nihms505094.pdf

Bacterial colonization factors control specificity and stability of

the gut microbiota

S. Melanie Lee

Gregory P. Donaldson

Zbigniew Mikulski

Silva Boyajian

Klaus Ley

and

Sarkis K. Mazmanian

1,*

Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA

91125, USA

Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA

92037, USA

Abstract

Mammals harbor a complex gut microbiome, comprised of bacteria that confer immunologic,

metabolic and neurologic benefits

. Despite advances in sequence-based microbial profiling and

myriad studies defining microbiome composition during health and disease, little is known about

the molecular processes employed by symbiotic bacteria to stably colonize the gastrointestinal

(GI) tract. We sought to define how mammals assemble and maintain the

Bacteroides

, one of the

most numerically prominent genera of the human microbiome. While the gut normally contains

hundreds of bacterial species

, we surprisingly find that germ-free mice mono-associated with a

single

Bacteroides

are resistant to colonization by the same, but not different, species. To identify

bacterial mechanisms for species-specific saturable colonization, we devised an

in vivo

genetic

screen and discovered a unique class of Polysaccharide Utilization Loci (PUL) that are conserved

among intestinal

Bacteroides

. We named this genetic locus the

ommensal

olonization

actors

(

ccf

). Deletion of the

ccf

genes in the model symbiont,

Bacteroides fragilis

, results in colonization

defects in mice and reduced horizontal transmission. The

ccf

genes of

B. fragilis

are up-regulated

during gut colonization, preferentially at the colonic surface. When we visualize microbial

biogeography within the colon,

B. fragilis

penetrates the colonic mucus and resides deep within

crypt channels, while

ccf

mutants are defective in crypt association. Remarkably, the CCF system

is required for

B. fragilis

colonization following microbiome disruption with

Citrobacter

rodentium

infection or antibiotic treatment, suggesting the niche within colonic crypts represents a

reservoir for bacteria to maintain long-term colonization. These findings reveal that intestinal

Bacteroides

have evolved species-specific physical interactions with the host that mediate stable

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Correspondence and requests for materials should be addressed to S.K.M. sarkis@caltech.edu.

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Author Contributions.

S.M.L. and S.K.M. conceived the project. S.M.L. performed most of the experiments; G.P.D., Z.M. and S.B.

contributed data. S.M.L., G.P.D., Z.M., K.L. and S.K.M. interpreted the data. K.L. and S.K.M secured funding. S.M.L. and S.K.M.

wrote the manuscript, G.P.D., Z.M. and K.L. edited the manuscript. G.P.D. and Z.M. contributed equally to the work.

The authors declare no competing financial interests.

Readers are welcome to comment on the online version of this article at

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Author manuscript

Nature

. Author manuscript; available in PMC 2014 March 19.

Published in final edited form as:

Nature

. 2013 September 19; 501(7467): 426–429. doi:10.1038/nature12447.

Author Manuscript

and resilient gut colonization, and the CCF system represents a novel molecular mechanism for

symbiosis.

International microbiome sequencing initiatives are revealing detailed inventories of diverse

bacterial communities across various body sites, diets and human populations

. Complex

ecosystems have been forged by co-adaptation over millennia between animals and

microbes to create stable and specific microbiomes

, suggesting the evolution of molecular

mechanisms that establish and maintain symbiotic microbial colonization.

Bacteroidetes

one of the most numerically abundant Gram-negative phyla in the mammalian GI tract

Studies in the genus

Bacteroides

have revealed species that induce glycosylation of the

intestinal epithelium

, produce glycoside hydrolases that digest carbohydrates for host

nutrient utilization

, direct host immune maturation

and protect animals from

inflammation in experimental models of IBD and multiple sclerosis

. To explore the

dynamics of microbiome assembly, we sequentially introduced

Bacteroides

species to germ-

free mice and monitored colonization via colony-forming units (CFU) in feces. Animals are

readily colonized with

Bacteroides fragilis

followed by

Bacteroides thetaiotaomicron

(Fig.

1a) or

Bacteroides vulgatus

(Fig. 1b), and altering the sequence of microbial exposure does

not affect results (Supplementary Fig. 1a). Remarkably however, animals colonized with

fragilis

and then exposed to the same species (marked by an antibiotic resistance gene) are

resistant to super-colonization and clear the challenging strain (Fig. 1c). This novel

observation of ‘colonization resistance’ by the same species is conserved in three other

Bacteroides

(Supplementary Fig. 1b-d), regardless of the antibiotic resistance markers used

(Supplementary Fig. 1e), but not in

Escherichia coli

(Supplementary Fig. 1f). As

conventional mice typically harbor 10

-10

CFU per gram of cecal content

(100-fold

greater than

Bacteroides

in mono-association), there appears to be no shortage of space or

nutrients under these conditions, using a nutrient rich standard diet. We thus hypothesized

that individual

Bacteroides

species colonize the gut by saturating a limited and unique niche.

