Biology subjects

Melnyk, R.

Publications and source records attributed to Melnyk, R..

6 recordsLinked to original sources

bioRxiv2026

Bile acid dependent attenuation of toxin mediated disease is independent of colonization resistance against C. difficile

Clostridioides difficile infection (CDI) is a severe antibiotic associated disease and a major cause of morbidity and mortality worldwide. CDI is thought to arise from the loss of protective gut microbes that mediate functions such as secondary bile acid metabolism and nutrient competition, yet the relative contributions of these mechanisms remain unclear. To determine how these processes influence C. difficile growth, virulence, and disease, we performed in vitro and in vivo experiments using two Clostridia strains previously associated with colonization resistance against C. difficile. Neither organism prevented colonization or growth through nutrient competition alone. In contrast, secondary bile acid metabolism significantly reduced toxin-mediated disease in vivo in a strain dependent manner. These findings demonstrate that secondary bile acid modulation is an important component of CDI prevention independent of nutrient competition and suggest that attenuating virulence, in addition to limiting colonization, may represent a key strategy for next-generation CDI therapeutics.

microbiology↗

bioRxiv2024

De novo designed inhibitor confers protection against lethal toxic shock

Paeniclostridium sordellii causes a toxic shock syndrome with a mortality rate of nearly 70%, primarily affecting postpartum and post-abortive women. This disease is driven by the production of the P. sordellii lethal toxin, TcsL, for which there are currently no effective treatments. We used a protein diffusion model, RFdiffusion, to design high affinity TcsL inhibitors. From a very small set of 48 starting designs and 48 additional sequence optimized designs, we developed a potent inhibitor with <100 pM affinity that protects mice prophylactically and therapeutically (post exposure) from lung edema and death in a stringent lethal challenge model. This inhibitor, which can be lyophilized without any loss of activity, is a promising therapeutic candidate for this rare but deadly disease, and our results highlight the ability of deep learning-based protein design to rapidly generate biologics with potential clinical utility.

microbiology↗

bioRxiv2024

Enhanced delivery of protein therapeutics with a diphtheria toxin-like platform that evades pre-existing neutralizing immunity

Targeted intracellular delivery of therapeutic peptides and proteins remains an important but unresolved goal in biotechnology. A promising approach is to engineer bacterial exotoxins that deliver their cytotoxic enzymes into cells and can be engineered to target cancer cells as is the case with immunotoxins. The well-studied diphtheria toxin translocation domain is ideally suited as a delivery platform as it has been shown to be capable of delivering a wide range of macromolecular cargo. Widespread deployment of DT-based therapeutics in humans, however, is complicated by the prevalence of pre-existing anti-DT antibodies from childhood vaccinations that reduce the exposure, efficacy and safety of this important class of protein drugs. Thus, there is a great need for delivery platforms with no pre-existing immunity in humans. Here, we describe the discovery and characterization of a distant diphtheria toxin homolog from the ancient reptile pathogen Austwickia chelonae that we have named Chelona Toxin (CT). We show that CT is comparable to DT structure and function in all respects except that it is not recognized by pre-existing anti-DT antibodies present in human sera. Moreover, we demonstrate that the CT translocase is superior to the DT translocase at delivering therapeutic protein cargo into target cells. These findings highlight CT as a potentially class-enabling new chassis for developing safer and more efficacious immunotoxins and intracellular protein delivery platforms for cancer therapy.

biochemistry↗

bioRxiv2022

Tauroursodeoxycholic acid inhibits Clostridioides difficile toxin induced apoptosis

C. difficile infection (CDI) is a highly inflammatory disease mediated by the production of two large toxins that weaken the intestinal epithelium and cause extensive colonic tissue damage. Antibiotic alternative therapies for CDI are urgently needed as current antibiotic regimens prolong the perturbation of the microbiota and lead to high disease recurrence rates. Inflammation is more closely correlated with CDI severity than bacterial burden, thus therapies that target the host response represent a promising yet unexplored strategy for treating CDI. Intestinal bile acids are key regulators of gut physiology that exert cytoprotective roles in cellular stress, inflammation and barrier integrity, yet the dynamics between bile acids and host cellular processes during CDI have not been investigated. Here we show that several bile acids are protective against apoptosis caused by C. difficile toxins in Caco-2 cells and that protection is dependent on conjugation. Out of 20 tested bile acids, taurine conjugated ursodeoxycholic acid (TUDCA) was the most potent inhibitor yet unconjugated UDCA did not alter toxin-induced apoptosis. TUDCA treatment decreased expression of genes in lysosome associated and cytokine signaling pathways. TUDCA did not affect C. difficile growth or toxin activity in vitro whereas UDCA significantly reduced toxin activity in a Vero cell assay and decreased tcdA gene expression. These results demonstrate that bile acid conjugation can have profound effects on C. difficile as well as the host and that conjugated and unconjugated bile acids may exert different therapeutic mechanisms against CDI.

microbiology↗

bioRxiv2022

Dual inhibition of vacuolar ATPase and TMPRSS2 is required for complete blockade of SARS-CoV-2 entry into cells

An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using non-specific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.

cell biology↗

bioRxiv2019

Identification of the C. sordellii lethal toxin receptor elucidates principles of receptor specificity in clostridial toxins

Clostridium sordellii lethal toxin (TcsL) is responsible for an almost invariably lethal toxic shock syndrome associated with gynecological C. sordellii infections. Here, using CRISPR/Cas9 screening, we identify semaphorins SEMA6A and SEMA6B as the cellular receptors for TcsL and demonstrate that soluble extracellular SEMA6A can protect mice from TcsL-induced edema. A 3.3 [A] cryo-EM structure shows that TcsL binds SEMA6A with the same region that the highly related C. difficile TcdB toxin uses to bind structurally unrelated Frizzled receptors. Remarkably, reciprocal mutations in this evolutionarily divergent surface are sufficient to switch receptor specificity between the toxins. Our findings establish semaphorins as physiologically relevant receptors for TcsL, and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

molecular biology↗