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Objective: The goal of this study is to purify inerolysin produced by Lactobacillus iners and vaginolysin produced by Gardnerella vaginalis, and to investigate the cytotoxic effects of the combination of these purified toxins on VK2 and HeLa cells. Methods: Out of 592 specimens from Baghdad teaching hospitals, 101 isolates were identified as L. iners and 165 isolates as G. vaginalis. The toxins were purified using ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The cytotoxic effects of the combined inerolysin and vaginolysin on VK2 and HeLa cells were studied. Results: For inerolysin, the optimal precipitation saturation ratio was 70%, with ion exchange chromatography yielding a specific activity of 14 mg and gel filtration increasing the specific activity to 40 U/mg. For vaginolysin, the optimal precipitation saturation ratio was 50%, with ion exchange chromatography yielding a specific activity of 4 mg and gel filtration increasing the specific activity to 10 U/mg. The combination of toxins showed a viability ratio of 38% for VK2 cells and 31% for HeLa cells at higher toxin concentrations. Conclusion: Inerolysin and vaginolysin cause lysis of vaginal and cervical cells, which may lead to abortion. The killing ratio increases with higher concentrations of the toxins. [ABSTRACT FROM AUTHOR]

Background: Gardnerella vaginalis (G. vaginalis) has been recognised as the characteristic microorganism in bacterial vaginosis (BV), but its contribution to the pathogenesis of BV remains poorly understood and is largely understudied in South Africa. This study investigated the diversity of the virulence factors (i.e. vaginolysin [vly], sialidase [sld] and phospholipase C [pho]) of G. vaginalis in pregnant South African women across the BV positive, BV intermediate, and BV negative states to determine their relation to BV status. Methods: This study included 150 pregnant women recruited from a public hospital in Durban, South Africa. Enrolled women provided two self-collected vaginal swabs for BV diagnosis by Nugent score and cultivation of G. vaginalis for further laboratory testing. Amplification, sequencing, and phylogenetic analysis were performed to detect the presence of vly, sld and pho genes of G. vaginalis. Results: A total of 16 G. vaginalis isolates were successfully cultured from the study population. Of these 16 isolates, 14 (87.5%) were positive for vly, 15 (93.8%) were positive for sld, and 13 (81.3%) were positive for pho. There were 11 (68.8%) isolates that harboured all three virulence factors. Phylogenetic analysis revealed cluster heterogeneity for all three virulence factors, indicative of little to no diversity across the BV states. Conclusion: This study was the first to report on the virulence factors of G. vaginalis isolates found in the different BV states from pregnant South African women. Understanding the pathogenic mechanisms of G. vaginalis is vital to combat BV infection and will aid in prevention and treatment strategies for BV. [ABSTRACT FROM AUTHOR]

Bacterial vaginosis (BV) is a dysbiotic condition of the vaginal microbiome associated with higher risk of infection by Neisseria gonorrhoeae— the cause of gonorrhea. Here we test if one known facet of BV—the presence of bacterial cytolysins—leads to mobilization of intracellular contents that enhance gonococcal virulence. We cloned and expressed recombinant vaginolysin (VLY), a cytolysin produced by the BV-associated bacterium Gardnerella , verifying that it liberates contents of cervical epithelial (HeLa) cells, while vector control preparations did not. We tested if VLY mediates a well-known gonococcal virulence mechanism—the molecular mimicry of host glycans. To evade host immunity, N. gonorrhoeae caps its lipooligosaccharide (LOS) with α2-3-linked sialic acid. For this, gonococci must scavenge a metabolite made inside host cells. Flow cytometry-based lectin-binding assays showed that gonococci exposed to vaginolysin-liberated contents of HeLa cells displayed greater sialic acid capping of their LOS. This higher level of bacterial sialylation was accompanied by increased binding of the complement regulatory protein factor H, and greater resistance to complement attack. Together these results suggest that cytolytic activities present during BV may enhance the ability of N. gonorrhoeae to capture intracellular metabolites and evade host immunity via glycan molecular mimicry. [ABSTRACT FROM AUTHOR]

