Genetic variation in the human leukocyte antigen region confers susceptibility to Clostridioides difficile infection.

Clostridioides difficile (C. diff.) infection (CDI) is a leading cause of hospital acquired diarrhea in North America and Europe and a major cause of morbidity and mortality. Known risk factors do not fully explain CDI susceptibility, and genetic susceptibility is suggested by the fact that some patients with colons that are colonized with C. diff. do not develop any infection while others develop severe or recurrent infections. To identify common genetic variants associated with CDI, we performed a genome-wide association analysis in 19,861 participants (1349 cases; 18,512 controls) from the Electronic Medical Records and Genomics (eMERGE) Network. Using logistic regression, we found strong evidence for genetic variation in the DRB locus of the MHC (HLA) II region that predisposes individuals to CDI (P > 1.0 × 10-14; OR 1.56). Altered transcriptional regulation in the HLA region may play a role in conferring susceptibility to this opportunistic enteric pathogen.

Dual RNA-seq identifies genes and pathways modulated during Clostridioides difficile colonization.

IMPORTANCE: The initial interactions between the colonic epithelium and the bacterium are likely critical in the establishment of Clostridioides difficile infection, one of the major causes of hospital-acquired diarrhea worldwide. Molecular interactions between C. difficile and human gut cells have not been well defined mainly due to the technical challenges of studying cellular host-pathogen interactions with this anaerobe. Here we have examined transcriptional changes occurring in the pathogen and host cells during the initial 24 hours of infection. Our data indicate several changes in metabolic pathways and virulence-associated factors during the initial bacterium-host cell contact and early stages of infection. We describe canonical pathways enriched based on the expression profiles of a dual RNA sequencing in the host and bacterium, and functions of bacterial factors that are modulated during infection. This study thus provides fresh insight into the early C. difficile infection process.

Transcriptomic analysis of biofilm formation in strains of Clostridioides difficile associated with recurrent and non-recurrent infection reveals potential candidate markers for recurrence.

The transcriptomic profile in a biofilm model of ribotypes (RT) 001 and 027 associated with recurrent Clostridioides difficile infection (R-CDI) and not associated with recurrent (NR)-CDI was analyzed to identify genes that may favor the recurrence. Twenty strains were selected, 10 RT001 and 10 RT027. From each ribotype, 5 were R-CDI and 5 NR-CDI. Biofilm and nonadherent cells were prepared from each clinical isolate, and the RNA was extracted. RNA samples were pooled in 8 combinations implying ribotype, recurrence, and biofilm formation. Each pool was separately labeled with Cy3 dye and hybridized on a microarray designed for this study. Slides were scanned, analyzed, and gene expression was compared between unique and grouped pools using the Student's t-test with Benjamini-Hochberg correction when appropriate. Validation was carried out by qRT-PCR for selected genes. Results: After comparisons of differentially expressed genes from both ribotypes of R-CDI strains (nonadherent cells vs. biofilm) and both ribotypes in biofilm (R-CDI vs. NR-CDI), we found 3 genes over-expressed and 1 under-expressed in common (adj. p ≤ 0.05). Overexpressed genes were CAJ70148 (a putative dehydrogenase), CAJ68100 (a secretion type II system protein from the GspH (pseudopilins) family), and CAJ69725 (a putative membrane protein); under-expressed was CAJ68151 (a segregation and condensation protein A). Because CAJ70148, CAJ68100, CAJ69725 and CAJ68151 were differentially expressed in biofilm in strains associated with R-CDI, they may support the biofilm favoring the recurrence of CDI. However, further studies will be needed for poorly studied genes.

G Protein-Coupled Receptor 120 Mediates Host Defense against Clostridium perfringens Infection through Regulating NOD-like Receptor Family Pyrin Domain-Containing 3 Inflammasome Activation.

