Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 May 13.
Published in final edited form as: Cell Host Microbe. 2015 May 13;17(5):672–680. doi: 10.1016/j.chom.2015.04.002

Colitogenic Bacteroides thetaiotaomicron antigens access host immune cells in a sulfatase-dependent manner via outer membrane vesicles

Christina A Hickey 1,2,†, Kristine A Kuhn 1,†,#, David L Donermeyer 1, Nathan T Porter 3, Chunsheng Jin 4, Elizabeth A Cameron 3, Haerin Jung 1, Gerard E Kaiko 1, Marta Wegorzewska 1, Nicole P Malvin 1, Robert W P Glowacki 3, Gunnar C Hansson 4, Paul M Allen 1,*, Eric C Martens 3,*, Thaddeus S Stappenbeck 1,*
PMCID: PMC4432250  NIHMSID: NIHMS680150  PMID: 25974305

Summary

Microbes interact with the host immune system via several potential mechanisms. One essential step for each mechanism is the method by which intestinal microbes or their antigens access specific host immune cells. Using genetically-susceptible mice (dnKO) that develops spontaneous, fulminant colitis, triggered by Bacteroides thetaiotaomicron (B. theta), we investigated the mechanism of intestinal microbial access under conditions that stimulate colonic inflammation. B. theta antigens localized to host immune cells through outer membrane vesicles (OMVs) that harbor bacterial sulfatase activity. We deleted the anaerobic sulfatase maturating enzyme (anSME) from B. theta, which is required for post-translational activation of all B. theta sulfatase enzymes. This bacterial mutant strain did not stimulate colitis in dnKO mice. Lastly, access of B. theta OMVs to host immune cells was sulfatase-dependent. These data demonstrate that bacterial OMVs and associated enzymes promote inflammatory immune stimulation in genetically susceptible hosts.

Introduction

Host-microbial interactions play a vital role in systemic physiology and pathophysiology; however, such contact, especially in the colon, is greatly limited by host barriers. The colonic epithelium provides important key barriers against a dense (~1011–12 bacteria/ml) and diverse population of bacteria (Human Microbiome Project, 2012a, b). Epithelial stem cells at the base of the crypts of Lieberkühn drive rapid epithelial cell turnover of differentiated cell lineages that include absorptive colonocytes and goblet cells, which in turn form two major barriers (Kuhnert et al., 2004; Lee et al., 2009). First, colonocytes migrate and exit crypts to form a sheet of cells, creating a cellular barrier. Overlying this layer of cells is a second barrier composed of a stratified~50 µm inner mucus layer that lines the apical surface of the epithelium (Johansson et al., 2008). This mucus layer acts as a physical obstruction as bacteria cannot enter the net-like sheaths formed by the MUC2 polymers (Ambort et al., 2012; Johansson et al., 2013). The function of these two epithelial-based barriers have important reciprocal interactions with indigenous microbes (Kaiko and Stappenbeck, 2014).

Overall, microbes interact with the host immune system and bypass these barriers using several potential mechanisms. One simple mechanism involves capture of bacteria near the mucosal surface by host antigen presenting cells (APCs). In the small intestine, APCs can directly engulf bacteria within the intestinal lumen (Niess et al., 2005). Another mechanism involves diffusion of soluble microbial antigens or products that can be detected by the host through specific receptors (i.e. lipopolysaccharide through TLR4) (Tannahill et al., 2013). Lastly, bacteria, especially Gram negative microbes, can release enzyme-containing outer membrane vesicles (OMVs) that perform a variety of activities to benefit the parent microbe (Elhenawy et al., 2014; Ellis and Kuehn, 2010; Kulp and Kuehn, 2010). OMVs have been proposed to mediate microbial interactions with the host (Shen et al., 2012) though the mechanisms by which they traverse host barriers are unclear.

Bacteroidaceae is a prominent family of intestinal symbiotic organisms. The extent to which these diverse organisms influence host physiology and disease models is unclear beyond a few examples (Bloom et al., 2011; Housseau and Sears, 2010; Shen et al., 2012), and precise mechanisms are still elusive. One member of this family, Bacteroides thetaiotaomicron (B. theta), is fully sequenced, genetically manipulable, and has a well-defined role in complex carbohydrate metabolism, including interactions with host mucin glycans (Benjdia et al., 2011; Martens et al., 2008; Sonnenburg et al., 2005), making it an appropriate experimental model to elucidate both beneficial and detrimental host interactions among members of this family. Specifically, B. theta is well suited to interact with at least one of the barriers, the host mucin glycans, because of polysaccharide utilization loci (PULs) in B. theta’s genome that encode several mucin-degrading enzymes such as glycosidase hydrolases (GHs) and sulfatases (Benjdia et al., 2011; Martens et al., 2008). Here, we used B. theta (strains dnlkv9 and VPI-5482, thereby referred to in the text as B. theta) in an experimental mouse model of intestinal inflammation to decipher the mechanism by which B. theta traverses host barriers and contacts the host. We have developed a highly reproducible system, dnKO mice (CD4-dnTgfb2; IL10rb−/−), in which exposure to B. theta is sufficient to trigger disease (Bloom et al., 2011; Kang et al., 2008). In this study, we demonstrate that B. theta require sulfatases to cause colitis in dnKO mice, and that B. theta OMVs gain access to host immune cells in a sulfatase-dependent manner.

