iation in the mouse model of Borrelia burgdorferi
infection. PLOS Pathog. 2009;5(2):e1000293. doi: 10.
1371/journal.ppat.1000293
[217] Bankhead T, Chaconas G. The role of VlsE antigenic
variation in the Lyme disease spirochete: persistence
through a mechanism that differs from other
pathogens. Mol Microbiol. 2007;65(6):1547–1558. doi:
10.1111/j.1365-2958.2007.05895.x
[218] Glöckner G, Schulte-Spechtel U, Schilhabel M, et al.
Comparative genome analysis: selection pressure on
the Borrelia vls cassettes is essential for infectivity.
BMC Genomics. 2006;7(1):211. doi: 10.1186/1471-
2164-7-211
[219] Zhang JR, Hardham JM, Barbour AG, et al. Antigenic
variation in Lyme disease borreliae by promiscuous
recombination of VMP-like sequence cassettes. Cell.
1997;89(2):275–285. doi: 10.1016/S0092-8674(00)
80206-8
[220] Wang D, Botkin DJ, Norris SJ. Characterization of the
vls antigenic variation loci of the Lyme disease spiro­
chaetes Borrelia garinii Ip90 and Borrelia afzelii ACAI.
Mol Microbiol. 2003;47(5):1407–1417. doi: 10.1046/j.
1365-2958.2003.03386.x
[221] Verhey TB, Castellanos M, Chaconas G. Antigenic
variation in the Lyme spirochete: insights into recom­
binational switching with a suggested role for
error-prone repair. Cell Rep. 2018;23(9):2595–2605.
doi: 10.1016/j.celrep.2018.04.117
[222] Lin T, Gao L, Edmondson DG, et al. Central role of the
holliday junction helicase RuvAB in vlsE recombina­
tion and infectivity of Borrelia burgdorferi. PLOS
Pathog. 2009;5(12):e1000679. doi: 10.1371/journal.
ppat.1000679
[223] Lone AG, Bankhead T. The Borrelia burgdorferi VlsE
lipoprotein prevents antibody binding to an
arthritis-related surface Antigen. Cell Rep. 2020;30
(11):3663–3670.e5. doi: 10.1016/j.celrep.2020.02.081
[224] Kumar D, Ristow LC, Shi M, et al. Intravital imaging of
vascular transmigration by the Lyme spirochete:
requirement for the integrin binding residues of the
B. burgdorferi P66 protein. PLOS Pathog. 2015;11(12):
e1005333. doi: 10.1371/journal.ppat.1005333
[225] Ebady R, Niddam AF, Boczula AE, et al. Biomechanics of
Borrelia burgdorferi Vascular Interactions. Cell Rep.
2016;16(10):2593–2604. doi: 10.1016/j.celrep.2016.08.013
[226] Tan X, Lin Y-P, Pereira MJ, et al. VlsE, the nexus for
antigenic variation of the Lyme disease spirochete, also
mediates early bacterial attachment to the host micro­
vasculature under shear force. PLOS Pathog. 2022;18
(5):e1010511. doi: 10.1371/journal.ppat.1010511
[227] Tan X, Castellanos M, Chaconas G, et al.
Choreography of Lyme disease spirochete adhesins to
promote vascular escape. Microbiol Spectr. 2023;11(4):
e0125423. doi: 10.1128/spectrum.01254-23
[228] Sultan SZ, Manne A, Stewart PE, et al. Motility is
crucial for the infectious life cycle of Borrelia
burgdorferi. Infect Immun. 2013;81(6):2012–2021.
doi: 10.1128/IAI.01228-12
[229] Ge Y, Old I, Saint Girons I, et al. FliH and fliI of
Borrelia burgdorferi are similar to flagellar and viru­
lence factor export proteins of other bacteria. Gene.
