Hao-Sen Chiang

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in piglets, imposing significant economic burdens on swine production worldwide. Despite its impact, detailed characterization of intestinal colonization and comprehensive genomic profiling of virulent neonatal porcine ETEC strains remain limited. In this study, we isolated a β‐hemolytic ETEC strain, PT4357, from a diarrheic neonatal piglet and conducted whole‐genome sequencing (WGS) coupled with experimental infection in neonatal piglets to define its pathogenicity. The PT4357 belonged to serotype O8:H7 and sequence type ST2521 and harbored multiple virulence and antimicrobial resistance (AMR) genes, including the plasmid‐borne colistin resistance gene mcr-1 . Interestingly, a novel chaperone‐usher (CU) assembly pathway pilus has a homolog of the α family usher gene that was identified in the chromosome of PT4357. Compared with the CS1 pilus on ETEC, structural superposition of the CU pathway pilus of PT4357, namely, CS33, showed strong conservation in the major subunit and chaperone and was relatively divergent in the N‐terminal of usher and minor subunits while retaining essential core folds. Oral inoculation confirmed villi attachment of the ETEC PT4357, causing watery diarrhea in neonatal piglets without mucosal damage. Furthermore, the adherence of the ETEC PT4357 on porcine IPEC cells but not human Caco‐2 cells suggests limited human tropism. Our results indicate that Taiwan porcine ETECs carried AMR genes and alternative CU pathway pilus. The role of the CS33 pilus on the pathogenicity or virulence of the pathogen requires further investigation and surveillance.

Candida albicans is a major human fungal pathogen; however, limited antifungal agents, undesirable drug side effects, and ineffective prevention of drug-resistant strains have become serious problems. Chitosan is a nontoxic, biodegradable, and biocompatible linear polysaccharide made from the deacetylation of chitin. In this study, a ZCF31 putative transcription factor gene was selected from a previous mutant library screen, as zcf31Δ strains exhibited defective cell growth in response to chitosan. Furthermore, chitosan caused notable damage to zcf31Δ cells; however, ZCF31 expression was not significantly changed by chitosan, suggesting that zcf31Δ is sensitive to chitosan could be due to changes in the physical properties of C. albicans. Indeed, zcf31Δ cells displayed significant increases in cell wall thickness. Consistent with the previous study, zcf31Δ strains were resistant to calcofluor white but highly susceptible to SDS (sodium dodecyl sulfate). These results implied that chitosan mainly influences membrane function, as zcf31Δ strengthens the stress resistance of the fungal cell wall but lessens cell membrane function. Interestingly, this effect on the cell surface mechanics of the C. albicans zcf31Δ strains was not responsible for the virulence-associated function. RNA-seq analysis further revealed that six mannosyltransferase-related genes were upregulated in zcf31Δ. Although five mannosyltransferase-related mutant strains in the zcf31Δ background partially reduced the cell wall thickness, only zcf31Δ/mnn45Δ showed the recovery of chitosan resistance. Our findings suggest that Zcf31 mediates a delicate and complicated dynamic balance between the cell membrane and cell wall architectures through the mannosyltransferase genes in C. albicans, leading to altered chitosan susceptibility.

Parkinson’s disease (PD) is a neurodegenerative disorder primarily characterized by motor impairment, resulting from the accumulation of α-synuclein and neuronal cell death in the substantia nigra of the midbrain. Emerging evidence suggests that α-synuclein aggregation may originate in the enteric nervous system (ENS) and subsequently propagate to the brain via the vagus nerve. Clinical observations, such as prodromal gastrointestinal dysfunction in PD patients and the increased incidence of PD among individuals with inflammatory bowel disease, support the hypothesis that abnormal intestinal inflammation may contribute to the onset of motor dysfunction and neuropathology in PD. This review examines recent findings on the interplay between intestinal immune cells and α-synuclein aggregation within the framework of gut-originated PD pathogenesis. It begins by discussing evidence linking dysbiosis and intestinal inflammation to α-synuclein aggregation in the ENS. Additionally, it explores the potential role of intestinal immune cells in influencing enteric neurons and α-synuclein aggregation, furthering the understanding of PD development.