Results

Extracellular antigens from WT B. theta localize to the host peri-cryptal mesenchyme in dnKO mice

Since B. theta is sufficient to trigger colitis in dnKO mice, we first determined B. theta’s localization with a panel of monoclonal antibodies. Multiple clones were reactive and specific to B. theta by ELISA (Figure 1A–B), but the two most promising candidates were selected by in situ luminal staining of intestinal tissue sections from WT mice: 3H2 and 6E9. We found that 3H2 labeled the periphery of bacterial cells in colonic sections of dnKO and control (IL10rb+/−) mice that were pretreated with antibiotics followed by gavage with WT B. theta (Figure 1C–D, Figure S1A) (Bloom et al., 2011). Based on this staining pattern, we surmised that the target of 3H2 was a highly expressed surface antigen such as one of the eight capsules for B. theta (Martens et al., 2009). We found that 3H2 recognized capsule 3 of B. theta (Figure S1B–C), and thereby stains a subset of whole B. theta. However, despite the robust signal in the dnKO colonic lumens (Figure 1C–D), we did not detect any staining in the mucosa or in the lumen of crypts (Figure 1F, Figure S1D for additional controls). In contrast, 6E9 labeled abundant small particles in the lumen that were not directly associated with Bacteroides DNA (Figure 1C, E; Figure S1A), providing us different antibodies to identify whole bacteria versus bacterial particles. Interestingly, 6E9-positive particles were present in mesenchymal cells around the crypt base of dnKO mice where inflammation is typically initiated in this model (Figure 1G, Figure S1E for additional controls) (Bloom et al., 2011; Kang et al., 2008).

Figure 1.

Figure 1

Open in a new tab

Extracellular bacterial antigen from WT B. theta localizes to the host peri-cryptal mesenchyme in dnKO mice. (A–B) Specificity of (A) 3H2 and (B) 6E9 antibody to different Bacteroides species (B. uniformis, B. vulgatus, B. TP5, B. theta) measured via ELISA at OD of 450 nm at increasing doses of antibody. Controls include (A) acapsular B. theta mutant and (A–B) E. coli. (C) Cartoon of the mouse colon indicating the location of images in (D–G). D,E= lumen, F,G=mucosa. (D–F) Sections of the colonic lumen (D, E) and mucosa (F, G) from dnKO mice 3 weeks after gavage with WT B. theta stained with 3H2 conjugated to Alexa 647 (D, F) and 6E9 conjugated to Alexa 594 antibodies, both in red (E, G). DAPI indicates nuclei in blue. Bars=3 µm (D, E). Bars=50 µm (F,G). See also Figure S1.

Host-penetrant B. theta antigen localizes to outer membrane vesicles (OMVs) with sulfatase activity

With immunogold electron microscopy, we found that 6E9 recognized numerous 10–80 nm vesicles that were not directly associated with B. theta (Figure 2A, Figure S2A for the isotype control). The size and shape of these vesicles were consistent with expelled OMVs that are classically produced by the physiological process of pinching off a portion of the outer membrane of gram-negative bacteria (Kulp and Kuehn, 2010). Immunogold labeling of cryo-sectioned fecal pellets from dnKO mice gavaged with WT B. theta revealed that both B. theta and its OMVs had 6E9-positive membranes (Figure 2B).

Figure 2.

Figure 2

Open in a new tab

Host-penetrant B. theta antigen localizes to outer membrane vesicles (OMVs) with sulfatase activity. (A–C) Transmission electron microscopy (TEM) image of cultured (A) WT or (C) ΔanSME B. theta strains on grids without sectioning. Arrows=OMVs. (B) Cryo-sections of pelleted fecal material obtained from a dnKO mouse gavaged with WT B. theta stained with mouse 6E9 antibody followed by secondary goat anti-mouse IgG antibody conjugated to18 nm colloidal gold. Image shows a transverse cross-sectional view of B. theta parent microbe with 6E9 staining on the bacterial membrane and a budding OMV also with 6E9 staining. Arrows= OMVs, Arrowhead=B. theta. (A and C) Bars=100nm. (B) Bar=500nm. (D) Immunoblot of WT and ΔanSME B. theta sonicates and OMV preps stained with the 6E9 antibody. (E) Sulfatase activity (mol.min.mg−1) of WT and ΔanSME B. theta lysates and OMVs grown in tryptone glucose yeast (TYG) and chondroitin media. Control is TYG media alone. One representative experiment shown of n=2. See also Figure S2 and Table S1.

