SUMMARY PARAGRAPH Lack of reproducibility is a prominent problem in biomedical research. An important source of variation in animal experiments is the microbiome, but little is known about specific changes in the microbiota composition that cause phenotypic differences. Here we show that genetically similar laboratory mice obtained from four different commercial vendors exhibited marked phenotypic variation in their susceptibility to Salmonella infection. Fecal microbiota transplantation into germ-free mice replicated donor susceptibility, revealing that variability was due to changes in the gut microbiota composition. Co-housing of mice only partially transferred protection against Salmonella infection, suggesting that minority species within the gut microbiota might confer this trait. Consistent with this idea, we identified endogenous Enterobacteriaceae, a low abundance taxon, as keystone species responsible for variation in the susceptibility to Salmonella infection. Protection conferred by endogenous Enterobacteriaceae could be modeled by inoculating mice with probiotic Escherichia coli, which conferred resistance by using its aerobic metabolism to compete with Salmonella for resources. We conclude that a mechanistic understanding of phenotypic variation can accelerate development of strategies for enhancing the reproducibility of animal experiments.

Microbial species often exist in complex communities where they must avoid predation and compete for favorable niches. The type VI secretion system (T6SS) is a contact-dependent bacterial weapon that allows for direct killing of competitors through the translocation of proteinaceous toxins. Vibrio cholerae is a Gram-negative pathogen that can use its T6SS during antagonistic interactions with neighboring prokaryotic and eukaryotic competitors. The T6SS not only promotes V. cholerae's survival during its aquatic and host life cycles, but also influences its evolution by facilitating horizontal gene transfer. This review details the recent insights regarding the structure and function of the T6SS as well as the diverse signals and regulatory pathways that control its activation in V. cholerae.

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SUMMARYA balanced gut microbiota contributes to health, but the mechanisms maintaining homeostasis remain elusive. Microbiota assembly during infancy is governed by competition between species and by environmental factors, termed habitat filters, that determine the range of successful traits within the microbial community. These habitat filters include the diet, host-derived resources, and microbiota-derived metabolites, such as short-chain fatty acids. Once the microbiota has matured, competition and habitat filtering prevent engraftment of new microbes, thereby providing protection against opportunistic infections. Competition with endogenous Enterobacterales, habitat filtering by short-chain fatty acids, and a host-derived habitat filter, epithelial hypoxia, also contribute to colonization resistance against Salmonella serovars. However, at a high challenge dose, these frank pathogens can overcome colonization resistance by using their virulence factors to trigger intestinal inflammation. In turn, inflammation increases the luminal availability of host-derived resources, such as oxygen, nitrate, tetrathionate, and lactate, thereby creating a state of abnormal habitat filtering that enables the pathogen to overcome growth inhibition by short-chain fatty acids. Thus, studying the process of ecosystem invasion by Salmonella serovars clarifies that colonization resistance can become weakened by disrupting host-mediated habitat filtering. This insight is relevant for understanding how inflammation triggers dysbiosis linked to noncommunicable diseases, conditions in which endogenous Enterobacterales expand in the fecal microbiota using some of the same growth-limiting resources required by Salmonella serovars for ecosystem invasion. In essence, ecosystem invasion by Salmonella serovars suggests that homeostasis and dysbiosis simply represent states where competition and habitat filtering are normal or abnormal, respectively.

Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity.

The clinical spectra of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) intersect to form a scantily defined overlap syndrome, termed pre-IBD. We show that increased Enterobacteriaceae and reduced Clostridia abundance distinguish the fecal microbiota of pre-IBD patients from IBS patients. A history of antibiotics in individuals consuming a high-fat diet was associated with the greatest risk for pre-IBD. Exposing mice to these risk factors resulted in conditions resembling pre-IBD and impaired mitochondrial bioenergetics in the colonic epithelium, which triggered dysbiosis. Restoring mitochondrial bioenergetics in the colonic epithelium with 5-amino salicylic acid, a PPAR-g (peroxisome proliferator-activated receptor gamma) agonist that stimulates mitochondrial activity, ameliorated pre-IBD symptoms. As with patients, mice with pre-IBD exhibited notable expansions of Enterobacteriaceae that exacerbated low-grade mucosal inflammation, suggesting that remediating dysbiosis can alleviate inflammation. Thus, environmental risk factors cooperate to impair epithelial mitochondrial bioenergetics, thereby triggering microbiota disruptions that exacerbate inflammation and distinguish pre-IBD from IBS.

The presence of the Lon protease in all three domains of life hints at its biological importance. The prokaryotic Lon protease is responsible not only for degrading abnormal proteins but also for carrying out the proteolytic regulation of specific protein targets. Posttranslational regulation by Lon is known to affect a variety of physiological traits in many bacteria, including biofilm formation, motility, and virulence. Here, we identify the regulatory roles of LonA in the human pathogen Vibrio cholerae. We determined that the absence of LonA adversely affects biofilm formation, increases swimming motility, and influences intracellular levels of cyclic diguanylate. Whole-genome expression analysis revealed that the message abundance of genes involved in biofilm formation was decreased but that the message abundances of those involved in virulence and the type VI secretion system were increased in a lonA mutant compared to the wild type. We further demonstrated that a lonA mutant displays an increase in type VI secretion system activity and is markedly defective in colonization of the infant mouse. These findings suggest that LonA plays a critical role in the environmental survival and virulence of V. cholerae. IMPORTANCEBacteria utilize intracellular proteases to degrade damaged proteins and adapt to changing environments. The Lon protease has been shown to be important for environmental adaptation and plays a crucial role in regulating the motility, biofilm formation, and virulence of numerous plant and animal pathogens. We find that LonA of the human pathogen V. cholerae is in line with this trend, as the deletion of LonA leads to hypermotility and defects in both biofilm formation and colonization of the infant mouse. In addition, we show that LonA regulates levels of cyclic diguanylate and the type VI secretion system. Our observations add to the known regulatory repertoire of the Lon protease and the current understanding of V. cholerae physiology.

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