Significance The microbial community in the human gut represents one of the densest known ecosystems. Community composition has broad impacts on health, and metabolic competition and host selection have both been implicated in shaping these communities. Here, we report that contact-dependent bacterial antagonism also determines the ability of human gut symbionts to persist in the microbiome. Simplified microbiomes, assembled in gnotobiotic mice, reveal effector transmission rates exceeding 1 billion events per minute per gram of colonic contents. Together, these results suggest that human gut symbionts define their closest competitors not only metabolically but also spatially. Moreover, strains within a single species can encode diverse effectors that may provide new avenues for shaping the microbiome to improve human health.

Abstract The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes—one of two major phyla in the gut—also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.

Interpersonal variation in gut microbial community composition, even at the species or strain level, can determine the contribution of these communities to cancer risk, drug metabolism, caloric extraction from diet, infectious disease resistance, and other responses (1⇓⇓⇓–5). However, the rules that determine community membership, especially at the species or strain level, are largely undefined. The gut environment is characterized by constant peristalsis and extensive niche heterogeneity, and factors previously implicated in shaping these communities (metabolites, vitamins, dietary polysaccharides, host IgA, bacteriocins) are freely diffusible (6⇓–8). In this context, the recent identification of genes encoding type VI secretion systems (T6SSs) in the genomes of many prominent human gut symbionts was unexpected because the ability of these dynamic machines to inject toxic effectors into adjacent cells is strictly dependent on direct cell-to-cell contact (9⇓⇓–12).

The T6SSs of Bacteroidetes share many subunits with those of Proteobacteria; these include the Hcp and TssB–TssC proteins, which interact and assemble into a contractile bacteriophage tail-like structure that is required for effector translocation from donor to recipient cells (9, 10). Bacteria with T6SSs lack a means for self-/non–self-discrimination; thus, sister cells inject one another with effectors. To nullify the antibacterial properties of these toxins, T6SS+ strains produce immunity proteins that directly bind cognate effectors. Despite our growing understanding of the mechanism and activity of T6S in vitro, little is known about the ecological role of this pathway in natural environments where direct encounters between microorganisms occur.

Here we report that the human gut symbiont Bacteroides fragilis NCTC 9343 (B. fragilisN) targets other members of the microbiome in a species- and strain-specific manner in the mammalian gut. We identify strain-specific effector/immunity (E/I) repertoires and show that the presence or absence of these genes can accurately predict interstrain dynamics in the gut. Furthermore, we combine microbial genetics, mathematical modeling, and gnotobiotic studies to determine the frequency of T6S-mediated events in live animals. Together, these results define a significant role for contact-mediated bacterial antagonism between human gut symbionts.

