Biology subjects

Carothers, K. E.

Publications and source records attributed to Carothers, K. E..

4 recordsLinked to original sources

bioRxiv2022

A superoxide reductase contributes to Clostridioides difficile resistance to oxygen

Clostridioides difficile causes a serious diarrheal disease and is a common healthcare-associated bacterial pathogen. Although it has a major impact on human health, mechanistic details of C. difficile intestinal colonization remain undefined. C. difficile is highly sensitive to oxygen and requires anaerobic conditions for in vitro growth. However, the mammalian gut is not entirely devoid of oxygen, and C. difficile tolerates moderate oxidative stress in vivo. The C. difficile genome encodes several antioxidant proteins, including a predicted superoxide reductase (SOR) that is upregulated upon exposure to antimicrobial peptides. The goal of this study was to establish SOR enzymatic activity and assess its role in protecting C. difficile against oxygen exposure. Insertional inactivation of sor rendered C. difficile more sensitive to superoxide indicating that SOR contributes to antioxidant defense. Heterologous C. difficile sor expression in Escherichia coli conferred protection against superoxide-dependent growth inhibition, and the corresponding cell lysates showed superoxide scavenging activity. Finally, a C. difficile SOR mutant exhibited global proteome changes under oxygen stress when compared to its parent strain. Collectively, our data establish the enzymatic activity of C. difficile SOR, confirm its role in protection against oxidative stress, and demonstrate its broader impacts on the vegetative cell proteome. ImportanceClotridioides difficile is an important pathogen strongly associated with healthcare settings and capable of causing severe diarrheal disease. While considered a strict anaerobe in vitro, C. difficile has been shown to tolerate low levels of oxygen in its mammalian host. Among other well-characterized antioxidant proteins, the C. difficile genome includes a predicted superoxide reductase (SOR), an understudied component of antioxidant defense in pathogens. The significance of the research reported herein is the characterization of the enzymatic activity of the putative SOR protein, including confirmation of its role in protection of C. difficile against oxidative stress. This furthers our understanding of C. difficile pathogenesis and presents a potential new avenue for targeted therapies.

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↗

bioRxiv2019

Discovery of genes encoding a Streptolysin S-like toxin biosynthetic cluster in a select highly pathogenic methicillin resistant Staphylococcus aureus JKD6159 strain

BackgroundStaphylococcus aureus (S. aureus) is a major human pathogen owing to its arsenal of virulence factors, as well as its acquisition of multi-antibiotic resistance. Here we report the identification of a Streptolysin S (SLS) like biosynthetic gene cluster in a highly virulent community-acquired methicillin resistant S. aureus (MRSA) isolate, JKD6159. Examination of the SLS-like gene cluster in JKD6159 shows significant homology and gene organization to the SLS-associated biosynthetic gene (sag) cluster responsible for the production of the major hemolysin SLS in Group A Streptococcus.\n\nResultsWe took a comprehensive approach to elucidating the putative role of the sag gene cluster in JKD6159 by constructing a mutant in which one of the biosynthesis genes (sagB homologue) was deleted in the parent JKD6159 strain. Assays to evaluate bacterial gene regulation, biofilm formation, antimicrobial activity, as well as complete host cell response profile and comparative in vivo infections in Balb/Cj mice were conducted.\n\nConclusionsAlthough no significant phenotypic changes were observed in our assays, we postulate that the SLS-like toxin produced by this strain of S. aureus may be a highly specialized virulence factor utilized in specific environments for selective advantage; studies to better understand the role of this newly discovered virulence factor in S. aureus warrant further investigation.

microbiology↗

bioRxiv2018

Novel antimicrobial peptide discovery using machine learning and biophysical selection of minimal bacteriocin domains

Bacteriocins are ribosomally produced antimicrobial peptides that represent an untapped source of promising antibiotic alternatives. However, inherent challenges in isolation and identification of natural bacteriocins in substantial yield have limited their potential use as viable antimicrobial compounds. In this study, we have developed an overall pipeline for bacteriocin-derived compound design and testing that combines sequence-free prediction of bacteriocins using a machine-learning algorithm and a simple biophysical trait filter to generate minimal 20 amino acid peptide candidates that can be readily synthesized and evaluated for activity. We generated 28,895 total 20-mer peptides and scored them for charge, -helicity, and hydrophobic moment, allowing us to identify putative peptide sequences with the highest potential for interaction and activity against bacterial membranes. Of those, we selected sixteen sequences for synthesis and further study, and evaluated their antimicrobial, cytotoxicity, and hemolytic activities. We show that bacteriocin-based peptides with the overall highest scores for our biophysical parameters exhibited significant antimicrobial activity against E. coli and P. aeruginosa. Our combined method incorporates machine learning and biophysical-based minimal region determination, to create an original approach to rapidly discover novel bacteriocin candidates amenable to rapid synthesis and evaluation for therapeutic use.

microbiology↗