Background: Brucellosis is a global zoonosis caused by Brucella. Histidine biosynthesis is essential for bacterial growth, but its role in Brucella melitensis virulence remains unclear. HisD catalyzes the final two steps of histidine synthesis and is absent in mammals, making it a potential drug target. Results: We constructed a hisD deletion mutant (ΔhisD) and complemented strain (ChisD) via homologous recombination. ΔhisD failed to grow in medium without histidine supplementation. It showed reduced survival under polymyxin B and SDS stress, and impaired outer membrane integrity under polymyxin B challenge, though no defect was observed under non-stressed conditions. Intracellularly, ΔhisD replicated poorly in HeLa and RAW264.7 cells, and this defect was rescued by exogenous histidine. In a mouse model, ΔhisD exhibited lower bacterial loads in liver and spleen, reduced splenomegaly, and attenuated hepatic granuloma formation. Conclusions: Histidine biosynthesis deficiency attenuates Brucella virulence by restricting nutritional acquisition and conditionally compromising outer membrane stability. HisD is a promising target for anti-brucellosis drug development, and ΔhisD holds potential as a live attenuated vaccine candidate.

Avian pathogenic Escherichia coli (APEC) can cause colibacillosis, which is economically devastating to poultry industries worldwide. The bacterial membrane is critical to its environment adaptability and virulence. The inner membrane protein TolA maintains membrane integrity, but its roles in the fitness and pathogenesis of APEC are not completely understood. Thus, a tolA gene mutant and complemented strains of APEC were constructed and characterized. Mutant strain ΔtolA showed damage in the inner and outer membranes, as well as altered morphology, impaired flagella production, reduced motility, increased outer membrane vesicle (OMV) production, and reduced resistance to antibiotics and environmental stress. Deletion of the tolA gene resulted in significant reductions in biofilm formation and interbacterial competition, due to the downregulated expression of biofilm-associated genes and type VI secretion system (T6SS) genes, respectively. In addition, the mutant strain exhibited reductions in serum bactericidal resistance, cell infection capacity, intracellular survival, consequently leading to attenuated bacterial survival and virulence in mice. Compared with the wild-type and complemented strains, the mutant strain induced less expression of inflammatory cytokine interleukin 1 beta (IL-1β) in HD-11 macrophages, consistent with the pathological damage in mice. In conclusion, inner membrane protein TolA contributes to the antibiotic resistance, environmental adaptability, biofilm formation and virulence of APEC.

Clostridioides difficile (C. difficile) is a leading cause of antibiotic-associated diarrhea and severe colitis, yet its genetic manipulation has long been constrained by low DNA transfer efficiency and limited recombination systems. Recent advances in CRISPR-based technologies have revolutionized the genetic toolkit for this pathogen, enabling precise genome editing and transcriptional regulation. Among CRISPR nucleases, Cas12a offers distinct advantages over Cas9 for bacterial applications, including a smaller size, T-rich PAM recognition, single-crRNA requirement, and reduced toxicity, which enhances conjugation efficiency in genetically recalcitrant organisms. AsCas12a-based platforms have enabled large fragment deletions, multiplex editing, and rapid generation of marker-free mutants in C. difficile. Complementing these nuclease-active systems, nuclease-deactivated variants (dCas9 or dAsCas12a) support CRISPR interference (CRISPRi)-a reversible, tunable approach for transcriptional repression without altering genomic sequences. Compared to traditional mutagenesis, CRISPRi greatly accelerates functional genomics by enabling high-throughput screening and drug target discovery. Together, our lab has independently developed CRISPR-AsCas12a-mediated genome editing and dAsCas12a-based CRISPRi tools, providing complementary strategies to overcome longstanding genetic barriers in C. difficile. These tools open new avenues for system-level interrogation of virulence, antibiotic resistance, and host-pathogen interactions.