Indeed, treatment of

B. fragilis

mono-associated mice with erythromycin to displace the

existing strain permits colonization by an erythromycin-resistant challenge strain (Fig. 1d).

These data suggest that

Bacteroides

colonize the gut in a species-specific and saturable

manner.

We developed a functional

in vivo

screen to identify genetic factor(s) from

B. fragilis

that

are sufficient to mediate species-specific colonization. Mice were mono-associated with

vulgatus

, then challenged with a library of

B. vulgatus

clones that each contained a fragment

B. fragilis

genomic DNA (schematic in Supplementary Fig. 2a). We reasoned that only

those clones containing genes that conferred stable gut colonization by

B. fragilis

would

persist, with the remainder being cleared. We screened 2,100 clones each containing 9-10

kilobases of DNA, providing a 3.8-fold coverage of the

B. fragilis

genome and 98%

probability that a given DNA sequence is present in the library (Supplementary Eq. 1).

Remarkably, 30 days after orally gavaging the library into animals, only two clones

sustained colonization. The inserts from both clones mapped to the same locus on the

fragilis

genome, BF3579-BF3583 (Supplementary Fig. 2b).

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Based on predicted protein sequences, BF3583 and BF3582 is a sigma (

) factor/anti-

factor gene pair. BF3581 is a member of the SusC family of outer membrane proteins.

BF3580 is a homolog of SusD, a lipoprotein often paired with SusC. These Sus-like systems

have been shown to bind and import a range of oligosaccharide molecules

. BF3579

encodes a putative chitobiase, suggesting a possible polysaccharide substrate for this

system

(Fig. 1e). Comparative genomic analysis using the Integrated Microbial Genomes

database (

http://img.jgi.doe.gov/cgi-bin/w/main.cgi

) reveals conservation of similar clusters

of genes among sequenced intestinal

Bacteroides

species (Supplementary Fig. 3). Sus-like

systems are numerous in

Bacteroides

within polysaccharide utilization loci (PULs), which

are gene cassettes used to harvest dietary sugars and/or forage host glycans during nutrient

deprivation

. As PULs have previously not been implicated in saturable niche

colonization, the locus we have identified encodes a unique pathway in

Bacteroides

for

species-specific gut association; we named the genes

ccfA-E,

for

ommensal

olonization

actors (Fig. 1e). Furthermore, as deletion of the most closely related genes from

B. fragilis

(BFΔ0227-0229; Supplementary Fig. 3) do not impact colonization dynamics

(Supplementary Fig. 4), we suggest that the CCF system represents a functionally unique

subset of PULs that evolved to promote long-term symbiosis.

To test if the putative structural genes (

ccfC-E

) are required for gut colonization, we

generated in-frame deletion mutants of

B. fragilis

; Δ

ccfC,

ccfD

and Δ

ccfE

. All strains

exhibit normal morphology on solid agar medium and unimpaired growth in laboratory

culture (data not shown). As previously shown, animals mono-colonized with WT

B. fragilis

completely clear the WT challenge strain after 30 days (Fig. 1f; 1

bars). However, animals

mono-associated with Δ

ccfC

or Δ

ccfD

are permissive to colonization by WT bacteria (Fig.

1f; 2

and 3

bars), unlike the

ccfE

mutant (Fig. 1f; 4

bars). A deletion mutant in all three

genes (

B. fragilis

ΔCCF) also allows WT

B. fragilis

to colonize (Fig. 1f; 5

bars). Trans-

complementation of the

B. fragilis

ΔCCF strain with

ccfA-E

restores colonization resistance

(Fig. 1f; 6

bars). Similarly, a mutant in the

B. vulgatus ccfC-E

orthologs (ΔBVU0946-

BVU0948) also permits WT

B. vulgatus

to colonize (Fig. 1g), demonstrating conservation in

Bacteroides

species. When we tested horizontal transmission of bacteria between WT

fragilis

and

B. fragilis

ΔCCF mono-associated mice, only WT bacteria cross-colonized (Fig.

1h). Thus, the CCF system is involved in colonization resistance by

Bacteroides

Building on the previous discovery that a population of

B. fragilis

associates with mucosal

tissues

, we show that

ccfB-E

are preferentially expressed by bacteria in contact with the

colon, with lower levels in cecal content and feces (Fig. 2a). There is virtually no expression

in laboratory culture. Thus,

in vivo

expression of

ccf

in gut tissue may be critical for

colonization. Indeed, in contrast to laboratory grown bacteria (see Fig. 1c), sustained

colonization is conferred to bacteria recovered directly from animals (Supplementary Fig.

