Phenylacetic acid, an anti-vaginitis metabolite produced by the vaginal symbiotic bacterium Chryseobacterium gleum.

The human microbiome contains genetic information that regulates metabolic processes in response to host health and disease. While acidic vaginal pH is maintained in normal conditions, the pH level increases in infectious vaginitis. We propose that this change in the vaginal environment triggers the biosynthesis of anti-vaginitis metabolites. Gene expression levels of Chryseobacterium gleum, a vaginal symbiotic bacterium, were found to be affected by pH changes. The distinctive difference in the metabolic profiles between two C. gleum cultures incubated under acidic and neutral pH conditions was suggested to be an anti-vaginitis molecule, which was identified as phenylacetic acid (PAA) by spectroscopic data analysis. The antimicrobial activity of PAA was evaluated in vitro, showing greater toxicity toward Gardnerella vaginalis and Candida albicans, two major vaginal pathogens, relative to commensal Lactobacillus spp. The activation of myeloperoxidase, prostaglandin E2, and nuclear factor-κB, and the expression of cyclooxygenase-2 were reduced by an intravaginal administration of PAA in the vaginitis mouse model. In addition, PAA displayed the downregulation of mast cell activation. Therefore, PAA was suggested to be a messenger molecule that mediates interactions between the human microbiome and vaginal health.

Resident microbes shape the vaginal epithelial glycan landscape.

Epithelial cells are covered in carbohydrates (glycans). This glycan coat or "glycocalyx" interfaces directly with microbes, providing a protective barrier against potential pathogens. Bacterial vaginosis (BV) is a condition associated with adverse health outcomes in which bacteria reside in direct proximity to the vaginal epithelium. Some of these bacteria, including Gardnerella, produce glycosyl hydrolase enzymes. However, glycans of the human vaginal epithelial surface have not been studied in detail. Here, we elucidate key characteristics of the "normal" vaginal epithelial glycan landscape and analyze the impact of resident microbes on the surface glycocalyx. In human BV, glycocalyx staining was visibly diminished in electron micrographs compared to controls. Biochemical and mass spectrometric analysis showed that, compared to normal vaginal epithelial cells, BV cells were depleted of sialylated N- and O-glycans, with underlying galactose residues exposed on the surface. Treatment of primary epithelial cells from BV-negative women with recombinant Gardnerella sialidases generated BV-like glycan phenotypes. Exposure of cultured VK2 vaginal epithelial cells to recombinant Gardnerella sialidase led to desialylation of glycans and induction of pathways regulating cell death, differentiation, and inflammatory responses. These data provide evidence that vaginal epithelial cells exhibit an altered glycan landscape in BV and suggest that BV-associated glycosidic enzymes may lead to changes in epithelial gene transcription that promote cell turnover and regulate responses toward the resident microbiome.

Rapid Point-of-Care Test Kit for Bacterial Vaginosis: Detection of Vaginolysin and Clue Cells Using Paper Strips and a Smartphone.

There is an unmet need for a point-of-care test that is accurate, affordable, and simple to diagnose bacterial vaginosis, the most common cause of vaginal symptoms among women. Bacterial vaginosis leaves patients with undesirable vaginal discharge, malodor, and discomfort. Currently, the diagnosis of bacterial vaginosis is inaccurate and complex, leading to high rates of misdiagnosis. Inaccurate diagnoses are unsafe as bacterial vaginosis increases the risks of acquiring sexually transmitted infections as well as the likelihood of miscarriages. To date, the most commonly identified bacteria associated with bacterial vaginosis is Gardnerella vaginalis. We developed a method for the expression, purification, and detection of vaginolysin, the most well-characterized virulence factor of G. vaginalis. Elevated levels of G. vaginalis have been shown to lead to a toxic vaginal environment, facilitating bacterial vaginosis. We have developed an enzyme-linked immunosorbent assay for the detection of vaginolysin, which was translated to a lateral flow assay for use in a rapid, straightforward, cost-effective paper-based diagnostic test for vaginolysin that does not require the use of instrumentation. In conjunction, we have employed a commercially available smartphone microscopy kit to visualize clue cells without the need for equipment or electricity. The combination of these methodologies allows for an accurate and easy approach to diagnose bacterial vaginosis with minimal resources for use in any setting.

Gardnerella vaginalis infection in pregnancy: Effects on placental development and neonatal outcomes.

