Review

Good Gone Bad: One Toxin Away

From Disease for Bacteroides fragilis

Ezequiel Valguarnera and Juliane Bubeck Wardenburg

Department of Pediatrics, Washington University School of Medicine, 660 S. Euclid Ave. Box 8208, St. Louis, MO 63110

Correspondence to Juliane Bubeck Wardenburg: jbubeck@wustl.edu

https://doi.org/10.1016/j.jmb.2019.12.003
Edited by John Ralston Brannon

Abstract
The human gut is colonized by hundreds of trillions of microorganisms whose acquisition begins during early
infancy. Species from the Bacteroides genus are ubiquitous commensals, comprising about thirty percent of
the human gut microbiota. Bacteroides fragilis is one of the least abundant Bacteroides species, yet is the
most common anaerobe isolated from extraintestinal infections in humans. A subset of B. fragilis strains carry
a genetic element that encodes a metalloprotease enterotoxin named Bacteroides fragilis toxin, or BFT.
Toxin-bearing strains, or Enterotoxigenic B. fragilis (ETBF) cause acute and chronic intestinal disease in
children and adults. Despite this association with disease, around twenty percent of the human population
appear to be asymptomatic carriers of ETBF. BFT damages the colonic epithelial barrier by inducing cleavage
of the zonula adherens protein E-cadherin and initiating a cell signaling response characterized by
inflammation and c-Myc-dependent pro-oncogenic hyperproliferation. As a consequence, mice harboring
genetic mutations that predispose to colonic inflammation or tumor formation are uniquely susceptible to toxin-
mediated injury. The recent observation of ETBF-bearing biofilms in colon biopsies from humans with colon
cancer susceptibility loci strongly suggests that ETBF is a driver of colorectal cancer. This article will address
ETBF biology from a host-pathobiont perspective, including clinical data, analysis of molecular mechanisms of
disease, and the complex ecological context of the human gut.
© 2019 Published by Elsevier Ltd.

Human beings are persistently colonized by
commensal microorganisms. The gut microbiota, in
particular, is composed of tens of trillions of micro-
organisms and is normally acquired from birth to
around 3 years of age [1]. Bacteroides fragilis is a
ubiquitous member of the human gut microbiota. It
belongs to the phylum Bacteroidetes, which together
with the phylum Firmicutes constitutes about 80% of
the total human gut microbiota [2,3]. B. fragilis is a
commensal organism that can become an opportu-
nistic pathogen in certain individuals. Although
B. fragilis comprises around 0.1e0.5% of total gut
bacteria, it is the most frequently isolated anaerobe
from peritoneal and abdominal abscesses, as well
as from samples of bloodstream infections [4]. This
species became notorious in the 1970s because
reports have shown its relevance in peritoneal,
abdominal, intestinal, and blood infections, as well
as its propensity to develop antibiotic resistance
[5e11]. Most B. fragilis strains will remain as a
commensal during the lifetime of an individual, with
colonization occurring during early childhood
[12e14]. Nevertheless, a subset of strains can
produce a proteolytic enterotoxin, named B.fragilis
toxin (BFT), or fragilysin, that causes secretory
diarrhea and colonic epithelial damage [15]. BFT is
among the most studied virulence factors of
B. fragilis, and current evidence suggests that this
toxin may be a driver for chronic colitis and colorectal
cancer [16e18]. Toxin-producing strains, or enter-
otoxigenic B. fragilis (ETBF), were discovered in the
1980s and were found to be associated with diarrhea
in lambs, calves, pigs, foals, and humans and also
found to be present in sewage waters [19e24]. Since
those initial reports, multiple studies have enhanced
our understanding of ETBF as a relevant pathogen in
humans and BFT as the key virulence factor in
disease. Humans are variably colonized with non-
toxigenic B. fragilis (NTBF) or ETBF strains, and
most frequently, a single B. fragilis strain type is

0022-2836/© 2019 Published by Elsevier Ltd. Journal of Molecular Biology (2020) 432, 765e785
766
found in fecal samples from individual healthy
donors [14]. Presently, knowledge of genetic deter-
minants of human colonization by B. fragilis is in its
infancy. The host and microbial factors that govern
temporal and spatial plasticity of the B. fragilis niche
are not understood; similarly, determinants respon-
sible for progression from asymptomatic carriage of
B. fragilis toward disease remain undefined. In this
work, we will review current knowledge on toxigenic
B. fragilis (ETBF) and discuss the dual nature of this
organism as both a commensal and pathogen in the
context of a complex ecological scenario.
Colonization of Humans by ETBF
ETBF has been identified in stool samples from
healthy individuals and patients with diarrhea.
Interestingly, the prevalence of ETBF is about
twice as high in patients (20e30%) than in healthy
individuals (10e20%) [25e27]. Reports from distinct
urban and underdeveloped geographic locations
show higher percentages of ETBF colonization in
children older than 1 year with diarrhea than in age-
matched controls [25,28e32]. Interestingly, in chil-
dren younger than 1 year, prevalence is the lowest in
the general population and is not associated with
diarrhea, suggesting that the developmental trajec-
tory is important for diarrheal association to occur
[28,32]. An association of ETBF with chronic
intestinal disease has been established for more
than 20 years, the first reported in patients with
inflammatory bowel disease (IBD) [33]. ETBF is also
positively associated with ulcerative colitis and
colonic neoplasia [34e41]. The association of
ETBF in patients with colorectal cancer (CRC)
includes sporadic and familial cases, indicating that
progression of this disease, regardless of the onset,
has physiologic commonalities. The disparity
between high asymptomatic carriage of ETBF in
human populations and the low number of cases of
ETBF-associated IBD and sporadic CRC suggests
that pathogenicity is not stochastic and depends on
unknown host susceptibility determinants. It is
currently not known if ETBF strains typically produce
enterotoxin in asymptomatic carriers [42]. Studies
measuring amounts of secreted BFT in both healthy
and diseased individuals will be required to establish
a functional association between carriage of ETBF
and specific manifestations of disease. Furthermore,
the fact that NTBF accounts for the vast majority of
B. fragiliseassociated infections increases the com-
plexity of studies that will be required to define both
epidemiologic and mechanistic contributions of
ETBF to human disease.
