Borrelia burgdorferi and Lyme Disease

History

The first case of the Lyme disease was in Lyme, Connecticut in 1975. The disease presented arthritic-like symptoms, and
was therefore referred to as Lyme arthritis. The deer tick, Ixodes scapularis, was associated with the transmission of the
disease in 1977, but the cause of the disease remained unknown until Willy Burgdorferi discovered Borrelia burgdorferi
in 1981. The disease is caused by three species of bacteria all belonging to Borrelia-Borrelia burgdorferi, Borrelia afzelii,
and Borrelia garinii. Borrelia burgdorferi is the main cause of Lyme disease in North America, where the other two
species affect Europe[1, 2].

Borrelia burgdorferi Description and Structure

Borrelia burgdorferi is a Gram-negative bacterium belonging to the class Spirochaetes [Figure 1] . This bacterium is
helical and has both an inner and outer membrane as well as a flexible cell wall. The cell is usually 1um wide, but can be
up to 10-25 um long. Bacteria of the class spirochaetes have flagella located on the inside of the periplasm in between
the inner and outer membranes. The flagellum along with the helical structure of the bacterium allows the bacterium to
migrate through viscous fluids and burrow through various tissues. As a result, this cell is highly invasive. B. burgdorferi
is also known for its outer surface proteins OspA and OspC have been studied extensively and have a role in
transmission of the bacteria into the host cell. The metabolism of this cell is limited, therefore; B. burgdorferi relies on
their host for energy precursors. Its genome encodes transport proteins such as ABC transporters. B. burgdorferi also
codes for enzymes and proteins that are used in the phosphotransferase system. The host serum as well as the
environment are targets for these transport systems. The genome of this bacterium is distinctive. It consists of one
linear chromosome of 910,725 base pairs long with at least 17 linear and circular plasmids that combine to a size of
more than 533,000 base pairs [2] . As a result of its large number of plasmids, the genetic organization of B. burgdorferi
is specialized.

What is Lyme Disease?

Lyme disease is an infectious disease caused by the spirochete Borrelia burgdorferi that is carried in deer tick Ixodes
scapularis. This disease is the most common vector borne disease in the United States, affecting far more individuals
than West Nile virus. It has been estimates that there were nearly 40,000 cases of Lyme reported in 2009, a large
underestimate. The Center for Disease Control estimates the true number of cases to be as much as 12 times higher,
making the estimated total as high as 480,000. This estimate makes Lyme disease more prevalent than AIDS. Moreover,
national surveillance began in 1982, which increased the number of cases reported nearly 25-fold [3]. Lyme disease is
most commonly transmitted to humans through the bite of an infected tick as it bites [ Figure 2]. Without treatment, B.
burgdorferi is able to migrate through the bloodstream and many connective tissues. In it’s early stages, Lyme disease
affects the skin through skin rash and in later stages spreads to the joints, nervous system, and other organ systems in
the later stages. If caught and treated early, Lyme disease is almost always cured. However, this disease has a high rate
of progression and if it is not treated early, symptoms may last for months or even years. Unfortunately, thousands of
patients go undiagnosed or misdiagnosed with doctors telling them their symptoms are in their head due to the
difficulty in testing. Part of how this disease goes untreated and misdiagnosed is due to the characteristics of the
burrowing bacterium, Borrelia burgdorferi. This bacterium has become an expert at hiding and surviving in human
tissues, and its ability to take on different forms such as cysts, which allows the bacterium to escape the immune
system, avoid antibiotics, and hide from detection by blood tests. The mechanism of transmission is poorly understood,
but for many bacterial pathogens such as B. burgdorferi, the initial step in colonizing host tissues involves the expression
of adhesive molecules that facilitate bacterial adherence to host cells or to the extracellular matrix.

Symptoms

Lyme disease almost always first presents itself as a skin rash called erythema migrans [Figure 3]. The rash most likely
originates from the site of the tick bite and spreads. The rash can be a solid expanding red spot, or it can be a single red
spot surrounded by lighter red skin resembling a bulls-eye. The skin rash is visible 1 to 2 weeks after the disease has
been transmitted from the tick, and lasts for 3 to 5 weeks. Ticks are able to attach anywhere on the body, but-armpit,
groin, back of the knee, nape-body creases are what is most common. Symptoms that can present themselves at the
time of the rash include, joint pain, chills, fever, and fatigue. As the spirochete spreads throughout the body, more sever
symptoms begin to surface. The more serious symptoms include tingling or numbness in the extremities, paralysis,
severe fatigue and a stiff neck. Severe headaches, chronic arthritis, swelling of joints, cardiac abnormalities and mental
disorders can appear weeks, months, and in some cases, years after being bitten.

Treatment

When Lyme disease is caught early, within the first few weeks of initial infection, the treatment is straightforward and it
is highly likely that treatment will end in a cure. However, once it has been longer than three weeks since being infected,
the likelihood of finding a cure decreases as long as the patient goes untreated. According to the American Lyme Disease
Foundation, the most common antibiotics recommended for most symptoms of Lyme disease are Doxycycline,
amoxicillin, and ceftin. Treatment for Lyme disease can be frustrating and be seen as inconclusive due to the sporadic
progression of the disease. Some patients may experience symptoms of Lyme disease for years . The documentary,
Under Our Skin, tells the story of several patients whose lives have been destroyed by this disease. After being
misdiagnosed, undiagnosed, and untreated for so long, these patients experience extreme symptoms preventing them
from walking and carrying out a normal lifestyle.

