Streptococcus gallolyticus Pil3 Pilus Is Required for Adhesion to Colonic Mucus and for Colonization of Mouse Distal Colon
Streptococcus gallolyticus Pil3 Pilus Is Required for Adhesion to Colonic Mucus and for Colonization of Mouse Distal Colon
Mariana Martins 1 2
Laetitia Aymeric 0
Laurence du Merle 1 2
Camille Danne 1 2
Catherine Robbe-Masselot 3
Patrick Trieu-Cuot 1 2
Philippe Sansonetti 0
Shaynoor Dramsi 1 2
0 Department of Cell Biology and Infection, Molecular Microbial Pathogenesis Unit, Institut Pasteur , Paris
1 Department of Microbiology, Centre National de la Recherche Scientifique (CNRS) ERL3526
2 Department of Microbiology, Biology of Gram-Positive Pathogens Unit, Institut Pasteur
3 University Lille Nord, USTL, UGSF , Villeneuve d'Ascq , France
Streptococcus gallolyticus is an increasing cause of bacteremia and infective endocarditis in the elderly. Several epidemiological studies have associated the presence of this bacterium with colorectal cancer. We have studied the interaction of S. gallolyticus with human colonic cells. S. gallolyticus strain UCN34, adhered better to mucus-producing cells such as HT-29-MTX than to the parental HT-29 cells. Attachment to colonic mucus is dependent on the pil3 pilus operon, which is heterogeneously expressed in the wild-type UCN34 population. We constructed a pil3 deletion mutant in a Pil3 overexpressing variant (Pil3+) and were able to demonstrate the role of Pil3 pilus in binding to colonic mucus. Importantly, we showed that pil3 deletion mutant was unable to colonize mice colon as compared to the isogenic Pil3+ variant. Our findings establish for the first time a murine model of intestinal colonization by S. gallolyticus.
adhesion; colon; colonization; mucins; Pil3 pilus; S; bovis; S; gallolyticus
Streptococcus gallolyticus, formerly classified as S. bovis
biotype I, is an emerging opportunistic pathogen
responsible for septicemia and infective endocarditis in
the elderly with underlying diseases [1–3]. This
grampositive coccus is one of the few intestinal bacteria
consistently linked to colorectal cancer (CRC) [4–10].
Numerous studies have shown that a colon tumor or
polyp was detected upon full bowel examination in
33% to 90% of patients diagnosed with an S. gallolyticus
infection. While fecal carriage of S. gallolyticus in the
healthy population is relatively low, it increases about
5-fold in patients with CRC . However, whether
S. gallolyticus is a cause or a consequence of CRC
remains to be determined [11, 12].
The first genome of S. gallolyticus strain UCN34
isolated from a patient suffering from endocarditis and
thereafter diagnosed for colon cancer provided
important insights on the adaptation and virulence strategies
evolved by this bacterium . It revealed the presence
of 3 pilus operons, pil1, pil2, and pil3 . We
previously showed that Pil1 pilus is important for binding to
collagen through the pilus-associated adhesin PilA and
colonization of heart valves in a rat model of
experimental endocarditis .
How S. gallolyticus interacts with the host colon is
not known. Mucus is the first physical barrier that
protects intestinal cells against microbial infection but is
also used by commensals to interact with the host .
The pil3 locus of S. gallolyticus strain UCN34 is a
likely candidate in this process because a bioinformatic
analysis revealed the existence of a putative
mucusbinding domain in the Pil3 associated adhesin encoded
by gallo_2040. Pilus-like fibers on the surface of UCN34
were characterized by immunoblotting and immune
electron microscopy using polyclonal antibodies against
the two structural Pil3 pilin subunits (Gallo_2040 and
Gallo_2039). Like pilus Pil1, Pil3 is heterogeneously
expressed in the UCN34 population, with a majority
of cells weakly piliated and a minority of cells highly
piliated. We constructed a pil3 deletion mutant in a
Pil3-overexpressing variant (Pil3+) and showed that Pil3 is
involved in S. gallolyticus attachment to colonic mucus and that
the adhesin Pil3A plays an essential role in this interaction.
Furthermore, we demonstrated the requirement of Pil3 pilus for the
colonization of mice colon in vivo, establishing for the first time
a murine model of intestinal colonization by S. gallolyticus.