The gut microbiota and its metabolites are important regulators of mucosal immunity against enteric pathogens. However, the host pathways that coordinate intestinal microbial ecology, bile acid metabolism, and antibacterial defense remain incompletely understood. STING is a key adaptor in nucleic acid sensing, but its role in shaping the intestinal microbial-metabolic environment during bacterial infection is unclear. Wild-type and STING-deficient mice were compared in an oral Salmonella enterica serovar Typhimurium infection model. Disease severity was assessed by survival, body weight change, histopathology, bacterial burden, serum cytokines, and flow cytometry. The microbiota and metabolite profiles of colonic luminal contents, together with host transcriptional responses in distal colon tissue, were analyzed using 16 S rRNA gene sequencing, untargeted LC-MS/MS metabolomics, RNA sequencing, and integrative multi-omics analyses. STING-deficient mice showed increased susceptibility to Salmonella infection, including reduced survival, greater body weight loss, higher bacterial burdens, more severe intestinal pathology, along with impaired intestinal barrier integrity, and increased inflammatory cytokine production. STING deficiency was associated with reduced microbial diversity and altered gut microbial composition, including enrichment of infection-associated taxa such as Parabacteroides and depletion of commensal genera such as Turicibacter and Lactobacillus. Metabolomic profiling revealed remodeling of the intestinal metabolic landscape, with prominent alterations in bile acid- and lipid-related metabolites. Host transcriptomic analysis showed reduced expression of genes and pathways related to MHC class II antigen presentation and T cell activation. Cross-omics correlation analyses linked altered microbial taxa and bile acid metabolites with impaired mucosal immune gene expression. These findings identify STING as a host factor associated with intestinal microbial and bile acid metabolic homeostasis during Salmonella infection. STING deficiency is associated with coordinated alterations in gut microbial composition, bile acid metabolism, mucosal immune transcriptional programs, and host susceptibility to enteric infection.

Avian pathogenic Escherichia coli (APEC) is a major poultry pathogen that causes colibacillosis and imposes a substantial economic burden on global poultry production. Human extraintestinal pathogenic Escherichia coli (ExPEC) and APEC exhibit comparable serotypes, phylogenetic lineages, and virulence-associated gene profiles. Moreover, APEC acts as a source of virulence determinants and antimicrobial resistance genes that are also relevant to human ExPEC infections. We identified a pentatricopeptide repeat (PPR) protein EspX1 in APEC, but its function is unknown. The espX1 gene mutant and complemented strains of APEC were constructed and characterized. The results showed that EspX1 did not affect APEC growth or motility. The espX1 deletion mutant exhibited enhanced biofilm formation and cell adhesion but reduced serum resistance and intracellular survival. Furthermore, the mutant strain showed decreased colonization capacity in the liver and spleen during systemic infection and attenuated virulence in vivo. Additionally, EspX1 was found to inhibit the expression of inflammatory cytokines TNF-α, IL-1β, and IL-8 in host cells. These findings suggest that EspX1 plays a crucial role in the pathogenicity of APEC by modulating multiple virulence-related phenotypes and host immune responses. Moreover, EspX1 enhanced APEC resistance to ciprofloxacin. This study provides novel insights into PPR proteins functions in prokaryotes and lays a theoretical foundation for the development of new strategies to control APEC infections.

Conjugated linoleic acid (CLA) is a dietary lipid that modulates host-microbiota-immune interactions, yet its mechanistic impact on mucosal defense remains unclear. Here, we show that oral CLA supplementation enhances resistance to Salmonella Typhimurium infection and is associated with coordinated changes in gut microbial composition and mucosal immune responses. CLA-enriched commensals, including Dubosiella and Lactobacillus, were associated with increased production of CLA-derived oxylipins and activation of immune surveillance genes. Functionally, CLA pretreatment reduced Salmonella colonization, preserved epithelial integrity, and decreased neutrophilic inflammation without direct antibacterial effects. Single-cell RNA sequencing of ileal intraepithelial lymphocytes revealed that CLA predominantly reprogrammed intestinal CD8⁺ T cells toward an oxidative phenotype and enhanced effector activity. ATAC-seq revealed increased chromatin accessibility at loci associated with metabolic regulation, consistent with transcriptional reprogramming toward oxidative fitness. Mechanistically, CLA directly activated PPARγ signaling to promote mitochondrial biogenesis, oxidative phosphorylation, and the production of IFN-γ and granzyme B in CD8⁺ T cells; pharmacologic inhibition of PPARγ attenuated these effects both in vitro and in vivo. Notably, depletion of CD8⁺ T cells eliminated CLA-mediated protection and abolished early restriction of bacterial dissemination at Peyer's patches and mesenteric lymph nodes. Although CLA enhanced CD8⁺ T-cell effector programs, antibiotic depletion and fecal microbiota transplantation experiments demonstrated that an intact gut microbiota is necessary for effective protection in vivo. Together, these findings identify CLA as a dietary modulator that strengthens mucosal resistance to Salmonella by promoting PPARγ-mediated metabolic reprogramming and enhanced effector fitness in intestinal CD8⁺ T cells.

