Biology subjects

Zackular, J. P.

Publications and source records attributed to Zackular, J. P..

8 recordsLinked to original sources

bioRxiv2026

Generation and characterization of a novel MHC-II tetramer for tracking and characterization of toxin B-specific CD4+ T cell responses

The gastrointestinal pathogen Clostridioides difficile, is a major burden for health systems due to high rates of recurrence. C. difficile pathogenesis is mediated by two virulence factors, toxin A (TcdA) and Toxin B (TcdB). Antibodies specific for TcdA and TcdB are correlated with protection from symptomatic recurrence, however, the role for CD4+ T cells is poorly understood in part due to the lack of tools to study the toxin-specific CD4+ T cell response. Our group recently demonstrated the antibody and CD4+ T cell response to C. difficile toxins is impaired via the glucosyltransferase activity of the toxins; however, tools do not exist to study the protective capacity and the phenotype of toxin-specific CD4+ T cells. Therefore, we developed an MHC-II tetramer to identify TcdB-specific CD4+ T cells via flow cytometry. Herein, we identified an immunodominant epitope (TcdB1961-1975) in the CROPs region of TcdB and optimized an MHC-II tetramer for use in tracking and phenotyping TcdB-specific CD4+ T cell responses following multiple different immunization strategies in mice. Utilizing the tetramer, TcdB-specific T follicular helper (Tfh) cells were detected following TcdB-CROPs mRNA-LNP vaccination validating the advantage of the tetramer. Furthermore, using a modular mRNA vector expressing the TcdB1961 peptide covalently bound to the beta chain of MHC-II (MHC-II{beta}) we were able to generate a robust population of TcdB-specific CD4+ T cells. These data outline the generation of new tools for the C. difficile field and lay the groundwork for future studies of toxin-specific CD4+ T cell responses.

immunology↗

bioRxiv2025

Pharmacological reduction of neutrophil infiltration reduces Clostridioides difficile infection severity

Clostridioides difficile is the leading cause of nosocomial infections and an urgent public health threat. This bacterial pathogen is challenging to treat due to antibiotic resistance and high recurrence rates, highlighting the need for additional therapeutic strategies. The host inflammatory response is a major driver of C. difficile-associated disease and associated with worse clinical outcomes. Currently, few strategies targeting the inflammatory response have been leveraged to treat CDI. Here, we show that administration of the prostaglandin E1 (PGE1) analog misoprostol markedly reduces CDI severity by modulating host immune responses. During CDI, misoprostol decreases circulating neutrophils and limits infiltration into the colon, reducing epithelial damage, intestinal pathology, and infection severity. Additionally, misoprostol reduces serum granulocyte colony-stimulating factor (G-CSF), an important cytokine in neutrophil mobilization, controlling neutrophil levels during CDI. Together, these findings highlight neutrophil infiltration as a key driver of C. difficile-associated disease and identify innate immune modulation as a potential host-directed therapeutic strategy.

immunology↗

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↗

bioRxiv2025

Enterococcus faecalis modulates phase variation in Clostridioides difficile

To adapt and persist in the gastrointestinal tract, many enteric pathogens, including Clostridioides difficile, employ strategies such as phase variation to generate phenotypically heterogeneous populations. Notably, the role of the gut microbiota and polymicrobial interactions in shaping population heterogeneity of invading pathogens has not been explored. Here, we show that Enterococcus faecalis, an opportunistic pathogen that thrives in the inflamed gut during C. difficile infection, can impact the phase variable CmrRST signal transduction system in C. difficile. The CmrRST system controls multiple phenotypes including colony morphology, cell elongation, and cell chaining in C. difficile. Here we describe how interactions between E. faecalis and C. difficile on solid media lead to a marked shift in C. difficile phenotypes associated with phase variation of CmrRST. Specifically, E. faecalis drives a switch of the C. difficile population to the cmr-ON state leading to chaining and a rough colony morphology. This phenomenon preferentially occurs with E. faecalis among the enterococci, as other enterococcal species do not show a similar effect, suggesting that the composition of the polymicrobial environment in the gut is likely critical to shaping C. difficile population heterogeneity. Our findings shed light on the complex role that microbial ecology and polymicrobial interactions can have in the phenotypic heterogeneity of invading pathogens. IMPORTANCE STATEMENTClostridioides difficile is an enteric pathogen with critical implications for public health. The microbial ecosystem in which C. difficile resides shapes the behavior and fitness of C. difficile; however, the mechanisms underlying these interactions are not well defined. Here, we demonstrate that Enterococcus faecalis, an opportunistic pathogen known to co-colonize the gut with C. difficile, influences phase variation and downstream growth phenotypes in C. difficile. This phenomenon represents a new paradigm by which co-residing bacteria can modulate phase variation dynamics in C. difficile or other enteric pathogens. Understanding factors that influence C. difficile behavior may elucidate new therapeutic strategies, especially in complex polymicrobial infections.

microbiology↗

bioRxiv2025

Carbohydrate consumption drives adaptive mutations in Escherichia coli associated with increased risk for systemic infection