INTRODUCTION: Gardnerella vaginalis (GV)-associated bacterial vaginosis is recognised for its detrimental effects on pregnancy resulting in poor obstetric and neonatal outcomes. There is limited knowledge of the effects on placental histomorphology following GV infection in pregnancy. We investigated the effects of GV infection on the placenta, particularly with regards to the syncytiotrophoblasts and vascular development, and related these to neonatal outcomes. METHODS: A prospective cohort study involving GV-positive pregnant women presented with abnormal vaginal discharge, with gestational age-matched healthy pregnant women controls. Placental sampling was performed upon delivery and examined histologically. Vascular endothelial growth factor-A (VEGF-A) and hypoxia-inducible factor-1α (HIF-1α) mRNA and protein expression were analysed by real-time PCR and immunohistochemistry respectively. The standard measures in neonatal outcomes were recorded. RESULTS: Placentas from GV-positive mothers were found to have significant histological evidence of maternal and/or fetal inflammatory response compared with the controls (17/28: 60.7% vs 2/20: 10%) (p = 0.0011). There was an increase in the percentage of syncytial nuclear aggregates (SNAs) per villus (47.4 ± 11.09%) in placentas from GV-positive mothers (p < 0.0001). VEGF-A was significantly increased in specifically, the villous endothelial cells of placentas with GV infection, but no difference in the immunoexpression of HIF-1α in these cells between groups. However, these were not associated with adverse neonatal outcomes. DISCUSSION: Increased placental VEGF-A expression associated with increased SNAs in pregnant women with GV infection of the genital tract may be an intrauterine response towards placental vascular remodeling, that may also serve as a protective role in moderating birth outcomes.

Vaginal sialoglycan foraging by Gardnerella vaginalis: mucus barriers as a meal for unwelcome guests?

Bacterial vaginosis (BV) is a condition of the vaginal microbiome in which there are few lactobacilli and abundant anaerobic bacteria. Members of the genus Gardnerella are often one of the most abundant bacteria in BV. BV is associated with a wide variety of poor health outcomes for women. It has been recognized since the 1980s that women with BV have detectable and sometimes markedly elevated levels of sialidase activity in vaginal fluids and that bacteria associated with this condition produce this activity in culture. Mounting evidence collected using diverse methodologies points to the conclusion that BV is associated with a reduction in intact sialoglycans in cervicovaginal secretions. Here we review evidence for the contributions of vaginal bacteria, especially Gardnerella, in the processes of mucosal sialoglycan degradation, uptake, metabolism and depletion. Our understanding of the impacts of vaginal sialoglycan degradation is still limited. However, the potential implications of sialic acid depletion are discussed in light of our current understanding of the roles played by sialoglycans in vaginal physiology.

Glycan cross-feeding supports mutualism between Fusobacterium and the vaginal microbiota.

Women with bacterial vaginosis (BV), an imbalance of the vaginal microbiome, are more likely to be colonized by potential pathogens such as Fusobacterium nucleatum, a bacterium linked with intrauterine infection and preterm birth. However, the conditions and mechanisms supporting pathogen colonization during vaginal dysbiosis remain obscure. We demonstrate that sialidase activity, a diagnostic feature of BV, promoted F. nucleatum foraging and growth on mammalian sialoglycans, a nutrient resource that was otherwise inaccessible because of the lack of endogenous F. nucleatum sialidase. In mice with sialidase-producing vaginal microbiotas, mutant F. nucleatum unable to consume sialic acids was impaired in vaginal colonization. These experiments in mice also led to the discovery that F. nucleatum may also "give back" to the community by reinforcing sialidase activity, a biochemical feature of human dysbiosis. Using human vaginal bacterial communities, we show that F. nucleatum supported robust outgrowth of Gardnerella vaginalis, a major sialidase producer and one of the most abundant organisms in BV. These results illustrate that mutually beneficial relationships between vaginal bacteria support pathogen colonization and may help maintain features of dysbiosis. These findings challenge the simplistic dogma that the mere absence of "healthy" lactobacilli is the sole mechanism that creates a permissive environment for pathogens during vaginal dysbiosis. Given the ubiquity of F. nucleatum in the human mouth, these studies also suggest a possible mechanism underlying links between vaginal dysbiosis and oral sex.

Inerolysin and vaginolysin, the cytolysins implicated in vaginal dysbiosis, differently impair molecular integrity of phospholipid membranes.