Because we demonstrated that B. theta produced OMVs in vivo, we next wanted to determine the molecular mechanism by which the OMVs accessed the host. OMVs have multiple properties that would facilitate passage through host barriers including small size (Elhenawy et al., 2014), enzymes such as sulfatases and glycoside hydrolases (Elhenawy et al., 2014) that may assist in glycan breakdown, and the ability to be endocytosed by host cells (Irving et al., 2014). Desulfation is necessary for the degradation of sulfated mucin glycans, which are abundant in the colon. Of the nearly 5,000 genes present in the B. theta genome, 28 of these genes encode sulfatases (Benjdia et al., 2011). Thus, we hypothesized that the sulfatase activity of B. theta may be important for the development of colitis in dnKO mice.

Fortuitously, a single gene in B. theta, the anaerobic sulfatase maturating enzyme (anSME) gene, is required for post-translational activation of all 28 of B. theta's sulfatase enzymes (Berteau et al., 2006). An engineered mutant strain of B. theta (ΔanSME strain) abrogates sulfatase activity (Benjdia et al., 2011). The ΔanSME strain produced 6E9 positive OMVs that are similar to WT B. theta OMVs in appearance (Figure 2C, Figure S2B for isotype control) and concentration (Figure S1D, Figure S2C) (Chutkan et al., 2013). 6E9 also recognized a single 19-kD Ag in OMVs from both strains (Figure 2D, see Figure S2D for additional controls). Immunoprecipitation followed by mass spectroscopy analyses showed this protein was homologous to a predicted membrane protein, BT3901a (Figure S2E) that is widely conserved in Bacteroides and highly expressed by B. theta both in vivo and in vitro (Sonnenburg et al., 2005). BT3901a was confirmed as the 6E9 antigen on an immunoblot comparing WT B. theta and a BT3901a expressing E. coli (Figure S2F). As expected, the ΔanSME strain of B. theta showed reduced growth using a sole energy source of gastric mucin O-glycans (Figure S2G) that contained sulfated glycan structures (Table S1). Also, WT B. theta OMVs but not ΔanSME OMVs contained detectable sulfatase activity when comparing equivalent amounts of protein (Figure 2E, Figure S2H for bacterial comparisons).

In addition to this in vitro validation of B. theta anSME function, we next determined the feasibility of testing its function in vivo. First, we examined the populations of Lactobacillus, Enterococcacceae, and Lachnospiraceae/Ruminicoccaceae via 16s qPCR (Bloom et al., 2011) in six different groups: both dnKO and IL10rb+/− mice gavaged with either PBS, WT B. theta or the ΔanSME strain of B. theta. No significant differences were noted in levels of any of these families between any groups (Figure S2I–L). Second, B. theta sulfatase genes are found within polysaccharide utilization loci (PULs) that also encode glycoside hydrolases (GHs) and other enzymes involved in glycan degradation (Benjdia et al., 2011; Martens et al., 2008). Because previous transcriptional studies of B. theta were isolated to the mouse cecum and revealed low expression of many sulfatase-containing PULs (Sonnenburg et al., 2005), we confirmed that multiple of these B. theta sulfatase-containing PULs were highly expressed in the distal colon (Figure S3A–K). Also, because germ free mice produce varying amounts of sulfated mucin glycans in different regions of the colon (Holmen Larsson et al., 2013), we measured the thickness of the inner sulfated mucus layer which is Alcian blue positive at pH=1, (Lev and Spicer, 1964) in the distal colons of dnKO and control IL10rb+/− mice (Figure S3L–R). All groups showed similar thickness regardless genotype or gavage inoculum (Figure S3L–R).

The B. theta anSME gene is necessary for causing colitis in dnKO mice

To determine if the anSME gene was required for B. theta’s colitogenic potential, we pretreated dnKO and littermate controls (IL10rb+/−) with antibiotics for three weeks beginning at weaning as previously described (Kang et al., 2008). We gavaged the mice with ΔanSME mutant and WT B. theta strains two days after the cessation of antibiotics and performed analysis three weeks thereafter. Compared with WT B. theta, the ΔanSME strain was unable to elicit disease in dnKO mice as determined by histologic analysis of the colons (Figure 3A, Figure S3S for additional controls) and quantification of crypt loss and epithelial hyperproliferation (Fig. 3B–C). As a control, when the ΔanSME B. theta mutant was genetically complemented (ΔanSME::anSME), it stimulated colitis similarly to the WT parent strain (Figure 3A–C). All B. theta strains colonized control and dnKO mice to similar levels demonstrating that the observed colitogenic differences between WT B. theta and the ΔanSME strains were not due to quantitative differences in overall colonization (Figure 3D; Figure S3T–U and Figure S1D for additional controls). These data support a role for bacterial sulfatases in B. theta’s colitogenic potential.

Figure 3.