Genetic Organization and Expression of the Pil3 Pilus in the
In this study, the pil3 locus of S. gallolyticus UCN34 was
characterized at the molecular level. As shown in Figure 1A, this
locus is composed of 5 genes encoding 2 structural pilin subunits
(gallo_2040 and gallo_2039), 1 sortase C enzyme (gallo_2038), 1
homolog of type 1 signal peptidase (gallo_2041), and 1 small
open-reading frame (gallo_2042) of unknown function. This
organization is reminiscent of many pilus operons described in
gram-positive bacteria . The structural proteins Gallo2040
(Pil3A) and Gallo2039 (Pil3B) are typical LPXTG proteins of
1664 and 478 amino acids, respectively; Pil3A being the putative
adhesin and Pil3B the major pilin composing the pilus fiber. A
search for conserved domains using Simple Modular
Architecture Research Tool (SMART) software (EMBL) revealed the
presence of a putative mucus-binding domain within the Pil3A protein
(amino acids 1154 to 1238).
Organization of the pil3 promoter region is similar to that of
pil1, with a putative leader peptide encoding gene containing 13
GCAGA tandem repeats followed by a stem-loop transcription
terminator (Figure 1A). We previously showed that this
structure is involved in the heterogeneous expression of pil1 by a
phase variation mechanism combined with transcriptional
Pil3 pilus heterogeneity in S. gallolyticus strain UCN34 was
first visualized by immunofluorescence using specific antibodies
directed against the pilins Pil3A and Pil3B. As shown in
Figure 1B, only a small proportion of the bacteria expressed the
Pil3 pilus at a high level. An overexpressing variant (Pil3+)
selected from the wild-type strain UCN34  exhibited a higher
proportion of cells expressing Pil3 pilus at a high level. Pil3
heterogeneous expression was further analyzed by flow cytometry
and illustrated by the broad peak profile in the wild-type strain
using specific anti-Pil3B antibody (Supplementary Figure 1A).
Quantification of Pil3B levels in wild-type UCN34 indicated
that approximately 70%–90% of the cells are weakly piliated
(Pil3low) and 10%–30% highly piliated (Pil3high). The Pil3+
strain exhibited a sharper peak in flow cytometry experiment
and a higher number of bacteria (approximately 95%) displayed
high levels of Pil3 pilus. As negative control, we constructed a strain
deleted for the entire pil3 locus (Δpil3) in the Pil3+ background.
Pil3 pilus biogenesis was then assessed by Western blotting of
cell wall protein extracts from S. gallolyticus UCN34, Pil3+, and
Δpil3 using the specific antibodies against Pil3A and Pil3B
pilins. As shown in Figure 1C, a typical laddering profile of
highmolecular-weight polymers can be observed in the Pil3+ variant
but not in the control Δpil3. Very low amount of Pil3 polymers
was detected in UCN34, in agreement with the
immunofluorescence and flow cytometry data. Antibodies produced against
both Pil3A and Pil3B are highly specific, as demonstrated by
the absence of high-molecular-weight reactive bands in Δpil3
protein extracts. Of note, a nonspecific band of 25 kDa was
detected with both antibodies serving as a loading control. Finally,
immunoelectron microscopy revealed the presence of typical
pilus fiber structures in the Pil3+ variant that were not observed
with the isogenic Δpil3 mutant (Figure 1D).