As non-toxigenic (NTBF readily colonizes the
human colon and is suggested to benefit the
development of the host T-cell response through
studies conducted in murine model systems [43,44],
Good gone bad
the following question arisesdwhat is the benefit of
toxin production by B. fragilis in an organism that
exhibits a commensal existence? Several plausible
answers to this question can be put forth: (1)
intoxication facilitates colonic niche acquisition and
survival. A report from our group showed that a
particular strain of ETBF (43859) can colonize a
niche previously occupied by NTBF (TM4000) in a
toxin-dependent manner [45]. The presence of the
toxin was not, however, a universal determinant of
intraluminal niche acquisition or competition, which
also relies in part on the B. fragilis type VI secretion
system (T6SS) and other genetic determinants
[46e54]. A precedent for toxin-mediated modulation
of the bacterial niche to facilitate survival has
recently been demonstrated for Vibriocholerae [55].
(2) Intoxication facilitates transmission of ETBF. As
previously mentioned, in children aged between 1
and 5 years, the ETBF count is increased in patients
with diarrhea. Toxin production and the associated
diarrhea may represent a key strategy for transmis-
sion of the pathogen from human to human, by
increasing fecal-oral contamination. Human intest-
inal pathogens such as V.cholerae and Salmonella
enterica use diarrhea as a means to transmit
infection between human hosts, so this could also
be the case for ETBF [56,57]. (3) Intoxication permits
an extraintestinal lifestyle for B. fragilis. As a leading
cause of anaerobic infection, B. fragilis exhibits the
ability to invade multiple tissue sites within the body
[58]. Studies that have investigated the presence of
ETBF in extraintestinal infections have not reached a
clear consensus; however, they suggest that ETBF
may be more represented in bloodstream isolates
and samples of vaginal infections than in other
isolation sites [59e65]. BFT has been shown to be
toxic in vitro to kidney and lung epithelial cells as well
as to the endothelium, suggesting a potential role in
extraintestinal infections [66,67]. Although other
genetic determinants of colonization, including cap-
sular polysaccharide, have been implicated in
extraintestinal infections and abscess formation
[68], such roles have not been clearly defined for
BFT. A considerable amount of epidemiologic
investigation focused within the human population
and paired mechanistic analyses in model systems
will be needed to provide clarity on the potential
evolutionary advantage of maintenance of BFT and
its associated pathogenicity island within the
B. fragilis genome.
Discovery of BFT and Toxin Activation
The first report of enterotoxicity by B. fragilis was
presented in 1984, describing diarrhea in lambs [19].
At that time, there was a clear distinction between
enterotoxigenic and nonenterotoxigenic strains, but
the etiology of the diarrhea was unknown. In 1992,
Good gone bad
Weikel et al. [69] were the first to demonstrate that a
component in culture supernatants from enterotoxi-
genic strains was causing the secretory phenotype
previously observed when injecting lamb ileal loops
(LILs) with bacteria. The authors developed an in
vitro assay of BFT cell damage using colon
carcinoma HT29 cell lines, allowing for simultaneous
screening of multiple enterotoxigenic strains [69].
The toxin was initially purified from culture super-
natants as a 20-KDa protein and characterized as
enterotoxic and cytotoxic in LIL and HT29 models,
respectively. Partial cloning and expression of the
toxin revealed that it is a heat-labile metalloprotease
[70,71]. Diagnostics for detection of the toxin were
also developed, allowing for confirmation that the
enterotoxin is present in ETBF strains isolated from
humans with ETBF-associated diarrhea [72,73]. The
complete sequence of the bft gene was cloned, and
characterization of the amino acid sequence and
biochemical analysis suggested that BFT is pro-
duced as a protoxin that is processed releasing the
active C-terminal domain into the extracellular milieu
[74,75]. Further studies showed that cleavage of the
protoxin is not dependent on the zinc-binding motif of
the metalloprotease domain and that the whole C-
terminal domain is essential for toxin activity [76,77].
Detailed structural analysis determined that the N-
terminal prodomain is likely involved in the secretion
of the protoxin through the cell envelope and in the
inhibition of toxin activity within the bacterial cell [78].
Interestingly, the N-terminal domain contains a
lipoprotein signal peptide representing a unique
fold, whereas the C-terminal metalloprotease
domain is a xenolog of eukaryotic A Disintegrin
and Metalloprotease (ADAM) proteases, suggesting
horizontal acquisition of bft by B. fragilis [78]. As
shown in Fig. 1, cleavage of the protoxin can occur in
vivo by host proteases from the intestinal lumen.
Interestingly, in bloodstream, BFT can only be
activated by a single endogenous bacterial cysteine
protease named fragipain (Fpn) [67,79]. An ETBF
fpn mutant strain was unable to cause lethality in
mice when bacteria were administered intrave-
nously, showing the importance of Fpn in the context
of ETBF-mediated sepsis [67]. The fpn gene is
present in most NTBF and ETBF strains, suggesting
an additional role of Fpn beyond BFT activation.
Genomic Context and Transcriptional
Regulation
Studies on the genetic locus that encodes BFT have
been crucial to understand its origin and distribution
within strains. The bft gene is part of the ~6-kbp
B. fragilis pathogenicity island (BfPAI). The BfPAI is
absent in NTBF strains, and it also encodes another
metalloprotease (mpII), whose role in pathogenicity is
unclear [80,81]. Three bft variants have been identi-
767
Fig. 1. Known molecular mechanisms that govern
secretion of BFT. The RprX/RprY two-component system
is activated under certain as-yet-unidentified conditions,
with concomitant phosphorylation of RprY (RprY-P) and
binding to the bft promoter, repressing gene expression. In
a noneRprY-P situation, bft is expressed and translated
as a signal peptide-containing protein into the periplasmic
space. Pre-BFT is most likely a lipoprotein that gets
transported to the outer membrane. Surface-exposed pre-
BFT can be cleaved by Fragipain or host gut proteases,
releasing the active metalloprotease C-terminal domain
into the extracellular milieu. BFT, Bacteroides fragilis toxin;
Fpn, fragipain.
fied, bft-1, bft-2, and bft-3 [62,82,83]. Most ETBF
strains carry a single bft variant; each variant
demonstrates different degrees of potency in vitro
and in germ-free mice. Among these, BFT-2 exhibits
the greatest potential to elicit tissue damage [84e86].
