BackgroundBacterial vaginosis (BV) is the most common vaginal disorder of reproductive-age women. Yet the cause of BV has not been established. To uncover key determinants of BV, we employed a multi-omic, systems-biology approach, including both deep 16S rRNA gene-based sequencing and metabolomics of lavage samples from 36 women. These women varied demographically, behaviorally, and in terms of health status and symptoms.Principal Findings16S rRNA gene-based community composition profiles reflected Nugent scores, but not Amsel criteria. In contrast, metabolomic profiles were markedly more concordant with Amsel criteria. Metabolomic profiles revealed two distinct symptomatic BV types (SBVI and SBVII) with similar characteristics that indicated disruption of epithelial integrity, but each type was correlated to the presence of different microbial taxa and metabolites, as well as to different host behaviors. The characteristic odor associated with BV was linked to increases in putrescine and cadaverine, which were both linked to Dialister spp. Additional correlations were seen with the presence of discharge, 2-methyl-2-hydroxybutanoic acid, and Mobiluncus spp., and with pain, diethylene glycol and Gardnerella spp.ConclusionsThe results not only provide useful diagnostic biomarkers, but also may ultimately provide much needed insight into the determinants of BV.

Bacterial communities colonizing the reproductive tracts of primates (including humans) impact the health, survival and fitness of the host, and thereby the evolution of the host species. Despite their importance, we currently have a poor understanding of primate microbiomes. The composition and structure of microbial communities vary considerably depending on the host and environmental factors. We conducted comparative analyses of the primate vaginal microbiome using pyrosequencing of the 16S rRNA genes of a phylogenetically broad range of primates to test for factors affecting the diversity of primate vaginal ecosystems. The nine primate species included: humans (Homo sapiens), yellow baboons (Papio cynocephalus), olive baboons (Papio anubis), lemurs (Propithecus diadema), howler monkeys (Alouatta pigra), red colobus (Piliocolobus rufomitratus), vervets (Chlorocebus aethiops), mangabeys (Cercocebus atys) and chimpanzees (Pan troglodytes). Our results indicated that all primates exhibited host-specific vaginal microbiota and that humans were distinct from other primates in both microbiome composition and diversity. In contrast to the gut microbiome, the vaginal microbiome showed limited congruence with host phylogeny, and neither captivity nor diet elicited substantial effects on the vaginal microbiomes of primates. Permutational multivariate analysis of variance and Wilcoxon tests revealed correlations among vaginal microbiota and host species-specific socioecological factors, particularly related to sexuality, including: female promiscuity, baculum length, gestation time, mating group size and neonatal birth weight. The proportion of unclassified taxa observed in nonhuman primate samples increased with phylogenetic distance from humans, indicative of the existence of previously unrecognized microbial taxa. These findings contribute to our understanding of host-microbe variation and coevolution, microbial biogeography, and disease risk, and have important implications for the use of animal models in studies of human sexual and reproductive diseases.

Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal microbial populations have not been investigated using molecular methods. Here, we evaluated site-specific microbial composition within the vaginal ecosystem and examined the influence of sampling technique in detection of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is not homogenous throughout the vaginal tract but differs significantly within an individual with regard to anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical predictor of vaginal health.The vaginal microbiota is important for maintaining vaginal health and preventing infections of the reproductive tract (10, 25, 34). However, studies using 16S rRNA gene clone libraries for identifying vaginal microbes have revealed considerably more diversity in the vaginal microbial communities of healthy premenopausal women than previously realized (14,17,31,33,35,36), thereby calling into question currently existing models for a healthy vaginal ecosystem and how it might be assessed. Although the vaginal microbiota in healthy individuals was traditionally thought to be dominated by Lactobacillus species, more recent studies have demonstrated that Lactobacillus is not the predominant bacterial genus within the vaginal tracts of a significant number of healthy women (1,17,31,35,36). Although 60 to 70% of the women had Lactobacillus-dominated vaginal microbiota, there were also individuals who lacked Lactobacillus altogether and instead had Gardnerella, Atopobium, Prevotella, Pseudomonas, or Streptococcus as the predominant bacteria in the vagina (1,17,31,35,36). The significant bacterial diversity observed among these individuals suggests that defining a healthy vaginal environment is more complex than originally thought.Although the most common method of collecting vaginal samples for clinical analysis is to swab the middle or deep vaginal canal (6,14,31,35), the extent to which this approach yields samples representative of the entire vaginal microbiota is unclear. Indeed, the size and anatomical complexity of the vaginal tract suggest the possibility that distinct microbial populations may reside at discrete sites (e.g., cervix, fornix, outer vaginal canal). However, within individuals, the presence or absence of vaginal microniches capable of supporting discrete microbial populations has not been evaluated. Differences in microbial population distributions within individuals may impede identification of the specific bac...

Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which while effective cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single domain VHH antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast displayed VHH with equilibrium dissociation constants (K D ) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 by BoNT/A Lc. The most potent VHH (Aa1) had a solution K D for BoNT/A Lc of 1.47 × 10 -10 M, an IC 50 of 4.7 × 10 -10 M, and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, the X-ray crystal structure of the BoNT/A Lc -Aa1 VHH complex was solved at 2.6 Å resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

The potent mitogenic toxin from Pasteurella multocida (PMT) is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH4Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn) and cholera toxin (CT), the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity.