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 α2-3- and α2-6-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.
Highlights
Gardnerella vaginalis is abundant in bacterial vaginosis (BV), a condition associated with adverse reproductive health
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
Health complications that can be reproduced in murine models by administering G. vaginalis include ascending uterine infection with G. vaginalis and other potential pathogens as well as recurrent urinary tract infection caused by Escherichia coli [33, 35]
Summary
We set out to identify the genetic basis for sialidase activity in G. vaginalis. Bacterial sialidase domains typically contain an N-terminal RIP (Arg-Ile/Leu-Pro) motif, four or five aspartate box repeats (Ser/Thr-X-Asp-X-Gly-X-Thr-Trp/Phe), and seven conserved active site residues [44] These features are present in the amino acid sequences of all three G. vaginalis JCP8151B NanH proteins, with the exception of NanH3, which lacks the C-terminal auxiliary glutamate residue (Fig. 1B). To determine whether NanH2 or NanH3 could cleave O-acetylated sialic acids, we incubated each sialidase with BSM or intact GBS cells, both of which contain sialoglycan chains modified with 7-O- and 9-O-acetyl esters For these experiments, we used a GBS strain with high levels of O-acetylation resulting from an active site mutation in the neuA esterase gene [54]. Primers G. vag tuf F1 G. vag tuf R1 G. vag sia f G. vag sia R 8151B nanH1 F Nco 8151B nanH1 his R Bam 8151B nanH2 F Nco 8151B nanH2 his R Bgl2 8151B nanH3 F Nco 8151B nanH3 his R Pst 8151B nanH2 A51F Nco 8151B nanH2 S908 his R Bgl2 8151B nanH3 T702 his R Pst G. vag sia universal F3 G. vag sia universal R1 G. vag nanH2 qPCR F G. vag nanH2 qPCR R G. vag nanH3 qPCR F G. vag nanH3 qPCR R G. vag nanH2 MP F1 G. vag nanH2 MP R1
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