Biology subjects

Topf, M. A.

Publications and source records attributed to Topf, M. A..

3 recordsLinked to original sources

bioRxiv2024

Impacts of perR on oxygen sensitivity, gene expression, and murine infection in Clostridioides difficile 630Δerm

Clostridioides difficile infection (CDI), characterized by colitis and diarrhea, afflicts approximately half a million people in the United States every year, burdening both individuals and the healthcare system. C. difficile 630{Delta}erm is an erythromycin-sensitive variant of the clinical isolate C. difficile 630 and is commonly used in the C. difficile research community due to its genetic tractability. 630{Delta}erm possesses a point mutation in perR, an autoregulated transcriptional repressor that regulates oxidative stress resistance genes. This point mutation results in a constitutively de-repressed PerR operon in 630{Delta}erm. To address the impacts of perR on phenotypes relevant for oxygen tolerance and relevant to a murine model of CDI, we corrected the point mutant to restore PerR function in 630{Delta}erm (herein, 630{Delta}erm perRWT). We demonstrate that there is no difference in growth between 630{Delta}erm and a 630{Delta}erm perRWT under anaerobic conditions or when exposed to concentrations of O2 that mimic those found near the surface of the colonic epithelium. However, 630{Delta}erm perRWT is more sensitive to ambient oxygen than 630{Delta}erm, which coincides with alterations in expression of a variety of perR-dependent and perR-independent genes. Finally, we show that 630{Delta}erm and 630{Delta}erm perRWT do not differ in their ability to infect and cause disease in a well-established murine model of CDI. Together, these data support the hypothesis that the perR mutation in 630{Delta}erm arose as a result of exposure to ambient oxygen and that the perR mutation in 630{Delta}erm is unlikely to impact CDI-relevant phenotypes in laboratory studies. ImportanceClostridioides difficile is a diarrheal pathogen and a major public health concern. To improve humans understanding of C. difficile, a variety of C. difficile isolates are used in research, including C. difficile 630{Delta}erm. 630{Delta}erm is a derivative of the clinical isolate 630 and is commonly studied because it is genetically manipulable. Previous work showed that a mutation in perR in 630{Delta}erm results in a dysregulated oxidative stress response, but no work has been done to characterize perR-dependent effects on the transcriptome or to determine impacts of perR during infection. Here, we identify transcriptomic differences between 630{Delta}erm and 630{Delta}erm perRWT exposed to ambient oxygen and demonstrate that there is no strain-based difference in burdens in murine C. difficile infection.

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↗

bioRxiv2022

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests utilizing molecularly intact heme. Here we describe the mechanism underlying this trait, which we discovered using molecular genetics, phylogenetics, and population analyses. Most metronidazole-resistant strains evolved a T to G mutation, we term PnimBG, in the -10 regulatory promoter of the 5-nitroimidazole reductase nimB, resulting in the gene being constitutively transcribed. Silencing or deleting nimB eliminated metronidazole resistance. We identified the protein as a heme-dependent nitroreductase that degraded nitro-drugs to an amine lacking antimicrobial activity. We further discovered that the metronidazole-resistant PnimBGmutation was strongly associated with the Thr82Ile substitution conferring fluoroquinolone resistance in epidemic strains. Re-analysis of published genomes from global isolates confirmed that all but one encoding PnimBG also carried the Thr82Ile mutation. Our findings suggest that fluoroquinolone and metronidazole resistance co-mediated the pandemic of healthcare-associated C. difficile that are associated with poorer treatment outcomes in CDI patients receiving metronidazole.

microbiology↗