Adhesion Mechanisms

The pathogenic bacterium Borrelia burgdorferi is the spirochete that causes Lyme disease. These spirochetes have
distinctive characteristics that allow them to cause chronic infection and remain in an infected human. Many
researchers have focused on the outer surface being proteins of B. burgdorferi as these proteins mediate the interaction
between the bacteria and the human host [Figure 5.] Adhesion to mammalian and tick substrates may have a role in the
host response to the bacteria as well as evasion from the host immune system [4]. The following agents of Borrelia
burgdorferi have been studied in hopes of understanding the mechanisms that allow these bacteria to cause infection,
and in some cases disease. There are a few characteristics of this bacterium that caught the attention of many
researchers. First of all, this bacterium has a large number of adhesion genes for its small (1.5 Mbp) genome. 7.8% of the
genome encodes known or predicted lipoproteins, where some have been classified as adhesions [5]. Within the small
genome, many proteins that bind to either mammalian or tick cells or extracellular matrix have been discovered and
contribute to the regime of these bacteria.

Borrelia burgdorferi has numerous proteins that support an interaction with the extracellular components of the host
cell. Different adhesions of Borrelia burgdorferi will be reviewed here to understand the characteristics of this bacterium
and the disease it causes.

Interaction with Extracellular Matrix

Fibronectin (Fn) is a large, dimeric glycoprotein and is produced by numerous cell types. It can exist as a soluble
molecule as well as an insoluble molecule, and can therefore reside in body fluids as well as in the extracellular matrix.
Pertaining to its structure, fibronectin is made up of three types of protein modules that are arranged in different
domains. Through these multiple domains, Fn is able to bind to many trans-membrane receptors as well as other
extracellular components including, collagen, fibrinogen, and proteoglycans. However, Fn specifically encourages cell
adhesion, growth, migration, and differentiation. Moreover, Fn plays an important role in wound healing and embryonic
development. Borrelia burgdorferi synthesizes multiple Fn-binding adhesions, with BBK32 being the binding protein best
characterized. BBK32 gene has been isolated from B. burgdorferi and has an Fn-binding region that has been localized to
32 amino acids and was found in B. burgdorferi sense stricto, B. garinii, and B. afzelii, which are strains known to cause
disease in humans. In the isolate, BBK32 remained on the outer surface and the adhesion specifically interacted with the
collagen-binding domain of fibronectin. The binding of BBK32 to Fn relates B. burgdorferi to Fn-binding adhesions of
gram-positive pathogens Staphylococcus aureus and Staphylococcus pyogenes. In relation to Lyme disease, in B.
burgdorferi BBK32 is expressed as the tick feeds and while the bacteria are in the mammalian host [6,2].

Adhesion to Host cell

Decorin is a proteoglycan with a protein core that consists primarily of leucine rich repeats and a glycosaminoglycan
protein. These proteins function as structural components of the extracellular matrix as well as signaling mediators. The
borrelial decorin-binding proteins, DbpA and DbpB, have been discovered and identified as targets for host cell decorin.
These surface lipoproteins are coded by the dbpB/A operon, Decorin binds collagen, which is found in many tissues as a
part of the connective tissue. This relationship allows B. burgdorferi to interact with connective tissues. Studies have
shown that the expression of both proteins is increased in response to a reduced pH and by a shift in temperature from
23 to 37 ° C, which illustrates their role in the mammalian environment. When DbpA and DbpB are expressed in B.
burgdorferi strain lacking the gene that codes for the dbpB/A operon, lp54, the strain was able to bind mammalian
epithelial cells. However, when the proteins are not expressed, no binding can occur [2].

Many studies have researched the role of DbpA and DbpB in the life cycle of B. burgdorferi and their contribution to
Lyme disease. Brown et. Al. reported the multiplication of bacterial organisms to be significantly lower in decorin-
deficient mice compared to wild-type mice [7]. Moreover, Shi et al. found that these proteins are not necessary for
causing an infection, but are critical for the overall virulence of the bacteria [8]. In regards to Lyme disease, decorin
binding proteins play a strong role in the later stages of disease-during dissemination and while establishing chronic
infections in decorin rich tissues.
In addition to decorin, there has been evidence of binding activity with more generalized proteoglycans (PGs). Even
though PG binding accounts for some of B. burgdorferi binding activity, it does not account for all. With PG binding,
recognizing glycosaminoglycan (GAG) chains are essential. Parveen et al. discovered a borrelia glycosaminoglycan
binding protein as it presented itself on the surface of the bacteria [9]. The different strains of Borrelia have different cell
and GAG binding preferences, which allows a variety in the cell types the bacteria can bind as well as the GAG proteins
expressed.

Persistence in ticks

Outer surface proteins (Osp) A and OspB are two lipoproteins that have a role in the persistence of B. burgdorferi in
ticks. Interestingly, these two proteins were discovered when antibodies specific to recognize OspA were reactive with
isolates from Lyme disease patients. The antigenic characteristic of this protein was also discovered. These proteins are
transcribed together from a single promoter and are encoded on the linear plasmid of B. burgdorferi [10]. As a result,
these two proteins have a high percentage of similarity in sequence and structure [11]. OspA and OspB are expressed in
the gut of starving ticks and their expression is down regulated once they begin feeding. OspA specifically facilitates the
binding of B. burgdorferi to the gut of the tick through the binding receptor TROSPA. Moreover, the expression of OspA
is down regulated in order to allow the spirochete to travel from the gut into the salivary glands. On the other hand,
OspB has its role in the bacterium’s ability to colonize and live in the gut of the tick. When working together, these
proteins allow for persistence of B. burgdorferi in the gut of a tick soon to be transferred to the human host.

Transmission from Tick to Host

OspC, like the previous outer surface proteins, is a lipoprotein that lies on the surface of the bacterium and is encoded
by a circular plasmid. As discussed earlier, the expression of OspA and OspB is down regulated soon after the tick is done
feeding. While, the expression of these proteins is down regulated, the expression of OspC is increased [12,13]. Osp
binds a salivary protein in ticks, Salp15, which accounts for its role in transmission from the tick to the host [14]. Grimm
et al. showed through their research that OspC is an essential component of B. burgdorferi to cause infection [15]. OspC
deletion mutants remain a virulent when infected through needle and tick [16]. Overall, OspC is required for
transmission and infection in the host cell.