1996;168(1):73–75. doi: 10.1016/0378-1119(95)00743-1
[230] Li C, Xu H, Zhang K, et al. Inactivation of a putative
flagellar motor switch protein FliG1 prevents Borrelia
burgdorferi from swimming in highly viscous media
and blocks its infectivity. Mol Microbiol. 2010;75
(6):1563–1576. doi: 10.1111/j.1365-2958.2010.07078.x
[231] Charon NW, Goldstein SF. Genetics of motility and
chemotaxis of a fascinating group of bacteria: the
spirochetes. Ann Rev Genet. 2002;36(1):47–73. doi:
10.1146/annurev.genet.36.041602.134359
[232] Motaleb MA, Corum L, Bono JL, et al. Borrelia burg­
dorferi periplasmic flagella have both skeletal and
motility functions. Proc Natl Acad Sci, USA. 2000;97
(20):10899–10904. doi: 10.1073/pnas.200221797
[233] DeHart TG, Kushelman MR, Hildreth SB, et al. The
unusual cell wall of the Lyme disease spirochaete
Borrelia burgdorferi is shaped by a tick sugar. Nat
Microbiol. 2021;6(12):1583–1592. doi: 10.1038/s41564-
021-01003-w
[234] Chen Y, Vargas SM, Smith TC, et al. Borrelia peptido­
glycan interacting protein (BpiP) contributes to the
fitness of Borrelia burgdorferi against host-derived fac­
tors and influences virulence in mouse models of Lyme
disease. PLOS Pathog. 2021;17(4):e1009535. doi: 10.
1371/journal.ppat.1009535
[235] Liang L, Wang J, Schorter L, et al. Rapid clearance of
Borrelia burgdorferi from the blood circulation.
Parasites Vectors. 2020;13(1):191. doi: 10.1186/
s13071-020-04060-y
[236] Hodzic E, Feng S, Freet KJ, et al. Borrelia burgdorferi
population dynamics and prototype gene expression
during infection of immunocompetent and immuno­
deficient mice. Infect Immun. 2003;71(9):5042–5055.
doi: 10.1128/IAI.71.9.5042-5055.2003
[237] Tilly K, Rosa PA, Stewart PE. Biology of infection with
Borrelia burgdorferi. Infect Dis Clin North Am.
2008;22(2):217–234. doi: 10.1016/j.idc.2007.12.013
[238] Cabello FC, Godfrey HP, Newman SA. Hidden in plain
sight: borrelia burgdorferi and the extracellular matrix.
Trends Microbiol. 2007;15(8):350–354. doi: 10.1016/j.
tim.2007.06.003
[239] Paulsen F, Tillmann B. Composition of the extracellu­
lar matrix in human cricoarytenoid joint articular
cartilage. Arch Histol Cytol. 1999;62(2):149–163. doi:
10.1679/aohc.62.149
[240] Vechtova P, Sterbova J, Sterba J, et al. A bite so sweet:
the glycobiology interface of tick-host-pathogen
interactions. Parasites Vectors. 2018;11(1):594. doi:
10.1186/s13071-018-3062-7
[241] Salo J, Pietikäinen A, Söderström M, et al. Flow-
tolerant adhesion of a bacterial pathogen to human
endothelial cells through interaction with biglycan.
J Infect Dis. 2016;213(10):1623–1631. doi: 10.1093/
infdis/jiw003
[242] Brissette CA, Bykowski T, Cooley AE, et al. Borrelia
burgdorferi RevA antigen binds host fibronectin. Infect

Immun. 2009;77(7):2802–2812. doi: 10.1128/IAI.
00227-09
[243] Hallström T, Haupt K, Kraiczy P, et al. Complement
Regulator–Acquiring Surface Protein 1 of Borrelia
burgdorferi Binds to Human Bone Morphogenic
Protein 2, Several Extracellular Matrix Proteins, and
Plasminogen. J Infect Dis. 2010;202(3):490–498. doi:
10.1086/653825
[244] Zhi H, Weening EH, Barbu EM, et al. The BBA33
lipoprotein binds collagen and impacts Borrelia burg­
dorferi pathogenesis. Mol Microbiol. 2015;96(1):68–83.
doi: 10.1111/mmi.12921
[245] Guo BP, Norris SJ, Rosenberg LC, et al. Adherence of
Borrelia burgdorferi to the proteoglycan decorin. Infect
Immun. 1995;63(9):3467–3472. doi: 10.1128/iai.63.9.
3467-3472.1995
[246] Guo BP, Brown EL, Dorward DW, et al. Decorin-
binding adhesins from Borrelia burgdorferi. Mol
Microbiol. 1998;30(4):711–723. doi: 10.1046/j.1365-
2958.1998.01103.x
[247] Fischer JR, LeBlanc KT, Leong JM. Fibronectin binding
protein BBK32 of the Lyme disease spirochete pro­
motes bacterial attachment to glycosaminoglycans.