5). To examine regulation of gene expression, we deleted the

factor

ccfA

, which led to

highly reduced expression of all five genes during animal colonization (Supplementary Fig.

6a). Accordingly, germ-free mice mono-colonized with

B. fragilis

ccfA

mutant are

permissive of super-colonization by WT

B. fragilis

, demonstrating a functional defect in the

saturable niche occupancy (Supplementary Fig. 6b). Based on the tissue-associated

expression pattern, we tested if the CCF system promotes bacterial localization to mucosal

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tissue. Mice were mono-associated with either the WT or the

ccf

deletion strain, and both

groups were subsequently challenged with WT

B. fragilis

. 24 hours after challenge, we

observe the same numbers for challenge strains in feces of both groups (Fig. 2b; 1

and 2

bars, and Supplementary Fig. 7a, b). In contrast,

ccf

mutant-associated animals show higher

levels of challenge strain in colon tissue, suggesting a colonization defect by the mutant

strain specifically at the mucosal surface (Fig. 2b; 3

and 4

bars, and Supplementary Fig.

7a, b). Therefore, CCF-mediated colonization fitness appears to involve physical association

with the gut.

Recent studies have revealed microbial communities that colonize intestinal crypts of

conventional mice in the absence of disease

and we have shown that

B. fragilis

occupies

the colonic crypts of mono-associated mice

. Discovering a role for

ccf

genes near mucosal

tissue led us to explore the intriguing hypothesis that the CCF system mediates crypt

occupancy. We mono-colonized mice with

B. fragilis

and visualized bacterial localization in

colon tissue by whole-mount confocal microscopy. Indeed, WT

B. fragilis

co-localize with

crypts from the ascending colon, appearing to be located in the center of crypt opening.

Remarkably,

B. fragilis

ΔCCFmono-associated mice display virtually no crypt occupancy

(Fig. 2c and Supplementary Fig. 8). Colon cross-section imaging also reveals that only WT

bacteria are crypt associated (Supplementary Fig. 9). Two-photon imaging of colon explants

clearly demonstrates presence of WT

B. fragilis

on the surface of the epithelium and inside

the crypt. While both WT and mutant strains of

B. fragilis

associate with the surface of the

epithelium, only WT bacteria are able to penetrate deep into the colonic crypts of mice (Fig.

2d and Supplementary video 1). Measuring the distance from the surface of the epithelium

to bacterial signals in a survey of crypts reveals significantly greater tissue penetration by

WT bacteria (Fig. 2e). Collectively, these data reveal that the CCF system allows

B. fragilis

to reside in a specific niche within crypts during steady-state colonization.

We next investigated the effects of the CCF system in the context of a complex microbiota.

Wild-type

Bacteroides

species do not readily colonize most strains of specific pathogen-free

(SPF) mice, namely BALB/c, Swiss Webster and C57BL/6, despite oral administration of

high inoculums (Supplementary Fig. 10a, c and data not shown). Furthermore, transfer of an

SPF microbiota to mono-colonized mice leads to clearance of WT

B. fragilis

(Supplementary Fig. 10b, d). To overcome this obstacle, we tested various additional genetic

backgrounds and empirically determined that C57BL/6 Rag-/- mice (which lack an adaptive

immune system) and non-obese diabetic (NOD) mice can be stably colonized by

B. fragilis

with a single oral gavage. We introduced either WT or

ccf

mutant

B. fragilis

at equal

inoculums into separate groups of animals, and measured colonization. Only WT

B. fragilis

stably colonizes SPF Rag-/- mice, whereas

B. fragilis

ΔCCF establishes a significantly lower

colonization in the gut (Fig. 3a). Co-inoculation of equal numbers of WT and

ccf

mutant

bacteria into Rag-/- mice also results in rapid clearance of the mutant strain from the gut

(Fig. 3b), demonstrating a cell intrinsic defect that could not be complemented in

trans

WT bacteria. NOD animals are also preferentially colonized by WT

B. fragilis

compared to

ccf

mutants in separate groups of animals (Fig. 3c) or in equal co-inoculation (Fig. 3d).

These data show that deletion of the

ccf

genes compromises

B. fragilis

colonization of hosts

with a complex microbiota.