Pil3 Promotes Bacterial Attachment to Colonic Mucus In Vitro
Pili have long been considered important players in bacterial
attachment to host tissues, an essential step in the pathogenic
process. The presence of a putative mucus-binding domain in
Pil3A led us to uncover the functional binding properties of
this pilus. The capacity of S. gallolyticus UCN34, Pil3+, and
Δpil3 mutant to adhere to the mucus recovered from
HT29MTX was tested in vitro (Figure 2A). This human colonic cell
line, upon differentiation for 15 to 20 days, is known to
constitutively produce mucus [18, 19]. As shown in Figure 2A, the
Pil3+ variant adheres more efficiently to mucus as compared
to the parental UCN34 strain or the Δpil3 mutant. A similar
result was obtained when using purified porcine gastric mucins
(Figure 2B). No significant binding was observed when using
bovine maxillary mucins (data not shown), suggesting some
specificity in host mucins serving as Pil3 ligand. Furthermore,
we analyzed the binding capacity of the same strains to purified
human MUC5AC mucin. Likewise, the Pil3+ strain efficiently
adhered to MUC5AC when compared to the Δpil3 mutant
To test the role of Pil3A putative adhesin in bacterial
attachment to mucus, we overexpressed gallo_2040 in the plasmid
pTCV-erm using a strong constitutive promoter. Expression
of the adhesin Pil3A in the Δpil3 (Pil3A) strain was demonstrated
by flow cytometry (Supplementary Figure 1B) and
immunofluorescence (Supplementary Figure 1C). As shown in Figure 2C,
overexpression of Pil3A alone in S. gallolyticus Δpil3 mutant
partially restored bacterial adhesion to HT29-MTX mucus as
compared to the control Δpil3 strain harboring the empty vector
(Δpil3(vector)). Furthermore, heterologous expression of Pil3A in
Lactococcus lactis strain NZ9000 also conferred to the
recombinant bacteria an enhanced capacity for binding to colonic
mucus, as compared to the control L. lactis strain harboring
the empty vector (Figure 2C). To further demonstrate the role
of Pil3A in mucus binding, we tested several clinical strains of S.
gallolyticus from our collection, both for expression of Pil3A
and binding to mucus derived from HT29-MTX cells. Because
Pil3 expression is heterogeneous and subjected to phase
variation, the experiments were carried out in parallel. A robust
correlation (r = 0.97; P <.0001) was observed between the level
of Pil3A expression measured by flow cytometry and the
binding capacity of the various S. gallolyticus strains to HT29-MTX
mucus (Figure 2D).
Role of Pil3 in Primary Attachment to Human Mucus-Producing
We next investigated the role of Pil3 pilus in adhesion to colonic
epithelial cells in the presence or absence of mucus. We
compared adhesion of S. gallolyticus UCN34, Pil3+, and Δpil3
strains to the mucus-producing HT29-MTX cells and to the
parental HT29 cell line that does not produce mucus. Both types
of cells were grown on coverslips for 2 weeks before infection
with the 3 strains. Preliminary adhesion experiments on
Caco-2 cells indicated that efficient adhesion of S. gallolyticus
occurred at 4 hours postinfection with a significant increase at
6 hours. We therefore chose to image S. gallolyticus adhesion to
HT29-MTX and HT29 at 6 hours postinfection by confocal
microscopy using an antibody raised against the whole bacterium
UCN34. We first verified that the 3 strains of S. gallolyticus,
UCN34, Pil3+, and Δpil3, grew similarly in the conditioned cell
culture medium from both cell types (data not shown).
Interestingly, both UCN34 and Pil3+ variant attached very efficiently to
HT29-MTX cells, whereas the Δpil3 mutant was unable to bind
these cells (Figure 3A). Strikingly, all 3 S. gallolyticus strains
bound very weakly to the HT29 parental cells (Figure 3A,
lower panel). Quantification of the fluorescence signal from
adherent bacteria is shown in Figure 3B. The results unambiguously
demonstrate the role of Pil3 in the attachment of S. gallolyticus to
colonic mucus-producing cells. All together, these in vitro results
strongly point to the importance of Pil3 pilus in bacterial
adhesion to colonic mucus and subsequently in the establishment of a
direct interaction with mucus-producing cells.
Pil3 Pilus Is Critical for Colonization of the Mouse
We next wondered whether S. gallolyticus Pil3 pilus could
contribute to colonization of the colon in vivo. To address this
question, we established for the first time an in vivo model of
gastrointestinal colonization for S. gallolyticus in mice.
Following oral inoculation of S. gallolyticus, we were able to quantify
living bacteria in intestinal tissues using selective culture media.
