Biology subjects

Pike, C. M.

Publications and source records attributed to Pike, C. M..

8 recordsLinked to original sources

bioRxiv2025

Systematic Discovery of Novel Phosphoinositide-Binding Effectors in Legionella Reveals Conserved α-Helical Folds

Intracellular bacterial pathogens deploy effector proteins to hijack host membranes by targeting specific lipids. Phosphoinositides are key eukaryotic signaling molecules that govern membrane identity and dynamics, making them powerful levers for microbial interference. However, the bacterial repertoire of phosphoinositide-binding effectors remains largely undefined. In this study, we systematically identified such proteins using an expression library of 241 Legionella pneumophila effectors. We applied a combination of lipid bead pulldowns, mass spectrometry, colocalization with phosphoinositide biosensors, and protein-lipid overlay assays, leading to the validation of 18 new phosphoinositide-binding effectors. Structural predictions revealed that these proteins share compact alpha-helical folds distinct from canonical eukaryotic lipid-binding domains. Guided by this structural signature, we performed a computational screen across L. pneumophila, Coxiella burnetii, and Burkholderia pseudomallei. This analysis identified 15 additional effectors, which were experimentally validated as phosphoinositide binders. Altogether, we report 33 previously unrecognized bacterial phosphoinositide-binding effectors. While their lipid-binding profiles were diverse, many effectors showed a preference for compartments enriched in phosphatidylinositol 3-phosphate. These findings expand the known diversity of lipid-binding effectors produced by intracellular pathogens, identify new structural modules bacteria use for phosphoinositide recognition, and broaden our understanding of how pathogens exploit phosphoinositide signaling to manipulate host membranes. ImportanceIntracellular bacterial pathogens hijack host membrane trafficking by targeting phosphoinositides (PIPs), essential eukaryotic signaling lipids. Despite their importance in virulence, bacterial PIP-binding effectors remain largely uncharted because they lack recognizable sequence motifs. Using Legionella pneumophila as a model system, we systematically identified 30 novel PIP-binding effectors, doubling the known repertoire, and discovered that they employ compact -helical folds structurally distinct from canonical eukaryotic PIP-binding domains. Remarkably, these -helical modules are conserved in Coxiella burnetii and Burkholderia pseudomallei, revealing a convergent evolutionary strategy for PIP recognition across phylogenetically diverse pathogens. This work expands our understanding of bacterial lipid-targeting mechanisms and provides a structural blueprint for identifying virulence factors in emerging pathogens.

microbiology↗

bioRxiv2025

Clostridioides difficile Toxins Alter Host Metabolic Pathway and Bile Acid Homeostasis Gene Expression In Colonic Epithelium

A major risk factor for acquiring Clostridioides difficile is antibiotic usage that disrupts a healthy microbial gut community facilitating establishment of infection. Once established, C. difficile secretes endotoxins (TcdA and TcdB) that are internalized into host colonic epithelial cells where they disrupt gut barrier function and induce hyper inflammation resulting in severe diarrhea and possibly leading to death. We employed three different platforms to explore gene expression of cells in the gut when exposed to C. difficile or its toxins, TcdA and TcdB. An antibiotic treated mouse model of C. difficile infection (CDI) was used to identify differential gene expression with a NanoString Technologies mouse inflammatory gene panel consisting of 770 genes including a subset of bile acid (BA) homeostasis and nuclear receptor genes. In the cecal tissue of mice with CDI, significant down expression was observed for genes involved in PPAR signaling, cholesterol and glucose metabolism, while a significant increase in expression was observed for IL-17 related inflammatory genes. Similarly, Caco-2 cell culture and primary human colonocytes (hCE) exposed to toxins for 24 hours showed altered expression in several PPAR regulated and cholesterol metabolic genes similar to those found in mice. These cell culture experiments also revealed significant alterations in gene expression of the FXR BA regulatory pathway. Together these data suggest that exposure to C. difficile and its toxins may alter host cholesterol metabolic processes, including BA transport and synthesis.

