Characterization of sialidase enzymes of Gardnerella spp.
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Bacterial Vaginosis (BV) is a condition that occurs when the healthy, Lactobacillus spp. dominated vaginal microbiota is replaced by BV related bacteria. BV is highly prevalent in women in their reproductive age and known to be associated with preterm delivery and increased susceptibility to sexually transmitted diseases. An abundance of Gardnerella spp. is often found in cases of symptomatic BV, although they are also found in healthy women without manifesting any signs of BV. Recently, the description of Gardnerella vaginalis was amended and three new species were defined within the genus Gardnerella: G. leopoldii, G. swidsinskii, and G. piotii. Sialidase activity is recognised as an important virulence factor that contributes to degradation of vaginal mucus and is only found in G. piotii and Gardnerella genome species 3. For years, nanH1 was assumed to be the gene responsible for sialidase activity, but the intact open reading frame (ORF) is also found in sialidase negative strains. Our lab group discovered a gene (nanH3) in Gardnerella piotii and Gardnerella genome sp. 3 that is predicted to encode a cell wall attached, extra cellular sialidase. Interestingly, the ORF of nanH3 contains a homopolymeric tract of about 12 cytosine residues. Genomic regions that contain short, homogenous or heterogenous repeats are susceptible for slipped-strand mispairing (SSM) and may change the length of the repeat region at each replication. Here we attempt to characterize nanH3, determine if the homopolymer region of nanH3 varies in length in G. piotii and localize sialidases encoded by nanH1 and nanH3. Since previous attempts to express the entire nanH3 failed, a truncated version of nanH3 was expressed as a GST fusion protein (GST+TN3) in E. coli. Although expression of GST+TN3 was successful, the catalytic activity of the recombinant protein was not confirmed due to its poor solubility. The length of the homopolymer region of nanH3 varied from 8-14 cytosine residues within and among strains Gardnerella piotii, W11, VN014, VN015 and NR032 indicating that the expression of nanH3 may be regulated by SSM. Sialidase activity was more associated with the intact cells and the sonicated cell pellet than the respective supernatants. This suggests that sialidase activity of the four strains of Gardnerella piotii is more likely to be localized in the cell wall. The results of this study contribute to knowledge of characteristics that differentiate Gardnerella spp. and to the future development of preferable diagnostics for identifying high risk microbiomes
DegreeMaster of Science (M.Sc.)
SupervisorHill, Janet E
CommitteeRuzzini, Antonio; Rubin, Joseph; Deneer, Harry
Copyright DateApril 2020
Gardnerella spp., Sialidase, Bacterial vaginosis, slipped-strand mispairing, homopolymer