To increase gastrointestinal colonization by S. gallolyticus,
specific pathogen-free mice were first treated with broad-spectrum
antibiotics to reduce mice endogenous gut microbiota, as
described previously , thus enhancing UCN34 colonization
by 3 logs (data not shown). We then compared colonization
efficiency of S. gallolyticus reference strain UCN34 in 2 different
mice strains: BALB/c and C57BL/6J. The colonization was 10
times more efficient in the C57BL/6 mouse background (data
not shown). Using this optimized protocol in C57BL/6 mice,
we compared colonization efficiencies of S. gallolyticus strain
UCN34, Pil3+ variant, and Δpil3 deletion mutant in different
parts of the gastrointestinal tract, including the ileum and distal
colon. A representative experiment with 5 mice in each group,
analyzed for colony-forming unit (CFU) quantification
(Figure 4A) and by immunohistochemistry (Figure 4B), is shown
in Figure 4. We found that the Δpil3 mutant was significantly
impaired, with a 2-log difference, in distal colon colonization
compared to the Pil3+ variant. UCN34 was able to colonize
at an intermediate level between Pil3+ and Δpil3, in agreement
with the smaller proportion of bacteria expressing pil3 in
To determine the localization of S. gallolyticus in vivo,
immunohistochemistry was performed using a polyclonal
antibody raised against UCN34 to label bacteria and wheat germ
agglutinin (WGA) to visualize the colonic mucus layer. As
shown in Figure 3B, most Pil3+ bacteria were found both in
the lumen and also tightly associated to the colonic mucus
layer. In contrast, the Δpil3 mutant was only found in the
lumen of the gastrointestinal (GI) tract (data not shown)
and in much lower number (Figure 4B, right lower panel),
in agreement with the CFU counts. These results demonstrate
the role of Pil3 pilus in the colonization of mouse distal colon
by S. gallolyticus.
Streptococcus gallolyticus belongs to the Streptococcus bovis/
Streptococcus equinus complex, a highly diverse group of
nonhemolytic streptococci that are intestinal commensals,
opportunistic pathogens, and food fermentation associates . It is an
emerging human pathogen that causes infective endocarditis
and is consistently associated with colorectal carcinomas .
Current medical recommendations advise to perform a
colonoscopy for any patient diagnosed with S. gallolyticus–
In the present study, we carried out the molecular and
functional characterization of the pil3 locus (gallo2042-2038)
encoding a putative mucus binding protein. The pil3 locus
is highly conserved and present in all 31 clinical strains of
S. gallolyticus analyzed previously . Basic Local Alignment
Search Tool (BLAST) analyses revealed the presence
of the pil3 operon in S. pasteurianus and in S. macedonicus
Pil3 pilus of S. gallolyticus strain UCN34 consists of 2
structural subunits—Pil3B, the major pilin, and Pil3A, the pilus
associated adhesin—that are covalently assembled by a class C
sortase (encoded by gallo_2038). Single-cell analyses
demonstrated that Pil3 is expressed heterogeneously in the UCN34
population by a mechanism of phase variation previously
characterized . By selecting a phenotypic variant expressing
higher amount of pili (Pil3+) and by constructing a pil3
deletion mutant in this variant, we were able to demonstrate the
role of Pil3 pilus in the attachment to colonic mucus of
HT29MTX cells. Overexpression of Pil3A in the S. gallolyticus pil3
mutant or in the heterologous L. lactis enhanced bacterial
binding to the colonic mucus in vitro, indicating that Pil3A
contributes to the mucus-binding function. Of note, Pil3 also conferred
binding to pig gastric mucins (Figure 2B), to purified MUC5AC
mucin (Figure 2C), but not to bovine maxillary mucins (data
HT29-MTX mucus is highly enriched in MUC5AC mucin
 and devoid of MUC2 (data not shown). While MUC2 is
the predominant mucin in healthy colon, MUC5AC is not
detected in normal conditions, but is frequently overexpressed in
adenomas and colon cancers . Interestingly, pig gastric
mucins, which act as ligand for Pil3, react with monoclonal
antibodies against human MUC5AC but not with MUC2. In
contrast, bovine maxillary mucins, which are not permissive
for Pil3 binding, did not react with MUC5AC monoclonal
antibody and only gave a faint signal with MUC2 (data not
shown). Finally, we demonstrated that Pil3 binds specifically
to purified MUC5AC mucin (Figure 2C), which may explain
the low carriage rate of S. gallolyticus in healthy colon of the
human population, and the higher carriage in the presence of
a colon tumor.
Importantly, we established for the first time a mouse model
to study gut colonization by S. gallolyticus and were able to show
that Pil3 promotes bacteria attachment to the distal colon.