Population-wide carriage has been studied, and BFT-
1 was found to be the most widely distributed toxin
variant within ETBF human isolates, whereas BFT-3
seems to be geographically restricted to southeast
Asia [31,62,87,88].
The BfPAI contains 12-bp repeats on both ends,
and it is always inserted in the same genomic
localization. A 17-bp GC-rich sequence is also found
at the putative insertion site in NTBF strains [80]. The
BfPAI is present within the conjugative transposon
CTn86, supporting the hypothesis of horizontal
acquisition of the BfPAI by ETBF strains [89e92].
Genome sequencing and comparison indicates high
genetic diversity between ETBF strains [92,93].
Some ETBF strains are more closely related to
NTBF strains than to other ETBF strains, further
supporting that the BfPAI has been acquired through
multiple independent horizontal transfer events [92].
Thus, the only known genetic determinant that
allows for differentiation between ETBF and NTBF
strains is the presence or absence of the BfPAI,
respectively.
The BfPAI contains a promoter sequence
upstream of the bft gene that controls transcription
768
[94]. A key mechanism for control of toxin production
is regulation of expression by the two-component
system (TCS) RprX/RprY. The TCS is a common
strategy to couple environmental stimuli with gene
regulation in bacteria, in which a sensor histidine
kinase (RprX) detects specific stimuli and phosphor-
ylates a response regulator (RprY) that binds to
promoter regions in the bacterial genome inducing or
repressing gene expression [95]. Phosphorylated
RprY binds to the promoter upstream of the bft gene
and represses expression (Fig. 1). Overexpression
of RprY abrogates toxin expression, and deletion of
rprY causes increased nonregulated expression of
bft [96]. Hence, ETBF strains could be sensing
environmental cues to determine the optimal situa-
tion for toxin expression. BFT can be repressed by
fermentable sugars and induced by heat and
oxidative stress [45]. Although RprX/Y seems the
key to bft regulation in vivo, stimuli for this TCS
inside the host remain unknown.
Toxin-Host Interactions
Since the discovery of BFT, the molecular
mechanisms that govern damage of host cells
have been described. When the HT29 cell model
was established, it was observed that BFT caused
cell rounding and shedding, presumed to result from
damage to the intercellular junction [66,71]. Morpho-
logical changes in BFT-treated HT29 cells were
found to be a product of F- and G-actin rearrange-
ment [97,98]. BFT binds to an unidentified cell
receptor in a protease-dependent manner and
induces cleavage of the extracellular domain of the
zonula adherens protein E-cadherin; this cleavage
event occurs only in the context of intact cells (Fig. 2)
[99e101]. Given the similarity of BFT to ADAM10, it
is possible that BFT contributes directly to E-
cadherin cleavage [78]. As there is no evidence for
direct processing of cellular E-cadherin by BFT yet,
an alternative hypothesis is that activity of BFT
toward its receptor or another host protein could
trigger a signaling pathway responsible for the loss
of E-cadherin (Fig. 2) [100]. A study using human
colonic biopsies incubated with BFT on the luminal
or serosal side of the epithelium showed more
damage on the latter, suggesting BFT receptor
polarization toward the basal cell region [102].
BFT-dependent cleavage of E-cadherin causes
loss of cell-cell contacts and cell rounding, which
requires the intramembrane protease presenilin-1/
gamma secretase complex [103]. The intracellular
domain of E-cadherin is normally bound to a- and b-
catenins [104]. When b-catenin is dissociated from
E-cadherin, it can function as a transcription factor in
a Wnt-dependent manner, inducing cell proliferation
through activation of the c-Myc pathway [104]
(Fig. 3). BFT-mediated cleavage of E-cadherin
Good gone bad
Fig. 2. BFT-host cell interactions. BFT binds to colonic
epithelial cells (CECs) through an unknown receptor and
triggers cleavage of E-cadherin. BFT, Bacteroides fragilis
toxin; ETBF, enterotoxigenic Bacteroides fragilis.
promotes migration of beta-catenin to the nucleus
[105]. In addition, E-cadherin cleavage by BFT
triggers induction of mitogen-activated protein
kinases (MAPKs) and the NF-kappa B pathway,
thus increasing secretion of interleukin (IL)-8, a
chemokine that attracts polymorphonuclear cells
[106e111]. NF-kappa B activation controls fluid
secretion of intestinal cells through induction of
COX2 and an increase in prostaglandin E2 levels
[112]. COX2 and heme oxygenase-1 induction by
BFT is related to a delay of apoptosis in intestinal
epithelial cells [113,114]. BFT can also induce
mechanisms of host defense such as beta-defensin
2 and expression of the siderophore-binding anti-
microbial protein lipocalin-2 [115,116] and also
increases autophagy in human umbilical vein
endothelial cells through the MAPK, AP-1 [117].
Signaling pathways affected by the toxin cause
differential gene expression and epigenetic changes
in HT29 cells [118]. Variations in the host cell
transcription profile and epigenetic marks are lost
when toxin stimulation is withdrawn, suggesting that
continuous toxin secretion by ETBF could be a
component of disease progression [118].
The relevance of mechanistic findings in cell
culture systems has been elaborated in animal
models of ETBF-associated disease. Rabizadeh
et al [119] and Rhee et al [120] showed for the first
time in specific pathogenefree (SPF) C57BL/6 mice
that ETBF causes acute and persistent colitis
in mice, driven by cleavage of E-cadherin by BFT
in vivo. Activation of STAT3 in vivo can be observed
Good gone bad
Fig. 3. Pro-oncogenic signaling by BFT. CECs
induce cMyc through b-catenin and STAT3 activating a
cell proliferation program. cMyc also induces spermine
oxidase (SMOX), in a BFT-dependent manner, generating
a source of ROS (Reactive Oxygen Species) that can
contribute to DNA damage. BFT, Bacteroides fragilis toxin;
CEC, colonic epithelial cell.
at 24 h after infection in mucosal immune cells and
colonic epithelial cells [121], inducing c-myc expres-
sion and concomitant cell proliferation (Fig. 3)
[122,123]. Colonic histology of acute colitis (2 days
after infection) in SPF mice shows rupture of cell-cell
adhesions and epithelial exfoliation, with the pre-
sence of immune cell infiltrates [121]. Conversely,
chronic colitis, observed between 7 days and up to
16 months after infection, shows progressive hyper-
plasia of the colonic crypts, consequent with BFT-
dependent induction of a cell hyperproliferation
program [120,121].