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During symbiosis with mammals, the microbiota may be confronted by rapid environmental

changes with potentially adverse consequences to bacteria, such as enteric infections or

antibiotic exposure. Gastroenteritis is commonly experienced by humans and is known to

perturb the microbiota. To test if resilience of

B. fragilis

colonization is CCF-dependent, we

used

Citrobacter rodentium

infection of mice to mimic human GI infection

. Employing an

antibiotic treatment protocol that does not sterilize the gut but promotes colonization of SPF

mice by

Bacteroides

, we were able to simultaneously colonize mice with equivalent levels

of WT and

ccf

mutant bacteria. Mice were subsequently infected orally with

C. rodentium

and colonization of

B. fragilis

was monitored. WT bacteria decline in number at first, but

return to maximal levels 3-4 weeks post infection (Fig. 3e). Importantly, the

B. fragilis

ΔCCF

strain is completely cleared from the mouse gut following gastroenteritis (Fig. 3e), but not

when animals are left uninfected (Supplementary Fig. 11a). Next, we challenged mice that

were co-colonized with WT and

B. fragilis

ΔCCF with oral antibiotics and observe a

colonization defect only in

ccf

mutant bacteria (Fig. 3f and Supplementary Fig. 11b). These

results reveal that the CCF system establishes resilient colonization by gut

Bacteroides

following disruption of the microbiome. Finally, when SPF mice colonized with WT

fragilis

were given an antibiotic treatment that cleared fecal bacteria, crypt-associated

microbial populations persisted (Supplementary Fig. 12), suggesting that symbiotic bacteria

occupy a protected niche that creates a reservoir for stable gut colonization.

Co-evolution has bound microbes and man in an inextricable partnership, resulting in

remarkable specificity and stability of the human microbiome

. Our findings reveal a novel

pathway required for persistent colonization of the mammalian gut by the

Bacteroides

Homology to the Sus family of proteins suggests a role for CCF in uptake and utilization of

glycans. Although certain Sus-containing PULs in

B. thetaiotaomicron

mediate foraging of

host mucus

, their contributions to microbial colonization have been previously described

only during nutrient deprivation conditions

. Our discovery of CCF-dependent colonization

in mice fed a nutrient rich diet suggests a new role whereby

Bacteroides

evolved specific

Sus-like systems to utilize non-dietary glycans during homeostasis. Based on the findings

that

ccf

genes are preferentially expressed in proximity to mucosal tissues and

B. fragilis

associates with colonic crypts, we find it likely that host factors may promote expression of

the CCF system. In support of this notion, N-Acetyl-D-lactosamine (LacNAc)—a

component of host mucus—induces the

ccf

genes and its homologs in

B. fragilis

and

thetaiotaomicron

(Supplementary Fig. 13). But since a closely related PUL (BF0227-31)

responds to LacNAc but does not mediate saturable niche colonization (Supplementary Fig.

4), LacNAc alone may be an inducer but is not the substrate utilized by CCF systems. We

propose that specific glycan structures within colonic crypts serve as nutrient sources for

individual

Bacteroides

species, and that CCF systems provide a molecular mechanism for a

hypothesis proposed decades ago, “

that populations of most indigenous intestinal bacteria

are controlled by substrate competition, i.e., that each species is more efficient than the rest

in utilizing one or a few particular substrates and that the population level of that species is

controlled by the concentration of these few limiting substrates

”

. Future work will aim to

identify the precise glycan(s) for CCF systems from various

Bacteroides

. Finally, our data

suggests that the

ccf

genes encode for a specific subset of PULs that evolved the novel

activity of promoting stable and resilient colonization, and crypt-associated bacterial

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reservoirs may represent ‘founder’ cells that repopulate the gut following disruption of the

microbiome by enteric infections or antibiotic exposure. Discovery of a molecular

mechanism for colonization fitness by gut bacteria provides a glimpse into the evolutionary

forces that have shaped the assembly and dynamics of the human microbiome.

Methods Summary

All germ-free mice were bred and housed in flexible film isolators until 8 weeks of age, then

transferred to microisolator cages and maintained with autoclaved food, bedding and water

supplemented with gentamicin and erythromycin. Mice were mono-associated with

gentamicin and erythromycin resistant

Bacteroides

strains by single oral gavage.

Colonization level was determined over time by stool serial dilution plating on selective agar

media. For qRT-PCR, total RNA was extracted from laboratory bacterial culture, fecal and

cecal content (ZR Soil/Fecal RNA MicroPrep™, Zymo Research) and colon tissues (Trizol,

Invitrogen) from mono-associated animals, converted to first strand cDNA and analyzed by

qPCR using Power SYBR Green PCR Master Mix (Applied Biosystems). For colon whole-

mount imaging, tissues were harvested from germ-free or single strain mono-associated

animals, fixed with 4% PFA and stained with a

B. fragilis

specific antibody, DAPI and

phalloidin. The colon crypts were visualized by confocal microscopy and two-photon

microscopy. SPF mice were colonized with

B. fragilis

and/or

B. fragilis

ΔCCF by single oral

gavage and the colonization level was determined over time by stool DNA extraction (ZR

Fecal DNA MiniPrep™, Zymo Research) and qPCR using strain specific primers.