Histopathological analyses further demonstrated that S. gallolyticus
expressing a high level of Pil3 mainly colocalized with the mice
colonic mucus, whereas the Δpil3 mutant, present in much
lower numbers, was found mainly in the lumen and barely
associated to the mucus layer (Figure 4).
Other examples of pili from gram-positive
bacteriapromoting gut colonization are found in beneficial
commensals of the GI tract, such as lactobacilli and bifidobacteria .
In particular, the SpaCBA pilus of the probiotic Lactobacillus
rhamnosus GG is involved in binding to human mucus via the
adhesin SpaC . Interestingly, SpaC was found along
the whole pilus length, allowing both short- and long-distance
interactions with the host tissues, thus providing
mucusbinding strength to persist longer in the gastrointestinal
In conclusion, we identified and characterized the Pil3 pilus of
S. gallolyticus as a novel factor required for bacterial adhesion to
human colonic mucus and for colon colonization in the mouse model.
MATERIAL AND METHODS
Cell Culture and Bacterial Strains
The mucus-secreting HT29-MTX cell subpopulation  and
the parental HT29 cells were routinely grown in Dulbecco’s
modified Eagle medium supplemented with 10% heat-inactivated
fetal bovine serum. S. gallolyticus strains were grown at 37°C
in Todd-Hewitt (TH) broth in standing filled flasks. Lactococcus
lactis strains were grown in M17 medium containing 1%
glucose. Erythromycin and tetracycline were used at 10 µg/mL
for S. gallolyticus, and erythromycin was used at 150 µg/mL
for Escherichia coli. A Pil3 pilus-overexpressing strain (Pil3+)
was selected from the wild-type UCN34 by immunolabeling
screening as previously described . We have constructed a
pil3 deletion mutant in the Pil3+ strain background ( from
gallo_2042 to gallo_2038) as described previously . The
primers used are listed in Table 1.
Expression and Purification of Recombinant 6×His-Gallo2039
and 6×His-Gallo2040 N-terminus
DNA fragments internal to gallo_2039 and gallo_2040 were
produced by polymerase chain reaction using genomic DNA
of UCN34 as template and the primers gallo2039-NdeI and
gallo2039-BamHI, and gallo2040-NheI and gallo2040-BamHI,
respectively (Supplementary Table 1). These DNA fragments
were digested with the appropriate enzymes and cloned
into pET28-a(+) (Novagen). The resulting plasmids were
introduced into E. coli strain DH5α for sequence analysis
or BL21 (λDE3) for protein expression. Recombinant
6×HisGallo2039 and 6×His-Gallo2040 Nter were purified under
native conditions by affinity chromatography on nickel-charged
nitrilotriacetic acid (Ni-NTA) columns according to the
manufacturers’ recommendations (Novagen). Protein purity was
checked on sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE) and accurate protein concentrations
were determined with the bicinchoninic acid (BCA) system
Abbreviation: bp, base pair.
Generation of Rabbit and Mice Polyclonal Antibodies
Rabbit polyclonal antibodies against Gallo_2039 (Pil3B) and
Gallo_2040 (Pil3A) were generated by Covalab, Villeurbanne,
France (www.covalab.com). Rabbit serum samples were further
purified using the Melon Gel IgG Spin Purification Kit
(Thermo Scientific Pierce).
Immunofluorescence and Flow Cytometry Experiments
These experiments were performed exactly as described
previously  except that coverslips were mounted with ProLong
Gold Antifade (Life Technologies) and imaged on a Nikon
Eclipse Ni-U microscope.
HT29 and HT29-MTX were cultured in coverslips for 16 days
and infected with S. gallolyticus isogenic strains UCN34, Pil3+,
and Δpil3 at a multiplicity of infection of 5 bacteria per cell.
After 6 hours of infection, cells were washed once in
phosphate-buffered saline (PBS) and fixed with 4%
paraformaldehyde (PFA) for 10 minutes. Infected cells were then stained
with 1:200 anti-UCN34 antibody (Covalab, France), followed
by secondary DyLight488 conjugated antirabbit antibody (1:200)
to stain specifically S. gallolyticus. Hoescht 33 342 (1:2000) was
added to visualize cells nuclei and Alexa Fluor 647 phalloidin
(1:50) to stain the actin cytoskeleton. Samples were mounted
using the ProLong Gold Antifade reagent and visualized using a
Leica TCS SP5 confocal microscope. Maximum projections and
total fluorescence measurements were performed with Image J.