Avoiding host immune response

As mentioned in the introduction, B. burgdorferi is able to remain in patients for long periods of time and establish
chronic infection in the host tissues. This bacterium has developed strategies in order to escape death by the host
immune system. An example of this type of mechanism is antigenic variation. “Antigenic variation has been defined as
changes in the structure or expression of antigenic proteins that occur during infection at a frequency greater than the
usual mutation rate” [Seifert, 1988], [17]. In their study, Zhang and collogues identified the vmp-like sequence (vls) in B.
burgdorferi. A single vls expression site (vlsE) along with 15 additional silent, or non-expressed, vls cassettes were
discovered on a linear plasmid. Genetic variation at the vls locus leads to antigenic variation. The mechanism of evading
the host immune system includes parts of multiple silent vls cassette sequences recombining into the main vlsE cassette,
which causes antigenic variation of the lipoprotein being expressed and results in many vlsE sequence products [18].
Overall, vlsE is significant to the bacterium to cause infection, through colonization, dissemination, adherence, and
evasion of host immune systems [19]. Antigenic variation allows for the expression of vlsE without the protein being
eliminated by the host cell immune response. As a result, the bacterium is able to reside in the host tissues and cause
chronic illness.

Future Work

With the increasing research of surface proteins, there has been talk of generating a new Lyme disease vaccine. There
was a Lyme disease vaccine made based on the outer surface lipoprotein A (OspA) that was approved, but then taken off
the market about ten years ago and is no longer used [20]. As more outer surface proteins of Borrelia burgdorferi
increases, we can make advancements towards a new generation vaccine for Lyme disease. Being such a dangerous
disease that can evade the host immune system and avoid detection, a vaccine for Lyme disease would be highly
beneficial.

Concluding Remarks

Lyme disease is a tick borne disease caused by the spirochete Borrelia burgdorferi. Since it’s discovery in 1975, there
have been extensive research on the mechanisms of this bacterium. The last two decades have identified a number of
outer surface lipoproteins that are needed for the transmission of the disease in mammalian tissues. The diversity in
surface proteins allows for the bacterium to adapt to different environments, which is beneficial in infecting different
mammalian hosts. However, many of the adhesions of the bacterium overlap in function, which suggests that these
 functions are necessary in order for the bacterium to cause infection in its mammalian host. Further research on the
interaction between B. burgdorferi and its mammalian host will benefit those who are infected and can work towards
prevention though vaccines. Also, looking at the interaction of B. burgdorferi adhesions with host substrates at the
molecular level will be interesting and may lead to additional antibiotics for treatment.

yme disease is caused by three species of bacteria belonging to the genus Borrelia.[1][2] Borrelia burgdorferi, an obligate parasite,
is the most common cause of the disease in the United States and is transmitted via hard-bodied ticks of the Ixodidae family,
commonly known as the blacklegged or deer ticks. Borrelia spirochetes are motile, helical bacteria whose cell membranes have
many exposed, surface lipoproteins that are involved in both the pathogenesis and life cycle of the parasite. Two predominant
groups of the surface lipoproteins present are classified as outer surface proteins (Osps), which have been characterized as
Osps A through F, and the variable major protein-like sequence expressed (VlsE). Both of these groups of outer surface proteins
play important roles in both the pathogenesis and life cycle of Borrelia as well as roles in eliciting an immune response within the
host organism (Figure 1).[3]

In an introductory biology course at Stony Brook University, undergraduates are modeling and exploring B. burgdorferiouter
surface proteins and their respective antibodies, in which the host organism produces. This Proteopedia page is the product of
their efforts, with a focus on highlighted proteins from the following categories: OspC, OspB and the antibodies to OspB, OspA
and the antibodies to OspA, and VlsE.

The goal of this Proteopedia page is to describe Lyme disease from a structural biology perspective in order to answer key
questions regarding the relationship between the structure and function of B. burgdorferi proteins. Some of the key questions
answered on this page include, but are not limited to: What do B. burgdorferi outer surface proteins look like? How does the
structure/function of these proteins relate to the infection cycle of B. burgdorferi? What are the structural targets of the human
immune system and how have these targets evolved? What are the ideal structural targets for a vaccine to protect against Lyme
disease?

Contents
[hide]

 1 OspC and Lyme Disease

o 1.1 Structure of OspC

 1.1.1 OspC Structure and Antigenicity

o 1.2 OspC, Lyme Disease, and Ecology

 1.2.1 Using Ecological Models to Predict Lyme Disease Risk

 1.2.1.1 Ecological factors responsible for human Lyme disease risk

o 1.3 OspC-based vaccine

 2 OspB and Lyme Disease

o 2.1 Structure of the OspB-H6831 Complex

 2.1.1 Structural changes to OspB in the Complexed Form

o 2.2 Potential Mechanism of Lysis

 2.2.1 Potential for Proteolysis and the OspB Catalytic Triad

 3 OspA and Lyme Disease

o 3.1 Structure of OspA

o 3.2 Acute Lyme Neuroborreliosis (LNB)

o 3.3 Evasion and the Extracellular Matrix

o 3.4 Antibodies to OspA

 3.4.1 Interaction between OspA and LA-2

 3.4.1.1 Structural changes to OspA in the complexed form

o 3.5 OspA-based Vaccine

 4 VlsE and Lyme Disease

o 4.1 Structure of VlsE

 o 4.2 Antigenicity

o 4.3 Function in Immune System Evasion

o 4.4 C6 Diagnostic Testing

 5 References

 6 Teaching at Stony Brook University

OspC and Lyme Disease

Figure 2: The migration of B. burgdorferi from the midgut to the salivary glands in infected nymphs. Redrawn from Templeton 2004.

OspC, a highly variable B. burgdorferi outer surface protein, plays a pivotal role in the transmission of B. burgdorferi from the tick
vector to a mammalian host. This protein is upregulated when the tick feeds, allowing for it to adhere to the tick saliva, move to
the tick's mouth, and migrate into the mammalian host.[4] The upregulation of OspC is accompanied by a downregulation of two
other outer surface proteins, OspA and OspB, which is thought to be induced by changes in environmental temperature and pH
(Figure 2). [5]

Strains of B. burgdorferi are classified according to the sequence of the OspC locus into 19 outer surface major groups (oMGs),
denoted by type A through S, of which only four are invasive (disease causing).[6] Polymorphisms of OspC and the abundance of
invasive strains in a population of B. burgdorferi are driven by ecological factors, such as the mammalian host community
composition, and are determinants of human Lyme disease risk[7].