Tethered bilayer lipid membranes (tBLMs) are increasingly used in biosensor applications where electrochemical impedance spectroscopy (EIS) is the method of choice for amplifying and recording the activity of membrane-damaging agents such as pore-forming toxins or disrupting peptides. While the activity of these biological agents may depend on the pH of the analytes, there is increasing evidence that the sensitivity of tethered bilayer sensors depends on the pH of the solutions. In our study, we addressed the question of what are the fundamental reasons for the variability of the EIS signal of the tBLMs with pH. We designed an experiment to compare the EIS response of tBLMs with natural membrane defects and two different membrane disruptors: vaginolysin and melittin. Our experimental design ensured that the same amount of protein and peptide was present in the tBLMs, while the pH was varied by replacing the buffers with different pH values. Using a recently developed EIS data analysis algorithm from our research group, we were able to demonstrate that, in contrast to previous literature which relates the variability of tBLM, EIS response to the variation in defect density, the main reason for the observed variability in EIS response is the change in the sub-membrane properties of tBLMs with pH. Using surface-enhanced infrared absorption spectroscopy (SEIRAS), we have shown that pH changes from neutral to slightly acidic leads to an expulsion of water, presumably bound to ions, from the sub-membrane reservoir, resulting in a marked decrease in the carrier concentration and specific conductance of the sub-membrane reservoir. Such a decrease is recorded by the EIS as a decrease in the conductance of the tBLM complex and affects the sensitivity of a biosensor. Our data provide important evidence of pH-sensitive effects that should be considered in both the development and operation of biosensors. [ABSTRACT FROM AUTHOR]

Tethered bilayer lipid membranes (tBLMs) are increasingly used in biosensor applications where electrochemical impedance spectroscopy (EIS) is the method of choice for amplifying and recording the activity of membrane-damaging agents such as pore-forming toxins or disrupting peptides. While the activity of these biological agents may depend on the pH of the analytes, there is increasing evidence that the sensitivity of tethered bilayer sensors depends on the pH of the solutions. In our study, we addressed the question of what are the fundamental reasons for the variability of the EIS signal of the tBLMs with pH. We designed an experiment to compare the EIS response of tBLMs with natural membrane defects and two different membrane disruptors: vaginolysin and melittin. Our experimental design ensured that the same amount of protein and peptide was present in the tBLMs, while the pH was varied by replacing the buffers with different pH values. Using a recently developed EIS data analysis algorithm from our research group, we were able to demonstrate that, in contrast to previous literature which relates the variability of tBLM, EIS response to the variation in defect density, the main reason for the observed variability in EIS response is the change in the sub-membrane properties of tBLMs with pH. Using surface-enhanced infrared absorption spectroscopy (SEIRAS), we have shown that pH changes from neutral to slightly acidic leads to an expulsion of water, presumably bound to ions, from the sub-membrane reservoir, resulting in a marked decrease in the carrier concentration and specific conductance of the sub-membrane reservoir. Such a decrease is recorded by the EIS as a decrease in the conductance of the tBLM complex and affects the sensitivity of a biosensor. Our data provide important evidence of pH-sensitive effects that should be considered in both the development and operation of biosensors.

Gardnerella vaginalis produces vaginolysin (VLY), a cholesterol-dependent cytolysin, responsible of the cellular lysis and epithelial damage. In recently years, IgY antibodies produced in hens present several uses in diagnostic, treatment and research, however they present the disadvantage of unspecific recognition, which could be improved by the preselection of the epitope based on in silico analysis. Finally, multiantigenic peptides with eight ramification (MAP8) are groups of identical peptides synthesized on polylylisine core that increase immunogenicity, and allows its use to improve in vivo antibodies production and troubleshooting. In this work, we designed and administrated a MAP8 synthetic peptide in a hen to test its ability to induce the production of IgY antibodies. Further, we tested the reactivity of anti-VLY IgY antibody (derived of VLY-MAP8 peptide administration) in detecting the denaturing and native cognate antigen in the complete bacteria by ELISA, dot blot, western blot and inhibition of hemagglutination assay. Results showed the induction of IgY antibodies in the immunized hen, that were able to recognize the native and denatured cognate antigen in samples of culture supernatant and complete bacteria of Gardnerella vaginalis ATCC 14018 strain. Additionally, anti-VLY IgY antibody inhibit > 90% erythrocyte's lysis. In conclusion, the MAP8 peptide and the produced anti-VLY IgY antibody could serve as a potential diagnostic tool in bacterial vaginosis. Also, our study proposed a method for the improvement of IgY antibodies production supported by the administration of MAP8 peptides selected by in silico analysis of the complete antigen. [ABSTRACT FROM AUTHOR]

