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. 2015 May 13;17(5):672-80.
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 , Kristine A Kuhn  2 , David L Donermeyer  2 , Nathan T Porter  3 , Chunsheng Jin  4 , Elizabeth A Cameron  3 , Haerin Jung  2 , Gerard E Kaiko  2 , Marta Wegorzewska  2 , Nicole P Malvin  2 , Robert W P Glowacki  3 , Gunnar C Hansson  4 , Paul M Allen  5 , Eric C Martens  6 , Thaddeus S Stappenbeck  7
Affiliations

Colitogenic Bacteroides thetaiotaomicron Antigens Access Host Immune Cells in a Sulfatase-Dependent Manner via Outer Membrane Vesicles

Christina A Hickey et al. Cell Host Microbe. .

Abstract

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 develop 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 VSports手机版. 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. .

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Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
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.
Figure 3
Figure 3
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.
Figure 4
Figure 4
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.
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