Methods

Bacterial strains, plasmids and culture conditions

Bacterial strains and plasmids are described in Supplementary Table 1.

Bacteroides

strains

were grown anaerobically at 37°C for two days in brain heart infusion broth supplemented

with 5 μg/ml hemin and 0.5 μg/ml Vitamin K (BHIS), with gentamicin (200 μg/ml),

erythromycin (5 μg/ml), chloramphenicol (10 μg/ml) and tetracycline (2 μg/ml) added where

appropriate.

Escherichia coli

JM109 containing recombinant plasmids were grown in LB

with ampicillin (100 μg/ml) or kanamycin (30 μg/ml).

Citrobacter rodentium

DBS100 strain

was grown in LB at 37°C for 24 hours. For the induction of

susC/D

homologues,

B. fragilis

and

B. thetaiotaomicron

were grown in minimal medium (MM) with either glucose or N-

acetyllactosamine as the sole carbon source as described previously

Mice

8-10 week old male and female germ-free Swiss Webster mice were purchased from

Taconic Farms (Germantown, NY) and bred in flexible film isolators. For gnotobiotic

colonization experiments, germ-free mice were transferred to freshly autoclaved

microisolator cages, fed

ad libitum

with a standard autoclaved chow diet and given

autoclaved water supplemented with 10 μg/ml of erythromycin and 100 μg/ml of

gentamicin. Male SPF (Specific Pathogen-Free) C57BL/6 mice and Swiss Webster mice

were purchased from Taconic Farms. Male SPF NOD/ShiLtJ mice and Rag-/- C57BL/6

mice were purchased from the Jackson Laboratory. No randomization or blinding was used

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to allocate experimental groups. Sample size and standard deviation were based on empirical

data from pilot experiments. All procedures were performed in accordance with the

approved protocols using IACUC guidelines of the California Institute of Technology.

Construction of chromosomal library and screen

Genomic DNA was isolated from overnight culture of

B. fragilis

using a commercial kit

(Wizard

Genomic DNA Purification Kit, Promega). 20 μg of genomic DNA was incubated

with 4U of Sau3AI for 5, 10, 15, or 20 minutes at 37°C in 50 μl volume and the partially

digested genomic DNA was separated by electrophoresis on 0.7% agarose gel. 9-10 kb

fragment DNA was excised and recovered from the agarose gel (Zymoclean™ Gel DNA

Recovery Kit, Zymo Research). Insert DNA was ligated to

Bgl

II site of plasmid vector

(pFD340-

cat

BII, Supplementary Table 1), transformed into

E. coli

and amplified on LB-

ampicillin plate. Individual clones from the plasmid library were mobilized from

E. coli

B. vulgatus

by conjugal helper plasmid RK231 generating a library of

B. vulgatus

hosting

fragilis

chromosomal DNA fragments consisting of approximately

∼

2100 clones. To screen

the library

in vivo

, pools of 96 clones (10

CFU of each clone) were gavaged into 22 germ-

free Swiss Webster mice (10

CFU per animal) pre-colonized with

B. vulgatus

pFD340 for

1-2 weeks. Two weeks after gavage, fresh fecal samples were plated on BHIS agar plate

containing chloramphenicol to select for clones with colonization phenotype.

Generation of

ccfA, ccfC, ccfD, ccfE

ccfC-E

(ΔCCF) and ΔBF0227-0229 deletion mutants

∼

2 kb DNA segments flanking the region to be deleted were PCR amplified using primers

listed in Supplementary Table 2. Reverse primer of the left flanking DNA and forward

primer of the right flanking DNA were designed to be partially complementary at their 5

′

ends by 18-21 bp. Fusion PCR was performed using the left and right flanking DNA (

∼

300

ng each after gel purification) as DNA template and forward primer of the left flanking

DNA and reverse primer of the right flanking DNA

. The fused PCR product was cloned

into

Bam

HI or

Sal

I site of the

Bacteroides

conjugal suicide vector pNJR6 and mobilized into

B. fragilis

. Colonies selected for erythromycin resistance (Em

), indicating integration of the

suicide vector into the host chromosome were passaged for five days and then plated on

nonselective medium (BHIS). The resulting colonies were replica plated to BHIS containing

Em, and Em

(erythromycin sensitive) colonies were screened by PCR to distinguish wild-

type revertants from strains with the desired mutation. The same strategy was employed to

generate Δ

ccfC-E

deletion mutant from

B. vulgatus

Quantitative RT-PCR (qRT-PCR)

Total RNA was extracted from mid-log phase bacterial culture using ZR Fungal/Bacterial

RNA MiniPrep™ (Zymo Research), feces and cecal content from mice using ZR Soil/Fecal

RNA MicroPrep™ (Zymo Research), and mouse colon tissues after removing luminal

content by gently scraping the mucosal surface and PBS rinse using Trizol (Invitrogen).

cDNA was made using an iSCRIPT cDNA synthesis kit per manufacturer's instructions

(Bio-Rad). All qRT-PCR reactions were performed in ABI PRISM 7900HT Fast Real-Time

PCR System (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied

Biosystems). Gene-specific primers are described in Supplementary Table 2.