Objective: To update the status of Gardnerella vaginalis (G. vaginalis) as a causative agent of bacterial vaginosis (BV) in Malaysia and to define its epidemiology, metronidazole resistance and virulence properties. Methods: It is a single-centre (Gynaecology clinic at the Hospital Kuala Lumpur, Malaysia) prospective study with laboratory-based microbiological follow up and analyses. Vaginal swabs collected from the patients suspected for BV were subjected to clinical BV diagnosis, isolation and identification of G. vaginalis, metronidazole susceptibility testing, vaginolysin and sialidase gene PCR, Piot's biotyping and amplified ribosomal DNA restriction analysis genotyping. Results: Among the 207 patients suspected for BV, G. vaginalis was isolated from 47 subjects. G. vaginalis coexisted with Trichomonas vaginalis and Candida albicans in 26 samples. Three G. vaginalis isolates were resistant to metronidazole. Biotyping revealed 1 and 7 as the common types. Amplified ribosomal DNA restriction analysis genotype II was found to be more common (n = 22; 46%) than I (n = 12; 25.53%) and III (n = 13; 27.6%). All genotype I and III isolates carried the sialidase gene, while 91.6% and 84.6% contained the vaginolysin gene. Genotype I was significantly associated with post-gynaecological surgical complications and abortions (P = 0.002). Conclusions: The existence of pathogenic G. vaginalis clones in Malaysia including drug resistant strains should not be taken lightly and needs to be monitored as these may bring more complications especially among women of child bearing age and pregnant women.

Bacterial vaginosis (BV) is a vaginal anaerobic dysbiosis that affects women of reproductive age worldwide. BV is microbiologically characterized by the depletion of vaginal lactobacilli and the overgrowth of anaerobic bacterial species. Accumulated evidence suggests that Gardnerella spp. have a pivotal role among BV-associated bacteria in the initiation and development of BV. However, Gardnerella spp. often colonize healthy women. Lactobacillus iners is considered as a prevalent constituent of healthy vaginal microbiota, and is abundant in BV. Gardnerella spp. and L. iners secrete the toxins vaginolysin (VLY) and inerolysin (INY), which have structural and activity features attributed to cholesterol-dependent cytolysins (CDCs). CDCs are produced by many pathogenic bacteria as virulence factors that participate in various stages of disease progression by forming lytic and non-lytic pores in cell membranes or via pore-independent pathways. VLY is expressed in the majority of Gardnerella spp. isolates; less is known about the prevalence of the gene that encodes INY. INY is a classical CDC; membrane cholesterol acts a receptor for INY. VLY uses human CD59 as its receptor, although cholesterol remains indispensable for VLY pore-forming activity. INY-induced damage of artificial membranes is directly dependent on cholesterol concentration in the bilayer, whereas VLY-induced damage occurs with high levels of membrane cholesterol (>40 mol%). VLY primarily forms membrane-embedded complete rings in the synthetic bilayer, whereas INY forms arciform structures with smaller pore sizes. VLY activity is high at elevated pH, which is characteristic of BV, whereas INY activity is high at more acidic pH, which is specific for a healthy vagina. Increased VLY levels in vaginal mucosa in vivo were associated with clinical indicators of BV. However, experimental evidence is lacking for the specific roles of VLY and INY in BV. The interplay between vaginal bacterial species affects the expression of the gene encoding VLY, thereby modulating the virulence of Gardnerella spp. This review discusses the current evidence for VLY and INY cytolysins, including their structures and activities, factors affecting their expression, and their potential impacts on the progression of anaerobic dysbiosis.

