The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections. Conversely, disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens1–4. How pathogens capitalize upon the failure of microbiota-afforded protection is largely unknown. Here we show that two antibiotic-associated pathogens, Salmonella typhimurium and Clostridium difficile, employ a common strategy of catabolizing microbiota-liberated mucosal carbohydrates during their expansion within the gut. S. typhimurium accesses fucose and sialic acid within the lumen of the gut in a microbiota-dependent manner, and genetic ablation of the respective catabolic pathways reduces its competitiveness in vivo. Similarly, C. difficile expansion is aided by microbiota-induced elevation of sialic acid levels in vivo. Colonization of gnotobiotic mice with a sialidase-deficient mutant of the model gut symbiont Bacteroides thetaiotaomicron (Bt) reduces free sialic acid levels resulting in a downregulation of C. difficile’s sialic acid catabolic pathway and impaired expansion. These effects are reversed by exogenous dietary administration of free sialic acid. Furthermore, antibiotic treatment of conventional mice induces a spike in free sialic acid and mutants of both Salmonella and C. difficile that are unable to catabolize sialic acid exhibit impaired expansion. These data show that antibiotic-induced disruption of the resident microbiota and subsequent alteration in mucosal carbohydrate availability are exploited by these two distantly related enteric pathogens in a similar manner. This insight suggests new possibilities for therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.

Microbial communities associated with animals exert powerful influences on host physiology, regulating metabolism and immune function, as well as complex host behaviors. The importance of host–microbiome interactions for maintaining homeostasis and promoting health raises evolutionarily complicated questions about how animals and their microbiomes have coevolved, and how these relationships affect the ways that animals interact with their environment. Here, we review the literature on the contributions of host factors to microbial community structure and corresponding influences of microbiomes on emergent host phenotypes. We focus in particular on animal behaviors as a basis for understanding potential roles for the microbiome in shaping host neurobiology.

Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. IMPORTANCEDetermining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development. Further, we present evidence that this rotation releases apoptosis-triggering lipopolysaccharide in the form of outer membrane vesicles. Such release may also occur by pathogens but with different outcomes for the host.

Outer membrane vesicles (OMVs) are proteoliposome nanoparticles ubiquitously produced by Gram-negative bacteria. Typically bearing a composition similar to those of the outer membrane and periplasm of the cells from which they are derived, OMVs package an array of proteins, lipids, and nucleic acids. Once considered inconsequential by-products of bacterial growth, OMVs have since been demonstrated to mediate cellular stress relief, promote horizontal gene transfer and antimicrobial activity, and elicit metazoan inflammation. Recently, OMVs have gained appreciation as critical moderators of interorganismal dynamics. In this review, we focus on recent progress toward understanding the functions of OMVs with regard to symbiosis and ecological contexts, and we propose potential avenues for future OMV studies.

Summary Bacteroides is a dominant genus within the intestinal microbiota of healthy humans. Key adaptations of the Bacteroides to the dynamic intestinal ecosystem include a diverse repertoire of genes involved in sensing and processing numerous diet- and host-derived polysaccharides. One such adaptation is the carbohydrate-sensing hybrid two-component system (HTCS) family of signaling sensors, which has been widely expanded within the Bacteroides. Using Bacteroides thetaiotaomicron as a model, we have created a chimeric HTCS consisting of the well-characterized sensing domain of one HTCS, BT1754, and the regulatory domain of another HTCS, BT0366, to explore the regulatory capabilities of these molecules. We found that the BT0366 regulatory region directly binds to and mediates induction of the adjacent polysaccharide utilization locus (PUL) using whole genome transcriptional profiling after inducing signaling through our chimeric protein. We also found that BT0366 activation simultaneously leads to repression of distal PULs involved in mucus carbohydrate consumption. These results suggest a novel mechanism by which an HTCS enforces a nutrient hierarchy within the Bacteroides via induction and repression of multiple PULs. Thus, hybrid two-component systems provide a mechanism for prioritizing consumption of carbohydrates through simultaneous binding and regulation of multiple polysaccharide utilization loci.