Inflammatory bowel disease (IBD) is an autoimmune disorder characterized by chronic inflammation of the gut and compromised intestinal barrier function, resulting from aberrant immune responses targeting the intestinal microbiota. While the involvement of Candida albicans in IBD pathogenesis is well-documented, the role of non-albicans Candida species in IBD remains less understood. Recent studies have identified a correlation between elevated levels of Candida tropicalis, a notable non-albicans opportunistic fungus, and the development of IBD. However, the precise impact of C. tropicalis on intestinal barrier function is not well elucidated. To address this knowledge gap, we utilized a cell model comprising polarized Caco-2 monolayers, which mimic the intestinal epithelium, to investigate the interaction between C. tropicalis and intestinal barrier function. Our results showed that incubation with C. tropicalis influenced transepithelial electrical resistance and increased permeability to the small molecule lucifer yellow, but did not affect permeability to the larger molecule fluorescein isothiocyanate-dextran. In addition, we observed internalization of the tight junction protein claudin-1 in the Caco-2 monolayers. Further experiments using Caco-2 monolayers exposed to the dectin-1 ligand zymosan induced similar changes in the distribution of claudin-1 but did not alter monolayer permeability. These findings suggest that C. tropicalis specifically affects intestinal barrier integrity and permeability to smaller solutes in intestinal epithelial cells.

Candida tropicalis-a prevalent gut commensal fungus in healthy individuals – contributes to intestinal health and disease. However, how commensal C. tropicalis influences intestinal homeostasis and barrier function is poorly understood. Here, we demonstrated that the reference strain of C. tropicalis (MYA-3404) ameliorated intestinal inflammation in murine models of chemically induced colitis and bacterial infection. Intestinal colonization of C. tropicalis robustly upregulated the expression of IL-17A and IL-22 to increase barrier function and promote proliferation of intestinal epithelial cells in the mouse colon. Metabolomics analysis of fecal samples from mice colonized with C. tropicalis revealed alterations in vitamin B3 metabolism, promoting conversion of nicotinamide to nicotinic acid. Although nicotinamide worsened colitis, treatment with nicotinic acid alleviated disease symptoms and enhanced epithelial proliferation and Th17 cell differentiation. Oral gavage of C. tropicalis mitigated nicotinamide-induced intestinal dysfunction in experimental colitis. Blockade of nicotinic acid production with nicotinamidase inhibitors lowered the protective effects against colitis in mice treated with C. tropicalis. Notably, a clinical C. tropicalis strain isolated from patients with candidemia lacked the protective effects against murine colitis observed with the reference strain. Together, our results highlight a novel role for C. tropicalis in resolving intestinal inflammation through the modulation of vitamin B3 metabolism.

Background: Increased neutrophil extracellular trap (NET) formation and abundant NET-associated proteins are frequently found in the inflamed colon of patients with inflammatory bowel disease. Peptidyl arginine deiminase 4 (PAD4) activation is essential for the generation of NET and NET-mediated pathogenesis. However, the role of PAD4-dependent NET formation in murine inflammatory bowel disease models and the molecular mechanisms responsible for the altered gut barrier function are unknown. Methods: Wild-type and Pad4 knockout (Pad4-/-) mice were administrated 3% dextran sulfate sodium (DSS) in their drinking water. Caco-2 monolayers were used to test the effect of NETs on intestinal barrier function and cytotoxicity. Histones were intrarectally administrated to wild-type mice to determine their effects on intestinal barrier function and cytotoxicity in vivo. Results: PAD4 deficiency reduced the severity of DSS-induced colitis with decreased intestinal NET formation and enhanced gut barrier function and integrity in mice. NETs disrupted the barrier function in intestinal epithelial Caco-2 monolayers through their protein, rather than DNA, components. Pretreatment of NETs with histone inhibitors abrogated the effects on epithelial permeability. Consistent with these observations, adding purified histone proteins to Caco-2 monolayers significantly damaged epithelial barrier function, which was associated with the abnormal distribution and integrity of tight junctions as well as with increased cell death. Furthermore, intrarectal administration of histones damaged the intestinal barrier integrity and induced cytotoxicity in the mouse colon epithelium. Conclusions: PAD4-mediated NET formation has a detrimental role in acute colitis. NET-associated histones directly inhibit intestinal barrier function, resulting in cytotoxicity in vitro and in vivo.