Researchers are attempting to take advantage of the upregulation of OspC on B. burgdorferi's outer surface while the bacteria is
in the host to develop an OspC-based vaccine. However, development of an OspC-based vaccination has been presented with
difficulties due to its highly variable nature. [8]

Structure of OspC

The OspC three-dimensional model presented to the right is the B. burgdorferi B31 strain (residues 38-201) - also known as the
oMG A strain. This is one of the four invasive oMGs that are responsible for systematic Lyme disease in mammalian hosts. In
crystal structure, OspC exists as a dimer that coordinates a divalent ion, modeled here as magnesium. Each OspC subunit is
predominantly helical, consisting of five parallel α-helices , two short, antiparallel β-sheets and six random coils . The N and C
termini at the membrane proximal end of two long α-helices, α1 (residues 38-76) and α5 (residues 170-201), are in close
proximity to each other. At the membrane distal end, there are three remaining alpha helices, α2 (residues 95-112), α3 (residues
121-145), and a the final, short α4 (residues 152-159). At the end of the membrane surface, the connection between helices α1
and α2 forms two short, anti-parallel, β-strands, β1 (residues 79-80),and β2 (residues 88-89).

While most of the OspC locus is highly variable, the sequence alignment of all oMGs reveals that the surface-exposed residues
towards the membrane-proximal end of two of the helices, α1 and α5, are highly conserved and have a positively charged
surface. Other than those regions of the α1 and α5 helices, the surface-exposed residues on the remaining regions of the OspC
molecule are variable.[9]
OspC Structure and Antigenicity

At the membrane-distal region, the six loop regions, including two β-strands, illustrates the most antigenic sites of OspC with
the presence of variable surface-exposed residues. [10] Among these variable regions, the outer surface-exposed residues
connecting the helices α1 and α2, forming the loops, L1 (residues 74-78), L2 (residues 81-87), L3 (residues 90-93), two short
beta strands, β1 and β2, and L5 (residues 146-150) are more variable than those present in the loops, L4(residues 115-
119)and L6 (residues 161-169). The surface potential of the red region that projects away from the membrane is negatively
charged and primarily involved in the protein-protein or protein-ligand interactions.[9] Only four types of invasive oMG strains (A,
B, I and K), whose surface potential in the red region is highly negative relative to non-invasive strains, play a major role in
pathogenesis of human Lyme disease. [6]The His82 residue, located in the red region at the distal, membrane end, is unique in
that replacing this residue with other residues, with the exception of Lys82 and Gln82, which are only present in four invasive
oMG strains, enhances the possibility of turning invasive strains into non-invasive strains. Thus, the stronger the negative
electrostatic potential in the red region, the higher the chance OspC will to bind with positively charged host ligands; therefore,
the altering of an amino acid residue at the 82nd position in the red region determines OspC polymorphism and demonstrates
how this is connected to virulence and invasiveness.[6]

OspC, Lyme Disease, and Ecology

Figure 3: A Diagram of the Life Cycle of the Blacklegged Tick.[[1]]

Ticks are born with the absence of the B. burgdorferi parasites and acquire them while feeding on the blood of natural, reservoir
hosts, such as mice, squirrels, shrews, and other small vertebrates (Figure 3). After growth and development, the infected
nymphal and adult ticks can transmit B. burgdorferi to incidental vertebrates, including humans. The ecological interaction
between the competence of reservoir hosts and the ticks is an underlying measure of human Lyme disease risk. [11]The
occurrence of Lyme disease is dependent upon the abundance of ticks that are infected with B. burgdorferi in natural
ecosystems, and the number of reported cases of Lyme disease continues to increase annually in highly focused geographic
locations of the United States (Figure 4).
Using Ecological Models to Predict Lyme Disease Risk

Figure 4: Illustrated Prevalence of Lyme Disease in the United States (Generated by the CDC).[[2]]

Researchers have developed conceptual and mathematical models to characterize the ecological interactions between the
community of vertebrate hosts and distribution frequency of invasive oMGs and predict the distribution of Lyme disease. In one
effective model, the principal reservoir host used to calculate the risk of Lyme disease in northeastern and central United States
is of the white-footed mouse (Peromyscus leucopus). The white-footed mouse has both a high frequency distribution in all four
human infectious oMGs and high transmission probabilities of oMGs A, B, I and K. [7] It has been found that decreasing the
abundance of mammalian hosts with high transmission probabilities, such as the white-footed mouse, and increasing other
mammals with lower transmission probabilities drastically decreases the human Lyme disease risk. Many studies have found
support for this "dilution-effect model," indicating that maintaining a high diversity of the vertebrate host community may be
helpful in decreasing the incidence of Lyme disease. This model strongly demonstrates the relationship between species
diversity in the community of hosts and the risk of human exposure to Lyme disease, and the ecological driving forces described
in the model are useful tools in predicting the prevalence and risk of human Lyme disease.
Ecological factors responsible for human Lyme disease risk

The following are some factors used as parameters in ecological Lyme disease models:

 Vertebrate Community Composition[12]: Both the abundance and diversity of the mammalian hosts living in the community
strongly affects the proportion of infected nymphal ticks that can cause human Lyme disease.

 Distribution frequency of particular oMGs[7]: As only four types of oMGs (A, B, I and K) are responsible for systemic
human Lyme disease, the host-seeking nymphs that have a high distribution frequency of the four invasive oMGs is one of
the standard measures of human Lyme disease risk.