microbiology↗

bioRxiv2024

High-Throughput Assay for Predicting Diarrhea Risk Using a 2D Human Intestinal Stem Cell-Derived Model

Gastrointestinal toxicities (GITs) are the most prevalent adverse events (AE) reported in clinical trials, often resulting in dose-limitations that reduce drug efficacy and delay development and treatment optimization. Preclinical animal models do not accurately replicate human GI physiology, leaving few options for early detection of GI side effects prior to human studies. Development of an accurate model that predicts GIT earlier in drug discovery programs would better support successful clinical trial outcomes. Chemotherapeutics, which exhibit high rates of clinical GIT, frequently target mitotic cells. Therefore, we hypothesized that a model utilizing proliferative cell populations derived from human intestinal crypts would predict the occurrence of clinical GITs with high accuracy. Here, we describe the development of a multiparametric assay utilizing the RepliGut(R) Planar system, an intestinal stem cell-derived platform cultured in an accessible high throughput Transwell format. This assay addresses key physiological elements of GIT by assessing cell proliferation (EdU incorporation), cell abundance (DAPI quantification), and barrier function (TEER). Using this approach, we demonstrate that primary proliferative cell populations reproducibly respond to marketed chemotherapeutics at physiologic concentrations. To determine the ability of this model to predict clinical diarrhea risk, we evaluated a set of 30 drugs with known clinical diarrhea incidence in three human donors, comparing results to known plasma drug concentrations. This resulted in highly accurate predictions of diarrhea potential for each endpoint (balanced accuracy of 91% for DAPI, 90% for EdU, 88% for TEER) with minimal variation across human donors. In vitro toxicity screening using primary proliferative cells may enable improved safety evaluations, reducing the risk of AEs in clinical trials and ultimately lead to safer and more effective treatments for patients.

pharmacology and toxicology↗

bioRxiv2023

Characterization and optimization of variability in a human colonic epithelium culture model

Animal models have historically been poor preclinical predictors of gastrointestinal (GI) directed therapeutic efficacy and drug-induced GI toxicity. Human stem and primary cell-derived culture systems are a major focus of efforts to create biologically relevant models that enhance preclinical predictive value of intestinal efficacy and toxicity. The inherent variability in stem-cell-based complex cultures makes development of useful models a challenge; the stochastic nature of stem-cell differentiation interferes with the ability to build and validate robust, reproducible assays that query drug responses and pharmacokinetics. In this study, we aimed to characterize and reduce potential sources of variability in a complex stem cell-derived intestinal epithelium model, termed RepliGut(R) Planar, across cells from multiple human donors, cell lots, and passage numbers. Assessment criteria included barrier formation and integrity, gene expression, and cytokine responses. Gene expression and culture metric analyses revealed that controlling for stem/progenitor-cell passage number reduces variability and maximizes physiological relevance of the model. After optimizing passage number, donor-specific differences in cytokine responses were observed in a case study, suggesting biologic variability is observable in cell cultures derived from multiple human sources. Our findings highlight key considerations for designing assays that can be applied to additional primary-cell derived systems, as well as establish utility of the RepliGut(R) Planar platform for robust development of human-predictive drug-response assays.

cell biology↗

bioRxiv2023

The microbial derived bile acid lithocholate and its epimers inhibit Clostridioides difficile growth and pathogenicity while sparing members of the gut microbiota