Cell Wall Protein Extraction
Bacteria were grown overnight in TH broth and harvested for
protein analysis during the late-exponential phase of cultures
as described previously . When necessary, cell wall extracts
were concentrated using trichloroacetic acid (TCA) protein
precipitation. Samples were incubated with 7% TCA for 30 minutes
on ice washed with acetone and resuspended in PBS for
quantification. Protein extracts were boiled in Laemmli buffer and
Sequence 5′ → 3′
further analyzed on SDS-PAGE. Midi Criterion XT Precast gel
(4%–12% Bis-Tris; Bio-Rad) were used and transferred to
nitrocellulose membrane using the Trans-Blot Turbo transfer Pack,
Bio-Rad. Membrane was blocked in TBS–skimmed milk
5% and incubated for 1 hour with rabbit primary Pil3B and
Pil3A antibodies and then with the secondary
Dylight800coupled goat antirabbit antibody (Thermo Scientific Pierce).
Far-red fluorescence was detected using the LI-COR Odyssey
Infrared Imaging System (LI-COR Biosciences).
Mucus Adhesion Assay
Colonic mucus was recovered from HT29-MTX cells upon 16 to
20 days in culture and quantified using BCA assay (Thermo
Scientific, Pierce). Polystyrene Maxisorp (NUNC) plates (96-wells)
were precoated overnight with 5 µg/well of either MTX mucus or
porcine stomach mucins (Sigma Ref. M1778). Overnight cultures
grown in TH were washed once with 1× PBS and 100 µL of cell
suspension at optical density (600 nm) of two were added to each
well. Two were added to each well with further incubation at 37°C
for 2 hours. After 2 consecutive washes, the bacteria were stained
with 0.1% crystal violet for 30 minutes, washed twice, and
airdried for 15 minutes. Stained bacteria were resuspended for
quantification in ethanol/acetone solution (80:20) and
absorbance was measured at 595 nm.
Mice Colonization Experiments
All animal experiments were carried out under approval by the
Use Committee of Pasteur Institute and by the French Ministry
of Agriculture (Ethic committee protocol number: 2013-0030).
BALB/c and C57BL/6J Rj mice were obtained from Janvier Labs
(Le Genest-Saint-Isle, France) and maintained in a
pathogenfree area. Based on a published protocol , 8-week-old mice
were treated with broad-spectrum antibiotics, including
vancomycin, metronidazole, neomycin, and ampicillin, for 8 days.
Using a straight feeding cannula (Bioseb N-020), mice were
orally inoculated with 2 × 109 exponentially growing bacteria
on 3 consecutive days. Bacterial colonization was determined
7 days postinfection by CFU counts. Briefly, mice were
euthanized, and tissues were harvested, weighted, and homogenized
using Precellys homogenizer (Ozyme) for 2 × 15 seconds at a
frequency of 5000 rpm. Samples were diluted in saline and
plated on Enterococcus agar selective media (BD Difco) for specific
counting of S. gallolyticus exhibiting a typical pink color as
previously described . This experiment was repeated twice with
a minimum of 5 mice per group each time.
Immunocytochemistry of Mouse Tissues
Mouse tissues recovered 7-day postinfection were fixed for 48
hours in PBS-PFA 4% and embedded in paraffin following
routine procedures. Serial sections were permeabilized with 0.1%
Triton X-100 for 30 minutes and blocked for 5 minutes with
Ultra V block (Thermo Scientific). Samples were incubated
for 1 hour in PBS-10% Ultra V block with rabbit anti-UCN34
(1:200). Secondary Alexa Fluor 568-conjugated goat antirabbit
antibody (1:200, Life Technologies) and WGA-coupled to Alexa
Fluor 488 (1:200, Life Technologies) in PBS-10% Ultra V block
were added and samples were incubated for 45 minutes at room
temperature. The tissue preparations were then incubated with
4’,6-diamidino-2-phenylindole (1:1000) in PBS for 3 minutes
and mounted with ProLong Gold Antifade reagent. Sections
were imaged on a Cell Voyager CV1000 confocal scanner box
and fluorescence images were processed using Fiji software.