The pore-forming toxins, inerolysin (INY) and vaginolysin (VLY), produced by vaginal bacteria Lactobacillus iners and Gardnerella vaginalis were studied using the artificial cholesterol-rich tethered bilayer membranes (tBLMs) by electrochemical techniques. The electrochemical impedance spectroscopy (EIS) of tBLMs attested for the toxin-induced impairment of the integrity of phospholipid membranes. This observation was in line with the atomic force microscopy data demonstrating formation of oligomeric protein assemblies in tBLMs. These assemblies exhibited different morphologies: VLY mostly formed complete rings, whereas INY produced arciform structures. We found that both EIS (membrane damage) and the surface plasmon resonance (protein binding) data obtained on tBLMs are in-line with the data obtained in human cell lysis experiments. EIS, however, is capable of capturing effects inaccessible for biological activity assays. Specifically, we found that the INY-induced damage of tBLMs is nearly a linear function of membrane cholesterol content, whereas VLY triggered significant damage only at high (50 mol%) cholesterol concentrations. The observed differences of INY and VLY activities on phospholipid membranes might have clinical importance: both toxin-producing bacteria have been found in healthy vagina and dysbiosis, suggesting the need for adaptation at different vaginal conditions. Our results broaden the possibilities of application of tBLMs in medical diagnostics.

Interaction of Gardnerella vaginalis and Vaginolysin with the Apical versus Basolateral Face of a Three-Dimensional Model of Vaginal Epithelium.

Studies have implicated Gardnerella vaginalis as an important etiological agent in bacterial vaginosis (BV). It produces a cholesterol-dependent cytolysin, vaginolysin (VLY). In this study, we sought to characterize the interaction between vaginal epithelium, G. vaginalis, and VLY using EpiVaginal tissues from MatTek. These tissues are three-dimensional and have distinct apical and basolateral sides, enabling comparison of the effects of G. vaginalis and VLY following exposure to either side. We measured cytotoxicity, cytokine production, and bacterial growth, following apical versus basolateral exposure. G. vaginalis exhibited more-rapid growth in coculture with the tissue model when it was exposed to the apical side. VLY permeabilized cells on the basolateral side of the tissues but failed to permeabilize apical epithelial cells. Cytokine secretion in response to VLY and G. vaginalis also depended on the polarity of exposure. VLY did not cause significant changes in cytokine levels when exposed apically. Apical tissue challenge by G. vaginalis appeared to dampen the inflammatory response, as decreases in granulocyte-macrophage colony-stimulating factor (GM-CSF) (6.6-fold), RANTES (14.8-fold), and interferon gamma inducible protein 10 kDa (IP-10) (53-fold) and an increase in interleukin-1 receptor antagonist (IL-1ra) (5-fold) were observed. In vivo, G. vaginalis normally colonizes the apical face of the vaginal epithelium. Results from this study suggest that while G. vaginalis may grow on the apical face of the vaginal epithelium, its VLY toxin does not target these cells in this model. This phenomenon could have important implications regarding colonization of the vagina by G. vaginalis and may suggest an explanation for the lack of an overt immune response to this organism.

A Lactobacillus-Deficient Vaginal Microbiota Dominates Postpartum Women in Rural Malawi.

The bacterial community found in the vagina is an important determinant of a woman's health and disease status. A healthy vaginal microbiota is associated with low species richness and a high proportion of one of a number of different Lactobacillus spp. When disrupted, the resulting abnormal vaginal microbiota is associated with a number of disease states and poor pregnancy outcomes. Studies up until now have concentrated on relatively small numbers of American and European populations that may not capture the full complexity of the community or adequately predict what constitutes a healthy microbiota in all populations. In this study, we sampled and characterized the vaginal microbiota found on vaginal swabs taken postpartum from a cohort of 1,107 women in rural Malawi. We found a population dominated by Gardnerella vaginalis and devoid of the most common vaginal Lactobacillus species, even if the vagina was sampled over a year postpartum. This Lactobacillus-deficient anaerobic community, commonly labeled community state type (CST) 4, could be subdivided into four further communities. A Lactobacillus iners-dominated vaginal microbiota became more common the longer after delivery the vagina was sampled, but G. vaginalis remained the dominant organism. These results outline the difficulty in all-encompassing definitions of what a healthy or abnormal postpartum vaginal microbiota is. Previous identification of community state types and associations among bacterial species, bacterial vaginosis, and adverse birth outcomes may not represent the complex heterogeneity of the microbiota present. (This study has been registered at ClinicalTrials.gov as NCT01239693.)IMPORTANCE A bacterial community in the vaginal tract is dominated by a small number of Lactobacillus species, and when not present there is an increased incidence of inflammatory conditions and adverse birth outcomes. A switch to a vaginal bacterial community lacking in Lactobacillus species is common after pregnancy. In this study, we characterized the postpartum vaginal bacterial community of a large group of women from a resource-poor, undersampled population in rural Malawi. The majority of women were found to have a Lactobacillus-deficient community, and even when sampled a year after delivery the majority of women still did not have Lactobacillus present in their vaginal microbiota. The effect of becoming pregnant again for those who do not revert to a Lactobacillus-dominant community is unknown, and this could suggest that not all Lactobacillus-deficient community structures are adverse. A better understanding of this complex community state type is needed.