Clostridium perfringens is a major cause of infectious foodborne disease, frequently associated with the consumption of raw and undercooked food. Despite intensive studies on clarifying C. perfringens pathogenesis, the molecular mechanisms of host-pathogen interactions remain poorly understood. In soft tissue and mucosal infection models, Gpr120-/- mice, G protein-coupled receptor 120 (GPR120), are more susceptible to C. perfringens infection. Gpr120 deficiency leads to a low survival rate (30 and 10%, p < 0.01), more bacterial loads in the muscle (2.26 × 108 ± 2.08 × 108 CFUs/g, p < 0.01), duodenum (2.80 × 107 ± 1.61 × 107 CFUs/g, p < 0.01), cecum (2.50 × 108 ± 2.05 × 108 CFUs/g, p < 0.01), and MLN (1.23 × 106 ± 8.06 × 105 CFUs/g, p < 0.01), less IL-18 production in the muscle (8.54 × 103 ± 1.20 × 103 pg/g, p < 0.01), duodenum (3.34 × 103 ± 2.46 × 102 pg/g, p < 0.01), and cecum (3.81 × 103 ± 5.29 × 102 pg/g, p < 0.01), and severe organ injury. Obviously, GPR120 facilitates IL-18 production and pathogen control via potassium efflux-dependent NOD-like receptor family pyrin domain-containing 3 (NLRP3) signaling. Mechanistically, GPR120 interaction with NLRP3 potentiates the NLRP3 inflammasome assembly. Thus, this study uncovers a novel role of GPR120 in host protection and reveals that GPR120 may be a potential therapeutic target for limiting pathogen infection.

Make It Less difficile: Understanding Genetic Evolution and Global Spread of Clostridioides difficile.

Clostridioides difficile is an obligate anaerobic pathogen among the most common causes of healthcare-associated infections. It poses a global threat due to the clinical outcomes of infection and resistance to antibiotics recommended by international guidelines for its eradication. In particular, C. difficile infection can lead to fulminant colitis associated with shock, hypotension, megacolon, and, in severe cases, death. It is therefore of the utmost urgency to fully characterize this pathogen and better understand its spread, in order to reduce infection rates and improve therapy success. This review aims to provide a state-of-the-art overview of the genetic variation of C. difficile, with particular regard to pathogenic genes and the correlation with clinical issues of its infection. We also summarize the current typing techniques and, based on them, the global distribution of the most common ribotypes. Finally, we discuss genomic surveillance actions and new genetic engineering strategies as future perspectives to make it less difficile.

Gut microbiota features associated with Clostridioides difficile colonization in dairy calves.

Diarrheal disease, a major cause of morbidity and mortality in dairy calves, is strongly associated with the health and composition of the gut microbiota. Clostridioides difficile is an opportunistic pathogen that proliferates and can produce enterotoxins when the host experiences gut dysbiosis. However, even asymptomatic colonization with C. difficile can be associated with differing degrees of microbiota disruption in a range of species, including people, swine, and dogs. Little is known about the interaction between C. difficile and the gut microbiota in dairy calves. In this study, we sought to define microbial features associated with C. difficile colonization in pre-weaned dairy calves less than 2 weeks of age. We characterized the fecal microbiota of 80 calves from 23 different farms using 16S rRNA sequencing and compared the microbiota of C. difficile-positive (n = 24) and C. difficile-negative calves (n = 56). Farm appeared to be the greatest source of variability in the gut microbiota. When controlling for calf age, diet, and farm location, there was no significant difference in Shannon alpha diversity (P = 0.50) or in weighted UniFrac beta diversity (P = 0.19) between C. difficile-positive and-negative calves. However, there was a significant difference in beta diversity as assessed using Bray-Curtiss diversity (P = 0.0077), and C. difficile-positive calves had significantly increased levels of Ruminococcus (gnavus group) (Adj. P = 0.052), Lachnoclostridium (Adj. P = 0.060), Butyricicoccus (Adj. P = 0.060), and Clostridium sensu stricto 2 compared to C. difficile-negative calves. Additionally, C. difficile-positive calves had fewer microbial co-occurrences than C. difficile-negative calves, indicating reduced bacterial synergies. Thus, while C. difficile colonization alone is not associated with dysbiosis and is therefore unlikely to result in an increased likelihood of diarrhea in dairy calves, it may be associated with a more disrupted microbiota.

N6-Methyladenosine Methylation Analysis of Long Noncoding RNAs and mRNAs in IPEC-J2 Cells Treated With Clostridium perfringens beta2 Toxin.

