Biology subjects

Lacy, B.

Publications and source records attributed to Lacy, B..

6 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

Clostridioides difficile toxin A and toxin B inhibit toxin-specific adaptive immune responses through glucosyltransferase-dependent activity

Clostridioides difficile colonizes the gastrointestinal tract and secretes two virulence factors: toxin A (TcdA) and toxin B (TcdB). Protective immunity against C. difficile infection is limited as patients are susceptible to multiple rounds of recurrent infections. The factors determining whether immunity to TcdA and TcdB is generated remain incompletely defined. We determined that C. difficile-infected mice generate antibody and IL-17A-producing CD4+ T cell responses to TcdA, but not TcdB. To determine the mechanism of the failed anti-TcdB immunity, C. difficile mutant strains expressing glucosyltransferase inactive (GTX) TcdA, and/or glucosyltransferase inactive TcdB were used. Infection with TcdBGTX or dual mutant (TcdAGTX TcdBGTX) restored TcdB-specific antibody responses, while infection with TcdAGTX or TcdAGTX TcdBGTX led to an earlier induction of TcdA-specific antibodies. Finally, infection with the dual GTX mutant enhanced TcdA and TcdB-specific CD4+ T cell responses. These data demonstrate that the glucosyltransferase activity of TcdA and TcdB hinders the antigen-specific adaptive immune response to itself and may be a mechanism that underlies high recurrence rates following C. difficile infection in patients.

immunology↗

bioRxiv2021

Strain variation in Clostridioides difficile toxin activity associated with genomic variation at both PaLoc and non-PaLoc loci

Clinical disease from Clostridioides difficile infection can be mediated by two toxins and their neighboring regulatory genes encoded within the five-gene pathogenicity locus (PaLoc). We provide several lines of evidence that the toxin activity of C. difficile may be modulated by genomic variants outside of the PaLoc. We used a phylogenetic tree-based approach to demonstrate discordance between toxin activity and PaLoc evolutionary history, an elastic net method to show the insufficiency of PaLoc variants alone to model toxin activity, and a convergence-based bacterial genome-wide association study (GWAS) to identify correlations between non-PaLoc loci with changes in toxin activity. Combined, these data support a model of C. difficile disease wherein toxin activity may be strongly affected by many non-PaLoc loci. Additionally, we characterize multiple other in vitro phenotypes relevant to human infections including germination and sporulation. These phenotypes vary greatly in their clonality, variability, convergence, and concordance with genomic variation. Lastly, we highlight the intersection of loci identified by GWAS for different phenotypes and clinical severity. This strategy to identify the overlapping loci can facilitate the identification of genetic variation linking phenotypic variation to clinical outcomes. IMPORTANCEClostridioides difficile has two major disease mediating toxins, A and B, encoded within the pathogenicity locus (PaLoc). In this study we demonstrate via multiple approaches that genomic variants outside of the PaLoc are associated with changes in toxin activity. These genomic variants may provide new avenues of exploration in the hunt for novel disease modifying interventions. Additionally, we provide insight into the evolution of several additional phenotypes also critical to clinical infection such as sporulation, germination, and growth rate. These in vitro phenotypes display a range of responses to evolutionary pressures and as such vary in their appropriateness for certain bacterial genome wide association study approaches. We used a convergence-based association method to identify the genomic variants most correlated with both changes in these phenotypes and disease severity. These overlapping loci may be important to both bacterial function and human clinical disease.

microbiology↗

bioRxiv2021

E-cadherin redistribution mediated by Clostridioides difficile toxins increases spore-association to adherens junctions

Clostridioides difficile causes antibiotic-associated diseases in humans ranging from mild diarrhea to severe pseudomembranous colitis and death. A major clinical challenge is the prevention of disease recurrence, which affects nearly [~]20 - 30 % of the patients with a primary C. difficile infection (CDI). During CDI, C. difficile forms metabolically dormant spores that are essential for recurrence of CDI (R-CDI). In prior studies, we have shown that C. difficile spores interact with intestinal epithelial cells (IECs), which contributes to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore-adherence and internalization into IECs. C. difficile toxins TcdA/TcdB lead to adherens junctions opening and increase spore-adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA/TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By ELISA, immunofluorescence, and immunogold labelling, we observed that E-cadherin binds to C. difficile spores, specifically to the hair-like projections of the spore, reducing spore-adherence to IECs. Overall, these results expand our knowledge of how C. difficile spores bind to IECs by providing evidence that E-cadherin acts as a spore-adherence receptor to IECs and by revealing how toxin-mediated damage affects spore interactions with IECs.

microbiology↗

bioRxiv2020

Cryo-EM reveals species-specific components within the Helicobacter pylori Cag type IV secretion system core complex

The pathogenesis of Helicobacter pylori-associated gastric cancer is dependent on delivery of CagA into host cells through a type IV secretion system (T4SS). The H. pylori Cag T4SS includes a large membrane-spanning core complex containing 5 proteins, organized into an outer membrane cap (OMC), a periplasmic ring (PR) and a stalk. Here, we report cryo-EM reconstructions of a core complex lacking Cag3 and an improved map of the wild-type complex. We define the structures of two unique species-specific components (Cag3 and CagM) and show that Cag3 is structurally similar to CagT. Unexpectedly, components of the OMC are organized in a 1:1:2:2:5 molar ratio (CagY:CagX:CagT:CagM:Cag3). CagX and CagY are components of both the OMC and the PR and bridge the symmetry mismatch between these regions. These results reveal that assembly of the H. pylori T4SS core complex is dependent on incorporation of interwoven species-specific components.

microbiology↗

bioRxiv2020

Structural analysis of the Legionella pneumophila Dot/Icm Type IV Secretion System Core Complex

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia Legionnaires Disease. This infection and subsequent pathology require the Dot/Icm Type IV Secretion System (T4SS) to deliver effector proteins into host cells. Compared to prototypical T4SSs, the Dot/Icm assembly is much larger, containing ~27 different components including a core complex reported to be composed of five proteins: DotC, DotD, DotF, DotG, and DotH. Using single particle cryo-electron microscopy (cryo-EM), we report reconstructions of the core complex of the Dot/Icm T4SS that includes a symmetry mismatch between distinct structural features of the outer membrane cap (OMC) and periplasmic ring (PR). We present models of known core complex proteins, DotC, DotD, and DotH, and two structurally similar proteins within the core complex, DotK and Lpg0657. This analysis reveals the stoichiometry and contact interfaces between the key proteins of the Dot/Icm T4SS core complex and provides a framework for understanding a complex molecular machine.

microbiology↗