 Transmission Probability[7]: The transmission probability of each oMG between individual species differs. The higher the
transmission probability of a particular oMG from a vertebrate host, the higher the chances are that the ticks will carry that
particular oMG after receiving a blood meal from their hosts. Thus, it is one of the parameters that contributes to the
prevalence of human Lyme disease.
OspC-based vaccine

Researchers are currently developing an OspC-based vaccine against human Lyme disease, but because of the variability of
OspC, the recombinant OspC vaccine, targeting the antigenic site of one B. burgdorferi strain, may be ineffective for other
strains. Therefore, the development of a vaccine that recognizes the antigenic determinant on the variable regions of multiple
OspC strains is required in order to effectively activate the human immune response. Based on the mapping of epitope-
containing regions from oMGs A, B, K and D, the experiment-based tetravalent chimeric vaccine is being developed to trigger an
anti-ABKD response. [13] Taking advantage of the tetravalent ABKD construct, an octavalent chimeric vaccine, also known as
[8]
OspC-A8.1, recognizing additional epitopes of oMGs C, E, N and K has been tested in mice.

OspB and Lyme Disease

OspB and OspA comprise the most common proteins found on the surface of B. burgdorferi while residing in the tick vector,
during late-stage Lyme disease, and also during culture conditions.[14] OspB has been found to play a vital role in the adherence
of B. bugdorferi to the tick midgut whereas OspB-deficient B. burgdorferi are shown to bind poorly to tick gut extracts. While the
tick remains unfed, the expression of both OspB and OspA is upregulated to promote binding to the tick’s gut. However, during
transmission from the tick to a vertebrate host, OspB is downregulated while other proteins, such as OspC, are upregulated [15].
OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, which is known to
be largely invariant [16].

Figure 5: The separation of the Fab domains from the Fc domains of an antibody by Papain digestion.
A factor contributing to the severity of Lyme disease is the ability of B. burgdorferi to evade the host immune system. B.
burgdorferi is an extracellular pathogen, which is targeted by the humoral immune system of the host; the complement system
and antibodies produced by B-lymphocyte-derived plasma cells work together in order to fight off the infection. The complement
system consists of three pathways: the classical pathway, the alternative pathway, and the lectin pathway. Although each
pathway differs in the process of initiation, each pathway results in the amplification of an immune response and the formation of
the membrane attack complex (MAC), which ultimately leads to the pathogen's demise. In the lectin pathway and the alternative
complement pathway, complement is recruited to the pathogen surface directly, but in the classical complement pathway,
recruitment is dependent on the existence of an antibody-antigen immune complex; therefore, the traditional view of antibodies
functioning in a bactericidal capacity has required complement recruitment and MAC formation following antibody binding to
pathogens. Generally, antibodies are thought to be non-bactericidal in the absence of complement.

B. burgdorferi has developed resistance to a complement-dependent immune response by the evasion of the classical
complement pathway [17][18] and the alternative complement pathway by binding complement components C4b and CS,
respectively.[19] However, OspB is an important target of antibodies that can kill the bacteria without the help of the complement
system.[3] These antibodies are termed complement-independent bactericidal antibodies. One important complement
independent antibody whose bactericidal role has been well researched is CB2. [20] CB2 has been shown to kill bacteria in vitro in
the absence of complement [21]. Furthermore, it has been shown that a point mutation in OspB renders the epitope
unrecognizable by CB2, preventing CB2 from binding OspB and suggesting a possible mechanism for the evolution of the
bacteria to evade this aspect of the immune system[22].

Another complement independent antibody is H6831, an IgG antibody that targets the C-terminal of OspB (Figure 6). Studies on
both CB2 and H6831 have been conducted using the Fab (Fragment antigen binding) portion of these antibodies. A Fab
consists of a heavy chain and light chain, each chain containing both a variable region as well as a constant region (Figure 5).
The complementarity-determining regions (CDRs) are located at the N-terminal end of the variable region of the heavy and light
chains of the Fab and form unique tertiary and quaternary protein structures that determine the antigen binding specificity.
Binding of this region of the Fab to OspB of B. burgdorferi leads to the lysis of the bacteria, ruling out simple agglutination
(clumping) of the bacteria as the cause of the bactericidal effect.[3]

Structure of the OspB-H6831 Complex

The OspB-H6831 complex consists of two components: the outer surface protein B (OspB) and the region of an antibody
known as the Fab (Fragment Antigen-Binding) domain of H6831, which can then be further subdivided into the heavy
chain and the light chain of an antibody. Most hydrogen bonds and electrostatic interactions that are responsible for the
binding of H6831 to OspB are between the three adjacent, surface-exposed loops at the C-terminal of OspB and residues on
the Fab heavy chain variable region, that include tyrosine, tryptophan, glutamate, and histidine.[16]

The majority of hydrogen bonds and electrostatic interactions are between Loop2 (residues 250-254) and the Fab heavy chain
variable region. Lys253 in loop 2 of OspB has a critical role due to its central position in the surface-exposed loops. A mutation
at its position abrogates the binding interaction and causes the resistance of the bacteria to the bactericidal effect of either the
CB2 or H6831 Fab.[23][24] Lys253 interacts with two aromatic residues on the Fab heavy chain - tyrosine and tryptophan and also
forms and ionic bond and multiple hydrogen bonds with Glu50 in the heavy chain of the Fab. A carbonyl group in loop 1 of OspB
interacts with his52 in the Fab heavy chain, and loop 3 of OspB interacts with the Fab light chain variable region.[16]
Structural changes to OspB in the Complexed Form

The binding of CB2 or H6831 to OspB leads to some conformational changes within OspB - compared to its unbound form.
This was reflected in limited proteolysis experiments performed with recombinant OspB and CB2 in which unbound, recombinant
OspB was readily cleaved by trypsin and Arg-C.[25] Following CB2 binding, the rate of cleavage was significantly lowered,
suggesting a conformational change in OspB upon binding to CB2. Crystallography has shown that the most significant
difference is the loss of the central β-sheet strands 1-4 .[16] The loss of these β-sheets may be due to a conformational change
as a result of the binding or a disorder that could have occurred during the crystallization of the complex. Both small positional
shifts near the Fab binding site and a few larger structural changes away from the binding site were observed. The largest shifts
(7– 8 Å) correspond to the repositioning of a loop opposite the Fab-binding site at residues 218-220. In the free OspB structure,
all regions that exhibit shifts are adjacent to the central sheet; in the OspB-H6831 complex they all shift toward, and slightly
overlap, the position of the missing sheet.[16]
Aromatic residues tryptophan and tyrosine are also present in the OspB-H6831 interaction - a feature found in many antigen-
antibody complexes. Lys253 forms a trans conformation between these aromatic residues of H6831. In the complex structure of
the antibody binding site, the electron density is well defined and shows increased contact between Lys253 and the antigen-
binding sire of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain.