C. difficile infection (CDI) is associated with antibiotic usage, which disrupts the indigenous gut microbiota and causes the loss of microbial derived secondary bile acids that normally provide protection against C. difficile colonization. Previous work has shown that the secondary bile acid lithocholate (LCA) and its epimer isolithocholate (iLCA) have potent inhibitory activity against clinically relevant C. difficile strains. To further characterize the mechanisms by which LCA and its epimers iLCA and isoallolithocholate (iaLCA) inhibit C. difficile, we tested their minimum inhibitory concentration (MIC) against C. difficile R20291, and a commensal gut microbiota panel. We also performed a series of experiments to determine the mechanism of action by which LCA and its epimers inhibit C. difficile through bacterial killing and effects on toxin expression and activity. Here we show that epimers iLCA and iaLCA strongly inhibit C. difficile growth in vitro while sparing most commensal Gram-negative gut microbes. We also show that iLCA and iaLCA have bactericidal activity against C. difficile, and these epimers cause significant bacterial membrane damage at subinhibitory concentrations. Finally, we observe that iLCA and iaLCA decrease the expression of the large cytotoxin tcdA while LCA significantly reduces toxin activity. Although iLCA and iaLCA are both epimers of LCA, they have distinct mechanisms for inhibiting C. difficile. LCA epimers, iLCA and iaLCA, represent promising compounds that target C. difficile with minimal effects on members of the gut microbiota that are important for colonization resistance. ImportanceIn the search for a novel therapeutic that targets C. difficile, bile acids have become a viable solution. Epimers of bile acids are particularly attractive as they may provide protection against C. difficile while leaving the indigenous gut microbiota largely unaltered. This study shows that iLCA and iaLCA specifically are potent inhibitors of C. difficile, affecting key virulence factors including growth, toxin expression and activity. As we move toward the use of bile acids as therapeutics, further work will be required to determine how best to deliver these bile acids to a target site within the host intestinal tract.

microbiology↗

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↗

bioRxiv2021

Legionella pneumophila targets autophagosomes and promotes host autophagy during late infection

Autophagy is a fundamental eukaryotic process that mediates clearance of unwanted molecules and facilitates nutrient release. The bacterial pathogen Legionella pneumophila establishes an intracellular niche within phagocytes by manipulating host cellular processes, such as autophagy. Effector proteins translocated by L. pneumophilas Dot/Icm type IV secretion system have been shown to suppress autophagy. However evidence suggests that overall inhibition of autophagy may be detrimental to the bacterium. As autophagy contributes to cellular homeostasis and nutrient acquisition, L. pneumophila may translocate effectors that promote autophagy for these benefits. Here, we show that effector protein Lpg2411 binds phosphatidylinositol-3-phosphate lipids and preferentially binds autophagosomes. Translocated Lpg2411 accumulates late during infection and co-localizes with the autophagy receptor p62 and ubiquitin. Furthermore, autophagy is inhibited to a greater extent in host cells infected with a mutant strain lacking Lpg2411 compared to those infected with wild-type L. pneumophila, indicating that Lpg2411 stimulates autophagy to support the bacteriums intracellular lifestyle. SummaryLegionella pneumophila translocates several effector proteins that inhibit autophagic processes. In this study, we find that the effector protein Lpg2411 targets autophagosomes during late stages of infection and promotes autophagy.

microbiology↗

bioRxiv2020

Clostridioides difficile exploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota

Introductory paragraphClostridioides difficile is a bacterial pathogen that causes a range of clinical disease from mild to moderate diarrhea, pseudomembranous colitis, and toxic megacolon. Typically, C. difficile infections (CDIs) occur after antibiotic treatment, which alters the gut microbiota, decreasing colonization resistance against C. difficile. Disease is mediated by two large toxins and the expression of their genes is induced upon nutrient depletion via the alternative sigma factor TcdR. Using tcdR mutants in two strains of C. difficile, we defined how toxin-induced inflammation alters C. difficile metabolism, tissue gene expression, and the gut microbiota to determine how inflammation by the host may be beneficial to C. difficile. Here we show that C. difficile metabolism is significantly different in the face of inflammation, with changes in many carbohydrate and amino acid uptake and utilization pathways. Host gene expression signatures suggest that degradation of collagen and other components of the extracellular matrix by matrix metalloproteinases is a major source of peptides and amino acids that supports C. difficile growth in vivo. Lastly, the inflammation induced by C. difficile toxin activity alters the gut microbiota, excluding members from the genus Bacteroides that are able to compete against C. difficile for the same essential nutrients released from collagen degradation.

microbiology↗