Brucellosis caused by Brucella melitensis is a worldwide zoonotic disease, yet the role of the c-di-GMP phosphodiesterase BpdB in virulence remains incompletely understood. This study aimed to investigate the function of BpdB in B. melitensis pathogenicity and its involvement in host STING signaling. A bpdB deletion strain (ΔbpdB) and a complemented strain (CbpdB) were constructed in B. melitensis M5. Bacterial growth, stress tolerance, intracellular survival in macrophages, cytokine expression, and mouse virulence were evaluated. Deletion of bpdB did not affect in vitro growth but significantly attenuated virulence in BALB/c mice, reducing splenomegaly, splenic bacterial load, and hepatic granuloma formation. The ΔbpdB strain exhibited enhanced sensitivity to oxidative stress, whereas resistance to acid, polymyxin B, and SDS remained unchanged. Intracellular survival of ΔbpdB in RAW264.7 macrophages was reduced at 72 h post infection, a defect that was completely abrogated in STING-knockout RAW264.7 cells. ΔbpdB infection induced higher transcriptional levels of IFN-β and IL-1β, with IFN-β induction strictly dependent on STING. In C57BL/6J mice, the virulence attenuation of ΔbpdB was partially STING-dependent, as the reduction in splenic bacterial load was smaller in STING-knockout than in wild-type mice. These findings demonstrate that BpdB contributes to B. melitensis virulence by enhancing oxidative stress resistance and dampening STING-dependent host responses, providing new insights into c-di-GMP-mediated host-pathogen interactions.

Listeria monocytogenes (L. monocytogenes) is a significant zoonotic pathogen responsible for listeriosis, a foodborne infection with high mortality. The inflammasome, an innate immune complex, plays a critical role in controlling pathogenic infections through its rapid inflammatory output. During L. monocytogenes infection, pore-forming toxins such as listeriolysin-O and flagellin are quickly recognized by pattern recognition receptors (PRRs), triggering inflammatory responses and activating the host’s anti-infection immunity. However, excessive or chronic inflammasome activation and subsequent interleukin-1β (IL-1β) release are implicated in the pathogenesis of L. monocytogenes. Although inflammasome activation is an effective defense against L. monocytogenes, the bacterium has evolved multiple mechanisms to inhibit this immune pathway. Hence, research on inflammasomes activation is crucial for better understanding the pathogenic mechanism of L. monocytogenes. In this review, we highlight recent advances in the understanding of the molecular mechanisms of inflammasome activation by L. monocytogenes infection. We then discuss advances in the role of the inflammasome pathway in the pathogenesis of L. monocytogenes, along with an overview of the applications of inflammasome inhibitors. Extensive studies into the mechanisms by which L. monocytogenes activates the inflammasome could lead to the discovery of novel therapeutic targets and strategies to fight L. monocytogenes infections.

Avian pathogenic Escherichia coli (APEC) severely impedes the development of the poultry industry and poses a serious threat to public health. The LuxS affects physiological processes such as bacterial virulence, biofilm formation, environmental adaptability, and motility. However, the effects of the LuxS on the environmental adaptability of APEC are not fully understood. Our study aims to investigate the effects of the LuxS on the environmental adaptability of APEC. Thus, the luxS gene mutant and complemented strains of APEC were constructed and characterized. We found that the mutant strain ΔluxS exhibited impaired adaptability to environmental stress, including oxidative, acid, base, heat, and hyperosmotic stress, with corresponding downregulation of stress-responsive genes. For oxidative stress tolerance, marA, fldA, and the rseC gene in the SoxR reduction system showed reduced expression. Genes regulating membrane and acid-base homeostasis, pH-inducible proteins, and the EvgS/EvgA two-component system were downregulated for acid-base stress. Genes related to osmoprotectant synthesis, uptake, potassium regulation, and heat stress adaptability, including extra cytoplasmic stress response (ESR), also exhibited lower transcriptional levels. Furthermore, we found that the ΔluxS mutant strain was impaired in competition capability due to downregulation of type VI secretion system (T6SS) genes. In addition, the mutant strain exhibited lower outer membrane permeability and lower proton motive force (PMF) compared with the wild-type strain. In conclusion, these findings indicate that the LuxS plays a critical role in regulating environmental adaptability, competition capability, outer membrane permeability, and PMF maintenance in APEC.