Outer membrane vesicles (OMVs) are continuously produced by Gram-negative bacteria and are increasingly recognized as ubiquitous mediators of bacterial physiology. In particular, OMVs are powerful effectors in interorganismal interactions, driven largely by their molecular contents. These impacts have been studied extensively in bacterial pathogenesis but have not been well documented within the context of mutualism. Here, we examined the proteomic composition of OMVs from the marine bacterium Vibrio fischeri, which forms a specific mutualism with the Hawaiian bobtail squid, Euprymna scolopes. We found that V. fischeri upregulates transcription of its major outer membrane protein, OmpU, during growth at an acidic pH, which V. fischeri experiences when it transitions from its environmental reservoir to host tissues. We used comparative genomics and DNA pulldown analyses to search for regulators of ompU and found that differential expression of ompU is governed by the OmpR, H-NS, and ToxR proteins. This transcriptional control combines with nutritional conditions to govern OmpU levels in OMVs. Under a host-encountered acidic pH, V. fischeri OMVs become more potent stimulators of symbiotic host development in an OmpU-dependent manner. Finally, we found that symbiotic development could be stimulated by OMVs containing a homolog of OmpU from the pathogenic species Vibrio cholerae, connecting the role of a well-described virulence factor with a mutualistic element. This work explores the symbiotic effects of OMV variation, identifies regulatory machinery shared between pathogenic and mutualistic bacteria, and provides evidence of the role that OMVs play in animal-bacterium mutualism. IMPORTANCE Beneficial bacteria communicate with their hosts through a variety of means. These communications are often carried out by a combination of molecules that stimulate responses from the host and are necessary for development of the relationship between these organisms. Naturally produced bacterial outer membrane vesicles (OMVs) contain many of those molecules and can stimulate a wide range of responses from recipient organisms. Here, we describe how a marine bacterium, Vibrio fischeri, changes the makeup of its OMVs under conditions that it experiences as it goes from its free-living lifestyle to associating with its natural host, the Hawaiian bobtail squid. This work improves our understanding of how bacteria change their signaling profile as they begin to associate with their beneficial partner animals.

We screened a panel of R5X4 and X4 human immunodeficiency virus type 1 (HIV-1) strains for their sensitivities to AMD3100, a small-molecule CXCR4 antagonist that blocks HIV-1 infection via this coreceptor. While no longer under clinical development, AMD3100 is a useful tool with which to probe interactions between the viral envelope (Env) protein and CXCR4 and to identify pathways by which HIV-1 may become resistant to this class of antiviral agents. While infection by most virus strains was completely blocked by AMD3100, we identified several R5X4 and X4 isolates that exhibited plateau effects: as the AMD3100 concentration was increased, virus infection and membrane fusion diminished to variable degrees. Once saturating concentrations of AMD3100 were achieved, further inhibition was not observed, indicating a noncompetitive mode of viral resistance to the drug. The magnitude of the plateau varied depending on the virus isolate, as well as the cell type used, with considerable variation observed when primary human T cells from different human donors were used. Structure-function studies indicated that the V1/V2 region of the R5X4 HIV-1 isolate DH12 was necessary for AMD3100 resistance and could confer this property on two heterologous Env proteins. We conclude that some R5X4 and X4 HIV-1 isolates can utilize the AMD3100-bound conformation of CXCR4, with the efficiency being influenced by both viral and host factors. Baseline resistance to this CXCR4 antagonist could influence the clinical use of such compounds.

Summary Environmental pH can be an important cue for symbiotic bacteria as they colonize their eukaryotic hosts. Using the model mutualism between the marine bacterium Vibrio fischeri and the Hawaiian bobtail squid, we characterized the bacterial transcriptional response to acidic pH experienced during the shift from planktonic to host‐associated lifestyles. We found several genes involved in outer membrane structure were differentially expressed based on pH, indicating alterations in membrane physiology as V. fischeri initiates its symbiotic program. Exposure to host‐like pH increased the resistance of V. fischeri to the cationic antimicrobial peptide polymixin B, which resembles antibacterial molecules that are produced by the squid to select V. fischeri from the ocean microbiota. Using a forward genetic screen, we identified a homolog of eptA, a predicted phosphoethanolamine transferase, as critical for antimicrobial defense. We used MALDI‐MS to verify eptA as an ethanolamine transferase for the lipid‐A portion of V. fischeri lipopolysaccharide. We then used a DNA pulldown approach to discover that eptA transcription is activated by the global regulator H‐NS. Finally, we revealed that eptA promotes successful squid colonization by V. fischeri, supporting its potential role in initiation of this highly specific symbiosis.