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.

BackgroundGardnerella vaginalis is identified as the predominant colonist of the vaginal tracts of women diagnosed with bacterial vaginosis (BV). G. vaginalis can be isolated from healthy women, and an asymptomatic BV state is also recognised. The association of G. vaginalis with different clinical phenotypes could be explained by different cytotoxicity of the strains, presumably based on disparate gene content. The contribution of horizontal gene transfer to shaping the genomes of G. vaginalis is acknowledged. The CRISPR loci of the recently discovered CRISPR/Cas microbial defence system provide a historical view of the exposure of prokaryotes to a variety of foreign genetic elements.ResultsThe CRISPR/Cas loci were analysed using available sequence data from three G. vaginalis complete genomes and 18 G. vaginalis draft genomes in the NCBI database, as well as PCR amplicons of the genomic DNA of 17 clinical isolates. The cas genes in the CRISPR/Cas loci of G. vaginalis belong to the E. coli subtype. Approximately 20% of the spacers had matches in the GenBank database. Sequence analysis of the CRISPR arrays revealed that nearly half of the spacers matched G. vaginalis chromosomal sequences. The spacers that matched G. vaginalis chromosomal sequences were determined to not be self-targeting and were presumably neither constituents of mobile-element-associated genes nor derived from plasmids/viruses. The protospacers targeted by these spacers displayed conserved protospacer-adjacent motifs.ConclusionsThe CRISPR/Cas system has been identified in about one half of the analysed G. vaginalis strains. Our analysis of CRISPR sequences did not reveal a potential link between their presence and the virulence of the G. vaginalis strains. Based on the origins of the spacers found in the G. vaginalis CRISPR arrays, we hypothesise that the transfer of genetic material among G. vaginalis strains could be regulated by the CRISPR/Cas mechanism. The present study is the first attempt to determine and analyse the CRISPR loci of bacteria isolated from the human vaginal tract.

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Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO 2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application. KeywordsHuman carbonic anhydrase XII, inhibition of enzymatic activity, monoclonal antibodies History