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary
data are the sole responsibility of the authors. Questions or messages
regarding errors should be addressed to the author.
Acknowledgments. We sincerely thank Prof Alain Servin and Virginie
Liévin-Le Moal (Faculty of Pharmacy, Chatenay-Malabry, France) for
providing the cell lines HT29 and HT29-MTX. We are grateful to Adeline
Mallet from the Platform of Ultrastructural Microscopy for the electron
microscopy experiments. We also thank Jean-Yves Tivenez from the
Imagopole-Plateforme d’Imagerie Dynamique (PFID) France–BioImaging
infrastructure, supported by the French National Research Agency (ANR
10-INSB-04-01, Investments for the Future), for advice and access to
the CV1000 system. We thank Claire Poyart, head of the CNR-Strep for
providing clinical strains of S. gallolyticus.
Financial support. This work was supported by the French National
Research Agency (ANR) Blanc Glyco-Path (grant
n°ANR-10_BLANC1314), by the Foundation for Medical Research (FRM), and from the French
Government’s Investissement d’Avenir Program, Laboratoire d’Excellence
“Integrative Biology of Emerging Infectious Diseases” (grant
M. M. was supported by a stipend from the Pasteur–Paris University
(PPU) International PhD Program and by a 1-year extension fellowship
from the Association pour la Recherche sur le Cancer (ARC) Foundation
(01CA140068-ARC-MARTINS). L. A. was supported by “La Ligue
nationale contre le cancer.”
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
1. Watanakunakorn C. Streptococcus bovis endocarditis . Am J Med 1974 ; 56 : 256 - 60 .
2. Schlegel L. Reappraisal of the taxonomy of the Streptococcus bovis/Streptococcus equinus complex and related species: description of Streptococcus gallolyticus subsp. gallolyticus subsp . nov., S. gallolyticus subsp . macedonicus subsp. nov. and S. gallolyticus subsp. Int J Syst Evol Microbiol 2003 ; 53 : 631 - 45 .
3. Jans C , Meile L , Lacroix C , Stevens MJ . Genomics, evolution, and molecular epidemiology of the Streptococcus bovis/Streptococcus equinus complex (SBSEC). Infect Genet Evol 2015 ; 33 : 419 - 36 .
4. Klein RS , Recco RA , Catalano MT , Edberg SC , Casey JI , Steigbigel NH . Association of Streptococcus bovis with carcinoma of the colon . N Engl J Med 1977 ; 297 : 800 - 2 .
5. Ellmerich S , Schöller M , Duranton B , et al. Promotion of intestinal carcinogenesis by Streptococcus bovis . Carcinogenesis 2000 ; 21 : 753 - 6 .
6. Gupta A , Madani R , Mukhtar H. Streptococcus bovis endocarditis, a silent sign for colonic tumour . Colorectal Dis 2009 ; 12 : 164 - 71 .
7. Abdulamir A , Hafidh R , Bakar F. Molecular detection, quantification, and isolation of Streptococcus gallolyticus bacteria colonizing colorectal tumors: inflammation-driven potential of carcinogenesis via IL-1, COX-2, and IL-8. Mol Cancer 2010 ; 249 : 1 - 18 .
8. Boleij A , Muytjens CMJ , Bukhari SI , et al. Novel clues on the specific association of Streptococcus gallolyticus subsp gallolyticus with colorectal cancer . J Infect Dis 2011 ; 203 : 1101 - 9 .
9. Boleij A , Tjalsma H. The itinerary of Streptococcus gallolyticus infection in patients with colonic malignant disease . Lancet Infect Dis 2013 ; 13 : 719 - 24 .
10. Boleij A , van Gelder MMHJ , Swinkels DW , Tjalsma H. Clinical Importance of Streptococcus gallolyticus infection among colorectal cancer patients: systematic review and meta-analysis . Clin Infect Dis 2011 ; 53 : 870 - 8 .
11. zur Hausen H. Streptococcus bovis: causal or incidental involvement in cancer of the colon ? Int J Cancer 2006 ; 119:xi-xii .