Cyclic di-GMP (c-di-GMP) is a universal bacterial second messenger that orchestrates the transition between motile and sessile lifestyles, thereby shaping microbial physiology, virulence, and persistence. In Escherichia coli (E. coli), c-di-GMP functions as a central integrator of environmental cues, dynamically regulating motility, biofilm formation, energy metabolism, and pathogenicity. Here, we provide a comprehensive synthesis of recent advances in c-di-GMP signaling in E. coli, spanning local signaling modules that control curli and cellulose biosynthesis to global regulatory circuits linking central metabolism, stress adaptation, and virulence gene expression. We further highlight the emerging view of c-di-GMP as a pathogen-associated molecular pattern that interfaces with host immune sensors such as STING and DDX41, extending its biological relevance beyond bacterial physiology. Finally, we discuss the translational potential of targeting c-di-GMP signaling for biofilm disruption, enhancement of bacteriophage-mediated clearance, and vaccine adjuvant design. Collectively, this review positions E. coli as a powerful model to decode the multifaceted biology of c-di-GMP and to inspire novel antimicrobial and immunomodulatory strategies.

Abstract Symptoms of Clostridioides difficile infection (CDI) are primarily caused by two major protein toxins, toxin A (TcdA) and toxin B (TcdB). In addition, approximately 5-30% of C. difficile strains produce a third toxin, C. difficile binary toxin (CDT), which is has been associated with enhanced virulence and severe disease. CDT consists of an enzymatic component CDTa, and a binding and translocation component CDTb, which mediates the delivery of CDTa into host cells. CDTb contains two receptor-binding domains, RBD1 and RBD2. Recent structural studies suggest that RBD2 plays a critical role in the formation and stabilization of the di-heptameric CDTb assembly required for efficient intoxication of host cells. In this study, we evaluated the immunogenicity and protective potential of RBD1 and RBD2 using in silico, in vitro and in vivo approaches. Sequence analysis demonstrated that RBD2 is highly conserved among diverse CDT-producing C. difficile ribotypes and toxinotypes. Immunization of mice with RBD2, but not RBD1 conferred effective protection against direct CDT challenge. Moreover, RBD2 immunization protected hamsters against infection with a CDT-only-producing C. difficile strain (DSM 101085; TcdA⁻TcdB⁻CDT⁺). Mechanistically, anti-RBD2 serum, but not anti-RBD1 serum, effectively neutralized CDT-mediated cytotoxicity, as demonstrated by inhibition of cell rounding in Vero cells. Collectively, these findings identify RBD2 as a promising vaccine antigen targeting CDT and provide functional evidence supporting its critical role in CDT-mediated host-cell intoxication. Incorporation of RBD2 into multivalent C. difficile vaccines may broaden protection against hypervirulent, CDT-producing strains.

Brucella melitensis, a facultative intracellular pathogen, relies on membrane integrity and homeostasis to resist host defenses and establish infection. The plsC gene encodes 1-acyl-sn-glycerol-3-phosphate acyltransferase, a key enzyme in the glycerophospholipid pathway that catalyzes the synthesis of phosphatidic acid, an essential precursor for membrane lipid formation. However, its role in B. melitensis virulence remains poorly understood. Here, we constructed a plsC deletion mutant (ΔplsC) and a complemented strain (ΔplsC-Com) in B. melitensis strain M5 and characterized their phenotypes. Deletion of plsC impaired bacterial growth in nutrient-limited media, reduced tolerance to hydrogen peroxide and polymyxin B, and decreased lipid synthesis while increasing outer membrane permeability. Ultrastructural analysis revealed surface roughness, cytoplasmic voids, and nucleoid condensation in the mutant. Although ΔplsC retained normal adhesion and invasion capabilities in RAW264.7 macrophages and HeLa cells, its intracellular survival was specifically attenuated in macrophages at 48 h post-infection. In a mouse model, ΔplsC showed significantly reduced colonization of the spleen and liver and induced fewer and smaller liver granulomas as compared with the parental and complemented strains. These results demonstrate that PlsC is essential for maintaining membrane homeostasis and stress resistance in Brucella, which in turn supports its survival within professional phagocytes and full virulence in vivo. Our study suggests a critical link between phospholipid metabolism and Brucella pathogenicity.

