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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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Gardnerella 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., Results: The 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., Conclusions: The 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.

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., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Background: Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance., Results: Single-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G₄S)₄ were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells., Conclusions: Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis.

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]