Discussion Genome sequencing of strains isolated from individual human gut microbiomes over time has revealed that individual strains within these communities, particularly Bacteroidetes, can persist for years or decades in humans without significant strain replacement (20). Though metagenomic sequencing highlights broad (i.e., phylum-level) microbiome changes in response to diet, antibiotics, and other factors, the mechanisms that determine community membership at the species- or strain-level are largely unexplored. The human gut and its microbiome is one of the densest known ecosystems, and bacteria that occupy this habitat face stringent competition for dietary and host-derived nutrients localized to food particles or mucus (21); at the same time, they benefit from diffusible vitamins, metabolic by-products, and public goods produced by other species or strains (22, 23). Mechanisms for contact-dependent antagonism, including T6S, could allow cells to minimize local nutritional competition without impacting the fitness of spatially distant bacteria that provide these beneficial compounds. The distribution of T6SS+ strains within (and outside) the Bacteroidetes is not easily predicted due to extensive horizontal gene transfer (Fig. 2). Given that members of this phylum constitute 50–80% of the microbiome in many individuals (19), our results and others (15) suggest that contact-dependent antagonism is a general feature that shapes interbacterial interactions between human gut microbes. As expected from the contact-dependent nature of these interactions, mathematical modeling predicts that the frequency of T6S-mediated killing events reaches a maximum under conditions in which all cells are evenly mixed and donor and recipient cells each constitute half of the community (Fig. S6). Our experiments using defined microbial communities in germ-free mice support these predictions: B. fragilisN significantly reduces B. vulgatus abundance via T6S when the T6S-positive and -negative cells each represent ∼50% of the community (Fig. 1D), but this does not occur when the T6S-positive cells represent only 1% (Fig. S5). Similar dynamics may explain the discrepancy between in vitro and in vivo dynamics of T6S-mediated killing of B. thetaiotaomicron. Moreover, our model suggests that the decrease in the observed killing rate of B. fragilisN in a multispecies community (Fig. 5D) does not require an intrinsic reduction in the transmission rate (i.e., decreased β), but instead can be explained by changes in the population dynamics of the microbiome (e.g., altered ratios of donors and recipients over time, uneven mixing of species). Together, these studies highlight the importance of in vivo experiments for understanding the role of contact-mediated interactions between bacteria. Long-term studies could also reveal more subtle or indirect T6S interactions that manifest over one or more host generations. Fig. S6. Predicting the impact of evenness and mixing on T6S-mediated killing in vivo. (A) A mathematical model parameterized using time-series measurements from gnotobiotic mice predicts that the number of killing events peaks when donors and recipients each make up 50% of the community, maximizing the likelihood of encounters between the two cell types. (B) If the D and S populations each constitute 50% of the microbiome, the rate of killing is highest when there is complete spatial mixing, because this also maximizes encounters between donors and recipients. In A and B, the thick line is the model prediction based on the mean transmission rate β, and the dashed lines represent the 95% confidence intervals of the mean. Identification and genetic manipulation of E/I pairs in B. fragilis strains, combined with gnotobiotic animal models in which community composition can be controlled and measured, allows for the calculation of a transmission coefficient (β = 0.62 per D cell per day) for T6S activity in the gut. This calculation represents a lower bound for this activity in vivo for at least three reasons. First, uneven mixing would increase the number of transmission events required to mediate the observed killing rates (Fig. S6B). Second, the model assumes that a single effector transmission event into an S individual results in the death and subsequent removal of that individual from the population. However, if some proportion of transmission events into S cells does not result in the death of the recipient, then those individuals will not be removed from the population, representing transmission events that do not produce an observable change in S. Indeed, in vitro studies of the opportunistic proteobacterial pathogen Pseudomonas aeruginosa suggest that its T6SS kills recipient cells at rates of ∼5% or less per hour of contact (24). Third, our experiments used T6S-negative recipient populations. P. aeruginosa cells display an elevated propensity to activate T6S in response to a T6S attack (24, 25). Although this phenomenon has not been investigated in Bacteroides, the S strain and 14-species community in these experiments are T6S-negative and would not induce this increased transmission rate that may occur between T6S-positive (D) individuals. From our calculation of effector transmission rates between bacteria, we can predict that humans carrying B. fragilis at typical levels (19) host 60–600 billion effector transmission events per day from this species alone. A deeper understanding of the E/I repertoires present among human symbiont strains within an individual microbiome will help to predict the impact of T6S on other community members. Interestingly, our data show that human symbionts can accumulate immunity genes throughout their genomes that protect against T6S effectors that they do not encode, suggesting a selective advantage to maintaining the ability to evade contact-mediated antagonism. Because immunity genes are difficult to recognize by sequence alone (9, 10), many more may exist in the genomes of commensal bacteria. Unlike the immunity genes, B. fragilis effectors appear to be encoded in stereotyped positions inside the T6SS locus (Fig. 2). Because numerous human gut Bacteroidetes besides B. fragilis carry T6SS loci (11, 12, 15), additional effectors will be readily identifiable in other species. These effectors reveal strategies human gut symbionts themselves have evolved to target other members of this community, and may provide important new approaches for precision microbiome manipulation.

Acknowledgments We thank members of A.L.G.’s laboratory; J. Galán and E. Groisman for helpful discussions; and C. Sears for B. fragilisTB9 and B. fragilisDS. D.R.G. thanks the University of Maryland School of Pharmacy Mass Spectrometry Center (SOP1841-IQB2014). This work was supported NIH Grants GM103574 and GM105456 (to A.L.G.), AI080609 (to J.D.M.), GM101209 and GM108657 (to H.O.), and OD008440 (to J.B.X.); the Pew Scholars Program (A.L.G.); and the Burroughs Wellcome Fund (A.L.G. and J.D.M.). A.G.W. is supported by a fellowship from the Gruber Foundation, and J.C.W. is supported by a fellowship from the Canadian Institutes of Health Research.

Footnotes Author contributions: A.G.W. and A.L.G. designed research; A.G.W., Y.B., J.C.W., N.A.B., B.Q.T., and Y.A.G. performed research; A.G.W., L.-M.B., J.B.X., W.B.S., A.B.R., D.R.G., H.O., J.D.M., and A.L.G. analyzed data; and A.G.W. and A.L.G. wrote the paper.

The authors declare no conflict of interest.

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