Figure 3

Open in a new tab

The B. theta anSME gene is necessary and sufficient for causing colitis in dnKO mice. (A) Hematoxylin and eosin (H&E) stained rectal sections from dnKO and littermate controls (IL10rb+/−) 3 weeks after gavage with B. theta strains or PBS. dnKO mice were gavaged with PBS (A1), WT B. theta (A3), ΔanSME (A4), and ΔanSME::anSME (A5), and a littermate control (IL10rb+/−) was gavaged with WT B. theta (A2). For each low-power image (100×) shown per group, a high-power image (400×) is included (boxed region adjacent to the lowpower image). Bars=200 µm for 100× images. Bars=30 µm for 400× images. (B and C) Graphs of average (B) crypts per 400× field (0.55 mm) and (C) M-phase cells per 100 crypts are shown for different groups of gavaged dnKO mice. (D) Graph of colonization at day 4 of dnKO mice and littermate control by B. theta strains via qPCR. One-way ANOVA analysis: (B) F=15.70, P<0.0001, n≥7 per group; (C) F=21.79, P<0.0001, n≥7 per group; (D) F=116.4, P<0.0001, n≥9 per group. Means with different letters are significantly different by Tukey’s multiple comparisons test. See also Figure S3.

Outer membrane vesicles (OMVs) gain access to host immune cells in B. theta-colonized dnKO mice

We next tested if B. theta’s OMVs accessed host immune cells in a sulfatase-dependent manner. Immunogold localization in dnKO mice gavaged with WT B. theta showed 6E9-positive vesicles in mesenchymal cells that were morphologically consistent with macrophages (Figure 4A, Figure S4A–D). Then, to determine if OMVs required anSME to activate macrophages in the absence of the mucus barrier, we incubated bone marrow macrophages from dnKO mice with OMVs from either WT or ΔanSME B. theta strains in vitro and found that OMVs from both strains were internalized by these cells (Figure 4B–C; Figure S4E). As expected, macrophages from dnKO mice were more potently activated by an OMV stimulus when compared to macrophages from WT mice. However, OMVs from either bacterial strain were equally able to elicit TNFα production from either macrophage (Figure 4D). To determine the production of inflammatory modulators by colonic macrophages exposed in vivo to B. theta, we extracted colonic macrophages after gavage and then analyzed gene expression. mRNA for Ptgs2 (a.k.a. Cox-2), TNFα, and IL1-β were all significantly enriched in dnKO mice gavaged with WT B. theta as compared to PBS and ΔanSME B. theta groups (Figure S4F–H). Lastly, we performed double label immunofluorescence using 6E9 and F4/80 on dnKO mice gavaged with WT B. theta. In these mice, we readily identified macrophages that were 6E9 positive (Figure 4E–F). In contrast, in dnKOs gavaged with ΔanSME B. theta, we rarely observed macrophages with 6E9 staining (Figure 4E–F). Additionally, we performed double label immunofluorescence using 6E9 and CD11c on colonic sections of dnKOs gavaged with WT B. theta and did not see any colocalization, suggesting these cells are not dendritic cells. Together, these data show that only WT B. theta OMVs can efficiently access mucosal macrophages in vivo and suggest that B. theta accesses and activates the host immune system in a sulfatase-dependent manner.

Figure 4.

Figure 4

Open in a new tab

Outer membrane vesicles (OMVs) gain access to host macrophages in B. theta-colonized dnKO mice. (A) TEM image of 6E9 positive vesicle located within a cell consistent with a macrophage in the colon of a WT B. theta-gavaged dnKO mouse labeled with 6E9 mAb/goat anti-mouse IgG antibody conjugated to 18 nm colloidal gold. Bar=100nm. (B) Co-localization of macrophages (CFSE, green) derived from dnKO mice cultured with OMVs from WT B. theta (Dil Vybrant dye, red). Bar=50 µm. Bar=5 µm (inset). (C) Graph of the percent of CFSE+ macrophages that co-localized with OMVs. Unstimulated macrophages from IL10rb+/− and dnKO mice were used as a control. (D) Concentration of TNF-α (pg/ml) in the macrophage supernatant from IL10rb+/− or dnKO mice cultured with OMVs from WT or ΔanSME B. theta. (E) Staining of colonic mucosa from dnKOs gavaged with WT or ΔanSME B. theta with F4/80 (green) and 6E9 (red) antibodies. White dashed lines=outlined crypts. Bar=20 µm. Bar=2.5 µm (inset). (F) Graph of percentage of double positive F4/80+ and 6E9+ cells per crypt-associated mesenchyme in dnKO gavaged with PBS or B. theta strains. One-way ANOVA analysis: (C) F=10.65, P<0.0001, n=4 per group; (D) F=96.11, P<0.0001, n=4 per group; (F) F=5.86, P=0.01, n≥6 per group. Means with different letters are significantly different by Tukey’s multiple comparisons test. See also Figure S4.

Discussion

In this study, we showed that antigen from B. theta, a symbiotic bacterium, localizes to the mucosa of a host that is genetically susceptible to the development of colitis. We also determined that B. theta’s anSME gene, which activates all of its sulfatases, is necessary and sufficient for B. theta’s colitogenic behavior in dnKO mice. Furthermore, we found that B. theta OMVs localized to host immune cells in vivo in a sulfatase dependent manner. Taken together, these results suggest that B. theta OMVs can access the host’s immune cells in a sulfatase-dependent fashion.

