Vaginal ecosystem modeling of growth patterns of anaerobic bacteria in microaerophilic conditions.

The human vagina constitutes a complex ecosystem created through relationships established between host mucosa and bacterial communities. In this ecosystem, classically defined bacterial aerobes and anaerobes thrive as communities in the microaerophilic environment. Levels of CO2 and O2 present in the vaginal lumen are impacted by both the ecosystem's physiology and the behavior and health of the human host. Study of such complex relationships requires controlled and reproducible causational approaches that are not possible in the human host that, until recently, was the only place these bacterial communities thrived. To address this need we have utilized our ex vivo human vaginal mucosa culture system to support controlled, reproducible colonization by vaginal bacterial communities (VBC) collected from healthy, asymptomatic donors. Parallel vaginal epithelial cells (VEC)-VBC co-cultures were exposed to two different atmospheric conditions to study the impact of CO2 concentrations upon the anaerobic bacteria associated with dysbiosis and inflammation. Our data suggest that in the context of transplanted VBC, increased CO2 favored specific lactobacilli species defined as microaerophiles when grown as monocultures. In preliminary studies, the observed community changes also led to shifts in host VEC phenotypes with significant changes in the host transcriptome, including altered expression of select molecular transporter genes. These findings support the need for additional study of the environmental changes associated with behavior and health upon the symbiotic and adversarial relationships that are formed in microbial communities present in the human vaginal ecosystem.

The altered TBI fecal microbiome is stable and functionally distinct.

Introduction: Patients who suffer a traumatic brain injury (TBI) often experience chronic and sometimes debilitating sequelae. Recent reports have illustrated both acute and long-term dysbiosis of the gastrointestinal microbiome with significant alterations in composition and predicted functional consequences. Methods: Working with participants from past research, metagenomic stability of the TBI- associated fecal microbiome (FMB) was evaluated by custom qPCR array comparing a fecal sample from 2015 to one collected in 2020. Metatranscriptomics identified differently expressed bacterial genes and biochemical pathways in the TBI FMB. Microbiota that contributed the largest RNA amounts identified a set of core bacteria most responsible for functional consequences of the TBI FMB. Results: A remarkably stable FMB metagenome with significant similarity (two-tail Spearman nonparametric correlation p < 0.001) was observed between 2015 and 2020 fecal samples from subjects with TBI. Comparing the 2020 TBI FMB metagenome to FMBs from healthy controls confirmed and extended the dysbiotic genera and species. Abundance differences between average TBI and healthy FMBs revealed Bacteroides caccae, B. uniformis, Blautia spp., Collinsella spp., Dialister spp., and Ordoribacter spp. were significantly different. Functionally, the Parabacteroides genus contributed the highest percentage of RNA sequences in control FMBs followed by the Bacteroides genus as the second highest contributor. In the TBI FMB, the Corynebacterium genus contributed the most RNA followed by the Alistipes genus. Corynebacterium and Pseudomonas were distinct in the top 10 contributing genera in the TBI FMB while Parabacteroides and Ruminococcus were unique to the top 10 in controls. Comparing RNA profiles, TBI samples had ∼1.5 fold more expressed genes with almost 700 differently expressed genes (DEGs) mapped to over 100 bacterial species. Bioinformatic analysis associated DEGs with pathways led identifying 311 functions in the average TBI FMB profile and 264 in the controls. By average profile comparison, 30 pathways had significantly different abundance (p < 0.05, t-test) or were detected in >80% of the samples in only one of the cohorts (binary distinction). Discussion: Functional differences between TBI and healthy control FMBs included amino acid metabolism, energy and carbon source usage, fatty acid metabolism, bacterial cell wall component production and nucleic acid synthesis and processing pathways. Together these data shed light on the functional consequences of the dysbiotic TBI FMB decades after injury.

Development of a Novel ex vivo Nasal Epithelial Cell Model Supporting Colonization With Human Nasal Microbiota.

The nasal mucosa provides first line defense against inhaled pathogens while creating a unique microenvironment for bacterial communities. Studying the impact of microbiota in the nasal cavity has been difficult due to limitations with current models including explant cultures, primary cells, or neoplastic cell lines. Most notably, none have been shown to support reproducible colonization by bacterial communities from human donors. Therefore, to conduct controlled studies of the human nasal ecosystem, we have developed a novel ex vivo mucosal model that supports bacterial colonization of a cultured host mucosa created by immortalized human nasal epithelial cells (NEC). For this model, immortalized NEC established from 5 male and 5 female donors were cultured with an air-interfaced, apical surface on a porous transwell membrane. NEC were grown from nasal turbinate tissues harvested from willed bodies or from discarded tissue collected during sinonasal procedures. Immortalized cells were evaluated through molecular verification of cell type, histological confirmation of tissue differentiation including formation of tight junctions, NEC multilayer viability, metabolism, physiology and imaging of the luminal surface by scanning electron microscopy. Results showed proper differentiation and multilayer formation at seven to 10 days after air interface that was maintained for up to 3 weeks. The optimized mucosal cultures created an environment necessary to sustain colonization by nasal microbiomes (NMBs) that were collected from healthy volunteers, cryogenically preserved and characterized with customized quantitative polymerase chain reaction (qPCR) arrays. Polymicrobial communities of nasal bacteria associated with healthy and inflamed states were consistently reproduced in matured NEC co-cultures by transplant of NMBs from multiple community types. The cultured NMBs were stable after an initial period of bacterial replication and equilibration. This novel ex vivo culture system is the first model that supports controlled cultivation of NMBs, allowing for lab-based causation studies and further experimentation to explore the complexities of host-microbe and microbe-microbe interactions.

Cultivated Human Vaginal Microbiome Communities Impact Zika and Herpes Simplex Virus Replication in ex vivo Vaginal Mucosal Cultures.

The human vaginal microbiome (VMB) is a complex bacterial community that interacts closely with vaginal epithelial cells (VECs) impacting the mucosal phenotype and its responses to pathogenic insults. The VMB and VEC relationship includes nutrient exchange and regulation of signaling molecules that controls numerous host functions and defends against invading pathogens. To better understand infection and replication of sexually transmitted viral pathogens in the human vaginal mucosa we used our ex vivo VEC multilayer culture system. We tested the hypothesis that selected VMB communities could be identified that alter the replication of sexually transmitted viruses consistent with reported clinical associations. Sterile VEC multilayer cultures or those colonized with VMB dominated by specific Lactobacillus spp., or VMB lacking lactobacilli, were infected with Zika virus, (ZIKV) a single stranded RNA virus, or Herpes Simplex Virus type 2 (HSV-2), a double stranded DNA virus. The virus was added to the apical surface of the cultured VEC multilayer to model transmission during vaginal intercourse. Viral replication was measured 48 h later by qPCR. The results indicated that VEC cultures colonized by VMB containing Staphylococcus spp., previously reported as inflammatory, significantly reduced the quantity of viral genomes produced by ZIKV. HSV-2 titers were decreased by nearly every VMB tested relative to the sterile control, although Lactobacillus spp.-dominated VMBs caused the greatest reduction in HSV-2 titer consistent with clinical observations. To explore the mechanism for reduced ZIKV titers, we investigated inflammation created by ZIKV infection, VMB colonization or pre-exposure to selected TLR agonists. Finally, expression levels of human beta defensins 1-3 were quantified in cultures infected by ZIKV and those colonized by VMBs that impacted ZIKV titers. Human beta defensins 1-3 produced by the VEC showed no association with ZIKV titers. The data presented expands the utility of this ex vivo model system providing controlled and reproducible methods to study the VMB impact on STIs and indicated an association between viral replication and specific bacterial species within the VMB.

Early structural rearrangements in the photocycle of an integral membrane sensory receptor.

Sensory rhodopsins are the primary receptors of vision in animals and phototaxis in microorganisms. Light triggers the rapid isomerization of a buried retinal chromophore, which the protein both accommodates and amplifies into the larger structural rearrangements required for signaling. We trapped an early intermediate of the photocycle of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) in 3D crystals and determined its X-ray structure to 2.3 A resolution. The observed structural rearrangements were localized near the retinal chromophore, with a key water molecule becoming disordered and the retinal's beta-ionone ring undergoing a prominent movement. Comparison with the early structural rearrangements of bacteriorhodopsin illustrates how modifications in the retinal binding pocket of pSRII allow subtle differences in the early relaxation of photoisomerized retinal.