Bacterial Factors in Colonization and
Disease
Species from the Bacteroides genus are acquired
early in life and are commonly found in the lower
gastrointestinal tract [1]. There are many potential
determinants of success for a given strain to become
established in its niche, including host diet, devel-
opment, antibiotic usage, and interactions with other
members of the microbiota [12,124,125]. Although
external factors, such as the aforementioned ones,
are pivotal for colonization, B. fragilis carry genetic
determinants of colonization that contribute to niche
occupancy by interacting with the host and microbial
competitors. In this section, we will emphasize two
769
main aspects of colonization: (1) the relevance of the
B. fragilis capsule and other factors in host-patho-
biont relationships and (2) mechanisms that mediate
interbacterial competition in the gut, in particular
T6SSs and secreted antimicrobial toxins.
Capsular Polysaccharide
The B. fragilis capsule was described in the 1970s
in the studies by Kasper [126], Kasper et al [127,129]
and Lindberg et al [128] as a distinctive feature of this
organism relative to other species from the genus
Bacteroides. These observations, coupled with the
fact that B. fragilis is the most commonly isolated
Bacteroides species from anaerobic infections in
humans despite its low relative abundance in the
microbiome, led to the hypothesis that the B. fragilis
capsule is linked to disease pathogenesis. Indeed,
studies have indicated that capsule reduces phago-
cytosis by immune cells, thereby increasing bacterial
fitness outside the colonic lumen [130]. Consistent
with this finding, capsular polysaccharide is the main
contributor to extraintestinal abscess formation by
B. fragilis [128,130e132].
B. fragilis harbors 8 distinct genomic loci, each of
which encodes the required enzymes for synthesis
of a specific capsular polysaccharide variant.
Expression of these loci is regulated through
promoter inversion to “on” or “off” configurations
[133,134]. Capsular polysaccharide levels are
increased when B. fragilis is animal passaged,
indicating its key role in survival within the host
[130]. Monocolonization experiments in germ-free
mice show that any capsular polysaccharide is
sufficient for niche establishment [135]; however,
this finding does not indicate that all capsule variants
are immunologically equivalent within the host or that
a single capsule variant is sufficient for colonization
in a complex ecosystem [135,136]. B. fragilis cap-
sular polysaccharides are composed of repeating
units of zwitterionic glycans [132]. Polysaccharide A
(PSA), unlike most glycan antigens, can be pre-
sented within the context of MHCII antigen-present-
ing cells, inducing formation of regulatory T cells that
contribute to immune tolerance toward B. fragilis
[137e139]. PSA induces IL-10 production through
the Toll-like receptor (TLR) 2 pathway and represses
production of IL-17 in germ-free mice, promoting a
low inflammation environment [44]. The lack of PSA
however does not decrease IL-10 in SPF mice
colonized with NTBF [140,141]. For ETBF, recent
research using snap-frozen samples from human
colonic tissue biopsies showed a negative associa-
tion between PSA expression and carriage of bft
gene [140], suggesting the potential to exacerbate
inflammation. Together, these studies suggest there
are likely strain- and context-dependent regulatory
elements that modulate the host response to
B. fragilis polysaccharides.
770
Polysaccharide Utilization Loci
A genetic screening by Lee et al [142] identified the
“commensal colonization factor” (ccf) operon that is
conserved among many Bacteroides species and
confers stable niche acquisition in mice. The
structure of the ccf operon resembles typical
polysaccharide utilization loci (PULs) from Bacter-
oides [142]. PULs are specialized loci dedicated to
the breakdown and assimilation of complex glycans;
these constitute close to 20% of the genomic content
of some Bacteroides species [143]. Different PULs
can process distinct polysaccharides, allowing Bac-
teroides species to process both dietary and host
glycans [143e147]. Versatility in nutrient utilization
increases the chances of survival during host diet
changes; thus, PUL diversity within Bacteroides
species is the key to niche acquisition and stable
niche occupancy. Similar to most PULs, the ccf
operon consists of a transcriptional regulator sigma/
antisigma factor pair (ccfA and ccfB) that controls
gene expression. Downstream of ccfA/B is the
TonB-dependent receptor that transports breakdown
products into the cell periplasm (ccfC) and the SusD-
like accessory lipoprotein (ccfD) that is required for
control of nutrient transport by ccfC. The last gene in
the locus is a putative chitobiase (ccfE) that cleaves
a complex substrate polymer into oligomers for cell
acquisition. Functional characterization of ccf
showed that ccfA, ccfC, and ccfD are required for
stable colonization, precluding secondary coloniza-
tion events by Bacteroides from the same species.
On the contrary, ccfE is not required for ccf function,
most likely owing to enzymatic redundancy from
other B. fragilis gene products [142]. A recent study
showed that the ccf locus controls capsule variants,
repressing PSA and inducing polysaccharide C
(PSC) expression. ccf function allows IgA-depen-
dent niche establishment in response to PSC,
enabling B. fragilis cells to localize closer to the gut
epithelium [142,148]. The specific environmental
cues that induce ccf expression in vivo remain to
be defined.
B. fragilis Hemolysins
Many gram-negative and gram-positive bacteria
secrete enzymes that lyse red blood cells, known as
hemolysins [149e151]. B. fragilis is not an
exception as many strains carry hemolysin ortholog
genes [152]. Hemolysins A and B (HlyA and HlyB,
respectively) have been shown to be enzymatically
active in vitro against red blood cells [152]. B. fragilis
mutant strains lacking genes hlyA/B show reduced
fitness in vitro and in vivo, indicating that hemolysins
may be involved in colonization [153]. At present,
there is no clear evidence showing a specific role of
hemolysins in the pathogenesis of disease caused
by B. fragilis.