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Immunofluorescent staining of colon whole-mounts and frozen sections

For whole-mount staining, colons were fixed in buffered 4% paraformaldehyde, washed

with PBS and subjected to indirect immunofluorescence. Tissues were made permeable by

incubation with 0.5% (wt/vol) saponin, 2% (vol/vol) FBS, and 0.09% (wt/vol) azide in PBS

for at least 18 hours. The same buffer was used for subsequent incubations with antibodies.

Colon fragments were incubated with a primary polyclonal chicken IgY anti-

B. fragilis

antibodies for 12-16 hours at room temperature (RT) followed by 1-2 hour incubation at

37°C. Following PBS washes, samples were reacted with goat anti-chicken IgY secondary

antibodies (Alexa Fluor 488 or Alexa Fluor 633, 2 μg/ml, Molecular Probes), fluorescently

labeled phalloidin (fluorescein or AF568, 2 U/ml, Molecular Probes) and DAPI (2 μg/ml,

Molecular Probes) for 1 hour at RT. Tissues were mounted in Prolong Gold (Invitrogen) and

allowed to cure for at least 48 hours prior to imaging. In some experiments, anti-

B. fragilis

antibodies were pre-absorbed on tissue fragments derived from either germ-free mice (up to

18 hours at RT) or SPF mice (1 hour at RT).

For frozen sections, colon tissues were embedded in O.C.T. Compound (Sakura Finetek),

frozen on dry ice, and stored at -80°C. Frozen blocks were cut with a thickness of 10 μm

using a Microm HM505E cryostat, and sections were collected on positively charged slides

(Fisher Scientific) for staining. Slides were fixed with 4% buffered paraformaldehyde for 10

minutes and washed 2×10 minutes with PBS. Tissue sections were blocked with 10%

normal goat serum and 0.5% bovine serum albumin in PBS for 1 hour at RT. Sections were

incubated with anti-

B. fragilis

antibodies for at least 8 hours at 4°C, washed 2 times for 10

minutes with PBS, reacted with secondary reagents and mounted as described above. In

some experiments, anti-

B. fragilis

antibodies were pre-absorbed for 1 hour at RT on tissue

sections derived from germ-free mice.

Fluorescence microscopy

An SP5 resonant laser-scanning confocal and two-photon microscope (both scanning heads

mounted on the same DM 6000 upright microscope, Leica Microsystems) with a 40× oil

objective (numerical aperture 1.4) or 63× oil objective (numerical aperture 1.4) were used

for fluorescence microscopy. Images used for 3D reconstructions were acquired using dual

confocal – two-photon mode. For confocal imaging, 488-nm and 543-nm excitation

wavelengths were used for Alexa Fluor 488-labeled bacteria and Alexa Fluor 568-labeled

phalliodin, and signals were detected with internal photomultiplier tubes. 2-photon imaging

was performed with 4 nondescanned detectors (Leica Microsystems) and a Chameleon Ultra

Ti: Sapphire laser (Coherent) tuned at 700–800 nm for acquisition. Emitted fluorescence

was split with 3 dichroic mirrors (496 nm, 560 nm and 593 nm) and passed through an

emission filter (Semrock) at 585/40 nm. Images (512×512) acquired with a 0.5 μm Z step

were smoothed by median filtering at kernel size 3 × 3 pixels. 3D reconstructions of crypts

and bacteria were performed using Imaris software (version 7.5.1 ×64; Bitplane AG). Crypt

structures were visualized by DAPI and phalloidin signals. Images used for quantification

were acquired with FluoView FV10i confocal microscope (Olympus) using 60× (numerical

aperture 1.35) oil objective.

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Image analysis

For bacterial localization with respect to the epithelial layer, frames of 512×512 pixels were

acquired with 1 μm Z steps in the crypt length axis. Images were processed using ImageJ

software (NIH). Background was subtracted (rolling ball method), images were smoothed by

median filtering (3×3 pixels), segmented by threshold and position of the signal in the Z

stack was recorded. Data did not follow normal distribution and were analyzed by non-

parametric two-sided Mann-Whitney U test.