Infect Immun. 2006;74(1):435–441. doi: 10.1128/IAI.
74.1.435-441.2006
[248] Moriarty TJ, Shi M, Lin Y-P, et al. Vascular binding of
a pathogen under shear force through mechanistically
distinct sequential interactions with host
macromolecules. Mol Microbiol. 2012;86
(5):1116–1131. doi: 10.1111/mmi.12045
[249] Gaultney RA, Gonzalez T, Floden AM, et al. BB0347,
from the lyme disease spirochete Borrelia burgdorferi, is
surface exposed and interacts with the CS1
heparin-binding domain of human fibronectin. PLoS
One. 2013;8(9):e75643. doi: 10.1371/journal.pone.
0075643
[250] Coburn J, Magoun L, Bodary SC, et al. Integrins αvβ3
and α5β1 mediate attachment of Lyme disease spiro­
chetes to human cells. Infect Immun. 1998;66
(5):1946–1952. doi: 10.1128/IAI.66.5.1946-1952.1998
[251] Behera AK, Durand E, Cugini C, et al. Borrelia burgdor­
feri BBB07 interaction with integrin alpha3beta1 stimu­
lates production of pro-inflammatory mediators in
primary human chondrocytes. Cell Microbiol.
2008;10:320–331. doi: 10.1111/j.1462-5822.2007.01043.x
[252] Ristow LC, Bonde M, Lin Y, et al. Integrin binding by
B orrelia burgdorferi P66 facilitates dissemination but
is not required for infectivity. Cell Microbiol.
2015;17:1021–1036. doi: 10.1111/cmi.12418
[253] Wood E, Tamborero S, Mingarro I, et al. BB0172,
a Borrelia burgdorferi Outer Membrane Protein That
Binds Integrin α3β1. J Bacteriol. 2013;195
(15):3320–3330. doi: 10.1128/JB.00187-13
[254] Verma A, Brissette CA, Bowman A, et al. Borrelia burg­
dorferi BmpA is a laminin-binding protein. Infect Immun.
2009;77(11):4940–4946. doi: 10.1128/IAI.01420-08
[255] Brissette CA, Verma A, Bowman A, et al. The Borrelia
burgdorferi outer-surface protein ErpX binds mamma­
lian laminin. Microbiol (Reading). 2009;55:863–872.
[256] Bista S, Singh P, Bernard Q, et al. A novel
laminin-binding protein mediates microbial-endothelial
cell interactions and facilitates dissemination of Lyme
VIRULENCE 29
disease pathogens. J Infect Dis. 2020;221(9):1438–1447.
doi: 10.1093/infdis/jiz626
[257] Lin Y-P, Chen Q, Ritchie JA, et al. Glycosaminoglycan
binding by Borrelia burgdorferi adhesin BBK32 speci­
fically and uniquely promotes joint colonization. Cell
Microbiol. 2015;17(6):860–875. doi: 10.1111/cmi.12407
[258] Parveen N, Leong JM. Identification of a candidate
glycosaminoglycan-binding adhesin of the Lyme dis­
ease spirochete Borrelia burgdorferi. Mol Microbiol.
2000;35(5):1220–1234. doi: 10.1046/j.1365-2958.2000.
01792.x
[259] Fischer JR, Parveen N, Magoun L, et al. Decorin-
binding proteins a and B confer distinct mammalian
cell type-specific attachment by Borrelia burgdorferi,
the Lyme disease spirochete. Proc Natl Acad Sci U S A.
2003;100(12):7307–7312. doi: 10.1073/pnas.
1231043100
[260] Yang X, Lin Y-P, Heselpoth RD, et al. Middle region of
the Borrelia burgdorferi surface-located protein 1
(Lmp1) interacts with host chondroitin-6-sulfate and
independently facilitates infection. Cell Microbiol.
2016;18(1):97–110. doi: 10.1111/cmi.12487
[261] Dowdell AS, Murphy MD, Azodi C, et al.