Dissemination of organisms from the gut microbiota is a major contributor to sepsis and critical illness. Patients with cirrhosis are prone to systemic infections and are commonly prescribed the carbohydrate lactulose to manage hepatic encephalopathy (HE) 1. Commensal metabolism of lactulose is believed to reduce pathobiont colonization through short-chain fatty acid production, but its direct effects on gut pathobionts remain unexplored 2. Here, we show that lactulose consumption unexpectedly selects for mutations in Escherichia coli lactose (lac) operon regulation, enhancing its metabolic fitness and colonization capacity. This is mediated by selection for constitutive expression of the lac operon through mutations in its regulatory components. Using in vitro systems, murine models, and clinical samples, we demonstrate that these mutations enable E. coli to exploit lactulose as a carbon source, bypassing host carbohydrate metabolism and increasing its intestinal colonization. Despite its long-standing use in HE treatment, we find that lactulose has a paradoxical association with risk of infection hospitalization in patients with cirrhosis in a large epidemiologic study. The emergence of lactulose-adapted E. coli strains could be suppressed by a dietary oligosaccharide that competitively inhibits lactulose uptake. These findings reveal a mechanism by which dietary substrates exert selective pressure on the microbiome, with implications for diet-based strategies to modulate microbial evolution and infection risk.

microbiology↗

bioRxiv2025

A Novel Heme-Degrading Enzyme that Regulates Heme and Iron Homeostasis and Promotes Virulence in Enterococcus faecalis

Enterococcus faecalis, a gut commensal, is a leading cause of opportunistic infections. Its virulence is linked to its ability to thrive in hostile environments, which includes host-imposed metal starvation. We recently showed that E. faecalis evades iron starvation using five dedicated transporters that collectively scavenge iron from host tissues and other iron-deprived conditions. Interestingly, heme, the most abundant source of iron in the human body, supported growth of a strain lacking all five iron transporters ({Delta}5Fe). To release iron from heme, many bacterial pathogens utilize heme oxygenase enzymes to degrade the porphyrin that coordinates the iron ion of heme. Although E. faecalis lacks these enzymes, bioinformatics revealed a potential ortholog of the anaerobic heme-degrading enzyme anaerobilin synthase, found in Escherichia coli and a few other Gram-negative bacteria. Here, we demonstrated that deletion of OG1RF_RS05575 in E. faecalis ({Delta}RS05575) or in the {Delta}5Fe background ({Delta}5Fe{Delta}RS05575) led to intracellular heme accumulation and hypersensitivity under anaerobic conditions, suggesting RS05575 encodes an anaerobilin synthase, the first of its kind described in Gram- positive bacteria. Additionally, deletion of RS05575, either alone or in the {Delta}5Fe background, impaired E. faecalis colonization in the mouse gastrointestinal tract and virulence in mouse peritonitis and rabbit infective endocarditis models. These results reveal that RS05575 is responsible for anaerobic degradation of heme and identify this relatively new enzyme class as a novel factor in bacterial pathogenesis. Findings from this study are likely to have broad implications, as homologues of RS05575 are found in other Gram-positive facultative anaerobes. IMPORTANCEHeme is an important nutrient for bacterial pathogens, mainly for its ability to serve as an iron source during infection. While bacteria are known to release iron from heme using enzymes called heme oxygenases, a new family of anaerobic heme-degrading enzymes has been described recently in Gram-negative bacteria. Here, we report the first description of anaerobic heme degradation by a Gram-positive bacterium, the opportunistic pathogen Enterococcus faecalis, and link activity of this enzyme to their ability to colonize and infect the host. We also show that homologues of this enzyme are found in many Gram-positive facultative anaerobes, implying that the ability to degrade heme under anaerobic conditions may be an overlooked fitness and virulence factor of bacterial pathogens.

microbiology↗

bioRxiv2024

Amino acid competition shapes Acinetobacter baumannii gut carriage

Antimicrobial resistance is an urgent threat to human health. Asymptomatic colonization is often critical for persistence of antimicrobial-resistant pathogens. Gut colonization by the antimicrobial-resistant priority pathogen Acinetobacter baumannii is associated with increased risk of clinical infection. Ecological factors shaping A. baumannii gut colonization remain unclear. Here we show that A. baumannii and other pathogenic Acinetobacter evolved to utilize the amino acid ornithine, a non- preferred carbon source. A. baumannii utilizes ornithine to compete with the resident microbiota and persist in the gut in mice. Supplemental dietary ornithine promotes long-term fecal shedding of A. baumannii. By contrast, supplementation of a preferred carbon source--monosodium glutamate (MSG)-- abolishes the requirement for A. baumannii ornithine catabolism. Additionally, we report evidence for diet promoting A. baumannii gut carriage in humans. Together, these results highlight that evolution of ornithine catabolism allows A. baumannii to compete with the microbiota in the gut, a reservoir for pathogen spread.

microbiology↗

bioRxiv2018

Nonsteroidal anti-inflammatory drugs alter the microbiota and exacerbate Clostridium difficile colitis while dysregulating the inflammatory response

Clostridium difficile infection (CDI) is a major public health threat worldwide. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with enhanced susceptibility to and severity of nosocomial CDI; however, the mechanisms driving this phenomenon have not been elucidated. NSAIDs alter prostaglandin (PG) metabolism by inhibiting cyclooxygenase (COX) enzymes. Here, we found that treatment with the NSAID indomethacin prior to infection altered the microbiota and dramatically increased mortality and intestinal pathology associated with CDI in mice. We demonstrate that in C. difficile-infected animals, indomethacin lead to PG deregulation, an altered proinflammatory transcriptional and protein profile, and perturbed epithelial cell junctions. These effects were paralleled by an increased recruitment of intestinal neutrophils and CD4+ cells. Together, these data implicate NSAIDs in perturbation of the gut microbiota and disruption of protective COX-mediated PG production during CDI, resulting in altered epithelial integrity and associated immune responses.

microbiology↗