Bacterial vaginosis (BV) is a vaginal anaerobic dysbiosis that affects women of reproductive age worldwide. BV is microbiologically characterized by the depletion of vaginal lactobacilli and the overgrowth of anaerobic bacterial species. Accumulated evidence suggests that Gardnerella spp. have a pivotal role among BV-associated bacteria in the initiation and development of BV. However, Gardnerella spp. often colonize healthy women. Lactobacillus iners is considered as a prevalent constituent of healthy vaginal microbiota, and is abundant in BV. Gardnerella spp. and L. iners secrete the toxins vaginolysin (VLY) and inerolysin (INY), which have structural and activity features attributed to cholesterol-dependent cytolysins (CDCs). CDCs are produced by many pathogenic bacteria as virulence factors that participate in various stages of disease progression by forming lytic and non-lytic pores in cell membranes or via pore-independent pathways. VLY is expressed in the majority of Gardnerella spp. isolates; less is known about the prevalence of the gene that encodes INY. INY is a classical CDC; membrane cholesterol acts a receptor for INY. VLY uses human CD59 as its receptor, although cholesterol remains indispensable for VLY pore-forming activity. INY-induced damage of artificial membranes is directly dependent on cholesterol concentration in the bilayer, whereas VLY-induced damage occurs with high levels of membrane cholesterol (>40 mol%). VLY primarily forms membrane-embedded complete rings in the synthetic bilayer, whereas INY forms arciform structures with smaller pore sizes. VLY activity is high at elevated pH, which is characteristic of BV, whereas INY activity is high at more acidic pH, which is specific for a healthy vagina. Increased VLY levels in vaginal mucosa in vivo were associated with clinical indicators of BV. However, experimental evidence is lacking for the specific roles of VLY and INY in BV. The interplay between vaginal bacterial species affects the expression of the gene encoding VLY, thereby modulating the virulence of Gardnerella spp. This review discusses the current evidence for VLY and INY cytolysins, including their structures and activities, factors affecting their expression, and their potential impacts on the progression of anaerobic dysbiosis. [ABSTRACT FROM AUTHOR]

Gardnerella vaginalis has recently been split into 13 distinct species. In this study, we tested the hypotheses that species-specific variations in the vaginolysin (VLY) amino acid sequence could influence the interaction between the toxin and vaginal epithelial cells and that VLY variation may be one factor that distinguishes less virulent or commensal strains from more virulent strains. This was assessed by bioinformatic analyses of publicly available Gardnerella spp. sequences and quantification of cytotoxicity and cytokine production from purified, recombinantly produced versions of VLY. After identifying conserved differences that could distinguish distinct VLY types, we analyzed metagenomic data from a cohort of female subjects from the Vaginal Human Microbiome Project to investigate whether these different VLY types exhibited any significant associations with symptoms or Gardnerella spp.-relative abundance in vaginal swab samples. While Type 1 VLY was most prevalent among the subjects and may be associated with increased reports of symptoms, subjects with Type 2 VLY dominant profiles exhibited increased relative Gardnerella spp. abundance. Our findings suggest that amino acid differences alter the interaction of VLY with vaginal keratinocytes, which may potentiate differences in bacterial vaginosis (BV) immunopathology in vivo.

Using a chemically-defined medium simulating genital tract secretions, we have shown that pre-adhering Lactobacillus crispatus to Hela epithelial cells reduced cytotoxicity caused by Gardnerella vaginalis. This effect was associated to the expression of vaginolysin and was specific to L. crispatus interference, as other vaginal facultative anaerobes had no protective effect. [ABSTRACT FROM AUTHOR]

The present study aims to detection Genotyping of vaginolysin gene for Gardnerella vaginalis which isolated from preterm labor patients by PCR-RFLP in Hilla cityIn this study, 150 clinical sample were collected from patients with preterm labor submitted to Al-Hilla Teaching Hospital and Babylon Maternity and pediatric hospital. After extraction of DNA from high vaginal swabs, the DNA was subjected to PCR- RFLP method for identification of G.vginalis. results show It was found only (20%) of bacteria was give positive result. However, vagainolysin were carried out using specific primer and the result shows that 12 samples were positive amplification 1551bp then products were digested with EcoR I the results showed that vagainolysin exhibited three fragment 1000, 717, 100 bp respectively, assertion that G.vaginalis have four Genotyping this improving that it is had more virulent. [ABSTRACT FROM AUTHOR]