The Effect of Lysozyme on Reducing Biofilms by Staphylococcus aureus, Pseudomonas aeruginosa, and Gardnerella vaginalis: An In Vitro Examination.

Two basic questions about lysozyme activities on the selected microorganisms were investigated, namely whether lysozyme inhibits biofilm production and which concentrations of the enzyme have the ability to change the natural biofilm producing capacity of different strains of Staphylococcus aureus (methicillin sensitive and resistant), Streptococcus pyogenes, Pseudomonas aeruginosa, and Gardnerella vaginalis. The effect of lysozyme on biofilm formation capacities of 16 strains of selected microorganisms was investigated, whereby four testing replicates have been performed in vitro using the Test Tube method, and the potential of lysozyme to change biofilm forming capacities depending on its concentration, species, and strains of microorganisms is demonstrated. A lysozyme concentration of 30 µg/ml indicated to have the highest inhibiting effect on all tested microorganisms. Furthermore, G. vaginalis was the most sensitive of them all, as its biofilm formation was inhibited in the presence of as low as 2.5 µg/ml of lysozyme. At enzyme concentrations of 7.5-50 µg/ml (with the exception of 30 µg/ml) the biofilm forming capacities of P. aeruginosa were enhanced. Depending on the strain of P. aeruginosa, the total biofilm quantity was either reduced or unaffected at lysozyme concentrations of 2.5, 5, 7.5, and 30 µg/ml. In contrast, lysozyme concentrations below 15 or 20 µg/ml did not affect or increase the volume of biofilm formation, while higher concentrations (15, 20, 25 µg/ml) reduced biofilm formation by 50% (3/6) and 30 µg/ml of biofilm reduced biofilm forming capacity of S. aureus by 100% (6/6). The results of this study are a strong foundation for further research on lysozyme as a modulator of the biofilm forming capacity of different species with the potential to aid in the development of new drugs for the treatment of oral and vaginal infections.

The microbiome and HIV prevention strategies in women.

PURPOSE OF REVIEW: HIV prevention approaches that women can use and control are a priority. Results from topical and oral preexposure prophylaxis (PrEP) HIV prevention trials have produced inconsistent results in women. One of the main behavioural factors impacting effectiveness of PrEP has been suboptimal adherence. In this review, we examine biological factors that modulate topical PrEP efficacy, with particular focus on the vaginal microbiome. RECENT FINDINGS: Genital inflammation is an independent risk factor for HIV acquisition in women. Using 16S rRNA sequencing of the vaginal microbiota, anaerobic bacteria linked with bacterial vaginosis have been shown to be associated with both genital inflammation and HIV risk. Using proteomics, it was recently discovered that a dysbiotic vaginal microbiome, comprising less than 50% Lactobacillus spp., directly influenced topical PrEP efficacy. Gardnerella vaginalis, the dominant vaginal species in dysbiotic women, was able to directly degrade tenofovir, but not dapivirine, an antiretroviral also being developed for topical PrEP. SUMMARY: The link between bacterial vaginosis-associated organisms with HIV risk and altered tenofovir gel effectiveness underscores the importance of good vaginal health and good adherence for women to benefit maximally from topical PrEP. Altering the vaginal microbiome is one of the new directions being pursued for HIV prevention.

Association between statin use, the vaginal microbiome, and Gardnerella vaginalis vaginolysin-mediated cytotoxicity.