Good gone bad
Neuraminidase
Bacterial glycosidases are ubiquitous and are
used by many organisms to degrade complex
polysaccharides for nutritional and ecological pur-
poses [154]. B. fragilis and other Bacteroides sp.
Can cleave sialic acid from host glycoproteins by
secreting neuraminidase [155,156]. Sialic acid is
commonly present on host glycoproteins [157]. The
most studied neuraminidase from B. fragilis is
encoded by the nanH gene. Deletion of nanH
renders a mutant strain that is outgrown by the
wild-type strain in vitro and in vivo [158]. Sialic acid
release from host glycoproteins and utilization is
likely to function as an extra measure of nutrient
versatility, crucial to a gut commensal. Neuramini-
dase seems to also increase binding of B. fragilis to
mammalian epithelial cells, by releasing sialic acid
and uncovering other glycan moieties [159,160]. The
presence of the nanH gene has also been used as a
means of additional taxonomic information for
classification of B. fragilis isolates from infection
sites and stool samples [161].
Proteases
B. fragilis has been shown to produce other
proteases than BFT and fragipain. A subset of
cysteine proteases of the C10 family (BFP) was
found in genomes of B. fragilis strains [162]. Genes
bfp1e4 are present within mobile genetic elements,
which is indicative of genetic acquisition by B. fragilis
through horizontal transmission. bfp gene expres-
sion, particularly bfp4, is induced in vitro with
increased oxygen concentrations, suggesting a
putative role of BFP in adaptation to environmental
changes [163]. Bfp genes have been found both in
bacterial isolates from infected sites and in stool
samples from healthy donors [162]; however, iso-
genic bfp deletion strains have not been evaluated to
assess the role of BFP in pathogenicity. A fibrinogen-
degrading protease has also been identified in
B. fragilis, although its role in virulence is unknown
[164,165].
Adhesive Molecules
Bacterial cells adhere to substrates via adhesins,
proteins that bind specifically to receptors from host
cells [166,167]. Many adhesins are expressed as a
part of fimbriae or pili [168]. In commensal gut
bacteria such as Bacteroides, binding to intestinal
mucus and epithelial cells favors stable gut niche
colonization. In humans, piliated strains are more
commonly associated with abscess and healthy
stool samples, whereas nonpiliated strains are
enriched in isolates from blood infections [169].
Adhesive-piliated Bacteroides are more easily pha-
gocytosed by neutrophils; thus, loss of cell
Good gone bad
adhesiveness could promote extraintestinal disse-
mination and immune evasion [170]. Specific binding
to red blood cells, or hemagglutination, has been
observed more frequently in blood isolates than in
abscess or healthy stool isolates [171e173]. Sialic
acid has been shown to be required for hemagglu-
tinin phenotypes, and it is proposed as the receptor
for lectin-like adhesins from B. fragilis [159]. The
genetic basis for cell adhesiveness in B. fragilis still
remains unknown.
B. fragilis also presents extracellular matrix
(ECM)ebinding proteins, suggesting a role in extra-
intestinal survival. Binding to fibronectin, the most
abundant protein in the ECM, is mediated by a
protein similar to a TonB-dependent receptor,
BF1991 [174,175]. Surprisingly, a mutant strain
lacking bf1991 is more adhesive to fibronectin that
the wild-type strain, indicative of redundant fibro-
nectin-binding proteins. Bf1991 mutants are more
susceptible in vitro to phagocytosis by macrophages
[175]. Binding of B. fragilis to laminin-1 and collagen-
1 has also been reported [176e178]. Similar to other
human pathogens, B. fragilis can interact with
plasma proteins related to coagulation [179]. BF-
FBP is a 54-KDa protein that binds fibrinogen, the
major component of fibrin abscess formation [165].
Other proteins can bind plasminogen and high-
molecular-mass kininogen, yet the role in B. fragilis
manipulation of host coagulation is not clear
[180,181]. The diversity of host targets that
B. fragilis can potentially bind to allow us to
hypothesize that different binding patterns might be
induced in vivo as a response to environmental and
host cues.
Response to Oxidative Stress
Gut bacteria are exposed to oxygen outside of the
host and within the intestinal cavity owing to the
oxygen concentration gradient across the lumen
[182]. Bacteroides clinical isolates are more aero-
tolerant than nonclinical isolates, suggesting a role
for such adaptation in pathogenesis [183]. Oxidative
stress response (OSR) mechanisms might be
especially protective in the context of extraintestinal
infections, where tissues are more oxygenated than
the gut lumen [184,185]. The anaerobic/aerobic flux
can modify gene expression profiles, potentially
altering pathogenicity of the organism and its
association with abdominal and peritoneal infections
[186,187]. Although B. fragilis can grow in nanomolar
oxygen concentrations [188], exposure to higher
levels can arrest its growth [189]; hence, OSR is
required to avoid oxidative cellular damage [184].
OSR is achieved by a vast repertoire of proteins with
different functions, including superoxide dismutase
[190], catalase [191], peroxidases [185,192], iron
storage proteins [193e196], and thioredoxins [197].
The transcription factor OxyR is responsible for the
771
induction of many genes in the OSR pathway
[185,186,198]. Another transcription factor bmoR
has been reported as a component of OSR, inducing
genes required for the maintenance of the intracel-
lular redox state [199,200].
Response to Other Environmental Stimuli
B. fragilis has shown to be responsive to the
presence of bile salts, increasing cell adhesion and
coaggregation in vitro [201,202]. There is no
evidence of bile salt tolerance in Bacteroides as a
pathogenic trait, rather than part of an environmental
stress response. Transcriptional regulators such as
the mar system mediate resistance to antimicrobials
and other environmental stressors [199,203].
Lipopolysaccharide
In gram-negative bacteria, the outer layer of the
outer membrane is composed of phospholipids and
lipooligosaccharides or lipopolysaccharides (LOSs
or LPSs, respectively) [204]. LPS molecules are
composed of a lipid moiety knows as Lipid A, a core
glycan, and an outer glycan [205,206]. Lipid A, or
endotoxin, triggers an inflammatory response,
dependent on recognition and signaling by TLR4 of
immune host cells [206e208]. The endotoxin of
Bacteroidesinduces a much lower degree of LPS/
TLR4-mediated inflammation than Lipid A from
species of Enterobacteria owing to differences in
its chemical structure [208e210]. Monoclonal anti-
bodies against B. fragilis LPSs have been shown to
rescue from peritoneal infections and bacteremia in
nonimmune mice [211].
Mechanisms of Interbacterial
Competition
T6SS in Bacteroides
Bacteria that inhabit the gut are present in large
numbers, and competition for a stable place in such
an environment is commonly established [212,213].
Secretion of toxic proteins is a common mechanism
of competition [214e216], perhaps best exemplified
through multiple studies that have recently indicated
the importance of the T6SS in Bacteroides niche
establishment [46e48] and more broadly in inter-
bacterial competition within the intestine [217e220].
The T6SS resembles an inverted phage, with
sequence and structural analysis indicative of
orthology between these systems [221]. The T6SS
functions by secreting effectors (toxins) directly from
the cytosol of the bacterial cell into another cell in a
contact-dependent manner [222]. Secretion of T6SS
effectors by a bacterial cell is accompanied by
772
synthesis of specific immunity proteins that confer
resistance to attack by sister cells. Each effector
protein contains a cognate immunity protein, typi-
cally encoded by neighboring genes. Although it has
been shown that the T6SS can be used by certain
bacteria to inject toxins into eukaryotic cells, most
species use the T6SS as a mechanism of inter-
bacterial killing [223].
In the species of the order Bacteroidales, the
genes encoding the T6SS can be present in three
different genetic arrangements known as genomic
architectures (GAs), GA1, GA2, and GA3. While
GA1 and GA2 are found in many species of the order
Bacteroidales, GA3 is restricted to B. fragilis [49]. An
evolutionary explanation to B. fragilis GA3 restriction
is that GA1 and GA2 can be mobilized between
bacterial cells through genetic elements, but GA3
does not contain such features of transmission [49].
T6SS genes from Bacteroides sp. are present in up
to 75% of the population as per sequence analysis
from the Human Microbiome Project. This high
prevalence of T6SS-bearing Bacteroides in humans
suggests that this locus plays an important role in the
competition for colonic niche establishment. Most
adult human hosts are colonized by a single strain of
B. fragilis; however, during infancy, strain domi-
nance can shift, evidenced by differential relative
abundance of E-I pairs over time [14]. This finding
generates a compelling argument that the T6SS is a
critical feature for initial niche acquisition during early
childhood. The same study shows an association
between T6SS-bearing B. fragilis and an increase in
other species of Bacteroides in the same ecosys-
tem, suggesting that GA3 effector-immunity pairs are
used in vivo for competition mostly between
B. fragilis strains [14]. This could be due to other
Bacteroides species occupying different geographic
niches within the colon, limiting the negative effect of
the T6SS from B. fragilis [47]. In addition, other
Bacteroides species could be less sensitive in vivo to
B. fragilis T6SS-dependent killing than B. fragilis
strains carrying different GA3 E-I pairs [47]. A very
recent report has shed light on the mechanistic basis
of coexistence between B. fragilis and other species
from the same genus. Many Bacteroides species
have acquired additional immunity proteins against
GA3 from B. fragilis, gaining the capacity to defend
from GA3 attacks and potentially exploit
B. fragiliseoccupied niches [224]. The role of the
T6SS in bacterial competition makes it a potentially
useful tool to manipulate carriage of pathogens by
probiotic interventions. We have shown that coin-
oculation and competition between wild-type ETBF
(strain 43858) and NTBF (strain NCTC 9343) in
antibiotic-treated mice reduces the burden and
toxicity of ETBF in a T6SS-dependent manner.
When NTBF lacks an active T6SS or the effector
protein from the operon, ETBF is able to colonize
successfully and drive colonic disease [50]. This
Good gone bad
property of colonization resistance is strongly
dependent on the strain and temporal order of
colonization of both the NTBF and ETBF strains to
be tested [50].
Antimicrobial Proteins in Bacteroides
In addition to contact-dependent killing, Bacter-
oides can secrete soluble toxins. In particular,
Bacteroides genomes carry genes that encode
toxins that belong to a group named Bacteroi-
dales-secreted antimicrobial proteins (BSAPs)
[51e53]. BSAPs are orthologs of membrane attack
complex/perforin (MACPF) domainecontaining pro-
teins and have been shown to be important for
strain competition between Bacteroides species in
vitro and in vivo [51e53]. Bacteroides species can
also secrete a ubiquitin-like protein that mediates
interbacterial killing [54]. The wide variety of
contact-dependent and contact-independent
mechanisms and effector molecules for bacterial
competition reinforces the concept that a single
genetic determinant will not suffice to outcompete
many diverse organisms from a given niche. Multi-
ple genetic factors likely govern niche occupancy
by B. fragilis; hence, genomic diversity between
strains reduces the possibility of a single strain to be
used as a general probiotic to displace ETBF from
the gut ecosystem. It seems clear at this point that
curation of gene function is still one of the major
bottlenecks in understanding the role of the
15e20% strain-specific portion of B. fragilis gen-
omes in colonization.
Host Factors in Colonization and
Disease
When B. fragilis initially encounters the colon
during early infancy, it is immersed in an environ-
ment where constant and deep changes are
occurring to host and microbial cells [225,226].
Maturation of the colonic epithelial lining and mucus
layer are the key to avoid excessive exposure of the
host to microbes and prevent inflammation
[227,228]. The main structural component of the
mucus is the glycoprotein MUC2, secreted by
goblet cells [229]. MUC2 and other mucins form a
dense mucus layer in direct contact with epithelial
cells and a loose mucus layer around 50 mm apart
from the epithelium [230]. The loose mucus layer
allows penetration by many microorganisms,
whereas the dense layer restricts access to the
epithelium to a small group of bacteria [230,231].