For quantification of crypt associated bacterial signals from antibiotic treated animals, stacks

of 512×512 pixels by 8 frames (1 μm per frame) were flattened by maximum intensity

projection and filtered by median (3×3 kernel size). Images were segmented by

thresholding. Number of positive spots/1000 μm

and area occupied by individual spots

were analyzed. Data were not normally distributed and were analyzed by Mann-Whitney or

Kruskal-Wallis followed by Dunn's multiple comparisons test where appropriate. 11-13

stacks/group were examined. Total area that was analyzed within the group of stacks was

between 0.08-0.2 mm

Gnotobiotic animal colonization experiments

8-12 week old germ-free Swiss Webster mice were gavaged once with a 100 μl of bacterial

suspension for mono-association (

∼

CFU of each bacterial strain harvested from a log-

phase culture and resuspended in PBS with 1.5% NaHCO

). For sequential colonization,

germ-free mice were mono-associated with an initial strain for 6-7 days and subsequently

gavaged with a 100 μl suspension of a challenge strain. All

Bacteroides

strains used to

colonize germ-free animals were resistant to gentamicin inherently, and to erythromycin by

plasmid. Unless otherwise indicated, the initial strains carried pFD340-

cat

(chloramphenicol

resistant; Cm

) and the challenge strains, pFD340-

tetQ

(tetracycline resistant; Tet

). For

horizontal transfer by encounter experiment, two single-housed mice that were mono-

associated with either WT

B. fragilis

pFD340-

tetQ

B. fragilis

ΔCCF pFD340-

cat

for at

least 3 weeks were co-housed in a fresh sterile cage for 4 hours and then separated. At each

time point, fresh fecal samples were collected, weighed, homogenized and serially diluted in

PBS (or BHI broth) for plating on selective media to determine bacterial CFU per g of feces.

SPF animal colonization experiments

7-8 week old male SPF mice (C57BL/6, Swiss Webster, NOD, and Rag-/-) were given a

single inoculum of 1×10

CFU of either WT

B. fragilis

ΔCCF, or 1:1 mixture of

the two strains by oral gavage. At each time point, bacterial genomic DNA from fecal

samples were isolated using a commercial kit (ZR Fecal DNA MiniPrep™, Zymo Research)

following the manufacturer's instructions and the relative densities of bacteria were

determined by qPCR using strain-specific primers (Supplementary Table 2).

Citrobacter rodentium infection

8 week old female SPF Swiss Webster mice were treated with metronidazole (100 mg/kg)

by oral gavage every 24 hours and ciprofloxacin dissolved in drinking water (0.625 mg/ml;

Hikma Pharmaceuticals) for seven days; mice were transferred to a fresh sterile cage every

Lee et al.

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two days. Two days after the cessation of antibiotic treatment, mice were orally gavaged

with a single inoculum of 1:1 mixture of WT

B. fragilis

and

B. fragilis

ΔCCF (

∼

5×10

CFU

total per animal). 6-7 days after

B. fragilis

gavage, mice were either infected orally with

∼

5×10

CFU of overnight culture

C. rodentium

or PBS-gavaged as control. The relative

densities of bacteria were determined by fecal bacterial DNA extraction and qPCR.

Antibiotic treatment

8 week old female SPF Swiss Webster mice were treated with metronidazole (100 mg/kg)

by oral gavage every 24 hours and ciprofloxacin dissolved in drinking water (0.625 mg/ml)

for seven days; mice were transferred to a fresh sterile cage every two days. Two days after

the cessation of antibiotic treatment, mice were orally gavaged with a single inoculum of 1:1

mixture of WT

B. fragilis

and

B. fragilis

ΔCCF (

∼

5×10

CFU total per animal). 6-7 days

after

B. fragilis

gavage, one group of mice were treated with ciprofloxacin for 4 days

dissolved in drinking water (1 mg/ml) and another group were left untreated. The relative

densities of bacteria were determined by fecal bacterial DNA extraction and qPCR.