BACKGROUND: Bacterial vaginosis (BV) is the leading dysbiosis of the vaginal microbiome. The pathways leading towards the development of BV are not well understood. Gardnerella vaginalis is frequently associated with BV. G. vaginalis produces the cholesterol-dependent cytolysin (CDC), vaginolysin, which can lyse a variety of human cells and is thought to play a role in pathogenesis. Because membrane cholesterol is required for vaginolysin to function, and because HMG-CoA reductase inhibitors (statins) affect not only serum levels of cholesterol but membrane levels as well, we hypothesized that statins might affect the vaginal microbiome. METHODS: To investigate the relationship between use of the statins and the vaginal microbiome, we analyzed 16S rRNA gene taxonomic surveys performed on vaginal samples from 133 women who participated in the Vaginal Human Microbiome Project and who were taking statins at the time of sampling, 152 women who reported high cholesterol levels but were not taking statins, and 316 women who did not report high cholesterol. To examine the effect of statins on the cytolytic effect of vaginolysin, the cholesterol-dependent cytolysin (CDC) produced by Gardnerella vaginalis, we assessed the effect of simvastatin pretreatment of VK2E6/E7 vaginal epithelial cells on vaginolysin-mediated cytotoxicity. RESULTS: The mean proportion of G. vaginalis among women taking statins was significantly lower relative to women not using statins. Women using statins had higher mean proportions of Lactobacillus crispatus relative to women with normal cholesterol levels, and higher levels of Lactobacillus jensenii relative to women with high cholesterol but not taking statins. In vitro, vaginal epithelial cells pretreated with simvastatin were relatively resistant to vaginolysin and this effect was inhibited by cholesterol. CONCLUSIONS: In this cross-sectional study, statin use was associated with reduced proportions of G. vaginalis and greater proportions of beneficial lactobacilli within the vaginal microbiome. The negative association between statin use and G. vaginalis may be related to inhibition of vaginolysin function.

A multi-platform metabolomics approach identifies highly specific biomarkers of bacterial diversity in the vagina of pregnant and non-pregnant women.

Bacterial vaginosis (BV) increases transmission of HIV, enhances the risk of preterm labour, and is associated with malodour. Clinical diagnosis often relies on microscopy, which may not reflect the microbiota composition accurately. We use an untargeted metabolomics approach, whereby we normalize the weight of samples prior to analysis, to obtained precise measurements of metabolites in vaginal fluid. We identify biomarkers for BV with high sensitivity and specificity (AUC = 0.99) in a cohort of 131 pregnant and non-pregnant Rwandan women, and demonstrate that the vaginal metabolome is strongly associated with bacterial diversity. Metabolites associated with high diversity and clinical BV include 2-hydroxyisovalerate and γ-hydroxybutyrate (GHB), but not succinate, which is produced by both Lactobacillus crispatus and BV-associated anaerobes in vitro. Biomarkers associated with high diversity and clinical BV are independent of pregnancy status, and were validated in a blinded replication cohort from Tanzania (n = 45), where we predicted clinical BV with 91% accuracy. Correlations between the metabolome and microbiota identified Gardnerella vaginalis as a putative producer of GHB, and we demonstrate production by this species in vitro. This work illustrates how changes in community structure alter the chemical composition of the vagina, and identifies highly specific biomarkers for a common condition.

Vaginolysin drives epithelial ultrastructural responses to Gardnerella vaginalis.

Gardnerella vaginalis, the bacterial species most frequently isolated from women with bacterial vaginosis (BV), produces a cholesterol-dependent cytolysin (CDC), vaginolysin (VLY). At sublytic concentrations, CDCs may initiate complex signaling cascades crucial to target cell survival. Using live-cell imaging, we observed the rapid formation of large membrane blebs in human vaginal and cervical epithelial cells (VK2 and HeLa cells) exposed to recombinant VLY toxin and to cell-free supernatants from growing liquid cultures of G. vaginalis. Binding of VLY to its human-specific receptor (hCD59) is required for bleb formation, as antibody inhibition of either toxin or hCD59 abrogates this response, and transfection of nonhuman cells (CHO-K1) with hCD59 renders them susceptible to toxin-induced membrane blebbing. Disruption of the pore formation process (by exposure to pore-deficient toxoids or pretreatment of cells with methyl-ß-cyclodextrin) or osmotic protection of target cells inhibits VLY-induced membrane blebbing. These results indicate that the formation of functional pores drives the observed ultrastructural rearrangements. Rapid bleb formation may represent a conserved response of epithelial cells to sublytic quantities of pore-forming toxins, and VLY-induced epithelial cell membrane blebbing in the vaginal mucosa may play a role in the pathogenesis of BV.