Potential Mechanism of Lysis

CB2 Fab binding destabilizes the outer membrane (OM) of B. burgdorferi, with subsequent formation of spheroplasts. Through
the use of single chain variable fragment (scFv) of a related complement-independent bactericidal antibody, the bactericidal
activity of these antibodies has been shown to reside in the antibody variable region alone. [26] It has been observed that the
bactericidal action, but not the binding, requires the presence of divalent cations (Mg2+ and Ca2+), and the CB2-bound Fab is
unable to clear bacteria in the absence of these cations.[27] Once CB2 binds to OspB, it leads to the lysis of the bacterial cell (B.
burgdorferi) through membrane/vesicle removal.[28] Eventually, enough membrane is lost, leading to the creation of physical
openings in the OM of a defined size around the entire cell - increasing permeability and allowing for a rapid infusion of
electrolytes which then leads to osmotic lysis of the organisms.[29]

Interestingly, cholesterol and prokaryotic lipid rafts are critical for the bactericidal mechanism of CB2. [30] It is unusual for
prokaryotic organisms to have membrane cholesterol and Borrelia is one of the few that does have this sterol.[31][32]Indeed as is
the case in eukaryotic cells, the presence of cholesterol in the Borrelia membrane leads to the formation of distinct membrane
microdomains called lipid rafts.[33][34] The prokaryotic lipid rafts of Borrelia share the biochemical and biophysical characteristics
of eukaryotic lipid rafts.[35] In eukaryotes, lipid rafts are specialized membrane platforms that serve a critical role in cell
signaling.[36] The dependence on the presence of cholesterol for the bactericidal mechanism of CB2 suggests that the
prokaryotic lipid rafts of Borrelia are necessary for the bactericidal effect of complement-independent antibodies. It is speculated
that lipid rafts may contribute to this bactericidal mechanism by enhancing OspB coalescence and membrane blebbing/removal.
Additionally, enhance coalescence of OspB due to the presence of lipid raft interactions may trigger a cell-signaling pathway that
is required for the bactericidal effect of complement-independent antibodies.[37] This idea, however, is speculative. Interestingly,
the binding of CB2 to OspB results in changes in gene expression in B. burgdorferi[38] which could be suggestive of cell
signaling. In particular, there were dramatic changes in the expression of phage holins genes, which could conceivably result in
the assembly of bacteriophages that could attack the Borrelia membrane internally.

Due to its effective bactericidal actions, H6831 is used to generate escape variants of B. burgdorferi. [16] In the majority of the
mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C-terminus lead to
premature stop codons.[39] It has been suggested that OspB mutants are more sensitive to proteolysis due to missense
[16]
mutations that disturb the conformation of OspB .
Potential for Proteolysis and the OspB Catalytic Triad

Figure 6: Comparison of the Compositions of the OspB and a Serine Protease Catalytic Triads

The mechanism by which CB2 and H6831 Fab fragments destroy a spirochete appears to be a novel interaction. It is possible
that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or
aggregation. NMR methods have shown that the effects of binding can be sent to regions of the antigen distant from the epitope,
which is at the C-terminus shown in red (N-terminus in blue). OspB shows signs of truncation after interacting with Fab of
H6831 [40]. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a
beta sheet from OspB after binding, and cleavage may be a possible explanation for the conformational changes of OspB. [41]
It is quite possible that OspB performs an autoproteolysis. There is a set of three residues found on OspB that resembles the
catalytic triad of serine proteases. This "constellation" consists of Thr166, Arg162, and Glu184 - similar to the catalytic triad
residues of the serine protease trypsin, which are Ser195, His57, and Asp102.[42] Threonine and Glutamic acid are found in other
catalytic triads of the serine hydrolase family, but arginine seems unlikely to replace histidine as a base due to its higher pKa.
There have been studies that have shown that arginine is essential for other enzymatic functions, such as in the Ser-Arg-Asp
catalytic triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. Asn164 forms an hydrogen bond
with Thr166 and may rearrange to form a putative oxyanion hole with Thr166 and another unidentified atom if active in the
catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow arginine to function
in the catalytic triad of a protease.

OspA and Lyme Disease

Like OspC and OspB, the expression of OspA is regulated differently over the B. burgdorferi infection cycle. OspA is expressed
while the bacteria resides in the midgut of the tick, downregulated while the tick feeds on its host, and then upregulated in the
host's cerebrospinal fluid (CSF), which may induce an inflammatory response resulting in acute Lyme neuroborreliosis. Because
OspA is relatively highly expressed and relatively invariable it has been used as a target in the development of a vaccine
for Lyme disease.