Avian pathogenic Escherichia coli (APEC) induces avian colibacillosis and brings huge economic losses to global poultry production. The small alarmone (p)ppGpp mediates the bacterial stringent response, a vital pathway modulating microbial stress adaptation and pathogenic capacity. The functions of the (p)ppGpp synthase gene relA in APEC pathogenesis remain poorly characterized. In this study, we constructed a relA deletion mutant (ΔrelA) and its complemented strain (CΔrelA). The phenotypic and pathogenic characteristics of these strains were investigated. The results showed that deletion of relA did not significantly affect bacterial growth or motility. However, the ΔrelA strain showed increased susceptibility to aminoglycoside antibiotics. Furthermore, the enhanced interbacterial competition of the mutant was associated with the upregulation of core genes in the type VI secretion system (T6SS). Importantly, relA was essential for APEC adhesion to and invasion of avian DF-1 cells, as well as for colonization and virulence in ducklings, where ΔrelA exhibited significantly attenuated infectivity and reduced bacterial loads in the liver and spleen. Furthermore, transcriptomic analysis revealed that RelA deletion downregulated genes involved in integral components of the membrane, and further assays confirmed compromised membrane integrity in the mutant strain. These findings suggest that RelA maintains membrane integrity, which underpins its contributions to antibiotic resistance and virulence. These findings indicate that relA plays a key role in APEC virulence, antibiotic resistance, and membrane homeostasis, and could provide a theoretical basis for targeting the stringent response as a potential strategy to control avian colibacillosis.

Non-typhoidal salmonellosis, caused by Salmonella enterica, threatens poultry production and food safety in East Africa, where poultry is vital for livelihoods. This systematic review and meta-analysis assessed the pooled prevalence, serotype distribution, and antimicrobial resistance patterns of non-typhoidal Salmonella in East African poultry. Following PRISMA guidelines, we searched on PubMed, Web of Science, Scopus, African Journals Online, and Google Scholar for studies published up to December, 2025. A random-effects model was used to calculate pooled prevalence with 95% confidence intervals in R (Version 4.6.0). Subgroup analyses and I2 statistics were used to explore the source of heterogeneity, and a heatmap visualized the antibiotic resistance patterns. Thirteen studies comprising 422 positive isolates were included. The pooled prevalence of non-typhoidal Salmonella in East Africa was 6% (95% CI: 4-8%), with high heterogeneity (I2 = 95.3%). The subgroup analysis showed prevalence was highest in Uganda (11%, 95% CI: 7-16%) and lowest in Tanzania (1%, 95% CI: 0-6%). By diagnostic method, slide agglutination was higher (7%, 95% CI: 5-10%) than PCR (4%, 95% CI: 1-8%). By sample type, internal organs had the highest prevalence (17%, 95% CI: 12-21%) and environmental samples had the lowest (1%, 95% CI: 0-3%). By source, slaughterhouses recorded the highest rate (12%, 95% CI: 7-18%) and extensive farms the lowest rate (3%, 95% CI: 0-8%). The pooled antibiotic resistance was 65% (95% CI: 55-79%). Oxytetracycline is the most resistant antibiotic (84%, 95% CI: 70-98%). S. Typhimurium exhibited the highest pooled prevalence (18%, 95% CI: 15-22%), and S. Kentucky was the most multidrug-resistant serotype and had the highest number of resistance genes. In conclusion, non-typhoidal Salmonella is prevalent in East African poultry, with multidrug-resistant serotypes distributed across the region. Therefore, collaborative interventions are urgently needed to address this zoonotic threat.

