Alterations in the gut microbiome implicate key taxa and metabolic pathways across inflammatory arthritis phenotypes.

Musculoskeletal diseases affect up to 20% of adults worldwide. The gut microbiome has been implicated in inflammatory conditions, but large-scale metagenomic evaluations have not yet traced the routes by which immunity in the gut affects inflammatory arthritis. To characterize the community structure and associated functional processes driving gut microbial involvement in arthritis, the Inflammatory Arthritis Microbiome Consortium investigated 440 stool shotgun metagenomes comprising 221 adults diagnosed with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and 219 healthy controls and individuals with joint pain without an underlying inflammatory cause. Diagnosis explained about 2% of gut taxonomic variability, which is comparable in magnitude to inflammatory bowel disease. We identified several candidate microbes with differential carriage patterns in patients with elevated blood markers for inflammation. Our results confirm and extend previous findings of increased carriage of typically oral and inflammatory taxa and decreased abundance and prevalence of typical gut clades, indicating that distal inflammatory conditions, as well as local conditions, correspond to alterations to the gut microbial composition. We identified several differentially encoded pathways in the gut microbiome of patients with inflammatory arthritis, including changes in vitamin B salvage and biosynthesis and enrichment of iron sequestration. Although several of these changes characteristic of inflammation could have causal roles, we hypothesize that they are mainly positive feedback responses to changes in host physiology and immune homeostasis. By connecting taxonomic alternations to functional alterations, this work expands our understanding of the shifts in the gut ecosystem that occur in response to systemic inflammation during arthritis.

Accurate identification and quantification of commensal microbiota bound by host immunoglobulins.

BACKGROUND: Identifying which taxa are targeted by immunoglobulins can uncover important host-microbe interactions. Immunoglobulin binding of commensal taxa can be assayed by sorting bound bacteria from samples and using amplicon sequencing to determine their taxonomy, a technique most widely applied to study Immunoglobulin A (IgA-Seq). Previous experiments have scored taxon binding in IgA-Seq datasets by comparing abundances in the IgA bound and unbound sorted fractions. However, as these are relative abundances, such scores are influenced by the levels of the other taxa present and represent an abstract combination of these effects. Diversity in the practical approaches of prior studies also warrants benchmarking of the individual stages involved. Here, we provide a detailed description of the design strategy for an optimised IgA-Seq protocol. Combined with a novel scoring method for IgA-Seq datasets that accounts for the aforementioned effects, this platform enables accurate identification and quantification of commensal gut microbiota targeted by host immunoglobulins. RESULTS: Using germ-free and Rag1-/- mice as negative controls, and a strain-specific IgA antibody as a positive control, we determine optimal reagents and fluorescence-activated cell sorting (FACS) parameters for IgA-Seq. Using simulated IgA-Seq data, we show that existing IgA-Seq scoring methods are influenced by pre-sort relative abundances. This has consequences for the interpretation of case-control studies where there are inherent differences in microbiota composition between groups. We show that these effects can be addressed using a novel scoring approach based on posterior probabilities. Finally, we demonstrate the utility of both the IgA-Seq protocol and probability-based scores by examining both novel and published data from in vivo disease models. CONCLUSIONS: We provide a detailed IgA-Seq protocol to accurately isolate IgA-bound taxa from intestinal samples. Using simulated and experimental data, we demonstrate novel probability-based scores that adjust for the compositional nature of relative abundance data to accurately quantify taxon-level IgA binding. All scoring approaches are made available in the IgAScores R package. These methods should improve the generation and interpretation of IgA-Seq datasets and could be applied to study other immunoglobulins and sample types. Video abstract.

Interrogating the recognition landscape of a conserved HIV-specific TCR reveals distinct bacterial peptide cross-reactivity.

T cell cross-reactivity ensures that diverse pathogen-derived epitopes encountered during a lifetime are recognized by the available TCR repertoire. A feature of cross-reactivity where previous exposure to one microbe can alter immunity to subsequent, non-related pathogens has been mainly explored for viruses. Yet cross-reactivity to additional microbes is important to consider, especially in HIV infection where gut-intestinal barrier dysfunction could facilitate T cell exposure to commensal/pathogenic microbes. Here we evaluated the cross-reactivity of a 'public', HIV-specific, CD8 T cell-derived TCR (AGA1 TCR) using MHC class I yeast display technology. Via screening of MHC-restricted libraries comprising ~2×108 sequence-diverse peptides, AGA1 TCR specificity was mapped to a central peptide di-motif. Using the top TCR-enriched library peptides to probe the non-redundant protein database, bacterial peptides that elicited functional responses by AGA1-expressing T cells were identified. The possibility that in context-specific settings, MHC class I proteins presenting microbial peptides influence virus-specific T cell populations in vivo is discussed.

Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut.

The mammalian gastrointestinal tract is colonized by a high-density polymicrobial community where bacteria compete for niches and resources. One key competition strategy includes cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), a multiprotein needle-like apparatus that injects effector proteins into prokaryotic and/or eukaryotic target cells. However, the contribution of T6SS antibacterial activity during pathogen invasion of the gut has not been demonstrated. We report that successful establishment in the gut by the enteropathogenic bacterium Salmonella enterica serovar Typhimurium requires a T6SS encoded within Salmonella pathogenicity island-6 (SPI-6). In an in vitro setting, we demonstrate that bile salts increase SPI-6 antibacterial activity and that S Typhimurium kills commensal bacteria in a T6SS-dependent manner. Furthermore, we provide evidence that one of the two T6SS nanotube subunits, Hcp1, is required for killing Klebsiella oxytoca in vitro and that this activity is mediated by the specific interaction of Hcp1 with the antibacterial amidase Tae4. Finally, we show that K. oxytoca is killed in the host gut in an Hcp1-dependent manner and that the T6SS antibacterial activity is essential for Salmonella to establish infection within the host gut. Our findings provide an example of pathogen T6SS-dependent killing of commensal bacteria as a mechanism to successfully colonize the host gut.

Intraspecies competition for niches in the distal gut dictate transmission during persistent Salmonella infection.

In order to be transmitted, a pathogen must first successfully colonize and multiply within a host. Ecological principles can be applied to study host-pathogen interactions to predict transmission dynamics. Little is known about the population biology of Salmonella during persistent infection. To define Salmonella enterica serovar Typhimurium population structure in this context, 129SvJ mice were oral gavaged with a mixture of eight wild-type isogenic tagged Salmonella (WITS) strains. Distinct subpopulations arose within intestinal and systemic tissues after 35 days, and clonal expansion of the cecal and colonic subpopulation was responsible for increases in Salmonella fecal shedding. A co-infection system utilizing differentially marked isogenic strains was developed in which each mouse received one strain orally and the other systemically by intraperitoneal (IP) injection. Co-infections demonstrated that the intestinal subpopulation exerted intraspecies priority effects by excluding systemic S. Typhimurium from colonizing an extracellular niche within the cecum and colon. Importantly, the systemic strain was excluded from these distal gut sites and was not transmitted to naïve hosts. In addition, S. Typhimurium required hydrogenase, an enzyme that mediates acquisition of hydrogen from the gut microbiota, during the first week of infection to exert priority effects in the gut. Thus, early inhibitory priority effects are facilitated by the acquisition of nutrients, which allow S. Typhimurium to successfully compete for a nutritional niche in the distal gut. We also show that intraspecies colonization resistance is maintained by Salmonella Pathogenicity Islands SPI1 and SPI2 during persistent distal gut infection. Thus, important virulence effectors not only modulate interactions with host cells, but are crucial for Salmonella colonization of an extracellular intestinal niche and thereby also shape intraspecies dynamics. We conclude that priority effects and intraspecies competition for colonization niches in the distal gut control Salmonella population assembly and transmission.

Biogeographic patterns in genomic diversity among a large collection of Vibrio cholerae isolates.

Vibrio cholerae strains are capable of inhabiting multiple niches in the aquatic environment and in some cases cause disease in humans. However, the ecology and biodiversity of these bacteria in environmental settings remains poorly understood. We used the genomic fingerprinting technique enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) to profile 835 environmental isolates from waters and sediments obtained at nine sites along the central California coast. We identified 115 ERIC-PCR genotypes from 998 fingerprints, with a reproducibility of 98.5% and a discriminatory power of 0.971. When the temporal dynamics at a subset of sampling sites were explored, several genotypes provided evidence for cosmopolitan or geographically restricted distributions, and other genotypes displayed nonrandom patterns of cooccurrence. Partial Mantel tests confirmed that genotypic similarity of isolates across all sampling events was correlated with environmental similarity (0.04 < or = r < or = 0.05), temporal proximity (r = 0.09), and geographic distance (r = 0.09). A neutral community model for all sampling events explained 61% of the variation in genotype abundance. Cooccurrence indices (C-score, C-board, and Combo) were significantly different than expected by chance, suggesting that the V. cholerae population may have a competitive structure, especially at the regional scale. Even though stochastic processes are undoubtedly important in generating biogeographic patterns in diversity, deterministic factors appear to play a significant, albeit small, role in shaping the V. cholerae population structure in this system.