OMVs are pinched off fragments of the outer membrane and periplasm of Gram negative bacteria such as B. theta that are 20–250 nm in diameter (Kulp and Kuehn, 2010). Most studies of OMVs to date have been performed using E. coli and Pseudomonas, which are relatively poor producers of OMVs as evidenced by the fact that OMVs make up <1% of the outer membrane material in bacterial cultures (Bauman and Kuehn, 2006; Gankema et al., 1980). In contrast, B. theta is very efficient in producing OMVs, making it an ideal model organism for further studies (Elhenawy et al., 2014). OMVs have three general features that highlight B. theta’s mechanism of access. First, OMVs enable bacterial products to spread to locations that are inaccessible to bacteria (Kulp and Kuehn, 2010). Second, OMVs can be readily engulfed by host cells (Irving et al., 2014). Third, OMVs contain a variety of functional bacterial products including protein toxins, adhesins, and enzymes (Elhenawy et al., 2014; Ellis and Kuehn, 2010). Specifically, B. theta produces a variety of enzymes that degrade host-derived carbohydrates (Elhenawy et al., 2014; Martens et al., 2008; Martens et al., 2009), and some of these enzymes have recently been shown to be contained in OMVs produced by this species (Elhenawy et al., 2014). These properties of OMVs likely contribute to the ability of B. theta OMVs to access the host’s immune cells.

OMVs are an obvious candidate for mediating microbial-host interactions of certain symbiotic families. The challenge is how to visualize the localization of parent bacteria and their specific OMVs with respect to the host. For the parent microbe, a number of tools exist for denoting localization including 16S DNA probes (Swidsinski et al., 2005) and exogenous expression of fluorescent proteins (Round et al., 2011). However, there are few reagents to determine OMV localization. The monoclonal antibodies described in this study, 3H2 and 6E9, are efficient and specific to Bacteroides species and their OMVs, respectively. We anticipate that these reagents will be valuable tools in further studies of B. theta-host interactions.

OMVs can be taken up by a variety of host cells (Irving et al., 2014). In our study, we showed that F4/80 positive, CD11c negative immune cells, consistent with macrophages, engulfed B. theta's OMVs. The gut contains the highest number of macrophages in the body with the highest proportion in the colon (Hume et al., 1984; Lee et al., 1985). Intestinal macrophages typically reside in the lamina propria adjacent to the base of crypts (Hume et al., 1984). Interestingly, WT macrophages have been proposed to lack pro-inflammatory responses when primed by bacteria or their products (Bain et al., 2013; Smythies et al., 2005). In contrast, it has been suggested that macrophages in IL10RII deficient mice fail to respond to tolerogenic signals and become pro-inflammatory and potentially drive colitis (Shouval et al., 2014). Future studies are necessary to better understand how resident macrophages are involved in inflammatory diseases.

It is possible that B. theta sulfatases may be involved in disease in genetically susceptible human hosts. Normally, gastrointestinal mucus acts as a protective barrier, lubricant, and bacterial habitat (Johansson et al., 2008). Mucin glycans are protected from enzymatic breakdown by sulfate, sialic acid and ester modifications (Mian et al., 1979; Podolsky and Isselbacher, 1983; Tsai et al., 1992). Clinical studies of patients with inflammatory bowel disease suggest that alterations in sulfatase activity may be important in this disease. For example, human fecal samples from IBD patients have elevated sulfatase activity (Corfield et al., 1992). Also, colonic mucin in ulcerative colitis patients was shown to be degraded much more quickly than normal mucin (Corfield et al., 1996). Thus, the findings of our study emphasize how B. theta sulfatases may play a role in inflammatory diseases and have the potential as targets for drug research.

In conclusion, this work has opened up several avenues of research into microbial access to the host immune system. First, we have shown OMV’s to be a mode by which bacteria access the mucosa of a genetically susceptible host. Second, we have introduced antibodies that can identify Bacteroides in situ. Third, we have given credence to the idea that macrophages may be critical in the development of inflammatory diseases in a genetically susceptible host. Lastly, we have revealed a new potential microbial drug target for inflammatory intestinal diseases, (i.e., anSME), which is present in most anaerobic gut bacteria.

Experimental Procedures

For more details, please see Supplemental Experimental Procedures.

Mice

All experimental procedures were performed under approval by Washington University's Animal Studies Committee. Il10r2+/− and dnKO mice were co-housed in an enhanced specific pathogen-free facility. Antibiotic treatment with ciprofloxacin and metronidazole began at weaning.

anti-B. theta monoclonal antibody generation

C57BL/6 mice were immunized with killed B. theta (BT5482), boosted, then fused splenic B-cells with P3Ag8.6.5.3 myeloma cells to create hybridomas (Kearney et al., 1979). The monoclonal antibodies were fluorescently labeled with Alexa dyes.

Tissue harvest, fixation, and preparation for histology

Mouse tissue were fixed in methacarn, rinsed in methanol and ethanol, and then stained with hematoxylin and eosin (H+E) and pH 1.0 Alcian blue.