Aberrant neutrophil extracellular trap (NET) formation and the loss of barrier integrity in inflamed intestinal tissues have long been associated with inflammatory bowel disease (IBD). However, whether NETs alter intestinal epithelium permeability during colitis remains elusive. Here, we demonstrated that NETs promote the breakdown in intestinal barrier function for the pathogenesis of intestinal inflammation in mouse models of colitis. NETs were abundant in the colon of mice with colitis experimentally induced by dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS). Analysis of the intestinal barrier integrity revealed that NETs impaired gut permeability, enabling the initiation of luminal bacterial translocation and inflammation. Furthermore, NETs induced the apoptosis of epithelial cells and disrupted the integrity of tight junctions and adherens junctions. Intravenous administration of DNase I, an enzyme that dissolves the web-like DNA filaments of NETs, during colitis restored the mucosal barrier integrity which reduced the dissemination of luminal bacteria and attenuated intestinal inflammation in both DSS and TNBS models. We conclude that NETs serve a detrimental factor in the gut epithelial barrier function leading to the pathogenesis of mucosal inflammation during acute colitis.

Aberrant neutrophil extracellular trap (NET) formation and the loss of barrier integrity in inflamed intestinal tissues have long been associated with inflammatory bowel disease (IBD). However, whether NETs alter intestinal epithelium permeability during colitis remains elusive. Here, we demonstrated that NETs promote the breakdown in intestinal barrier function for the pathogenesis of intestinal inflammation in mouse models of colitis. NETs were abundant in the colon of mice with colitis experimentally induced by dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS). Analysis of the intestinal barrier integrity revealed that NETs impaired gut permeability, enabling the initiation of luminal bacterial translocation and inflammation. Furthermore, NETs induced the apoptosis of epithelial cells and disrupted the integrity of tight junctions and adherens junctions. Intravenous administration of DNase I, an enzyme that dissolves the web-like DNA filaments of NETs, during colitis restored the mucosal barrier integrity which reduced the dissemination of luminal bacteria, and attenuated intestinal inflammation in both DSS and TNBS models. We conclude that NETs serve a detrimental factor in the gut epithelial barrier function leading to the pathogenesis of mucosal inflammation during acute colitis.

Inflammasomes are intracellular multiple protein complexes that mount innate immune responses to tissue damage and invading pathogens. Their excessive activation is crucial in the development and pathogenesis of inflammatory disorders. Microtubules have been reported to provide the platform for mediating the assembly and activation of NLRP3 inflammasome. Recently, we have identified the microtubule-associated immune molecule guanine nucleotide exchange factor-H1 (GEF-H1) that is crucial in coupling microtubule dynamics to the initiation of microtubule-mediated immune responses. However, whether GEF-H1 also controls the activation of other immune receptors that require microtubules is still undefined. Here we employed GEF-H1-deficient mouse bone marrow-derived macrophages (BMDMs) to interrogate the impact of GEF-H1 on the activation of NLRP3 inflammasome. NLRP3 but not NLRC4 or AIM2 inflammasome-mediated IL-1β production was dependent on dynamic microtubule network in wild-type (WT) BMDMs. However, GEF-H1 deficiency did not affect NLRP3-driven IL-1β maturation and secretion in macrophages. Moreover, α-tubulin acetylation and mitochondria aggregations were comparable between WT and GEF-H1-deficient BMDMs in response to NLRP3 inducers. Further, GEF-H1 was not required for NLRP3-mediated immune defense against Salmonella typhimurium infection. Collectively, these findings suggest that the microtubule-associated immune modulator GEF-H1 is dispensable for microtubule-mediated NLRP3 activation and host defense in mouse macrophages.