Comprehensive spatial analysis of the Borrelia burg­
dorferi lipoproteome reveals a Compartmentalization
Bias toward the bacterial surface. J Bacteriol. 2017;199
(6):e00658–16. doi: 10.1128/JB.00658-16
[262] Setubal JC, Reis M, Matsunaga J, et al. Lipoprotein
computational prediction in spirochaetal genomes.
Microbiology (Reading). 2006;152(1):113–121. doi: 10.
1099/mic.0.28317-0
[263] Leong JM, Robbins D, Rosenfeld L, et al. Structural
requirements for glycosaminoglycan recognition by the
Lyme disease spirochete, Borrelia burgdorferi. Infect
Immun. 1998;66(12):6045–6048. doi: 10.1128/IAI.66.
12.6045-6048.1998
[264] Seshu J, Esteve-Gassent MD, Labandeira-Rey M, et al.
Inactivation of the fibronectin-binding adhesin gene
bbk32 significantly attenuates the infectivity potential of
Borrelia burgdorferi. Mol Microbiol. 2006;59
(5):1591–1601. doi: 10.1111/j.1365-2958.2005.05042.x
[265] Li X, Liu X, Beck DS, et al. Borrelia burgdorferi lacking
BBK32, a fibronectin-binding protein, retains full
pathogenicity. Infect Immun. 2006;74(6):3305–3313.
doi: 10.1128/IAI.02035-05
[266] Byram R, Gaultney RA, Floden AM, et al. Borrelia
burgdorferi RevA significantly affects pathogenicity
and host response in the mouse model of Lyme
disease. Infect Immun. 2015;83(9):3675–3683. doi: 10.
1128/IAI.00530-15
[267] Norman MU, Moriarty TJ, Dresser AR, et al. Molecular
mechanisms involved in vascular interactions of the
Lyme disease pathogen in a living host. PLOS Pathog.
2008;4(10):e1000169. doi: 10.1371/journal.ppat.
1000169
[268] Meriläinen L, Brander H, Herranen A, et al.
Pleomorphic forms of Borrelia burgdorferi induce dis­
tinct immune responses. Microbes Infect. 2016;18(7–
8):484–495. doi: 10.1016/j.micinf.2016.04.002
[269] Vancová M, Rudenko N, Vaněček J, et al.
Pleomorphism and viability of the Lyme disease patho­
gen Borrelia burgdorferi exposed to physiological stress
30 M. STRNAD ET AL.
conditions: a correlative cryo-fluorescence and
cryo-scanning electron microscopy study. Front
Microbiol. 2017;8:596. doi: 10.3389/fmicb.2017.00596
[270] Brorson O, Brorson SH. Transformation of cystic
forms of Borrelia burgdorferi to normal, mobile
spirochetes. Infection. 1997;25(4):240–246. doi: 10.
1007/BF01713153
[271] Brorson O, Brorson SH. In vitro conversion of Borrelia
burgdorferi to cystic forms in spinal fluid, and trans­
formation to mobile spirochetes by incubation in
BSK-H medium. Infection. 1998;26(3):144–150. doi:
10.1007/BF02771839
[272] Dunham-Ems SM, Caimano MJ, Eggers CH, et al.
Borrelia burgdorferi requires the alternative sigma fac­
tor RpoS for dissemination within the vector during
tick-to-Mammal transmission. PLOS Pathog. 2012;8
(2):e1002532. doi: 10.1371/journal.ppat.1002532
[273] Lantos PM, Auwaerter PG, Wormser GP. A systematic
review of Borrelia burgdorferi morphologic variants
does not support a role in chronic Lyme disease. Clin
Infect Dis. 2014;58(5):663. doi: 10.1093/cid/cit810
[274] Torres JP, Senejani AG, Gaur G, et al. Ex Vivo Murine
Skin Model for B. burgdorferi Biofilm. Antibiotics.
2020;9(9):528. doi: 10.3390/antibiotics9090528
[275] Sapi E, Bastian SL, Mpoy CM, et al. Characterization of
biofilm formation by Borrelia burgdorferi in vitro.
PLoS One. 2012;7(10):e48277. doi: 10.1371/journal.
pone.0048277
[276] Wu J, Weening EH, Faske JB, et al. Invasion of
Eukaryotic cells by Borrelia burgdorferi requires β1
Integrins and src kinase activity. Infect Immun.