Antibiotic treatment for colon whole-mount imaging

8 week old female SPF Swiss Webster mice were treated with metronidazole (100 mg/kg)

by oral gavage every 24 hours and ciprofloxacin dissolved in drinking water (0.625 mg/ml)

for seven days; mice were transferred to a fresh sterile cage every two days. Two days after

the cessation of antibiotic treatment, mice were orally gavaged with a 100 μl inoculum of

fragilis

(

∼

CFU) or PBS. 7 days after bacterial gavage (day 16), PBS or

B. fragilis

inoculated mice were treated with ciprofloxacin in drinking water (1 mg/ml) for 7 days and

one group of

B. fragilis

inoculated mice were left untreated. At the end of the ciprofloxacin

treatment (day 23), feces were collected for stool DNA extraction and colon tissues were

harvested and fixed with 4% PFA for whole-mount imaging.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We thank Taren Thron and Sara McBride (Caltech) for the maintenance of germ-free animals, Jane Selicha

(Caltech) for assisting with the experimental procedures, and Grzegorz Chodaczek (LIAI) for help with confocal

and two-photon microscopy. We are grateful to Dr. Eric C. Martens (Univ. of Michigan) and members of the

Mazmanian laboratory for critical review of the manuscript. S.M.L. and G.P.D. were supported by a pre-doctoral

training grant (GM007616). This work was supported by grants from the NIH (GM099535 and DK078938) and the

Crohn's and Colitis Foundation of America to S.K.M.

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Figure 1.

Bacteroides

species occupy species-specific niches in the gut via an evolutionarily

conserved genetic locus

a-c,

Germ-free mice were mono-associated with

B. fragilis

and challenged orally with (

)

thetaiotaomicron

; (

)

B. vulgatus

; or (

)

B. fragilis

, Mice were mono-associated with

erythromycin sensitive

B. fragilis

, and subsequently challenged with erythromycin resistant

B. fragilis

. Erythromycin was administered where indicated.

, Genomic organization of the

ccf

locus.

, Mice were mono-associated with either WT

B. fragilis

, mutant strains deleted in

ccfC

ccfD

ccfE

and

ccfC-E

(BFΔCCF), or complemented strain (BFΔCCF

∷

CCF) and

challenged with WT

B. fragilis.

CFUs were determined after 30 days.

, Mice were mono-

associated with WT

B. vulgatus

or a mutant strain deleted in

ccfC-E

genes (BVΔCCF), and

challenged with WT

B. vulgatus

. CFUs were determined after 30 days. In all sequential

colonization studies, results are representative of at least 2 independent trials (n=3-4

animals/group).

, Cross-colonization between WT

B. fragilis

and BFΔCCF mono-

associated mice at 7 days after encounter measured by CFUs of the initially colonizing and

the horizontally transmitted (challenge) strains. (n=2 animals/encounter, 5 independent

trials). All graphs: Dashed line indicates the limit of detection at 100 CFU/g feces, and error

bars indicate standard deviation (SD).

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Figure 2.

B. fragilis

colonization of the colonic crypts is mediated by the CCF system

, qRT-PCR of

ccf

gene expression levels normalized to 16S rRNA (n=3 animals, 2 trials).

Mice were mono-associated with either WT

B. fragilis

ΔCCF, and

challenged with WT

B. fragilis

. The percentage of challenge strain was determined in the

lumen (feces) and colon after 1 day (n=8 animals/group).

, Confocal micrographs of germ-

free, WT

B. fragilis

ΔCCF mono-associated mice colon whole-mount. Crypts

are visualized by DAPI (nuclei, blue) and phalloidin (F-actin, green). Bacteria (red) are

stained with IgY polyclonal antibody raised against

B. fragilis

. Images are representative of

7 different sites analyzed from at least 2 different colons. Scale bar: 5 μm.

, 3D

reconstructions of colon crypts from WT

B. fragilis

ΔCCF mono-associated

mice. Bacteria are detected on the apical surface of the epithelium (arrows) and in the crypt

space (arrowhead). Scale bar: 10 μm.

, Quantification of bacterial penetration, measured as

distance from the epithelial surface per crypt. Error bars indicate standard error of the mean

(SEM). NS: not significant. ND: not detected. **

<0.01.****

<0.0001.

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Figure 3.

B. fragilis

requires the

ccf

genes for stable and resilient colonization of mice

, Groups of SPF Rag-/- mice were gavaged with either WT

B. fragilis

ΔCCF.

, SPF Rag-/- mice were given a 1:1 co-inoculum of WT

B. fragilis

and

B. fragilis

ΔCCF by

single gavage.

, SPF NOD mice were gavaged with either WT

B. fragilis

fragilis

ΔCCF.

, SPF NOD mice were given a 1:1 co-inoculum of WT

B. fragilis

and

fragilis

ΔCCF by single gavage.

, SPF mice were co-associated with WT

B. fragilis

and

fragilis

ΔCCF, and infected with

Citrobacter rodentium

, SPF mice were co-associated with

B. fragilis

and

B. fragilis

ΔCCF, and given ciprofloxacin in drinking water for the time

period shown. For all analyses, bacterial colonization levels were assessed by real-time

qRT-PCR from stool DNA (n=4 animals/group). Results are representative of at least 2

independent trials per experiments. Error bars indicate SEM.*

<0.05. ***

<0.001.

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