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 ␣2-3and ␣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 Neu5,9Ac 2 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.

Glycan cross-feeding supports mutualism between Fusobacterium and the vaginal microbiota

“…In contrast, OsiaT was unable to deplete sialic acids and did not receive a growth benefit. While the sialoglycan substrate used here contains α2-3-linked sialic acids, Arthrobacter and Gardnerella sialidases are able to access both α2-3-and α2-6-linked sialic acids [34,55]. Together, these data demonstrate that F. nucleatum can access and consume sialic acids from sialo-glycoconjugates, only if sialidase activity from an exogenous source is available to first liberate sialic acids into the free form.…”

Human Neuraminidases Have Reduced Activity Towards Modified Sialic Acids on Glycoproteins

“…32,33 This strategy has been used to study sialic acid released by sialidase and sialate O-acetylesterase activity in bacteria. 14,34 The capacity for neuraminidase to hydrolyze acetylated sialic acids versus its non-acetylated variant is determined by comparing the amount of sialic acid released from control BSM to BSM pre-treated with NaOH to hydrolyze O-acetyl esters. 14 After enzyme treatment, the samples were again submitted to basic conditions to remove any O-acetyl groups to standardize the analysis to detection of only Neu5Ac and Neu5Gc across all samples (Figure 2A).…”

“…To account for the lower efficiency of the hNEU towards BSM we modified the assay to avoid pre-treatment of BSM with base, meaning we could not compare the amount of sialic acid released from BSM with or without O-acetyl modifications. 34 Instead, we calculated the ratio of modified sialic acids released by hNEU relative to…”

Gardnerella vaginalis as a Cause of Bacterial Vaginosis: Appraisal of the Evidence From in vivo Models

“…The authors conclude that increased mucin production in pregnancy may reflect enhanced protection against ascending infection. We note that the ATCC14018 strain used in these experiments is negative for sialidase and does not encode the genes recently shown to be responsible for sialidase activity in cultured isolates (Robinson et al, 2019). Thus, it is also possible that this strain lacks the ability to cause ascending infection due to an inability to engage in mucin degradation.…”