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. 2016 Aug 23;7(4):e01055-16.
doi: 10.1128/mBio.01055-16.

Bacteroidales Secreted Antimicrobial Proteins Target Surface Molecules Necessary for Gut Colonization and Mediate Competition In Vivo

Kevin G Roelofs  1 , Michael J Coyne  1 , Rahul R Gentyala  1 , Maria Chatzidaki-Livanis  1 , Laurie E Comstock  2
Affiliations

Bacteroidales Secreted Antimicrobial Proteins Target Surface Molecules Necessary for Gut Colonization and Mediate Competition In Vivo

Kevin G Roelofs et al. mBio. .

"VSports app下载" Abstract

We recently showed that human gut Bacteroidales species secrete antimicrobial proteins (BSAPs), and we characterized in vitro the first such BSAP produced by Bacteroides fragilis In this study, we identified a second potent BSAP produced by the ubiquitous and abundant human gut species Bacteroides uniformis The two BSAPs contain a membrane attack complex/perforin (MACPF) domain but share very little sequence similarity. We identified the target molecules of BSAP-sensitive cells and showed that each BSAP targets a different class of surface molecule: BSAP-1 targets an outer membrane protein of sensitive B. fragilis strains, and BSAP-2 targets the O-antigen glycan of lipopolysaccharide (LPS) of sensitive B. uniformis strains. Species-wide genomic and phenotypic analyses of B. fragilis and B. uniformis showed that BSAP-producing strains circumvent killing by synthesizing an orthologous nontargeted surface molecule VSports手机版. The BSAP genes are adjacent to the gene(s) encoding their target replacements, suggesting coacquisition. Using a gnotobiotic mouse competitive-colonization model, we found that the BSAP surface targets are important for colonization of the mammalian gut, thereby explaining why they are maintained in sensitive strains and why they were replaced rather than deleted in BSAP-producing strains. Using isogenic BSAP-producing, -sensitive, and -resistant strains, we show that a BSAP-producing strain outcompetes a sensitive strain but not a resistant strain in the mammalian gut. Human gut metagenomic datasets reveal that BSAP-1-sensitive strains do not cooccur with BSAP-1-producing strains in human gut microbiotas, further supporting the idea that BSAPs are important competitive factors with relevance to the strain-level composition of the human gut microbiota. .

Importance: We know relatively little about the ecology of the human intestinal microbiota and the combination of factors that dictate which strains and species occupy an individual's gut microbial community. Interference competition, mediated by bacterial factors that directly harm other members, is beginning to be appreciated as important in contributing to species- and strain-level dynamics of abundant gut bacteria. Here, we show that gut Bacteroidales secrete antimicrobial proteins (BSAPs) that antagonize strains of the same species. We show that BSAPs target molecules of sensitive cells that are important for gut colonization and therefore are maintained in sensitive cells. In an experimental animal model of gut colonization, a BSAP-1-producing strain antagonized and outcompeted an isogenic sensitive strain. Furthermore, metagenomic analyses showed that BSAP-1-producing and -sensitive strains are not found together in human gut microbiotas. These data suggest that BSAPs are strong ecological drivers shaping the strain-level composition of gut communities. V体育安卓版.

