Biology subjects

Klimowicz, A. K.

Publications and source records attributed to Klimowicz, A. K..

3 recordsLinked to original sources

bioRxiv2025

Genetic transformation of Gardnerella species and characterization of vaginolysin and sialidase mutants

Bacterial vaginosis (BV) is the most prevalent vaginal disorder in women of childbearing age and causes pregnancy complications including preterm birth. Species of Gardnerella increase just prior to the onset of symptoms and are considered to play major roles in the development and transmission of BV. However, Gardnerella species have remained genetically intractable, limiting investigations of their virulence mechanisms. Here we describe methods for genetic manipulation of Gardnerella. Through trial and error we optimized methods for electrotransformation of Gardnerella and created methods for making mutations and complements. We mutated the gene for the toxin vaginolysin (vly) in G. vaginalis and the gene for sialidase nanH3 in G. pickettii. A vly point mutant was tested in human cervix tissue and found to lack lytic activity. The nanH3 mutant lost sialidase and mucus degradation activity. Overall, this genetic toolkit opens a door for molecular characterization of Gardnerella and its mechanisms in BV. TeaserGenetic studies are now possible in a causative agent of bacterial vaginosis, and mutants were made lacking the toxin or mucus-degrading enzyme.

microbiology↗

bioRxiv2023

Genotypic and phenotypic differences among phase-variable colony variants conserved across Gardnerella spp.

The Gardnerella genus, now made up of more than 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly defined, and the contributions made by individual species are largely unknown. We report here that colony phenotypes characterized by size (large and small) and opacity (opaque and translucent) are phase variable and are conserved among all tested Gardnerella strains, representing at least ten different species. With the hypothesis that these different variants could be an important missing piece to the enigma of how BV develops in vivo, we characterized their differences. Beyond increased colony size, large colony variants (Lg) showed reduced vaginolysin secretion and faster growth rate relative to small colony variants (Sm). The ability to inhibit growth of Neisseria gonorrhoeae and commensal lactobacillus species varied by strain and in some instances differed between variants. Proteomics analyses indicate that 127-173 proteins are differentially expressed between variants. Further, whole genome sequencing analyses revealed an abundance of genes associated with variable poly-guanine tracts, implicating slipped strand mispairing in Gardnerella phase variation, and illuminating the potential for previously unrecognized variability within clonal populations. Collectively, these results suggest that colony variants may be primed to serve different roles in BV pathogenesis.

microbiology↗

bioRxiv2021

Transcriptional and translational responsiveness of the Neisseria gonorrhoeae type IV secretion system to conditions of host infections

The type IV secretion system of Neisseria gonorrhoeae translocates single-stranded DNA into the extracellular space, facilitating horizontal gene transfer and initiating biofilm formation. Expression of this system has been observed to be low under laboratory conditions, and multiple levels of regulation have been identified. We used a translational fusion of lacZ to traD, the gene for the type IV secretion system coupling protein, to screen for increased type IV secretion system expression. We identified several physiologically relevant conditions, including surface adherence, decreased manganese or iron, and increased zinc or copper, which increase gonococcal type IV secretion system protein levels through transcriptional and/or translational mechanisms. These metal treatments are reminiscent of the conditions in the macrophage phagosome. The ferric uptake regulator, Fur, was found to repress traD transcript levels, but to also have a second role, acting to allow TraD protein levels to increase only in the absence of iron. To better understand type IV secretion system regulation during infection, we examined transcriptomic data from active urethral infection samples from five men. These data demonstrated differential expression of 20 of 21 type IV secretion system genes during infection, indicating upregulation of genes necessary for DNA secretion during host infection.

microbiology↗