OspA is involved in the attachment of B. burgdorferi to the tick gut by binding to the tick receptor for OspA (TROSPA). TROSPA
is a tick gut protein that is required for colonization of the spirochetes in the midgut of the tick host [43]. When a tick feeds, OspA is
downregulated, releasing B. burgdorferi from the gut wall and allowing the bacteria to migrate into the tick's salivary glands and
ultimately into the host. The downregulation of OspA during transmission is evidenced by the fact that patients with Lyme
disease do not possess OspA antibodies in the early stages of the disease.[3][44]

Structure of OspA
OspA is made up of 273 residues over 21 anti-parallel β-sheets and a single α-helix. Its folded conformation is divided into three
main sections: an N-terminus "sandwich," a central region comprising of several β-sheets, and a C-terminus "barrel"
domain.[27] The folded regions at its ends are connected by a single β-sheet layer in the middle that gives the protein the unique
shape of a dumbbell.[45] There are three loops at the C-terminus of OspA that are important in binding with the LA-2 Fab
antibody (described below), whose interactions provide great insight into vaccine research and effectiveness. These three loops
are linearly arranged and form a protruding ridge at the C-terminus of OspA. Within these loops are three residues (show
residue R-groups) containing distinct variations between the different strains of B. burgdorferi and serve as potential targets for
the creation of a broader vaccine (display both the three loops and three residues together).[27]

Loop 1, (residues 203-220), is important in showing variation amongst the different strains of B. burgdorferi as well as being
optimally conformed for binding without steric hindrance. Loop 2 (residues 224-233) and Loop 3 (residues 246-257) are more
strongly conserved than Loop 1 but also help to show some variation amongst strains. The LA-2 Fab antibody readily recognizes
OspA from B. burgdorferi, but does not recognize that of B. afzelii or B. garinii. TheB. burgdorferi and B. afzelii genetic
sequences are generally invariant, but two residues change between the species: Ala208 in B. burgdorferiis a glutamine (Gln)
in B. afzelii, and Asn251 in B. burgdorferi is an alanine in B. afzelii. B. garinii has more variation in addition to the previous two
differences, having at least one more difference where Ala215 in B. burgdorferi is a lysine in B. garinii, and sometimes also has
a deletion at Ala208 of B. burgdorferi. LA-2 and OspA of B. burgdorferi form a tight interface when bound, and the longer
glutamine sidechain found in B. afzelii and B. garinii is more difficult to accommodate, reducing binding. A chimera that was
weakly recognized by LA-2 was made with parts of loop 1 from B. burgdorferi and loops 2 and 3 from B. garinii.[27] Recently, a
different kind of chimera has been made which combines the proximal region of B. burgdorferi and the distal region of B. afzelii;
it was able to successfully protect mice from both species.[46]

Acute Lyme Neuroborreliosis (LNB)

Acute Lyme Neuroborreliosis (LNB) is part of a later stage of Lyme disease in which the spirochete invades the peripheral and
central nervous systems (CNS). Symptoms of LNB include: meningoradiculitis with inflammation of the nerve roots
and radiculitis (Bannwarth’s syndrome), lymphocytic meningitis, and cranial and peripheral neuritis. In Europe, the strain
predominantly found in the cerebrospinal fluid (CSF) of patients with Bannwarth's syndrome is B. garinii. However, in the United
States, Bannwarth's syndrome is rare and the most common manifestations of Lyme neuroborreliosis is meningitis, caused by
the presence of B. burgdorferi OspA in the CSF, which leads to this complex inflammatory response. [44]
It is not fully understood how B. burgdorferi get past the blood-brain barrier composed of microvascular endohelial cells, among
other cells, though some researchers suggest a paracellular route, which involves a process using transient tether-type
associations involving OspA. Studies have shown that OspA adheres to brain microvascular cells by binding to
the CD40 receptors, followed by an induction of signaling cascades and adhesion to endothelial cells, ultimately resulting in the
movement of B. burgdorferi into the CNS. Similar cell signaling events are seen when leukocytes cross the blood-brain barrier,
and it has been proposed that B. burgdorferi may mimic this process, although it has been found that not all strains of B.
burgdorferi can utilize OspA to cross into the CNS. It has been found that OspA only contributes about 70% to adherence, and
other B. burgdorferi proteins are also needed in this process; it has also been seen that OspA mediates the adhesion of B.
burgdorferi to murine neural and glial cell lines. [47]

There are many steps involved in the host's inflammatory response to OspA. When B. burgdorferi enter the host’s CNS, they
encounter several different types of immune cells such as monocytes, macrophages, and dendritic cells. While in the CSF,
OspA is upregulated, and its increased expression promotes recognition by immune cells, such as monocytes. Upon recognition
of OspA, monocytes release proinflammatory cytokines (i.e. interferon), as well as chemokines, such as CXCL13. There is an
observed increase in the levels of these cytokines and chemokines in the CSF of LNB patients. The production of chemokines
leads to the recruitment of other immune cells to the site of infection. B-lymphocytes respond to the new concentration gradient
of CXCL13 and other chemokines between the blood and CSF which leads to their migration into the CSF. The B-lymphocytes
then differentiate and mature into antibody- producing plasma cells that create large quantities of anti-OspA antibodies specific
to this strain of B. burgdorferi and release them into the CSF to target the pathogen for destruction[44]. This process is two-sided
in the sense that the OspA aids in the pathogenesis of B. burgdorferi (neuroborreliosis) as well as eliciting the host immune
response to destroy the pathogens.

Evasion and the Extracellular Matrix

B. burgdorferi are able to hide in the extracellular matrix, allowing their survival by avoiding leukocytes circulating in the
bloodstream. OspA can rapidly bind to plasminogen, facilitating the spread of the bacteria [48][49]. By binding to plasminogen, B.
burgdorferi can exploit its function and utilize it to invade the extracellular matrix. However, due to the fact that OspA is
downregulated during feeding while staying unexpressed, a different mechanism may be used instead. Additionally, B.
burgdorferi induces the local upregulation of matrix metalloproteinase-9, causing the digestion of the surrounding extracellular
matrix, and B. burgdorferi can also bind to several proteins in the extracellular matrix, such as fibronectin, integrins or decorin,
which can aid in the spread and survival of the spirochetes in these tissues. [44]

Antibodies to OspA
Interaction between OspA and LA-2

LA-2 of the OspA-LA2 Complex is a murine, monoclonal IgG antibody that interacts with three exposed loops on the C-
terminal of OspA. These interactions include eight direct hydrogen bonds, four solvent-bridged hydrogen bonds, three ion pairs,
and numerous van der Waals interactions.[27] This particular antibody is being used in vaccine development, and it is important
to note that LA-2 depends on complement in order to create a bactericidal effect against B. burgdorferi.
Structural changes to OspA in the complexed form

