Altered Innate Immunity and Damaged Epithelial Integrity in Vaginal Microbial Dysbiosis.

The role of neutrophils relative to vaginal dysbiosis is unclear. We hypothesize that bacterial vaginosis (BV)-associated bacteria may induce the activation and accumulation of mucosal neutrophils within the female reproductive tract (FRT), resulting in epithelial barrier damage. We collected endocervical cytobrushes from women with and without BV and assessed bacteria community type and frequency/functional phenotypes of neutrophils. We performed in vitro whole blood co-cultures with BV-associated bacteria and healthy vaginal commensals and assessed their impact on epithelial integrity using transepithelial electrical resistance. We demonstrated increased neutrophil frequency (p < 0.0001), activation (p < 0.0001), and prolonged lifespan (p < 0.0001) in the cytobrushes from women with non-Lactobacillus dominant (nLD) communities. Our in vitro co-cultures confirmed these results and identified significant barrier damage in the presence of neutrophils and G. vaginalis. Here, we demonstrate that BV-associated bacteria induce neutrophil activation and increase lifespan, potentially causing accumulation in the FRT and epithelial barrier damage.

Simian Immunodeficiency Virus Susceptibility, Immunology, and Microbiome in the Female Genital Tract of Adolescent Versus Adult Pigtail Macaques.

In Sub-Saharan Africa, young women 15-24 years of age account for nearly 30% of all new HIV infections, however, biological and epidemiological factors underlying this disproportionate infection rate are unclear. In this study, we assessed biological contributors of SIV/HIV susceptibility in the female genital tract (FGT) using adolescent (n = 9) and adult (n = 10) pigtail macaques (PTMs) with weekly low-dose intravaginal challenges of SIV. Immunological variables were captured in vaginal tissue of PTMs by flow cytometry and cytokine assays. Vaginal biopsies were profiled by proteomic analysis. The vaginal microbiome was assessed by 16S rRNA sequencing. We were powered to detect a 2.2-fold increase in infection rates between age groups, however, we identified no significant differences in susceptibility. This model cannot capture epidemiological factors or may not best represent biological differences of HIV susceptibility. No immune cell subsets measured were significantly different between groups. Inflammatory marker MCP-1 was significantly higher (adj p = .02), and sCD40L trended higher (adj p = .06) in vaginal cytobrushes of adults. Proteomic analysis of vaginal biopsies showed no significant (adj p < .05) protein or pathway differences between groups. Vaginal microbiomes were not significantly different between groups. No differences were observed between age groups in this PTM model, however, these animals may not reflect biological factors contributing to HIV risk such as those found in their human counterparts. This model is therefore not appropriate to explore human adolescent differences in HIV risk. Young women remain a key population at risk for HIV infection, and there is still a need for comprehensive assessment and intervention strategies for epidemic control of this uniquely vulnerable population.

Increased mucosal neutrophil survival is associated with altered microbiota in HIV infection.

Gastrointestinal (GI) mucosal dysfunction predicts and likely contributes to non-infectious comorbidities and mortality in HIV infection and persists despite antiretroviral therapy. However, the mechanisms underlying this dysfunction remain incompletely understood. Neutrophils are important for containment of pathogens but can also contribute to tissue damage due to their release of reactive oxygen species and other potentially harmful effector molecules. Here we used a flow cytometry approach to investigate increased neutrophil lifespan as a mechanism for GI neutrophil accumulation in chronic, treated HIV infection and a potential role for gastrointestinal dysbiosis. We report that increased neutrophil survival contributes to neutrophil accumulation in colorectal biopsy tissue, thus implicating neutrophil lifespan as a new therapeutic target for mucosal inflammation in HIV infection. Additionally, we characterized the intestinal microbiome of colorectal biopsies using 16S rRNA sequencing. We found that a reduced Lactobacillus: Prevotella ratio associated with neutrophil survival, suggesting that intestinal bacteria may contribute to GI neutrophil accumulation in treated HIV infection. Finally, we provide evidence that Lactobacillus species uniquely decrease neutrophil survival and neutrophil frequency in vitro, which could have important therapeutic implications for reducing neutrophil-driven inflammation in HIV and other chronic inflammatory conditions.

A closely-related clade of globally distributed bloom-forming cyanobacteria within the Nostocales.

In order to better understand the relationships among current Nostocales cyanobacterial blooms, eight genomes were sequenced from cultured isolates or from environmental metagenomes of recent planktonic Nostocales blooms. Phylogenomic analysis of publicly available sequences placed the new genomes among a group of 15 genomes from four continents in a distinct ADA clade (Anabaena/Dolichospermum/Aphanizomenon) within the Nostocales. This clade contains four species-level groups, two of which include members with both Anabaena-like and Aphanizomenon flos-aquae-like morphology. The genomes contain many repetitive genetic elements and a sizable pangenome, in which ABC-type transporters are highly represented. Alongside common core genes for photosynthesis, the differentiation of N2-fixing heterocysts, and the uptake and incorporation of the major nutrients P, N and S, we identified several gene pathways in the pangenome that may contribute to niche partitioning. Genes for problematic secondary metabolites-cyanotoxins and taste-and-odor compounds-were sporadically present, as were other polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters. By contrast, genes predicted to encode the ribosomally generated bacteriocin peptides were found in all genomes.

Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

Here we report three complete bacterial genome assemblies from a PacBio shotgun metagenome of a co-culture from Upper Klamath Lake, OR. Genome annotations and culture conditions indicate these bacteria are dependent on carbon and nitrogen fixation from the cyanobacterium Aphanizomenon flos-aquae, whose genome was assembled to draft-quality. Due to their taxonomic novelty relative to previously sequenced bacteria, we have temporarily designated these bacteria as incertae sedis Hyphomonadaceae strain UKL13-1 (3,501,508 bp and 56.12% GC), incertae sedis Betaproteobacterium strain UKL13-2 (3,387,087 bp and 54.98% GC), and incertae sedis Bacteroidetes strain UKL13-3 (3,236,529 bp and 37.33% GC). Each genome consists of a single circular chromosome with no identified plasmids. When compared with binned Illumina assemblies of the same three genomes, there was ~7% discrepancy in total genome length. Gaps where Illumina assemblies broke were often due to repetitive elements. Within these missing sequences were essential genes and genes associated with a variety of functional categories. Annotated gene content reveals that both Proteobacteria are aerobic anoxygenic phototrophs, with Betaproteobacterium UKL13-2 potentially capable of phototrophic oxidation of sulfur compounds. Both proteobacterial genomes contain transporters suggesting they are scavenging fixed nitrogen from A. flos-aquae in the form of ammonium. Bacteroidetes UKL13-3 has few completely annotated biosynthetic pathways, and has a comparatively higher proportion of unannotated genes. The genomes were detected in only a few other freshwater metagenomes, suggesting that these bacteria are not ubiquitous in freshwater systems. Our results indicate that long-read sequencing is a viable method for sequencing dominant members from low-diversity microbial communities, and should be considered for environmental metagenomics when conditions meet these requirements.