Immnofluorescence Analysis

The 3H2 mAbs were directly conjugated to Alexa 647 and the 6E9 mAbs to Alexa 594. The M.O.M kit was the blocking agent for 3H2 staining; the SNIPER for 6E9.

B. theta ELISA

B. theta were grown to log phase, then fixed in Methacarn. Immulon 2 plates were coated with poly L-lysine. Plates were coated with dilutions of fixed bacteria O/N at 4°C. Plates were washed, blocked and incubated. The ELISA was developed with biotin-goat anti mouse IgG followed by streptavidin peroxidase and 1-Step Ultra TMB-Elisa.

Preparation of OMVs

B. theta outer membrane vesicles (OMV) were purified with multiple rounds of centrifugation and filtering (Chutkan et al., 2013).

Immunogold labeling

Samples were allowed to absorb onto formvar/carbon-coated copper grids, washed in dH2O, and stained with 1% aqueous uranyl acetate. Samples were viewed on a JEOL 1200EX transmission electron equipped with an AMT 8 megapixel digital camera.

Immunoblots for B. theta antigens

Bacteria or OMVs were resuspended in PBS and sonicated. Samples were loaded on gels for SDS page and western blotting using the Amersham ECL Prime Western Blotting Detection Reagent following manufacturer’s instructions. Primary antibody 6E9 was used at a 1:5000 dilution, and HRP goat anti-mouse IgG was used at 1:3000.

Sulfatase Assay

A sulfatase assay was performed (Benjdia et al., 2011). Briefly, sulfatase activity was measured at 25°C in 600 µl mixture of 10mM of the substrate p-nitrophenyl sulfate. One unit of activity was defined as the release of 1 µmol of product per min per mg of protein.

Development of bacterial B. theta mutants

The B. theta strain VPI-5482/ATCC 29148 was manipulated for generation of all mutants. Genetic deletion of the B. theta anSME gene was performed using allelic exchange (Benjdia et al., 2011; Cameron et al., 2014).

Colitis experiments

dnKO mice and littermate controls (IL10rb+/−) were given antibiotics for three weeks after weaning and then taken off antibiotics for two days prior to gavage and sacrified at 21 days (Bloom et al., 2011).

In vitro macrophage assays

Bone marrow cells were isolated from dnKO or Il10r2+/− littermate controls, and cultured in M-CSF (Hume et al., 1985). Macrophages were co-cultured with purified OMVs from either WT B. theta or ΔanSME. Supernatants were collected and assayed for the concentration of TNF-α by ELISA.

Statistical Analysis

Statistical analysis was performed using Prism v3.02 and v5.01 (GraphPad Software). Significance was determined with 1-way ANOVA and defined as p < 0.05.

Supplementary Material

1
2

Highlights.

  • B. thetaiotaomicron antigen localizes to the host’s mucosa in colitis-susceptible mice

  • Host-penetrant B. thetaiotaomicron antigen localizes to OMVs with sulfatase activity

  • A sulfatase-deficient B. thetaiotaomicron mutant strain is not colitogenic

  • B. thetaiotaomicron OMVs access host immune cells in a sulfatase-dependent manner

Acknowledgments

This work was supported by DK097079 (PMA, ECM, TSS) and the National Institute of Child Health and Human Development (NICHD) via the Pediatric Physician Scientist Program Award (CAH). We thank D. Kreamalmeyer for animal care and breeding, W. Beatty for electron microscopy, and Herbert W. Virgin and Emil R. Unanue for insights on the manuscript. Experimental support was provided by the Digestive Disease Research Core Center (NIH award number P30DK052574) of Washington University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The data reported in this paper are tabulated in the Supplementary Material.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Author Contributions: CAH and KAK designed the project and performed experiments. CAH and MW wrote the paper. NTP, RWPG and EAC created bacterial mutants and performed characterization. DLD, HJ, NPM and GEK performed cell and host experiments. GCH and CJ performed mass spectroscopy analysis. TSS, ECM and PMA assisted with project design and paper writing.