2011;79(3):1338–1348. doi: 10.1128/IAI.01188-10
[277] Klempner MS, Noring R, Rogers RA. Invasion of
human skin fibroblasts by the Lyme disease spirochete,
Borrelia burgdorferi. J Infect Dis. 1993;167
(5):1074–1081. doi: 10.1093/infdis/167.5.1074
[278] Aberer E, Surtov-Pudar M, Wilfinger D, et al. Co-
culture of human fibroblasts and Borrelia burgdorferi
enhances collagen and growth factor mRNA. Arch
Dermatol Res. 2018;310(2):117–126. doi: 10.1007/
s00403-017-1797-1
[279] Girschick HJ, Huppertz HI, Rüssmann H, et al.
Intracellular persistence of Borrelia burgdorferi in
human synovial cells. Rheumatol Int. 1996;16
(3):125–132. doi: 10.1007/BF01409985
[280] Ma Y, Sturrock A, Weis JJ. Intracellular localization of
Borrelia burgdorferi within human endothelial cells.
Infect Immun. 1991;59(2):671–678. doi: 10.1128/iai.
59.2.671-678.1991
[281] Livengood JA, Gilmore RD. Invasion of human neuro­
nal and glial cells by an infectious strain of Borrelia
burgdorferi. Microbes Infect. 2006;8(14–
15):2832–2840. doi: 10.1016/j.micinf.2006.08.014
[282] Kerstholt M, Netea MG, Joosten LAB. Borrelia burg­
dorferi hijacks cellular metabolism of immune cells:
Consequences for host defense. Ticks Tick Borne Dis.
2020;11(3):101386. doi: 10.1016/j.ttbdis.2020.101386
[283] Mayer KA, Stöckl J, Zlabinger GJ, et al. Hijacking the
supplies: metabolism as a novel facet of virus-host
interaction. Front Immunol. 2019;10:1533. doi: 10.
3389/fimmu.2019.01533
[284] Eisenreich W, Rudel T, Heesemann J, et al. How viral
and intracellular bacterial pathogens reprogram the
metabolism of host cells to allow their intracellular
replication. Front Cell Infect Microbiol. 2019;9:42.
doi: 10.3389/fcimb.2019.00042
[285] Gehre L, Gorgette O, Perrinet S, et al. Sequestration of
host metabolism by an intracellular pathogen. Elife.
2016;5:e12552. doi: 10.7554/eLife.12552
[286] Linder S, Heimerl C, Fingerle V, et al. Coiling pha­
gocytosis of Borrelia burgdorferi by primary human
macrophages is controlled by CDC42Hs and Rac1
and involves recruitment of wiskott-aldrich syn­
drome protein and Arp2/3 complex. Infect Immun.
2001;69(3):1739–1746. doi: 10.1128/IAI.69.3.1739-
1746.2001
[287] Siryaporn A, Kuchma SL, O’Toole GA, et al. Surface
attachment induces Pseudomonas aeruginosa
virulence. Proc Natl Acad Sci U S A. 2014;111
(47):16860–16865. doi: 10.1073/pnas.1415712111
[288] Carapeto AP, Vitorino MV, Santos JD, et al.
Mechanical properties of human bronchial epithelial
cells expressing wt- and mutant CFTR. Int J Mol Sci.
2020;21(8):E2916. doi: 10.3390/ijms21082916
[289] Patel NR, Bole M, Chen C, et al. Cell Elasticity
Determines Macrophage Function. PLoS One. 2012;7
(9):e41024. doi: 10.1371/journal.pone.0041024
[290] Costa TRD, Felisberto-Rodrigues C, Meir A, et al.
Secretion systems in Gram-negative bacteria: structural
and mechanistic insights. Nat Rev Microbiol. 2015;13
(6):343–359. doi: 10.1038/nrmicro3456
[291] Schulze RJ, Zückert WR. Borrelia burgdorferi lipopro­
teins are secreted to the outer surface by default. Mol
Microbiol. 2006;59(5):1473–1484. doi: 10.1111/j.1365-
2958.2006.05039.x
[292] Jan AT. Outer membrane vesicles (OMVs) of
gram-negative bacteria: a perspective update. Front
Microbiol. 2017;8:1053. doi: 10.3389/fmicb.2017.01053
[293] Anand D, Chaudhuri A. Bacterial outer membrane vesi­
cles: new insights and applications. Mol Membr Biol.