Multidrug-resistant (MDR) bacteria have reduced the effectiveness of antimicrobial agents and limited available treatment options in both human and animal settings. Antimicrobial resistance (AMR) in animal-derived Escherichia coli (E. coli) has become increasingly common, with many isolates showing resistance to multiple classes of antimicrobials. However, compared with other animal sources, resistance characteristics of E. coli from mink remain less well understood, particularly in terms of genomic features. In this study, we identified a MDR E. coli strain, EC0D1, isolated from the lung tissue of a farmed mink that succumbed to hemorrhagic pneumonia (HP) in China. Antimicrobial susceptibility testing indicated that EC0D1 exhibited resistance to β-lactams, fluoroquinolones, aminoglycosides, tetracyclines, and polymyxins but was sensitive to carbapenems and tigecycline. Whole genome sequencing showed that EC0D1 contains a single chromosome and eight plasmids. Among these, plasmid pEC0D1-3 coharbored blaCTX-M-55 and mcr-1.1, while pEC0D1-2 carried tet(A) and floR, together explaining the observed MDR phenotype. Phylogenetic analysis classified EC0D1 as sequence type ST457 and demonstrated a close genetic relationship with several human clinical isolates. Comparative genomic analysis further revealed that plasmids pEC0D1-2 and pEC0D1-3 shared high sequence similarity with plasmids previously identified in avian-derived E. coli and human-derived Klebsiella pneumoniae isolates. Conjugation assays confirmed that plasmids carrying blaCTX-M-55, mcr-1.1, tet(A), and floR were transferable to different bacterial recipients. The emergence of transferable resistance determinants in mink-associated E. coli suggests a potential role in the transmission of resistance genes between human and animal hosts.

Mycoplasma synoviae (MS) is considered to be one of the main mycoplasma pathogens of poultry, causing arthritis, airsacculitis, eggshell apex abnormalities and production drops in chickens and turkeys. Infection by MS usually results in considerable economic losses to the poultry industry worldwide. Therefore, it is essential to develop a highly sensitive and accurate diagnostic method in the livestock production. The MSLP53 was predicted as a highly conserved and specific membrane associated lipoprotein of MS by bioinformatics analysis. The His-tagged MSLP53 (rMSLP53) protein was expressed and purified using E. coli expression system, and was confirmed by Western blotting to react with each MS-positive serum, but not react with positive sera against other avian pathogens, suggesting that the rMSLP53 had strong immunoreactivity and specificity. An rMSLP53-based indirect ELISA was developed, compared to IDEXX kit with a pool of 277 chicken sera samples, and showed high sensitivity (85.54

Pullorum disease is a bacterial disease caused by Salmonella pullorum in chickens, which is characterized by gastrointestinal infection and diarrhea. In traditional perspectives, research on pullorum disease primarily focused on clinical symptoms, epidemiological characteristics, and the pathogenic sites. This study, however, approaches the subject from the standpoint of host genetic basis and gut microbiota. For the positive and negative offspring chicks, which are the offspring of positive roosters and hens and negative roosters and hens, respectively, we conducted whole-genome association analysis and identified 195 SNPs and 79 significant InDels on the host genome that were associated with susceptibility/resistance to pullorum disease. A total of 77 genes were annotated, among which MYH7, ATP2A3, and CACNA1S exhibited variations in the exons. After infection with S. pullorum, the diversity and community structure of the gut microbiota in the chicken also underwent significant changes. Lactobacillus, Escherichia_Shigella, and Klebsiella were dominant bacteria in the dead negative offspring chicks with significantly higher abundance compared to the survival negative offspring chicks. These significant changes in host genome and bacterial abundance suggest that they may be associated with the susceptibility/resistance of pullorum disease.IMPORTANCEPullorum disease can be transmitted vertically and horizontally. Population purification and antibiotic treatment are the main methods for preventing and treating this disease, but they are associated with issues, such as high cost, poor accuracy, bacterial resistance, and overused antibiotics. In traditional perspectives, research on pullorum disease primarily focused on clinical symptoms, epidemiological characteristics, and the pathogenic sites. This study, however, approaches the subject from the standpoint of host genetic basis and gut microbiota. Using the genome-wide association analysis and microbiome comparison analysis, with chicken death and survival following Salmonella pullorum infection as phenotypes, we identified significant genetic variations (e.g., MYH7, ATP2A3, and CACNA1S) and gut microbiota (e.g., Lactobacillus, Escherichia_Shigella, Bacillus, and Enterococcus_cecorum) that may relate to susceptibility/resistance of pullorum disease. These results indicate that the infection of chickens with S. pullorum and the achievement of vertical transmission may be related to the host genome and gut microbiota.