References

  1. Ambort D, Johansson ME, Gustafsson JK, Nilsson HE, Ermund A, Johansson BR, Koeck PJ, Hebert H, Hansson GC. Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:5645–5650. doi: 10.1073/pnas.1120269109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bain CC, Scott CL, Uronen-Hansson H, Gudjonsson S, Jansson O, Grip O, Guilliams M, Malissen B, Agace WW, Mowat AM. Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors. Mucosal immunology. 2013;6:498–510. doi: 10.1038/mi.2012.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bauman SJ, Kuehn MJ. Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response. Microbes and infection / Institut Pasteur. 2006;8:2400–2408. doi: 10.1016/j.micinf.2006.05.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benjdia A, Martens EC, Gordon JI, Berteau O. Sulfatases and a radical S-adenosyl-L-methionine (AdoMet) enzyme are key for mucosal foraging and fitness of the prominent human gut symbiont, Bacteroides thetaiotaomicron. The Journal of biological chemistry. 2011;286:25973–25982. doi: 10.1074/jbc.M111.228841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berteau O, Guillot A, Benjdia A, Rabot S. A new type of bacterial sulfatase reveals a novel maturation pathway in prokaryotes. The Journal of biological chemistry. 2006;281:22464–22470. doi: 10.1074/jbc.M602504200. [DOI] [PubMed] [Google Scholar]
  6. Bloom SM, Bijanki VN, Nava GM, Sun L, Malvin NP, Donermeyer DL, Dunne WM, Jr, Allen PM, Stappenbeck TS. Commensal Bacteroides species induce colitis in host-genotype-specific fashion in a mouse model of inflammatory bowel disease. Cell host & microbe. 2011;9:390–403. doi: 10.1016/j.chom.2011.04.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cameron EA, Kwiatkowski KJ, Lee BH, Hamaker BR, Koropatkin NM, Martens EC. Multifunctional nutrient-binding proteins adapt human symbiotic bacteria for glycan competition in the gut by separately promoting enhanced sensing and catalysis. mBio. 2014;5:e01441–e01414. doi: 10.1128/mBio.01441-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chutkan H, Macdonald I, Manning A, Kuehn MJ. Quantitative and qualitative preparations of bacterial outer membrane vesicles. Methods in molecular biology. 2013;966:259–272. doi: 10.1007/978-1-62703-245-2_16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Corfield AP, Myerscough N, Bradfield N, Corfield Cdo A, Gough M, Clamp JR, Durdey P, Warren BF, Bartolo DC, King KR, et al. Colonic mucins in ulcerative colitis: evidence for loss of sulfation. Glycoconj J. 1996;13:809–822. doi: 10.1007/BF00702345. [DOI] [PubMed] [Google Scholar]
  10. Corfield AP, Wagner SA, Clamp JR, Kriaris MS, Hoskins LC. Mucin degradation in the human colon: production of sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria. Infection and immunity. 1992;60:3971–3978. doi: 10.1128/iai.60.10.3971-3978.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Elhenawy W, Debelyy MO, Feldman MF. Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles. mBio. 2014;5:e00909–e00914. doi: 10.1128/mBio.00909-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ellis TN, Kuehn MJ. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiology and molecular biology reviews : MMBR. 2010;74:81–94. doi: 10.1128/MMBR.00031-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gankema H, Wensink J, Guinee PA, Jansen WH, Witholt B. Some characteristics of the outer membrane material released by growing enterotoxigenic Escherichia coli. Infection and immunity. 1980;29:704–713. doi: 10.1128/iai.29.2.704-713.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Holmen Larsson JM, Thomsson KA, Rodriguez-Pineiro AM, Karlsson H, Hansson GC. Studies of mucus in mouse stomach, small intestine, and colon. III. Gastrointestinal Muc5ac and Muc2 mucin O-glycan patterns reveal a regiospecific distribution. American journal of physiology Gastrointestinal and liver physiology. 2013;305:G357–G363. doi: 10.1152/ajpgi.00048.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Housseau F, Sears CL. Enterotoxigenic Bacteroides fragilis (ETBF)-mediated colitis in Min (Apc+/−) mice: a human commensal-based murine model of colon carcinogenesis. Cell cycle. 2010;9:3–5. doi: 10.4161/cc.9.1.10352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Human Microbiome Project, C. A framework for human microbiome research. Nature. 2012a;486:215–221. doi: 10.1038/nature11209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Human Microbiome Project, C. Structure, function and diversity of the healthy human microbiome. Nature. 2012b;486:207–214. doi: 10.1038/nature11234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hume DA, Allan W, Golder J, Stephens RW, Doe WF, Warren HS. Preparation and characterization of human bone marrow-derived macrophages. Journal of leukocyte biology. 1985;38:541–552. doi: 10.1002/jlb.38.4.541. [DOI] [PubMed] [Google Scholar]
  19. Hume DA, Perry VH, Gordon S. The mononuclear phagocyte system of the mouse defined by immunohistochemical localisation of antigen F4/80: macrophages associated with epithelia. The Anatomical record. 1984;210:503–512. doi: 10.1002/ar.1092100311. [DOI] [PubMed] [Google Scholar]
  20. Irving AT, Mimuro H, Kufer TA, Lo C, Wheeler R, Turner LJ, Thomas BJ, Malosse C, Gantier MP, Casillas LN, et al. The immune receptor NOD1 and kinase RIP2 interact with bacterial peptidoglycan on early endosomes to promote autophagy and inflammatory signaling. Cell host & microbe. 2014;15:623–635. doi: 10.1016/j.chom.2014.04.001. [DOI] [PubMed] [Google Scholar]