Gardnerella vaginalis produces cytolysin vaginolysin (VLY), which has been suggested to be a contributor to bacterial vaginosis pathogenesis. VLY along with intermedilysin (ILY) from Streptococcus intermedius have been attributed to a group of cholesterol-dependent cytolysins (CDCs) whose pore-forming activity depends on human CD59 (hCD59). Here, we show that different types of cells lacking hCD59 are susceptible to VLY-mediated lysis, albeit to different extents. We analyze the effects of both hCD59 and cholesterol on VLY cytolytic activity. We show that VLY binds to cholesterol-rich membranes of non-human cells, while VLY with an impaired cholesterol recognition site retains binding to the hCD59-containing cells. We further demonstrate that cholesterol binding by VLY is sufficient to trigger the formation of oligomeric complexes on cholesterol rich-liposomes lacking hCD59. Thus, VLY may induce cell lysis following two alternative pathways. One requires only cholesterol and does not depend on hCD59. The second pathway involves hCD59 contribution similarly to ILY. Apparently, under physiological conditions VLY acts in the most effective way by accepting the assistance of hCD59.

Objective To update the status of Gardnerella vaginalis ( G. vaginalis ) as a causative agent of bacterial vaginosis (BV) in Malaysia and to define its epidemiology, metronidazole resistance and virulence properties. Methods It is a single-centre (Gynaecology clinic at the Hospital Kuala Lumpur, Malaysia) prospective study with laboratory-based microbiological follow up and analyses. Vaginal swabs collected from the patients suspected for BV were subjected to clinical BV diagnosis, isolation and identification of G. vaginalis , metronidazole susceptibility testing, vaginolysin and sialidase gene PCR, Piot's biotyping and amplified ribosomal DNA restriction analysis genotyping. Results Among the 207 patients suspected for BV, G. vaginalis was isolated from 47 subjects. G. vaginalis coexisted with Trichomonas vaginalis and Candida albicans in 26 samples. Three G. vaginalis isolates were resistant to metronidazole. Biotyping revealed 1 and 7 as the common types. Amplified ribosomal DNA restriction analysis genotype II was found to be more common ( n = 22; 46%) than I ( n = 12; 25.53%) and III ( n = 13; 27.6%). All genotype I and III isolates carried the sialidase gene, while 91.6% and 84.6% contained the vaginolysin gene. Genotype I was significantly associated with post-gynaecological surgical complications and abortions ( P = 0.002). Conclusions The existence of pathogenic G. vaginalis clones in Malaysia including drug resistant strains should not be taken lightly and needs to be monitored as these may bring more complications especially among women of child bearing age and pregnant women. [ABSTRACT FROM AUTHOR]

Gardnerella species are associated with bacterial vaginosis (BV) and have been investigated as etiological agents of the condition. Nonetheless, the isolation of this taxon from healthy individuals has raised important questions regarding its etiological role. Recently, using advanced molecular approaches, the Gardnerella genus was expanded to include several different species that exhibit differences in virulence potential. Understanding the significance of these different species with respect to mucosal immunity and the pathogenesis and complications of BV could be crucial to solving the BV enigma. Here, we review key findings regarding the unique genetic and phenotypic diversity within this genus, virulence factors, and effects on mucosal immunity as they stand. We also comment on the relevance of these findings to the proposed role of Gardnerella in BV pathogenesis and in reproductive health and identify key gaps in knowledge that should be explored in the future., Competing Interests: The authors declare no conflict of interest.

Gardnerella vaginalis is a facultative anaerobic bacterium that inhabits the genitourinary tract of both healthy women and those with bacterial vaginosis. We report a case of G. vaginalis bacteremia associated with severe toxic encephalopathy in a young woman. Anaerobic blood cultures yielded pure growth of small gram-variable rods later identified as G. vaginalis by both rapid biochemical tests and 16S rRNA gene sequencing. The patient recovered after treatment with amoxicillin-clavulanate according to the in vitro susceptibility testing. The complete genome of G. vaginalis isolate from blood cultures was determined. In vitro G. vaginalis isolate produced elevated amounts of a pore-forming toxin vaginolysin compared to control G. vaginalis isolates. We hypothesize that this toxin, if produced in high amounts in blood, is able to disrupt the blood-brain barrier and exert a toxic activity on brain cells. [ABSTRACT FROM AUTHOR]