IntroductionTreatment options for C. difficile infection are limited, with very high rates of recurrence. Active vaccination provides an attractive opportunity to prevent C. difficile infection (CDI) and recurrence. In a search for potential surface-exposed antigens involved in C. difficile colonization, two putative lipoproteins, designated LP1 and LP2, were identified from C. difficile R20291.MethodsLipoprotein sequences were aligned, analyzed, and evaluated for their immune properties. The antigenic characteristics of both LP1 and LP2 were assessed in silico and in a mouse model of immunization and CDI.ResultsMultiple sequence alignments showed that the lipoprotein sequences were highly conserved among various ribotypes. In silico analysis predicted that LP1 and LP2 possess cytotoxic T-lymphocyte, helper T-lymphocyte, and B-cell epitopes with antigenic and immunogenic properties. Immune simulation provided insights into the ability of LP1 and LP2 to stimulate humoral and cellular immune responses. These properties were further examined in a mouse model of immunization and CDI. After three immunizations at 12-day intervals, significant amounts of IgG and IgA antibodies were detected in sera and feces. LP1 and LP2 immunizations provided mice with intermediate and higher levels of protection, respectively, against R20291 infection, and significantly reduced C. difficile spore and toxin levels in feces. Furthermore, anti-LP1 and anti-LP2 sera significantly inhibited adhesion of R20291 vegetative cells to HCT-8 gut epithelial cells.DiscussionThese results indicate that both lipoproteins play a significant role in C. difficile adhesion and that LP1 and LP2 are promising immunogens for preventing C. difficile colonization.

ABSTRACT Mutations affecting Clostridioides difficile flagellin (FliC) have been shown to be hypervirulent in animal models and display increased toxin production and alterations in central metabolism. The regulation of flagellin levels in bacteria is governed by a tripartite regulatory network involving fliC, fliW, and csrA, which creates a feedback system to regulate flagella production. Through genomic analysis of C. difficile clade 5 strains (non-motile), we identified they have jettisoned many of the genes required for flagellum biosynthesis yet retain the major flagellin gene fliC and regulatory gene fliW. We therefore investigated the roles of fliC, fliW, and csrA in the clade 5 ribotype 078 strain C. difficile 1015, which lacks flagella and is non-motile. Analysis of mutations in fliC, fliW, and csrA (and all combinations) on C. difficile pathogenesis indicated that FliW plays a central role in C. difficile virulence as animals infected with strains carrying a deletion of fliW showed decreased survival and increased disease severity. These in vivo findings were supported by in vitro studies showing that mutations impacting the activity of FliW showed increased toxin production. We further identified that FliW can interact with the toxin-positive regulator TcdR, indicating that modulation of toxin production via FliW occurs by sequestering TcdR from activating toxin transcription. Furthermore, disruption of the fliC-fliW-csrA network results in significant changes in carbon source utilization and sporulation. This work highlights that key proteins involved in flagellar biosynthesis retain their regulatory roles in C. difficile pathogenesis and physiology independent of their functions in motility.IMPORTANCEClostridioides difficile is a leading cause of nosocomial antibiotic-associated diarrhea in developed countries with many known virulence factors. In several pathogens, motility and virulence are intimately linked by regulatory networks that allow coordination of these processes in pathogenesis and physiology. Regulation of C. difficile toxin production by FliC has been demonstrated in vitro and in vivo and has been proposed to link motility and virulence. Here, we show that clinically important, non-motile C. difficile strains have conserved FliC and regulatory partners FliW and CsrA, despite lacking the rest of the machinery to produce functional flagella. Our work highlights a novel role for flagellin outside of its role in motility and FliW in the pathogenesis and physiology of C. difficile.