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FIG 1
FIG 1
Identification of BSAP-2. (A) ORF map of a B. uniformis CL03T00C23 (BuCL03) region showing a transposon (tn) insertion resulting in loss in antimicrobial activity against B. uniformis 8492 (Bu8492). (B to F) Agar overlay assays showing growth or inhibition of Bu8492 by (B) BuCL03 wild type (wt) and transposon mutant tn::1165. (C) The BUCL03 wt and deletion mutants of HMPREF1072_1165-7 (Δ1165-7) or HMPREF1072_1167 (Δ1167), (D) BuCL03 Δ1165-7 and Δ1167 mutants containing empty vector (vector) or plasmid-expressing HMPREF1072_01167 (1167), (E) B. fragilis (B.f.) CM11 with empty vector or 1167, (F) and a phosphate-buffered saline (PBS) control or purified His-tagged 1167 (His-1167).
FIG 2
FIG 2
BSAP-2 targets the O-ag of B. uniformis 8492. (A) Genetic region of Bu8492 encoding predicted LPS core and O-antigen biosynthesis loci. A transposon insertion into BACUNI_00969 causing BSAP-2 resistance is indicated. (B) Agar overlay assays showing sensitivity of Bu8492 strains to BuCL03 wt or the mutant lacking the gene encoding BSAP-2 (Δ1167). Overlay strains include BACUNI_00969 deletion mutant (Δ00969), Δ00969 with an empty vector (pVector), or BACUNI_00969 (p00969) in trans. (C) Western blot of whole-cell lysates of Bu8492 or tn::00969 probed with antiserum raised to Bu8492 and adsorbed against the tn::00969 mutant. (D) Silver stain of purified LPS from Bu8492 or Δ00969 with vector alone (−) or p00969 in trans. (E) Same as panel D except probed with the adsorbed serum used as described for panel C.
FIG 3
FIG 3
Correlation of O-ag with BSAP-2 in B. uniformis strains. (A) ORF maps showing the two types of O-ag loci (light and dark blue) in sequenced B. uniformis strains typified by Bu8492 and BuCL03. Surrounding genes are shown, including the BSAP-2-encoding gene colored red and a distinct MACPF domain protein-encoding gene colored yellow. Percent DNA identity is graphed below the open reading frames, with regions >95% identical colored in pink. (B) Ethidium bromide (EtBr)-stained agarose gel showing amplification products from various PCRs. Upper panel, Bu8492 O-ag region-specific gene; middle panel, BuCL03 O-ag region-specific gene; bottom panel, BSAP-2 gene. (C) Silver-stained gel and Western blot of purified LPS from various B. uniformis strains. The blot was probed with the adsorbed antiserum specific to the Bu8492 O-ag. (D) Agar overlays demonstrating the sensitivity or resistance of B. uniformis strains D20 (BuD20) CL03T00C23 (BuCL03), CL06T06C18 (BuCL06), CL07T00C16 (BuCL07), and CL14T09C07 (BuCL14) to BuCL03 or the BuCL03 mutant lacking the gene encoding BSAP-2. (E) Agar overlays showing zones of inhibition produced by BuCL03 and BuCL14 against Bu8492 but not the Bu8492 O-ag mutant (Δ00969).
FIG 4
FIG 4
BSAP-1 targets an outer surface protein of sensitive B. fragilis strains. (A) Genetic region of heterogeneity between BSAP-1 producer Bf638R and BSAP-1-sensitive strains BfCL05T12C13 (BfCL05) and B. fragilis 9343 (Bf9343). The BSAP-1-encoding gene is colored red. The gene encoding the BSAP-1-targeted outer membrane protein (OmpS) is colored light purple, while its nontargeted ortholog in Bf638R (OmpR) is colored dark purple. Percent DNA identity between BfCL05 and Bf638R is graphed below open reading frames, and regions >95% identical are colored pink. (B and C) Agar overlays showing zones of inhibition produced by the Bf638R wild type (wt) or the BSAP-1 mutant (Δ1646) against Bu8492 with vector control (pVector), HMPREF1080_01555 (p01555), HMPREF1080_01556 (p01556), BF638R_1645 (p1645), or BF9343_1563 (p1563) (B) or against Bf9343, a clean deletion of BF9343_1563 (Δ1563), and Δ1563 with either the vector alone (pVector) or p1563 in trans (C).
FIG 5
FIG 5
BSAP targets surface molecules critical for gut colonization. Data represent results of gnotobiotic mouse competitive gut colonization assays. (A) Bf9343 versus Bf9343Δ1563. (B) Bu8492 versus Bu8492Δ00969 (Bu8492ΔO-ag). Means and standard errors of the results of 2 separate trials of 3 mice each are graphed for each strain. Across both trials, the wt/mutant ratios in the inoculum compared to the day 7 feces samples were significantly different: for Bf9343 versus Bf9343Δ1563, P = 2 × 10−6; for Bu8492 versus Bu8492Δ00969, P = 3 × 10−5.
FIG 6
FIG 6
BSAP producers outcompete BSAP-sensitive strains in the mammalian gut. Isogenic strains that differed only in BSAP-1 production, sensitivity, or resistance were constructed by adding in to Bu8492 empty vector (pVector) or plasmids expressing BF638R_1646 (pBSAP-1), HMPREF1080_01556 (p01556), or BF638R_1645 (p1645). (A) Agar overlay assays of isogenic Bu8492 strains demonstrating BSAP production, sensitivity, and resistance in vitro. (B and C) Gnotobiotic mouse competitive-colonization assays between isogenic BSAP-1 producer and sensitive Omp (2 experiments, 3 mice each) (B) and BSAP-1 producer and resistant Omp (3 experiments, 3 mice each) (C). Means and standard errors are graphed. The ratios of strains in the inoculum versus day 7 feces samples were significantly different for BSAP-1 versus sensitive Omp (P = 1 × 10−10) but not for BSAP-1 versus resistant Omp (P > 0.05).
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