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Identification and characterization of NanH2 and NanH3, enzymes responsible for sialidase activity in the vaginal bacterium Gardnerella vaginalis.

ABSTRACT: Gardnerella vaginalis is abundant in bacterial vaginosis (BV), a condition associated with adverse reproductive health. Sialidase activity is a diagnostic feature of BV and is produced by a subset of G. vaginalis strains. Although its genetic basis has not been formally identified, sialidase activity is presumed to derive from the sialidase A gene, named here nanH1 In this study, BLAST searches predicted two additional G. vaginalis sialidases, NanH2 and NanH3. When expressed in Escherichia coli, NanH2 and NanH3 both displayed broad abilities to cleave sialic acids from ?2-3- and ?2-6-linked N- and O-linked sialoglycans, including relevant mucosal substrates. In contrast, recombinant NanH1 had limited activity against synthetic and mucosal substrates under the conditions tested. Recombinant NanH2 was much more effective than NanH3 in cleaving sialic acids bearing a 9-O-acetyl ester. Similarly, G. vaginalis strains encoding NanH2 cleaved and foraged significantly more Neu5,9Ac2 than strains encoding only NanH3. Among a collection of 34 G. vaginalis isolates, nanH2, nanH3, or both were present in all 15 sialidase-positive strains but absent from all 19 sialidase-negative isolates, including 16 strains that were nanH1-positive. We conclude that NanH2 and NanH3 are the primary sources of sialidase activity in G. vaginalis and that these two enzymes can account for the previously described substrate breadth cleaved by sialidases in human vaginal specimens of women with BV. Finally, PCRs of nanH2 or nanH3 from human vaginal specimens had 81% sensitivity and 78% specificity in distinguishing between Lactobacillus dominance and BV, as determined by Nugent scoring.

SUBMITTER: Robinson LS

PROVIDER: S-EPMC6462536 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Identification and characterization of NanH2 and NanH3, enzymes responsible for sialidase activity in the vaginal bacterium Gardnerella vaginalis.

Robinson Lloyd S LS Schwebke Jane J Lewis Warren G WG Lewis Amanda L AL

The Journal of biological chemistry 20190205 14

Gardnerella vaginalis is abundant in bacterial vaginosis (BV), a condition associated with adverse reproductive health. Sialidase activity is a diagnostic feature of BV and is produced by a subset of G. vaginalis strains. Although its genetic basis has not been formally identified, sialidase activity is presumed to derive from the sialidase A gene, named here nanH1 In this study, BLAST searches predicted two additional G. vaginalis sialidases, NanH2 and NanH3. ...[more]

PMID: 30723162

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Project description:Increased abundance of Gardnerella vaginalis and sialidase activity in vaginal fluid is associated with bacterial vaginosis (BV), a common but poorly understood clinical entity associated with poor reproductive health outcomes. Since most women are colonized with G. vaginalis, its status as a normal member of the vaginal microbiota or pathogen causing BV remains controversial, and numerous classification schemes have been described. Since 2005, sequencing of the chaperonin-60 universal target (cpn60 UT) has distinguished four subgroups in isolate collections, clone libraries and deep sequencing datasets. To clarify potential clinical and diagnostic significance of cpn60 subgroups, we undertook phenotypic and molecular characterization of 112 G. vaginalis isolates from three continents. A total of 36 subgroup A, 33 B, 35 C and 8 D isolates were identified through phylogenetic analysis of cpn60 sequences as corresponding to four "clades" identified in a recently published study, based on sequencing 473 genes across 17 isolates. cpn60 subgroups were compared with other previously described molecular methods for classification of Gardnerella subgroups, including amplified ribosomal DNA restriction analysis (ARDRA) and real-time PCR assays designed to quantify subgroups in vaginal samples. Although two ARDRA patterns were observed in isolates, each was observed in three cpn60 subgroups (A/B/D and B/C/D). Real-time PCR assays corroborated cpn60 subgroups overall, but 13 isolates from subgroups A, B and D were negative in all assays. A putative sialidase gene was detected in all subgroup B, C and D isolates, but only in a single subgroup A isolate. In contrast, sialidase activity was observed in all subgroup B isolates, 3 (9%) subgroup C isolates and no subgroup A or D isolates. These observations suggest distinct roles for G. vaginalis subgroups in BV pathogenesis. We conclude that cpn60 UT sequencing is a robust approach for defining G. vaginalis subgroups within the vaginal microbiome.