ANOVA-like differential expression (ALDEx) analysis for mixed population RNA-Seq.

Experimental variance is a major challenge when dealing with high-throughput sequencing data. This variance has several sources: sampling replication, technical replication, variability within biological conditions, and variability between biological conditions. The high per-sample cost of RNA-Seq often precludes the large number of experiments needed to partition observed variance into these categories as per standard ANOVA models. We show that the partitioning of within-condition to between-condition variation cannot reasonably be ignored, whether in single-organism RNA-Seq or in Meta-RNA-Seq experiments, and further find that commonly-used RNA-Seq analysis tools, as described in the literature, do not enforce the constraint that the sum of relative expression levels must be one, and thus report expression levels that are systematically distorted. These two factors lead to misleading inferences if not properly accommodated. As it is usually only the biological between-condition and within-condition differences that are of interest, we developed ALDEx, an ANOVA-like differential expression procedure, to identify genes with greater between- to within-condition differences. We show that the presence of differential expression and the magnitude of these comparative differences can be reasonably estimated with even very small sample sizes.

Comparative genomics of Gardnerella vaginalis strains reveals substantial differences in metabolic and virulence potential.

BACKGROUND: Gardnerella vaginalis is described as a common vaginal bacterial species whose presence correlates strongly with bacterial vaginosis (BV). Here we report the genome sequencing and comparative analyses of three strains of G. vaginalis. Strains 317 (ATCC 14019) and 594 (ATCC 14018) were isolated from the vaginal tracts of women with symptomatic BV, while Strain 409-05 was isolated from a healthy, asymptomatic individual with a Nugent score of 9. PRINCIPAL FINDINGS: Substantial genomic rearrangement and heterogeneity were observed that appeared to have resulted from both mobile elements and substantial lateral gene transfer. These genomic differences translated to differences in metabolic potential. All strains are equipped with significant virulence potential, including genes encoding the previously described vaginolysin, pili for cytoadhesion, EPS biosynthetic genes for biofilm formation, and antimicrobial resistance systems, We also observed systems promoting multi-drug and lantibiotic extrusion. All G. vaginalis strains possess a large number of genes that may enhance their ability to compete with and exclude other vaginal colonists. These include up to six toxin-antitoxin systems and up to nine additional antitoxins lacking cognate toxins, several of which are clustered within each genome. All strains encode bacteriocidal toxins, including two lysozyme-like toxins produced uniquely by strain 409-05. Interestingly, the BV isolates encode numerous proteins not found in strain 409-05 that likely increase their pathogenic potential. These include enzymes enabling mucin degradation, a trait previously described to strongly correlate with BV, although commonly attributed to non-G. vaginalis species. CONCLUSIONS: Collectively, our results indicate that all three strains are able to thrive in vaginal environments, and therein the BV isolates are capable of occupying a niche that is unique from 409-05. Each strain has significant virulence potential, although genomic and metabolic differences, such as the ability to degrade mucin, indicate that the detection of G. vaginalis in the vaginal tract provides only partial information on the physiological potential of the organism.

Drawing the line between commensal and pathogenic Gardnerella vaginalis through genome analysis and virulence studies.

BACKGROUND: Worldwide, bacterial vaginosis (BV) is the most common vaginal disorder. It is associated with risk for preterm birth and HIV infection. The etiology of the condition has been debated for nearly half a century and the lack of knowledge about its cause and progression has stymied efforts to improve therapy and prevention. Gardnerella vaginalis was originally identified as the causative agent, but subsequent findings that it is commonly isolated from seemingly healthy women cast doubt on this claim. Recent studies shedding light on the virulence properties of G. vaginalis, however, have drawn the species back into the spotlight. RESULTS: In this study, we sequenced the genomes of a strain of G. vaginalis from a healthy woman, and one from a woman with bacterial vaginosis. Comparative analysis of the genomes revealed significant divergence and in vitro studies indicated disparities in the virulence potential of the two strains. The commensal isolate exhibited reduced cytotoxicity and yet the cytolysin proteins encoded by the two strains were nearly identical, differing at a single amino acid, and were transcribed at similar levels. The BV-associated strain encoded a different variant of a biofilm associated protein gene and demonstrated greater adherence, aggregation, and biofilm formation. Using filters with different pore sizes, we found that direct contact between the bacteria and epithelial cells is required for cytotoxicity. CONCLUSIONS: The results indicated that contact is required for cytotoxicity and suggested that reduced cytotoxicity in the commensal isolate could be due to impaired adherence. This study outlines two distinct genotypic variants of G. vaginalis, one apparently commensal and one pathogenic, and presents evidence for disparate virulence potentials.