Gardnerella vaginalis produces cytolysin vaginolysin (VLY), which has been suggested to be a contributor to bacterial vaginosis pathogenesis. VLY along with intermedilysin (ILY) from Streptococcus intermedius have been attributed to a group of cholesterol-dependent cytolysins (CDCs) whose pore-forming activity depends on human CD59 (hCD59). Here, we show that different types of cells lacking hCD59 are susceptible to VLY-mediated lysis, albeit to different extents. We analyze the effects of both hCD59 and cholesterol on VLY cytolytic activity. We show that VLY binds to cholesterol-rich membranes of non-human cells, while VLY with an impaired cholesterol recognition site retains binding to the hCD59-containing cells. We further demonstrate that cholesterol binding by VLY is sufficient to trigger the formation of oligomeric complexes on cholesterol rich-liposomes lacking hCD59. Thus, VLY may induce cell lysis following two alternative pathways. One requires only cholesterol and does not depend on hCD59. The second pathway involves hCD59 contribution similarly to ILY. Apparently, under physiological conditions VLY acts in the most effective way by accepting the assistance of hCD59. [ABSTRACT FROM AUTHOR]

ABSTRACT Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins secreted mainly by pathogenic Gram-positive bacteria. CDCs generally recognize and bind to membrane cholesterol to create pores and lyse target cells. However, in contrast to typical CDCs such as streptolysin O, several atypical CDCs have been reported. The first of these was intermedilysin, which is secreted by Streptococcus intermedius and has human cell-specificity, human CD59 (huCD59) being its receptor. In the study reported here, the diversity of receptor recognition among CDCs was investigated and multi-receptor recognition characteristics were identified within this toxin family. Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) secreted by S. mitis strain Nm-65 isolated from a patient with Kawasaki disease was previously shown to hemolyze erythrocytes in a species-dependent manner, its maximum activity being in human cells. In the present study, it was found that Sm-hPAF recognizes both membrane cholesterol and huCD59 as receptors for triggering pore-formation. Moreover, vaginolysin (VLY) of Gardnerella vaginalis showed similar characteristics to Sm-hPAF regarding receptor recognition. On the basis of the results presented here, the mode of receptor recognition of CDCs can be categorized into the following three groups: (i) Group I, comprising typical CDCs with high affinity to cholesterol and no or very little affinity to huCD59; (ii) Group II, including atypical CDCs such as ILY, with no or very little affinity to cholesterol and high affinity to huCD59; and (iii) Group III, which contains atypical CDCs such as Sm-hPAF and VLY with affinity to both cholesterol and huCD59. [ABSTRACT FROM AUTHOR]

Gardnerella vaginalis is considered a substantial player in the progression of bacterial vaginosis ( BV). We analysed 17 G. vaginalis strains isolated from the genital tract of women diagnosed with BV to establish a potential link between genotypes/biotypes and the expression of virulence factors, vaginolysin ( VLY) and sialidase, which are assumed to play a substantial role in the pathogenesis of BV. Amplified ribosomal DNA restriction analysis revealed two G. vaginalis genotypes. Gardnerella vaginalis isolates of genotype 2 appeared more complex than genotype 1 and were subdivided into three subtypes. Biochemical typing allowed us to distinguish four different biotypes. A great diversity of the level of VLY production among the isolates of G. vaginalis may be related to a different cytotoxicity level of the strains. We did not find any correlation between VLY production level and G. vaginalis genotype/biotype. In contrast, a link between G. vaginalis genotype and sialidase production was established. Our findings on the diversity of VLY expression level in different clinical isolates and linking sialidase activity with the genotype of G. vaginalis could help to evaluate the pathogenic potential of different G. vaginalis strains. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

In this work, the assembly of functional tethered bilayer membranes (tBLMs) on silver surface was demonstrated for the first time. To avoid degradation of thin silver films the non-corrosive mixture of thiols composed of WC14 molecular anchor and 3-mercapto-1-propanol (3M1P) serving as a backfiller was used to self-assemble monolayers which allowed fusion of multilamellar vesicles and tBLM formation. tBLMs on silver films exhibited high stability and low residual defect densities as measured by the electrochemical impedance spectroscopy (EIS). Silver supported tBLMs demonstrated all features typical for interactions of pore-forming proteins and bilayer membranes. Specifically, the EIS spectral response was typical for the formation of water filled defects when tBLMs were exposed to the solution of vaginolysin (VLY), the cholesterol dependent cytolysin. Increase of defect density was observed only in case tBLMs contained significant amount, e.g., 50 %, of cholesterol, thus confirming functional reconstitution of vaginolysin into tBLMs. This observation is consistent with the biological nature of interaction between the bilayer and the cholesterol dependent cytolysins. The reconstitution of VLY was confirmed by the direct imaging of the protein entities in tBLMs by the atomic force microscopy (AFM). Notably, the quantitative estimate of the functional protein-pores in tBLMs measured by EIS, was found to be close to the number of VLY entities in tBLMs measured by the AFM. Our work demonstrates that silver can serve as less expensive alternative to gold in designing tBLM based devices, for example phospholipid biosensors, as well as the experimental platform for studying membrane protein interactions by electrochemical, and microscopy techniques. [ABSTRACT FROM AUTHOR]