Project description:ObjectivesTo examine the influence of sexual activity on the composition and consistency of the vaginal microbiota over time, and distribution of Gardnerella vaginalis clades in young women.MethodsFifty-two participants from a university cohort were selected. Vaginal swabs were self-collected every 3-months for up to 12 months with 184 specimens analysed. The vaginal microbiota was characterised using Roche 454 V3/4 region 16S rRNA sequencing, and G.vaginalis clade typing by qPCR.ResultsA Lactobacillus crispatus dominated vaginal microbiota was associated with Caucasian ethnicity (adjusted relative risk ratio[ARRR] = 7.28, 95%CI:1.37,38.57,p = 0.020). An L.iners (ARRR = 17.51, 95%CI:2.18,140.33,p = 0.007) or G.vaginalis (ARRR = 14.03, 95%CI:1.22,160.69, p = 0.034) dominated microbiota was associated with engaging in penile-vaginal sex. Microbiota dominated by L.crispatus, L.iners or other lactobacilli exhibited greater longitudinal consistency of the bacterial communities present compared to ones dominated by heterogeneous non-lactobacilli (p<0.030); sexual activity did not influence consistency. Women who developed BV were more likely to have clade GV4 compared to those reporting no sex/practiced non-coital activities (OR = 11.82, 95%CI:1.87,74.82,p = 0.009). Specimens were more likely to contain multiple G.vaginalis clades rather than a single clade if women engaged in penile-vaginal sex (RRR = 9.55, 95%CI:1.33,68.38,p = 0.025) or were diagnosed with BV (RRR = 31.5, 95%CI:1.69,586.87,p = 0.021).ConclusionsSexual activity and ethnicity influenced the composition of the vaginal microbiota of these young, relatively sexually inexperienced women. Women had consistent vaginal microbiota over time if lactobacilli were the dominant spp. present. Penile-vaginal sex did not alter the consistency of microbial communities but increased G.vaginalis clade diversity in young women with and without BV, suggesting sexual transmission of commensal and potentially pathogenic clades.

Project description:Bacterial vaginosis (BV) is a dysbiosis of the vaginal flora characterized by a shift from a Lactobacillus-dominant environment to a polymicrobial mixture including Actinobacteria and gram-negative bacilli. BV is a common vaginal condition in women and is associated with increased risk of sexually transmitted infection and adverse pregnancy outcomes such as preterm birth. Gardnerella vaginalis is one of the most frequently isolated bacterial species in BV. However, there has been much debate in the literature concerning the contribution of G. vaginalis to the etiology of BV, since it is also present in a significant proportion of healthy women. Here we present a new murine vaginal infection model with a clinical isolate of G. vaginalis. Our data demonstrate that this model displays key features used clinically to diagnose BV, including the presence of sialidase activity and exfoliated epithelial cells with adherent bacteria (reminiscent of clue cells). G. vaginalis was capable of ascending uterine infection, which correlated with the degree of vaginal infection and level of vaginal sialidase activity. The host response to G. vaginalis infection was characterized by robust vaginal epithelial cell exfoliation in the absence of histological inflammation. Our analyses of clinical specimens from women with BV revealed a measureable epithelial exfoliation response compared to women with normal flora, a phenotype that, to our knowledge, is measured here for the first time. The results of this study demonstrate that G. vaginalis is sufficient to cause BV phenotypes and suggest that this organism may contribute to BV etiology and associated complications. This is the first time vaginal infection by a BV associated bacterium in an animal has been shown to parallel the human disease with regard to clinical diagnostic features. Future studies with this model should facilitate investigation of important questions regarding BV etiology, pathogenesis and associated complications.

Dataset Information

Identification and characterization of NanH2 and NanH3, enzymes responsible for sialidase activity in the vaginal bacterium Gardnerella vaginalis.

Publications

Identification and characterization of NanH2 and NanH3, enzymes responsible for sialidase activity in the vaginal bacterium <i>Gardnerella vaginalis</i>.

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