Thickness and penetrability of the mucus is highly
influenced by microbiota composition, highlighting
the complex relationship between the variables
affecting gut health [232,233]. Challenge with ETBF
in Muc2 mucus-deficient mice leads to high
Good gone bad
lethality, indicating the key role of the mucus layer in
preventing ETBF-associated disease [96]. Con-
stant and dynamic communication between host
cells and microbiota during intestinal maturation
contributes to achievement of a symbiotic steady
state that can be disrupted by perturbation of either
the host or the microbiome constituents. In this
section, we will focus on the known host determi-
nants for progression of disease caused by ETBF.
Association of ETBF With Chronic Disease in
Humans
The association between colitis and CRC with
carriage of ETBF has led to the hypothesis that this
strain, because of its tissue-damaging toxin, is a
causal agent of CRC instead of a bystander such as
Streptococcus gallolyticus [234]. Most cases of
CRC (~90%) are sporadic whereas a minor
percentage are familial in nature. In both sporadic
and familial CRC, mutations in the adenomatous
polyposis coli (APC) gene are present in 70e80% of
cases [235,236]. The lack of functional APC is one
of the main drivers of polyp disease and early-onset
CRC [237]. APC forms a protein complex that
targets free cytosolic b-catenin to degradation
through ubiquitinylation [237]. When E-cadherin is
cleaved in a BFT-dependent manner, more soluble
b-catenin is available for Wnt/b-catenin signaling,
inducing a proliferative response through c-myc
(Fig. 3) [105]. Thus, mutations that abrogate
functional APC, together with the effect of BFT on
E-cadherin and the free cytosolic pool of b-catenin,
create the conditions for cellular hyperproliferation
and crypt hyperplasia. The use of multiple intestinal
neoplasia (Min) mouse models carrying mutations
in APC has promoted a clearer causal relationship
between ETBF and CRC [18,238,239]. These mice
spontaneously develop tumors in the small intestine
after 2e3 months in the presence of their endogen-
ous microbiota. However, after oral gavage with
ETBF, colonic disease appears ~4 weeks after
inoculation [239]. BFT-driven damage of colonic
cells in APC-mutant mice triggers a hyperprolifera-
tive response (Fig. 3) and inflammatory cascade
(Fig. 4) in a Stat3- and IL-17edependent manner
[16,18,239]. Secretion of IL-8 and other CXC
chemokines recruits immature polymorphonuclear
cells, leading to exacerbation of inflammation and
cell damage (Fig. 4) [16,18]. These experiments
indicate that ETBF can remodel the colonic epithe-
lium drastically in a predisposed model organism
toward a state of disease. A very recent study
looked at colon biopsies from human patients with
familial adenomatous polyposis and found evi-
dence of causal association between ETBF and
development of CRC [17]. In these individuals,
ETBF was present in biofilms together with pro-
773
Fig. 4. Effects of BFT on the immune response.
Activation of b-catenin/Wnt, STAT3, and NF-kb pathways
is required for cell damage and inflammation. Macro-
phages within the lamina propria secrete proinflammatory
cytokines that promote a Th17 cellular phenotype. The NF-
kB pathway requires IL-17 signaling in CECs. CECs
secrete CXC chemokines such as IL-8, promoting recruit-
ment of myeloid cells to the site of damage. BFT,
Bacteroides fragilis toxin; CEC, colonic epithelial cell; IL,
interleukin; MAPK, mitogen-activated protein kinase.
tease-producing (colibactin) pks and E. coli, restrict-
ing the presence of other microorganisms (Fig. 5)
[17]. Coinoculation of both bacteria in an azoxy-
methane-treated mice model of colon cancer
increased severity of the disease, dependent on
ETBF-driven secretion of IL-17 [17]. Most recently,
mucosal bacterial communities from both healthy
individuals and patients with colon cancer were
shown to induce tumors in three Apc mouse
models, implying that such communities would be
contributors to disease in predisposed individuals
[240]. Major perturbations of gut epithelial home-
ostasis seem to be accompanied of more severe
ETBF-dependent phenotypes.
The intrinsic predisposition of each individual to
colonic inflammation and dysbiosis is likely to
govern whether ETBF displays a phenotype con-
sistent with asymptomatic colonization or severe
colonic disease. As shown in Fig. 5, host suscept-
ibility to ETBF is determined by the state of the
mammalian host cells per se and the symbiotic
microorganisms that together form a complex and
dynamic ecosystem. During periods of gut home-
ostasis, either toxin production could be abolished
or become nondamaging in a host that presents a
774 Good gone bad

Fig. 5. Host and microbial factors that modulate degree of commensalism or pathogenicity of ETBF. ETBF
carriage can result in a risk of colonic disease owing to secretion of BFT and cell damage, but host predisposition is the key
to disease by ETBF. A healthy steady-state microbial community that is not permissive to colonization by ETBF should
contain NTBF bacteria that compete for the colonic niche with ETBF. NTBF requires the capsule to colonize and establishes
competition through the T6SS and BSAPs. In addition, colonization by bacterial commensals during early infancy drives
secretion of anti-inflammatory IL-10, contributing to immune tolerance and microbial symbiosis. On the other hand, damage
caused by BFT and pathogenicity can be worsened by stable colonization by ETBF through bactericidal mechanisms such
as T6SS and BSAPs and specific host susceptibility factors including APC mutations, defects in the epithelial mucus layer,
and colonization by other deleterious microbes such as protease-positive (pksþ) E. coli. BFT, Bacteroides fragilis toxin;
ETBF, enterotoxigenic Bacteroides fragilis; T6SS, type VI secretion system; BSAP, Bacteroidales-secreted antimicrobial
protein; APC, adenomatous polyposis coli; NTBF, non-toxigenic B. fragilis; IL, interleukin.

healthy mucus layer and a symbiotic microbiome.
Perturbations of the gut environment could trigger
pathogenicity of ETBF, resulting in inflammation
and dysbiosis. Changes in bacterial composition
and host cell metabolism could be fueling the
ecosystem with metabolites that can be hijacked
by ETBF, in a similar way to what has been reported
for S.enterica and V.cholerae [55,241]. A case for
that is polyamine metabolism, affected in mice
colonized with ETBF in a BFT-dependent manner.