Production and characterization of monoclonal antibodies against vaginolysin: mapping of a region critical for its cytolytic activity.

Vaginolysin (VLY) is a protein toxin released by Gardnerella vaginalis. VLY belongs to the group of cholesterol-dependent cytolysins (CDCs). We have generated a panel of novel monoclonal antibodies (MAbs) against VLY. For the generation of MAbs, we have used recombinant VLY expressed in Escherichia coli. The functional activity of recombinant VLY was confirmed by an in vitro hemolytic assay using human erythrocytes. The MAbs raised against recombinant VLY were reactive with VLY from G. vaginalis both by Western blot and ELISA. The cross-reactivity of MAbs with other CDCs was investigated. For this purpose, recombinant cytolysins perfringolysin, listeriolysin, intermedilysin, pneumolysin and streptolysin were expressed in E. coli. The MAbs were specific exclusively to VLY and did not react with other CDCs. All MAbs were studied for the ability to neutralize hemolytic activity of VLY in vitro and several neutralizing MAbs were identified. The MAb produced by clone 9B4 showed the most potent neutralizing activity. The epitope for this MAb was localized near the N-terminus of VLY, between amino acid (aa) residues 112 and 268. The region recognized by the neutralizing MAb 9B4 includes the conserved motif (VAARMQYD, aa 189-196) supposed to be involved in VLY oligomerization. Selected MAbs were employed to develop a sandwich ELISA for VLY quantification. The MAb-based immunoassay was suitable for the detection of VLY in the cultures of G. vaginalis. In conclusion, the MAbs described in the current study may be useful for structural and functional studies of VLY as well as immunodetection of VLY in biological specimens.

Antibody-based detection and inhibition of vaginolysin, the Gardnerella vaginalis cytolysin.

Bacterial vaginosis (BV) is the most common vaginal infection worldwide and is associated with significant adverse sequelae. We have recently characterized vaginolysin (VLY), the human-specific cytotoxin produced by Gardnerella vaginalis and believed to play a critical role in the pathogenesis of BV and its associated morbidities. We hypothesize that novel antibody-based strategies may be useful for detection of VLY and for inhibition of its toxic effects on human cells. Using purified toxin as an immunogen, we generated polyclonal rabbit immune serum (IS) against VLY. A western blot of G. vaginalis lysate was probed with IS and a single band (57 kD) identified. Immunofluorescence techniques using IS detected VLY production by G. vaginalis. In addition, we have developed a sandwich ELISA assay capable of VLY quantification at ng/ml concentrations in the supernatant of growing G. vaginalis. To investigate the potential inhibitory role of IS on VLY-mediated cell lysis, we exposed human erythrocytes to VLY or VLY pretreated with IS and determined the percent hemolysis. Pretreatment with IS resulted in a significant reduction in VLY-mediated lysis. Similarly, both human cervical carcinoma cells and vaginal epithelial cells exhibited reduced cytolysis following exposure to VLY with IS compared to VLY alone. These results confirm that antibody-based techniques are an effective means of VLY detection. Furthermore, VLY antiserum functions as an inhibitor of VLY-CD59 interaction, mitigating cell lysis. These strategies may have a potential role in the diagnosis and treatment of BV.

Prevotella bivia as a source of lipopolysaccharide in the vagina.

OBJECTIVES: To compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA). METHODS: Vaginal washes obtained from patients with (n=43) and without (n=59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts. RESULTS: The median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P<0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06+/-0.36 vs 5.4+/-2.2 log(10) CFU/ml, respectively, P<0.001). Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0+/-306.6 EU/ml) than either E. coli (4679.0+/-585.3 EU/ml) or G. vaginalis (0.07+/-0.01 EU/ml of LPS). The loss of DA neuron was 20-27% in cultures treated with vaginal washes from BV-negative patients and 58-97% in cultures treated with vaginal washes from patients with BV. CONCLUSION: P. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.