Abstract Background Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis. Results The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. Conclusions Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and allowed generation of multivalent scFv-Fc proteins with high VLY-neutralizing potency. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. The current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins.

Gardnerella vaginalis has recently been split into 13 distinct species. In this study, we tested the hypotheses that species-specific variations in the vaginolysin (VLY) amino acid sequence could influence the interaction between the toxin and vaginal epithelial cells and that VLY variation may be one factor that distinguishes less virulent or commensal strains from more virulent strains. This was assessed by bioinformatic analyses of publicly available Gardnerella spp. sequences and quantification of cytotoxicity and cytokine production from purified, recombinantly produced versions of VLY. After identifying conserved differences that could distinguish distinct VLY types, we analyzed metagenomic data from a cohort of female subjects from the Vaginal Human Microbiome Project to investigate whether these different VLY types exhibited any significant associations with symptoms or Gardnerella spp.-relative abundance in vaginal swab samples. While Type 1 VLY was most prevalent among the subjects and may be associated with increased reports of symptoms, subjects with Type 2 VLY dominant profiles exhibited increased relative Gardnerella spp. abundance. Our findings suggest that amino acid differences alter the interaction of VLY with vaginal keratinocytes, which may potentiate differences in bacterial vaginosis (BV) immunopathology in vivo. [ABSTRACT FROM AUTHOR]

Background Retrocyclins are cyclic antimicrobial peptides that have been shown to be both broadly active and safe in animal models. RC-101, a synthetic retrocyclin, targets important human pathogens and is a candidate vaginal microbicide. Its activity against microbes associated with bacterial vaginosis is unknown. Methods We investigated the effect of RC-101 on toxin activity, bacterial growth and biofilm formation of Gardnerella vaginalis in vitro. Results RC-101 potently inhibits the cytolytic activity of vaginolysin, the Gardnerella vaginalis toxin, on both erythrocytes and nucleated cells. RC-101 lacks inhibitory activity against planktonic G. vaginalis but markedly decreases biofilm formation. Conclusions These dual properties, toxin inhibition and biofilm retardation, justify further exploration of RC-101 as a candidate agent for bacterial vaginosis prevention. [ABSTRACT FROM AUTHOR]

Bacterial vaginosis (BV) is a common polymicrobial infection affecting women in the reproductive age and is associated with adverse obstetric and gynaecological outcomes. Gardnerella vaginalis is the most virulent anaerobic bacterial species predominantly associated with BV. However, a clear understanding of the mechanisms by which it contributes to the pathogenesis and persistence of BV is lacking. In this report, we demonstrate for the first time, the isolation of membrane vesicles (MVs) from G. vaginalis ATCC 14019. These MVs are approximately 120–260 nm in diameter. Proteomic characterization of the MVs by LC-MS/MS led to the identification of 417 proteins, including proteins involved in cellular metabolism as well as molecular chaperones and certain virulence factors. Immunoblot analysis of the MVs confirmed the presence of vaginolysin, the most well-characterized virulence factor of G. vaginalis. The exposure of the vaginal epithelial cells, VK2/E6E7 to the G. vaginalis MVs resulted in the internalization of the MVs. The MVs induced cytotoxicity and an increase in the levels of the pro-inflammatory cytokine, IL-8 in VK2 cells as well lysis of erythrocytes. The results of the study indicate that G. vaginalis MVs may be involved in the delivery of cytotoxic proteins and other virulence factors to the host cells and could thereby contribute towards enhancing the cellular damage associated with pathogenesis of BV. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2019 American Society for Microbiology.)