  21. Johansson ME, Phillipson M, Petersson J, Velcich A, Holm L, Hansson GC. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proceedings of the National Academy of Sciences of the United States of America. 2008;105:15064–15069. doi: 10.1073/pnas.0803124105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johansson ME, Sjovall H, Hansson GC. The gastrointestinal mucus system in health and disease. Nature reviews Gastroenterology & hepatology. 2013;10:352–361. doi: 10.1038/nrgastro.2013.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kaiko GE, Stappenbeck TS. Host-microbe interactions shaping the gastrointestinal environment. Trends in immunology. 2014 doi: 10.1016/j.it.2014.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kang SS, Bloom SM, Norian LA, Geske MJ, Flavell RA, Stappenbeck TS, Allen PM. An antibiotic-responsive mouse model of fulminant ulcerative colitis. PLoS medicine. 2008;5:e41. doi: 10.1371/journal.pmed.0050041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kearney JF, Radbruch A, Liesegang B, Rajewsky K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. Journal of immunology. 1979;123:1548–1550. [PubMed] [Google Scholar]
  26. Kuhnert F, Davis CR, Wang HT, Chu P, Lee M, Yuan J, Nusse R, Kuo CJ. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:266–271. doi: 10.1073/pnas.2536800100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kulp A, Kuehn MJ. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annual review of microbiology. 2010;64:163–184. doi: 10.1146/annurev.micro.091208.073413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee G, White LS, Hurov KE, Stappenbeck TS, Piwnica-Worms H. Response of small intestinal epithelial cells to acute disruption of cell division through CDC25 deletion. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:4701–4706. doi: 10.1073/pnas.0900751106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lee SH, Starkey PM, Gordon S. Quantitative analysis of total macrophage content in adult mouse tissues. Immunochemical studies with monoclonal antibody F4/80. The Journal of experimental medicine. 1985;161:475–489. doi: 10.1084/jem.161.3.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lev R, Spicer SS. Specific Staining of Sulphate Groups with Alcian Blue at Low Ph. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 1964;12:309. doi: 10.1177/12.4.309. [DOI] [PubMed] [Google Scholar]
  31. Martens EC, Chiang HC, Gordon JI. Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont. Cell host & microbe. 2008;4:447–457. doi: 10.1016/j.chom.2008.09.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Martens EC, Roth R, Heuser JE, Gordon JI. Coordinate regulation of glycan degradation and polysaccharide capsule biosynthesis by a prominent human gut symbiont. The Journal of biological chemistry. 2009;284:18445–18457. doi: 10.1074/jbc.M109.008094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mian N, Anderson CE, Kent PW. Effect of O-sulphate groups in lactose and N-acetylneuraminyl-lactose on their enzymic hydrolysis. The Biochemical journal. 1979;181:387–399. doi: 10.1042/bj1810387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Niess JH, Brand S, Gu X, Landsman L, Jung S, McCormick BA, Vyas JM, Boes M, Ploegh HL, Fox JG, et al. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science. 2005;307:254–258. doi: 10.1126/science.1102901. [DOI] [PubMed] [Google Scholar]
  35. Podolsky DK, Isselbacher KJ. Composition of human colonic mucin. Selective alteration in inflammatory bowel disease. The Journal of clinical investigation. 1983;72:142–153. doi: 10.1172/JCI110952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Round JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA, Mazmanian SK. The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota. Science. 2011;332:974–977. doi: 10.1126/science.1206095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shen Y, Giardino Torchia ML, Lawson GW, Karp CL, Ashwell JD, Mazmanian SK. Outer membrane vesicles of a human commensal mediate immune regulation and disease protection. Cell host & microbe. 2012;12:509–520. doi: 10.1016/j.chom.2012.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shouval DS, Biswas A, Goettel JA, McCann K, Conaway E, Redhu NS, Mascanfroni ID, Al Adham Z, Lavoie S, Ibourk M, et al. Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function. Immunity. 2014;40:706–719. doi: 10.1016/j.immuni.2014.03.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smythies LE, Sellers M, Clements RH, Mosteller-Barnum M, Meng G, Benjamin WH, Orenstein JM, Smith PD. Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity. The Journal of clinical investigation. 2005;115:66–75. doi: 10.1172/JCI19229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sonnenburg JL, Xu J, Leip DD, Chen CH, Westover BP, Weatherford J, Buhler JD, Gordon JI. Glycan foraging in vivo by an intestine-adapted bacterial symbiont. Science. 2005;307:1955–1959. doi: 10.1126/science.1109051. [DOI] [PubMed] [Google Scholar]
  41. Swidsinski A, Loening-Baucke V, Lochs H, Hale LP. Spatial organization of bacterial flora in normal and inflamed intestine: a fluorescence in situ hybridization study in mice. World journal of gastroenterology : WJG. 2005;11:1131–1140. doi: 10.3748/wjg.v11.i8.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tannahill GM, Curtis AM, Adamik J, Palsson-McDermott EM, McGettrick AF, Goel G, Frezza C, Bernard NJ, Kelly B, Foley NH, et al. Succinate is an inflammatory signal that induces IL-1beta through HIF-1alpha. Nature. 2013;496:238–242. doi: 10.1038/nature11986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tsai HH, Sunderland D, Gibson GR, Hart CA, Rhodes JM. A novel mucin sulphatase from human faeces: its identification, purification and characterization. Clin Sci (Lond) 1992;82:447–454. doi: 10.1042/cs0820447. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS680150-supplement-1.xlsx (20.4KB, xlsx)
2
NIHMS680150-supplement-2.pdf (16.9MB, pdf)

RESOURCES