12. Hensler ME . Streptococcus gallolyticus, infective endocarditis, and colon carcinoma: new light on an intriguing coincidence . J Infect Dis 2011 ; 203 : 1040 - 2 .
13. Rusniok C , Couvé E , Da Cunha V , et al. Genome sequence of Streptococcus gallolyticus: insights into its adaptation to the bovine rumen and its ability to cause endocarditis . J Bacteriol 2010 ; 192 : 2266 - 76 .
14. Danne C , Entenza JM , Mallet A , et al. Molecular characterization of a Streptococcus gallolyticus genomic island encoding a pilus involved in endocarditis . J Infect Dis 2011 ; 204 : 1960 - 70 .
15. Johansson M. The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial interactions . Proc Natl Acad Sci USA 2011 ; 108 (suppl): 4659 - 65 .
16. Danne C , Dramsi S. Pili of Gram-positive bacteria: roles in host colonization . Res Microbiol Elsevier Masson SAS 2012 ; 163 : 645 - 58 .
17. Danne C , Dubrac S , Trieu-Cuot P , Dramsi S. Single cell stochastic regulation of pilus phase variation by an attenuation-like mechanism . PLOS Pathog 2014 ; 10 :e1003860.
18. Lesuffleur T , Barbat A , Dussaulx E , Zweibaum A. Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus-secreting cells . Cancer Res 1990 ; 50 : 6334 - 43 .
19. Lesuffleur T , Violette S , Vasile-Pandrea I , et al. Resistance to high concentrations of methotrexate and 5-fluorouracil of differentiated HT-29 colon-cancer cells is restricted to cells of enterocytic phenotype . Int J Cancer 1998 ; 76 : 383 - 92 .
20. Reikvam DH , Erofeev A , Sandvik A , et al. Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression . PLOS One 2011 ; 6 : 1 - 13 .
21. Sillanpää J , Nallapareddy SR , Qin X , et al. A collagen-binding adhesin, Acb, and ten other putative MSCRAMM and pilus family proteins of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis group, biotype I) . J Bacteriol 2009 ; 191 : 6643 - 53 .
22. Lin I-H , Liu T-T , Teng Y-T , et al. Sequencing and comparative genome analysis of two pathogenic Streptococcus gallolyticus subspecies: genome plasticity, adaptation and virulence . PLOS One 2011 ; 6 : e20519 .
23. Jans C , Follador R , Hochstrasser M , Lacroix C , Meile L , Stevens MJA . Comparative genome analysis of Streptococcus infantarius subsp. infantarius CJ18, an African fermented camel milk isolate with adaptations to dairy environment . BMC Genomics 2013 ; 14 : 200 .
24. Papadimitriou K , Anastasiou R , Mavrogonatou E , et al. Comparative genomics of the dairy isolate Streptococcus macedonicus ACA-DC 198 against related members of the Streptococcus bovis/Streptococcus equinus complex . BMC Genomics 2014 ; 15 : 272 .
25. Gouyer V , Wiede A , Buisine M. Specific secretion of gel-forming mucins and TFF peptides in HT-29 cells of mucin-secreting phenotype . Biochem Biophys Acta 2001 ; 1539 : 71 - 84 .
26. Byrd J , Bresalier R. Mucins and mucin binding proteins in colorectal cancer . Cancer Metastasis Rev 2004 ; 23 : 77 - 99 .
27. Ventura M , Turroni F , van Sinderen D. Probiogenomics as a tool to obtain genetic insights into adaptation of probiotic bacteria to the human gut . Bioeng Bugs 2012 ; 3 : 73 - 9 .
28. Kankainen M , Paulin L , Tynkkynen S , et al. Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a humanmucus binding protein . Proc Natl Acad Sci USA 2009 ; 106 : 17193 - 8 .
29. Reunanen J , von Ossowski I , Hendrickx APA , Palva A , de Vosa WM . Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG . Appl Environ Microbiol 2012 ; 78 : 2337 - 44 .
30. Danne C , Guérillot R , Glaser P , Trieu-Cuot P , Dramsi S. Construction of isogenic mutants in Streptococcus gallolyticus based on the development of new mobilizable vectors . Res Microbiol 2013 ; 164 : 973 - 8 .