Spermine oxidase catalyzes formation of Reactive
Oxygen Species (ROS), and its expression is
increased in HT29 cells exposed to purified BFT,
and in C57BL/6 mice, causing DNA damage and
inflammation (Fig. 3) [242]. Probiotic interventions
could be a very valuable tool to delay progression of
disease caused by ETBF in predisposed indivi-
duals. It has recently been shown that in Min mice,
coinoculation of mice with NTBF (strain NCTC
9343) and ETBF (strain 86-5443-2-2, or 86), or
inoculation of NTBF after ETBF colonization, does
not rescue the progression of chronic colitis and
CRC [141]. Therefore, previous niche occupancy by
ETBF highly reduces the success of a second
acquisition event by a probiotic NTBF strain
[50,141]. In this study, dominance of ETBF over
NTBF is partly explained by the secretion of a
MACPF non-T6SS toxin, highlighting how poten-
tially complex probiotic interventions would be in
clinical settings, where genetic diversity is high, and
most niche acquisition determinants remain fairly
unexplored [141].
ETBF as Part of a Complex Ecological
Scenario
B. fragilis is one of the least abundant species of
Bacteroides spp. in humans, comprising around
0.5% of total gut bacteria, yet is the most commonly
isolated Bacteroides species from samples of infec-
tions. As previously mentioned, human metagen-
ome data in children show that this period of life is
pivotal for initial niche acquisition by B. fragilis and is
characterized by fierce strain competition through
the T6SS [14]. From an evolutionary perspective, it is
tempting to speculate that environmental pressure
for niche establishment could have positively
selected the presence of additional factors such as
BFT that allows ETBF to trespass the colonic
epithelial barrier and gain access to other niches. A
link between the site of infection by B. fragilis and its
original niche localization in the gut remains to be
found.
The generation of efficacious probiotic interven-
tions requires a detailed molecular understanding of
the repertoire of genetic determinants of niche
acquisition within many strains of B. fragilis. Geno-
mic acquisition of bft has occurred through multiple
independent events of horizontal transmission [92].
Good gone bad
Thus, diverse genetic backgrounds with different
capacities for niche establishment and strain com-
petition can produce BFT and potentially cause
disease. A desirable probiotic strain against ETBF
should attempt to outcompete various strains by
using “designed” combinations of genetic determi-
nants of colonization; given the complexity of host
colonization, these may require a personalized
medicine perspective to effectively treat those at
risk of ETBF-associated disease. To this end,
knowledge within the field should progress toward
understanding genetic determinants of colonization
and how these can be strategically manipulated to
subvert a given strain of ETBF. Moving toward such
an approach will require genomic analysis of multiple
human isolates and a comprehensive understanding
of the determinants of niche competition.
One of the major challenges to the field is to
understand the initial niche acquisition interactions
of B. fragilis with the human host and to define how
these interactions modulate the immune system
toward a state of tolerance that promotes niche
stability and symbiosis. While these events most
likely occur during infancy, further studies will be
necessary to understand the kinetics of niche
acquisition in the human population. In mouse
models, the time frame for early immunological
recognition and generation of tolerance to a Bacter-
oides antigen spans between 10 and 20 days after
birth [243]. Nonexposure to gut bacterial antigens
during that time frame or prolonged exposure
beyond weaning is detrimental to tolerance by the
immune system, even to gut bacteria that become
dominant later in life [243]. Longitudinal studies
involving large cohorts of infants will be necessary to
define ETBF prevalence and to be able to narrow
down the temporal aspect of initial host-pathobiont
interactions [225]. The current lack of such studies is
one of the major caveat in the comprehension of how
early childhood acquisition of ETBF impacts disease
progression in adults. Studies to date in mouse
models would suggest that ETBF-driven disease is a
chronic condition in predisposed individuals, result-
ing from loss of homeostasis of the colonic cells and
microbiota over long periods of time [244]. Hence,
understanding early events in stable niche occu-
pancy by ETBF is probably pivotal for understanding
the risk of progression of disease. We are still
unaware of how BFT expression and secretion is
regulated in vivo in humans. It is unknown whether
low BFT production or toxin resistance by the host is
responsible for such a high proportion of asympto-
matic carriers of ETBF. The process of long-term
and stable colonization of humans by B. fragilis
remains as uncharted territory.
The field is now poised with an opportunity to
develop and use methods with higher spatial
775
resolution than enumeration of colony-forming
units from stool samples to understand the rele-
vance of bacterial niche occupancy to human health
and disease. Recent research has clearly shown
that fecal matter does not necessarily reflect the
abundance and degree of colonization of specific
gut mucosal tissues by bacteria [245,246], high-
lighting the need for detailed investigation of the
microbial niche with a focus on anatomic resolution.
The use of new tools that enable analysis of in vivo
regulation of gene expression and visualization of
Bacteroides cells directly from intestinal tissue
sections will be crucial to dissect the process of
host colonization in a temporal, spatial, and genetic
manner [247,248]. A novel observation in humans
shows that B. fragilis continues adapting within
healthy individuals via de novo mutations. Long-
itudinal sampling from a human cohort has shown
the appearance of novel strain variants that coexist
in the same host, increasing our grasp on micro-
biome biodiversity within a single individual [249]. In
this review, we have focused on genetic determi-
nants from the host-pathobiont side of the B. fragilis/
ETBF story. There are other important factors for
colonization, such as host diet, antibiotics, and
bacteriophages, that are crucial in the development
and stability of the lower Gastrointestinal (GI)
ecosystem. A challenge for the field will therefore
be the use of emerging complex data analysis
platforms to integrate host and microbial factors in a
temporal manner and to better comprehend how
pathobionts such as ETBF can acquire a niche in
the gut, can become a stable member of the
microbiome, and can cause disease progression
in predisposed hosts.
Acknowledgments
This work was supported by the Department of
Pediatrics at Washington University and by the grant
R01 AI138565-A1 (to J.B.W.).
Received 18 April 2019;
Received in revised form 27 November 2019;
Accepted 5 December 2019
Available online 17 December 2019
Keywords:
Bacteroides fragilis;
BFT;
Fragilysin;
Metalloprotease;
Gut microbiota
776
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