2016;33(6–8):125–137. doi: 10.1080/09687688.2017.
1400602
[294] Kuehn MJ, Kesty NC. Bacterial outer membrane vesi­
cles and the host-pathogen interaction. Genes Dev.
2005;19:2645–2655. doi: 10.1101/gad.1299905
[295] Kudryashev M, Cyrklaff M, Baumeister W, et al.
Comparative cryo-electron tomography of pathogenic
Lyme disease spirochetes. Mol Microbiol. 2009;71
(6):1415–1434. doi: 10.1111/j.1365-2958.2009.06613.x
[296] Karvonen K Mechanisms and elimination of Borrelia
burgdorferi persistence in vitro. JYU Dissertations
[Internet]. 2022 [cited 2022 Oct 11]. Available from:
https://jyx.jyu.fi/handle/123456789/79467
[297] Davis MM, Brock AM, DeHart TG, et al. The
peptidoglycan-associated protein NapA plays an
important role in the envelope integrity and in the
pathogenesis of the lyme disease spirochete. PLOS
Pathog. 2021;17(5):e1009546. doi: 10.1371/journal.
ppat.1009546
[298] Cluss RG, Silverman DA, Stafford TR. Extracellular
secretion of the Borrelia burgdorferi Oms28 porin
and Bgp, a glycosaminoglycan binding protein. Infect

Immun. 2004;72(11):6279–6286. doi: 10.1128/IAI.72.
11.6279-6286.2004
[299] Skare JT, Shang ES, Foley DM, et al. Virulent strain
associated outer membrane proteins of Borrelia
burgdorferi. J Clin Invest. 1995;96(5):2380–2392. doi:
10.1172/JCI118295
[300] Parveen N, Cornell KA, Bono JL, et al. Bgp, a secreted
glycosaminoglycan-binding protein of Borrelia burg­
dorferi strain N40, displays nucleosidase activity and
is not essential for infection of immunodeficient mice.
Infect Immun. 2006;74(5):3016–3020. doi: 10.1128/IAI.
74.5.3016-3020.2006
[301] Garon CF, Dorward DW, Corwin MD. Structural fea­
tures of Borrelia burgdorferi–the Lyme disease spiro­
chete: silver staining for nucleic acids. Scanning
Microsc Suppl. 1989;3:109–115.
[302] Crowley JT, Toledo AM, LaRocca TJ, et al. Lipid
Exchange between Borrelia burgdorferi and Host
Cells. PLOS Pathog. 2013;9(1):e1003109. doi: 10.1371/
journal.ppat.1003109
[303] Groshong AM, McLain MA, Radolf JD, et al. Host-
specific functional compartmentalization within the
oligopeptide transporter during the Borrelia burgdor­
feri enzootic cycle. PLOS Pathogens. 2021;17(1):
e1009180. doi: 10.1371/journal.ppat.1009180
[304] LaRocca TJ, Crowley JT, Cusack BJ, et al. Cholesterol
lipids of Borrelia burgdorferi form lipid rafts and are
required for the bactericidal mechanism of a
complement-independent antibody. Cell Host Microbe.
2010;8(4):331–342. doi: 10.1016/j.chom.2010.09.001
[305] Jain S, Showman AC, Jewett MW, et al. Molecular
Dissection of a Borrelia burgdorferi In Vivo Essential
Purine Transport System. Infect Immun. 2015;83
(6):2224–2233. doi: 10.1128/IAI.02859-14
[306] Pappas CJ, Iyer R, Petzke MM, et al. Borrelia burgdor­
feri requires glycerol for maximum fitness during the
tick phase of the enzootic cycle. PLOS Pathog. 2011;7
(7):e1002102. doi: 10.1371/journal.ppat.1002102
[307] Corona A, Schwartz I, Conway T, et al. Borrelia burg­
dorferi: Carbon metabolism and the tick-Mammal
enzootic cycle. Microbiol Spectr. 2015;3(3):10.1128/
microbiolspec.MBP-0011–2014. doi: 10.1128/microbiol
spec.MBP-0011-2014
[308] Skare JT, Mirzabekov TA, Shang ES, et al. The Oms66
(p66) protein is a Borrelia burgdorferi porin. Infect
Immun. 1997;65(9):3654–3661. doi: 10.1128/iai.65.9.
