Biology subjects

Roxas, B. A. P.

Publications and source records attributed to Roxas, B. A. P..

3 recordsLinked to original sources

bioRxiv2022

Low-Toxin Clostridioides difficile RT027 Strains Exhibit Robust Virulence

Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. Currently, there is lack of consensus on a single optimal diagnostic method for C. difficile infection (CDI). Nucleic acid amplification tests (NAAT) that detect toxin genes are highly sensitive, but their specificity limitations could inflate CDI rates. Alternate multi-step diagnostic algorithms emphasize the detection of C. difficile toxins TcdA/TcdB, and are premised on the rationale that stool toxin-negative (Tox-) CDI patients have less severe disease, shorter diarrhea duration, and fewer disease complications. There have been no systematic assessments, however, of the virulence of C. difficile strains from Tox-/NAAT+ (discrepant) specimens. In our prospective analysis of 1243 C. difficile-positive patient stool specimens from three Southern Arizona hospitals, 31% were discrepant. Ribotype 027 (RT027) strains were recovered from 221 specimens; of these, 23% were discrepant. Post-culture, RT027 strains produced a range of toxin amounts including levels lower than that of the non-epidemic strain CD630. These low-toxin RT027 (LT-027) strains harbored both tcdA and tcdB genes, and their culture supernatants were cytotoxic to cultured fibroblasts. We confirmed robust colonization and virulence of a subset of LT-027 strains using multiple rodent models; lethality in animals infected with LT-027 strains was comparable to that potentiated by a high-toxin RT027 strain. Comparative genomics and proteomics analyses of several LT-027 strains identified unique genes and altered protein abundances relative to closely-related high-toxin strains. Collectively our data highlight the robust virulence and clinical relevance of low-toxin-producing, RT027 C. difficile isolates.

microbiology↗

bioRxiv2022

The Alternative Sigma Factor SigL Influences Clostridioides difficile Toxin Production, Sporulation, and Cell Surface Properties

The alternative sigma factor SigL (Sigma-54) facilitates bacterial adaptation to the extracellular environment by modulating the expression of defined gene subsets. A homolog of the gene encoding SigL is conserved in the diarrheagenic pathogen Clostridioides difficile. To explore the contribution of SigL to C. difficile biology, we generated sigL-disruption mutants (sigL::erm) in strains belonging to two phylogenetically distinct lineages - the human-relevant Ribotype 027 (strain BI-1) and the veterinary-relevant Ribotype 078 (strain CDC1). Comparative proteomics analyses of mutants and isogenic parental strains revealed lineage-specific SigL regulons. Concomitantly, loss of SigL resulted in pleotropic and distinct phenotypic alterations in the two strains. Sporulation kinetics, biofilm formation, and cell surface-associated phenotypes were altered in CDC1 sigL::erm relative to the isogenic parent strain, but remained unchanged in BI-1 sigL::erm. In contrast, secreted toxin levels were significantly elevated only in the BI-1 sigL::erm mutant relative to its isogenic parent. We also engineered SigL overexpressing strains and observed enhanced biofilm formation in the CDC1 background, and reduced spore titers as well as dampened sporulation kinetics in both strains. Thus, we contend that SigL is a key, pleiotropic regulator that dynamically influences C. difficiles virulence factor landscape, and thereby, its interactions with host tissues and co-resident microbes. Contribution to the FieldAlternative sigma factors modulate bacterial gene expression in response to environmental and metabolic cues. C. difficile encodes multiple alternative sigma factors which mediate critical cellular processes. A Sigma-54 ortholog, SigL, is conserved across all sequenced strains, yet its function remains to be elucidated. We demonstrate that SigL dynamically influences C. difficile biology in fundamental, but distinct, ways in two strains representing two clinically-relevant C. difficile phylogenetic lineages (ribotypes). Loss of SigL results in changes in sporulation kinetics, biofilm formation, alterations in cell surface properties, and secreted toxin levels in a strain-specific manner. SigL overexpression, however, uniformly impedes sporulation. Thus, SigL plays a fundamental role in regulating the expression of C. difficile virulence factors and likely profoundly impacts pathogenesis in a strain-specific manner.

microbiology↗

bioRxiv2020

Phylogenomic analysis of Clostridiodes difficile ribotype 106 strains reveals novel genetic islands and emergent phenotypes

BackgroundClostridioides difficile RT106 has emerged as a dominant molecular type in the USA in recent years, but the underlying factors contributing to its predominance remain undefined. As part of our ongoing C. difficile infection (CDI) surveillance in Arizona, we monitored RT106 frequency and characterized the genomic and phenotypic properties of the recovered isolates. ResultsFrom 2015-2018, RT106 was the second-most prevalent molecular type isolated from CDI patients in our surveillance. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed in any other microbial genome; however, smaller segments were detected in select Enterococcus faecium strains. Molecular clock analyses suggest that GI1 was horizontally acquired and sequentially assembled over time. Consistent with the presence of genes encoding homologs of VanZ and a SrtB-anchored collagen-binding adhesin in GI1, all tested RT106 strains had increased teicoplanin resistance and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands have features of mobile genetic elements and encode several putative virulence factors. ConclusionsConsistent with the known genetic plasticity of C. difficile, strains belonging to the RT106 clade harbor unique genetic islands. Correspondingly, emergent phenotypic properties may contribute to the relatively rapid shifts of strain distribution in patient populations.

genomics↗