Conformational changes upon the binding of OspA and LA-2 show that LA-2 recognition of OspA involves an induced fit
mechanism, where the conformations of loops 1-3 shift to optimize complementarity to the antigen-combining site.[27] The overall
structure of the OspA C-terminus is unchanged upon the binding of LA-2 with comparison to the free OspA. The maximum
atomic shift is 4.7Å at the site of Ser 206.[27]

OspA-based Vaccine
The membrane composition of B. burgdorferi is abundant in both OspA and OspB, and the two proteins share a 53% similarity in
their primary sequences, however, OspB has greater variability than OspA.[16]. Of the three exposed loops found on OspA, only
loop 1 is variable while loops 2 and 3 are conserved. This makes OspA a more consistent antigen (compared to OspB and
OspC) for the immune system to target and usable as a vaccine to Lyme disease.[27] The first vaccine developed against Lyme
disease, Lymerix, used a purified recombinant form of OspA and functioned in blocking transmission of the spirochetes
expressing OspA from tick to host during feeding, killing them while still attached to the tick's gut.[3][50] The vaccine was 76% to
92% effective in separate clinical trials in which patients were treated for two years following a three-dose schedule. However,
the vaccine was suspended from use in 2002 when opponents claimed the IgG antibodies for OspA were associated with the
onset of severe chronic arthritis, as well as other side effects affecting immunity. [3][51] This claim, in conjunction with the desire for
a more widespread vaccine treating multiple strains of B. burgdorferi, has spurred research towards a new vaccine. One goal is
to develop a vaccine with broader protection through the creation of a chimera by mixing the OspA of different strains of B.
burgdorferi.

Study of the epitope of OspA and its interactions with the murine monoclonal antibody LA-2 have proved useful in determining
effectiveness of a given vaccine trial as high levels of antibodies in test sera compete against LA-2 for binding with OspA. LA-2
makes direct contact with three exposed loops of the C-terminus of OspA. The recognition of OspA by LA-2 requires an induced
fit mechanism where these three loops undergo conformational changes to optimize their interaction in the complex. [27]

VlsE and Lyme Disease

Variable Major Protein (VMP)-like sequence Expressed (VlsE) is another surface lipoprotein of B. burgdorferi that is used for
Lyme disease diagnosis. It undergoes antigenic variation, and this representation of VlsE illustrates the only crystal PDB-
published structure, which is only one of the 1030 possible combinations of the VR.

Structure of VlsE
VlsE is composed of four similar subunits, each possessing two invariable domains and one variable domain. [52] The variable
domain contains six variable regions (VR1-VR6) and six invariable regions (IR1-IR6). Research suggests that the protein may
exist as a dimer in which each monomeric C & N termini neighbor each other and the variable regions neighbor each other -
forming the membrane proximal portion of the protein and the membrane distal portion, respectively. [53] [54] The invariable regions
are largely embedded in the protein and remain relatively unchanged within the host and across strains. The variable regions
encompass 37% of the exposed surface area of VlsE, despite comprising only 25% of the protein. [52] [53] However, 50% of the
surface area of the VR is exposed while IR6, a strong antigen, exposes just 13.7% of its surface area. This leaves only four
residues of the antigenic IR6 unprotected: Lys276, Gln279, Lys291, and Lys294. Thus, it is almost entirely embedded in the
protein and shielded by the variable regions.[53]

Antigenicity
The variable regions undergo a recombination event stimulated by the host’s cytokines, and in the absence of those cytokines, a
decreased bacterial burden results.[55] Recombination leads to variation with an estimated 1030 possible combinations, far
exceeding the number of antibodies found in the human immune system. While the VR does exhibit antigenicity, this
recombination makes it unlikely that a sufficient amount of a single VR variation will be present in large enough supply to lead to
an immunodominant variable region.[56] IR6, however, exhibits immunodominance while IR1-IR5are primarily non-antigenic in
humans. Thus, shielding of the immunodominant IR6 by VR regions not subject to antibody response allows for IR6 to elicit an
immune response while remaining inaccessible to antibody binding. Research suggests that the 26 amino residues of IR6 may
function as a single epitope with a central alpha helical core.[53] [55] [57]

Function in Immune System Evasion

VlsE is essential to the persistence and virulence of Lyme disease and is upregulated under humoral immune
pressure.[58][59] While the exact mechanism for immune evasion remains unknown, several theories have been put forth. One
popular theory maintains that VlsE masks other surface antigens by coating the surface of the bacteria, thereby sterically
hindering the antigens from antibody binding. This is similar to other pathogens with variable regions, such as Trypanosoma
brucei, the protozoa responsible for African sleeping sickness and Neisseria gonorrhea, the bacterial cause of the well-known
sexually transmitted infection (STI) gonorrhea. However, recent studies have cast doubt on this theory, and an alternate theory
provides that VlsE directly stimulates B-lymphocyte-derived plasma cell antibody production independent of T-lymphocytes, in
which the robust response elicited is thought to override antibody production against other antigens. [58]

C6 Diagnostic Testing
Throughout the course of the disease, IR6 produces a strong antibody response that can be identified from early to late phases.
Applications in diagnostic testing have been identified as a result of this strong immune response and the relative invariability of
IR6 across strains.[60] [57] A C6 ELISA test has been developed that uses a 26 amino acid synthetic peptide, C 6, containing the
IR6 sequence. Results show 99% specificity and 100% precision with high sensitivity. In fact, OspA vaccination does not
influence C6 specificity; therefore, C6 ELISA tests are valuable diagnostic tools.[60] The CDC currently recommends a two-step
test incorporating first a polyvalent, whole-cell sonicate (WCS) immunofluorescent assay. If results are positive, this is followed
by IgG and IgM WCS Western blots to eliminate false positives.[61]Therefore, this one-step ELISA test presents an accurate and
economical alternative to the current two-step model.[60]