3654-3661.1997
[309] Skare JT, Champion CI, Mirzabekov TA, et al. Porin activ­
ity of the native and recombinant outer membrane protein
Oms28 of Borrelia burgdorferi. J Bacteriol. 1996;178
(16):4909–4918. doi: 10.1128/jb.178.16.4909-4918.1996
[310] von Lackum K, Stevenson B. Carbohydrate utilization
by the Lyme borreliosis spirochete, Borrelia
burgdorferi. FEMS Microbiol Lett. 2005;243
(1):173–179. doi: 10.1016/j.femsle.2004.12.002
[311] Hoon-Hanks LL, Morton EA, Lybecker MC, et al.
Borrelia burgdorferi malQ mutants utilize
VIRULENCE 31
disaccharides and traverse the enzootic cycle. FEMS
Immunol Med Microbiol. 2012;66(2):157–165. doi: 10.
1111/j.1574-695X.2012.00996.x
[312] Görke B, Stülke J. Carbon catabolite repression in
bacteria: many ways to make the most out of
nutrients. Nat Rev Microbiol. 2008;6(8):613–624. doi:
10.1038/nrmicro1932
[313] Zhang J-J, Ranghunandanan S, Wang Q, et al.
Mechanism of repression of the glycerol utilization
operon in Borrelia burgdorferi Internet].
bioRxivAvailable from. 2022 cited 2023 Jun
12:2022.11.01.514788. 10.1101/2022.11.01.514788v1.
[314] Shaw DK, Hyde JA, Skare JT. The BB0646 protein
demonstrates lipase and hemolytic activity associated
with Borrelia burgdorferi, the aetiological agent of
Lyme disease. Mol Microbiol. 2012;83:319–334. doi:
10.1111/j.1365-2958.2011.07932.x
[315] Wang X-G, Scagliotti JP, Hu LT. Phospholipid synth­
esis in Borrelia burgdorferi: BB0249 and BB0721
encode functional phosphatidylcholine synthase and
phosphatidylglycerolphosphate synthase proteins.
Microbiology (Reading). 2004;150(2):391–397. doi: 10.
1099/mic.0.26752-0
[316] Belisle JT, Brandt ME, Radolf JD, et al. Fatty acids of
Treponema pallidum and Borrelia burgdorferi
lipoproteins. J Bacteriol. 1994;176(8):2151–2157. doi:
10.1128/jb.176.8.2151-2157.1994
[317] Drecktrah D, Hall LS, Crouse B, et al. The
glycerol-3-phosphate dehydrogenases GpsA and GlpD
constitute the oxidoreductive metabolic linchpin for
Lyme disease spirochete host infectivity and persistence
in the tick. PLOS Pathog. 2022;18(3):e1010385. doi: 10.
1371/journal.ppat.1010385
[318] Purser JE, Lawrenz MB, Caimano MJ, et al. A
plasmid-encoded nicotinamidase (PncA) is essential
for infectivity of Borrelia burgdorferi in a mammalian
host. Mol Microbiol. 2003;48(3):753–764. doi: 10.1046/
j.1365-2958.2003.03452.x
[319] Schüler W, Bunikis I, Weber-Lehman J, et al. Complete
genome sequence of Borrelia afzelii K78 and compara­
tive genome analysis. PLoS One. 2015;10(3):e0120548.
doi: 10.1371/journal.pone.0120548
[320] Schutzer SE, Fraser-Liggett CM, Qiu W-G, et al.
Whole-Genome Sequences of Borrelia bissettii,
Borrelia valaisiana, and Borrelia spielmanii.
J Bacteriol. 2012;194(2):545–546. doi: 10.1128/JB.
06263-11
[321] Kingry LC, Batra D, Replogle A, et al. Whole genome
sequence and Comparative genomics of the novel
Lyme borreliosis causing pathogen, Borrelia mayonii.
PLoS One. 2016;11(12):e0168994. doi: 10.1371/journal.
pone.0168994
[322] Rego ROM, Trentelman JJA, Anguita J, et al.
Counterattacking the tick bite: towards a rational
design of anti-tick vaccines targeting pathogen
transmission. Parasites Vectors. 2019;12(1):229. doi:
10.1186/s13071-019-3468-x