Biology subjects

Kordus, S. L.

Publications and source records attributed to Kordus, S. L..

5 recordsLinked to original sources

bioRxiv2025

A targeted cell lysis mechanism facilitates toxin release in Clostridioides difficile

Clostridioides difficile infection depends on the production of two large toxins, TcdA and TcdB, encoded within a pathogenicity locus alongside the phage-like holin TcdE. The mechanism of toxin secretion remains actively debated, with current models proposing either TcdE-dependent non-lytic secretion or TcdE-independent lytic release. Here, we provide evidence for a unifying model where TcdE drives lysis in a phenotypically distinct subpopulation of cells. We show that TcdE, TcdA, and TcdB expression is restricted to a small fraction of cells exhibiting markers of active lysis and establish this subpopulation as the driver of severe pathogenic outcomes in a mouse model of CDI. Overexpression of TcdR, the sigma factor regulating the pathogenicity locus, triggers TcdE-dependent lysis, even in strains previously reported to employ TcdE-independent secretion mechanisms. Correlative light and electron microscopy combined with cryo-ET reveal a distinctive ultrastructure in lytic cells. Membrane vesicles accumulate between a disrupted inner membrane and intact peptidoglycan, alongside electron-dense material containing TcdA. These observations reveal a population-level strategy in which a minority of bacteria sacrifice themselves through TcdE-mediated lysis to release toxins as a 'bet-hedging strategy'.

microbiology↗

bioRxiv2025

Essential Role of MHC II in the Antitubercular Efficacy of Pyrazinamide

Antibacterial drug mechanisms have traditionally been examined through a drug-pathogen lens, often overlooking the hosts role in shaping drug activity. However, growing evidence suggests that the host environment is crucial for antibacterial efficacy. Pyrazinamide (PZA), a key component of modern tuberculosis therapy, exemplifies this complexity--exhibiting potent in vivo activity despite its inability to reduce Mycobacterium tuberculosis viability in standard in vitro culture. Here, using macrophage and murine infection models, we identify a critical role for host cell-mediated immunity in PZAs antitubercular action. Through the use of MHC II knockout mice, we demonstrate that CD4 T cell help is essential for PZA efficacy. Notably, while IFN-{gamma} is required for PZA-mediated clearance of M. tuberculosis at extrapulmonary sites, bacterial reduction in the lungs occurs independently of IFN-{gamma} signaling. Additionally, we show that PZA leverages cell-mediated immunity in part through activation of the oxidative burst. Our findings underscore the need to incorporate host factors into antibacterial drug evaluation and highlight potential avenues for host-directed therapies and adjunctive antibiotics in first- and second-line tuberculosis treatment.

microbiology↗

bioRxiv2023

Nanobodies against C. difficile TcdA and TcdB reveal unexpected neutralizing epitopes and provide a toolkit for toxin quantitation in vivo

