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. 2014 Aug 13;16(2):227-236.
doi: 10.1016/j.chom.2014.07.007. Epub 2014 Jul 25.

A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism

Alistair B Russell  1 , Aaron G Wexler  2 , Brittany N Harding  1 , John C Whitney  1 , Alan J Bohn  1 , Young Ah Goo  3 , Bao Q Tran  3 , Natasha A Barry  2 , Hongjin Zheng  4 , S Brook Peterson  1 , Seemay Chou  1 , Tamir Gonen  4 , David R Goodlett  3 , Andrew L Goodman  5 , Joseph D Mougous  6
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A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism

Alistair B Russell et al. Cell Host Microbe. .

Abstract

Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway VSports手机版. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens. .

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Figure 1
Figure 1. T6S-like gene clusters are found within the Bacteroidetes
(A–C) Gene content and conservation both within and between selected representative members of T6SSi (A), T6SSii (B), and T6SSiii (C). Genes with commonly used tss nomenclature are abbreviated to a single letter. The Francisella tularensis FPI is depicted owing to its status as the only T6SSii to be characterized; however, this system lacks clear homologs of tssA and tssJ, which are present in representative T6SSii systems such the F. novicida gene cluster shown. Locus tags of the regions shown: B. thailandensis E264 BTH_I2705 and BTH_I2954-2968 (A); F. novicida U112 FTN_0037-0054, F. tularensis SCHU FTT_1344-1361c (B); F. johnsoniae UW101 Fjoh_3254-328, B. fragilis NCTC 9343 BF9343_1918-1943, P. veroralis F0319 HMPREF0973_02422-02423 and HMPREF0973_02441-02466. Genes encoding F. johnsoniae T6SSiii substrates identified in this study (dark grey) and a validated immunity locus (light grey) are labeled. For sequence alignments of T6SSiii TssB,C,E,F,G,K proteins, including those depicted, see File S1. (D) Comparison of domain organization of ClpV homologs from T6SSi (B. thailandensis, Bt) and T6SSiii (F. johnsoniae, Fj) pathways. Colors denote homologous domains: blue, Clp N; red, AAA+; purple, ClpB D2. Sequences highlight the conservation of motifs implicated in ATP binding and hydrolysis within the Walker A and B motifs. (E) SDS-PAGE analysis of purified Fjoh_3262 bearing a C-terminal hexahistidine tag (Fj Hcp1–H6). Proteins were visualized by Coomassie Blue staining. (F) F. johnsoniae Hcp1 is a ring-shaped molecule with dimensions similar to Proteobacterial Hcp proteins. Transmission electron micrograph of purified Fj Hcp1–H6 (E) negatively stained with uranyl formate. The arrowhead indicates a typical ring-like assembly. Scale bar, 40 nm.
Figure 2
Figure 2. T6SSiii is phylogenetically distinct from T6SSi and T6SSii
(A) Maximum likelihood (ML) phylogenetic tree generated from a partial alignment of 686 representative TssC sequences spanning the diversity present in T6SSi, T6SSii, and T6SSiii gene clusters. Phyla represented by each system are indicated. Branch support values derived from aBayes analysis for the T6SSiii clade are shown. Scale bar represents amino acid changes per site. A similar tree for TssF is provided in Figure S2a. (B) ML tree created from a partial alignment of TssC sequences found within T6SSiii gene clusters. The tree is rooted with Acidobacterial TssC sequences. Lengths do not reflect evolutionary distance. Colors trace the Class from which each sequence is derived. Nodes representing TssC sequences of organisms discussed in the text and those of particular significance are marked with an asterisk. Branch support values were generated by aBayes analysis. Partial sequence alignments and branch supports corresponding to phylogenetic trees in panels (A) and (B) and Figure S2 are provided in Files S1–4.
Figure 3
Figure 3. F. johnsoniae T6SSiii exports an antibacterial protein that is encoded adjacent to a cognate immunity determinant
(A) Domain organization of the putative substrates of F. johnsoniae T6SSiii. PAAR-like (blue), zinc-dependent metalloprotease (red), glycoside hydrolase (green), and zinc-dependent peptidoglycan amidase (purple) domains are indicated. Expanded amino acid sequences in each domain correspond to conserved motifs and invariant or critical catalytic residues (red). (B–C) Growth of E. coli strains harboring the indicated expression vectors. Empty vectors (control) and vectors that introduce an N-terminal Sec signal peptide (peri) are indicated. Cells were induced to express predicted immunity proteins (C) at time 0 and Fte1 at the indicated time point (arrow). Type VI secretion immunity protein 1 (Tsi1) is used as a non-cognate immunity control. Error bars represent ± standard deviation (SD) (n = 3). Expression data for (B) are provided in Figure S3. (D) Intercellular self-intoxication of the indicated F. johnsoniae strains as measured by propidium iodide staining. Liquid cultures were grown with vigorous shaking, which inhibits the formation of the prolonged cell–cell contacts required for T6-mediated interactions (Hood et al., 2010; Leroux et al., 2012). Error bars represent ± standard deviation (SD) (n = 3). Samples differing significantly from parent as measured by a two-tailed T-test are indicated by asterisks (p < 0.01).
Figure 4
Figure 4. F. johnsoniae utilizes a dynamic T6SSiii apparatus to target competitor organisms
(A–B) Growth experiments measuring fitness of the indicated F. johnsoniae (Fj) strains in co-culture for 20 hours with fluorescently-labeled competitors B. thailandensis (A, Bt) or P. putida (B, Pp). Competitive index is defined as the change in F. johnsoniae/competitor colony forming units between initiation and harvest of the co-culture. Experiments were performed under contact-promoting (solid) and contact-inhibiting (liquid) conditions. Qualitative analysis of competition outcome by photographic (P) and fluorescence (F) imaging is shown for corresponding samples grown under contact-promoting conditions after 48 hours of co-culture. Error bars represent ± SD (n = 3). (C) Micrograph series depicting a 10s time course of wild-type F. johnsoniae clpV–gfp. Phase and GFP channels are presented separately. Three dynamic foci were observed over the duration of the experiment (arrowheads), and the presence or absence of each focus in each frame is schematized in the phase micrographs with white or unfilled circles, respectively. Full-length movies that include the region represented are available as Supplemental Files (Movies S1 and S2). See also Figure S4.
Figure 5
Figure 5. The T6SSiii pathway is expressed and active in the genus Bacteroides
(A) Quantitative RT-PCR analysis of the in vivo expression of tssC for the indicated bacteria. Expression was measured in cecal samples from germfree mice colonized for one week with B. fragilis, B. eggerthii, and E. coli and normalized using species-specific primers for the housekeeping sigma factor, rpoD. (B) Growth competitions measuring viability of B. thetaiotaomicron after 24 h in the presence of the indicated B. fragilis strains on solid media. B. thetaiotaomicron growth was normalized to values obtained in the absence of B. fragilis. Viability was determined by colony forming units. Error bars represent ± SD (n = 3). Significance as indicated by asterisks was measured by a two-tailed T-test (p<0.002), n.s.; no statistical difference.
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