Clostridioides difficile is a leading cause of antibiotic-associated diarrhea and nosocomial infection in the United States. The symptoms of C. difficile infection (CDI) are associated with the production of two homologous protein toxins, TcdA and TcdB. The toxins are considered bona fide targets for clinical diagnosis as well as the development of novel prevention and therapeutic strategies. While there are extensive studies that document these efforts, there are several gaps in knowledge that could benefit from the creation of new research tools. First, we now appreciate that while TcdA sequences are conserved, TcdB sequences can vary across the span of circulating clinical isolates. An understanding of the TcdA and TcdB epitopes that drive broadly neutralizing antibody responses could advance the effort to identify safe and effective toxin-protein chimeras and fragments for vaccine development. Further, an understanding of TcdA and TcdB concentration changes in vivo can guide research into how host and microbiome-focused interventions affect the virulence potential of C. difficile. We have developed a panel of alpaca-derived nanobodies that bind specific structural and functional domains of TcdA and TcdB. We note that many of the potent neutralizers of TcdA bind epitopes within the delivery domain, a finding that could reflect roles of the delivery domain in receptor binding and/or the conserved role of pore-formation in the delivery of the toxin enzyme domains to the cytosol. In contrast, neutralizing epitopes for TcdB were found in multiple domains. The nanobodies were also used for the creation of sandwich ELISA assays that allow for quantitation of TcdA and/or TcdB in vitro and in the cecal and fecal contents of infected mice. We anticipate these reagents and assays will allow researchers to monitor the dynamics of TcdA and TcdB production over time, and the impact of various experimental interventions on toxin production in vivo. Author SummaryC. difficile (C. diff) is a leading cause of diarrhea and is recognized as an urgent threat by the Centers for Disease Control. Disease symptoms are caused by two large, similar, protein toxins, TcdA and TcdB. These toxins are drug targets and are also important for diagnosis. Despite their clear importance, the understanding of how to neutralize toxin activity is incomplete, and there are no freely available tools to quantify toxin concentration in research studies. To address these issues, we have developed nanobodies that bind and neutralize TcdA and TcdB and have also used these nanobodies to develop quantitative assays for TcdA and TcdB detection. Neutralization studies led us to discover that many of the potent neutralizers of TcdA bind epitopes within the delivery domain. This finding suggests either a role for the delivery domain in receptor binding or that the nanobodies block pore-formation and thereby inhibit delivery of the toxin enzyme domains to the cytosol. The availability of nanobody assays that can differentiate the quantities of TcdA from TcdB should permit a better understanding of toxin-specific effects and how toxin levels change over the course of infection.

microbiology↗

bioRxiv2020

Mechanism of selectivity reveals novel antifolate drug interactions

Antimicrobial agents that target a specific pathogen of interest is the gold standard in drug design. para-Aminosalicylic acid (PAS), remains a cornerstone therapy, in the treatment against Mycobacterium tuberculosis, owing to its high level of selectivity. Despite its high level of selectivity, PAS has been reassigned to treat drug-resistant strains of M. tuberculosis because it causes severe gastrointestinal (GI) distress that results in poor patient compliance. We have previously shown PAS inhibits the folate biosynthetic pathway specifically inhibiting dihydrofolate reductase1,2. In this study, we sought to determine the mechanistic basis of PAS selectivity and determined that PAS can be utilized in folate biosynthesis by other bacterial pathogens. The utilization of PAS ultimately led to the antagonism of key antibiotics, specifically the sulfonamides, used to prophylactically treat individuals with HIV-AIDS3. In addition, we found many bacteria in the GI tract could also utilize PAS to make a hydroxy-folate species which resulted in GI toxicity. Using sulfonamides as a tool to prevent PAS associated toxicity in the GI tract, we discovered that the sulfonamides antagonized the antimycobacterial activity of PAS. These findings indicate a new need for understanding the mechanisms of selective therapies and more important, that HIV-AIDS/M. tuberculosis co-infected individuals should avoid co-treatment of PAS and sulfonamides.

microbiology↗

bioRxiv2018

Methionine antagonizes para-aminosalicylic acid activity via affecting folate precursor biosynthesis pathway in Mycobacterium tuberculosis

para-Aminosalicylic acid (PAS) is a second-line anti-tubercular drug that is used for the treatment of drug-resistant tuberculosis (TB). PAS efficacy in the treatment of TB is limited by its lower potency against Mycobacterium tuberculosis relative to many other drugs in the TB treatment arsenal. It is known that intrinsic metabolites, such as para-aminobenzoic acid (PABA) and methionine, antagonize PAS and structurally related anti-folate drugs. While the basis for PABA-mediated antagonism of anti-folates is understood, the mechanism for methionine-based antagonism remains undefined. In the present study, we used both targeted and untargeted approaches to identify factors associated with methionine-mediated antagonism of PAS activity. We found that synthesis of folate precursors as well as a putative amino acid transporter play crucial roles in this process. We also discovered that intracellular biotin confers intrinsic PAS resistance in a methionine-independent manner. Collectively, our results demonstrate that methionine-mediated antagonism of anti-folate drugs occurs through sustained production of folate precursors.

microbiology↗