Background: The n6-methyladenosine (m6A) modification is present widely in mRNAs and long non-coding RNAs (lncRNAs), and is related to the occurrence and development of certain diseases. However, the role of m6A methylation in Clostridium perfringens type C infectious diarrhea remains unclear. Methods: Here, we treated intestinal porcine jejunum epithelial cells (IPEC-J2 cells) with Clostridium perfringens beta2 (CPB2) toxin to construct an in vitro model of Clostridium perfringens type C (C. perfringens type C) infectious diarrhea, and then used methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify the methylation profiles of mRNAs and lncRNAs in IPEC-J2 cells. Results: We identified 6,413 peaks, representing 5,825 m6A-modified mRNAs and 433 modified lncRNAs, of which 4,356 m6A modified mRNAs and 221 m6A modified lncRNAs were significantly differential expressed between the control group and CPB2 group. The motif GGACU was enriched significantly in both the control group and the CPB2 group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis showed that the differentially methylated modified mRNAs were mainly enriched in Hippo signaling pathway and Wnt signaling pathway. In addition, the target genes of the differentially m6A modified lncRNAs were related to defense response to virus and immune response. For example, ENSSSCG00000042575, ENSSSCG00000048701 and ENSSSCG00000048785 might regulate the defense response to virus, immune and inflammatory response to resist the harmful effects of viruses on cells. Conclusion: In summary, this study established the m6A transcription profile of mRNAs and lncRNAs in IPEC-J2 cells treated by CPB2 toxin. Further analysis showed that m6A-modified RNAs were related to defense against viruses and immune response after CPB2 toxin treatment of the cells. Threem6A-modified lncRNAs, ENSSSCG00000042575, ENSSSCG00000048785 and ENSSSCG00000048701, were most likely to play a key role in CPB2 toxin-treated IPEC-J2 cells. The results provide a theoretical basis for further research on the role of m6A modification in piglet diarrhea.

A new phenothiazine derivate is active against Clostridioides difficile and shows low cytotoxicity.

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matters of concern for public health. Thioridazine, a compound belonging to the phenothiazine group, has previous shown antimicrobial activity against C. difficile. The purpose of this present study was to investigate the potential of a novel phenothiazine derivative, JBC 1847, as an oral antimicrobial for treatment of intestinal pathogens and CDIs. The minimal inhibition concentration and the minimum bactericidal concentration of JBC 1847 against C. difficile ATCC 43255 were determined 4 µg/mL and high tolerance after oral administration in mice was observed (up to 100 mg/kg bodyweight). Pharmacokinetic modeling was conducted in silico using GastroPlusTM, predicting low (< 10%) systemic uptake after oral exposure and corresponding low Cmax in plasma. Impact on the intestinal bacterial composition after four days of treatment was determined by 16s rRNA MiSeq sequencing and revealed only minor impact on the microbiota in non-clinically affected mice, and there was no difference between colony-forming unit (CFU)/gram fecal material between JBC 1847 and placebo treated mice. The cytotoxicity of the compound was assessed in Caco-2 cell-line assays, in which indication of toxicity was not observed in concentrations up to seven times the minimal bactericidal concentration. In conclusion, the novel phenothiazine derivative demonstrated high antimicrobial activity against C. difficile, had low predicted gastrointestinal absorption, low intestinal (in vitro) cytotoxicity, and only induced minor changes of the healthy microbiota, altogether supporting that JBC 1847 could represent a novel antimicrobial candidate. The clinical importance hereof calls for future experimental studies in CDI models.

The Role of Toll-Like Receptor-2 in Clostridioides difficile Infection: Evidence From a Mouse Model and Clinical Patients.

Background: Clostridioides difficile is the leading cause of nosocomial infectious diarrhea. Toll-like receptors (TLRs) are the major components of innate immunity that sense pathogens. The relationship between TLRs and C. difficile infection (CDI) was analyzed in clinical patients and a mouse model. Materials and Methods: A prospective investigation was conducted in medical wards of Tainan Hospital, Ministry of Health and Welfare, Tainan, Taiwan, from January 2011 to January 2013. Adult patients were followed up for the development of CDI. Single nucleotide polymorphisms (SNPs) of TLR2 and TLR4 were analyzed to assess the relationship between genetic polymorphisms and the development of CDI. A mouse model of CDI was used to investigate the pathogenic role of TLRs in CDI, TLR2 and TLR4 knockout (Tlr2-/- and Tlr4-/-) mice. Results: In the prospective study, 556 patients were enrolled, and 6.5% (36) of patients, accounting for 3.59 episodes per 1000 patient-days, developed CDI. Of 539 patients with available blood samples, the TLR2 rs3804099 polymorphism was more often noted in those with CDI than in those without CDI (64.5% vs. 46.1%; P = 0.046) but was not significant in multivariate analysis. Because the TLR2 rs3804099 polymorphism was moderately associated with CDI, the role of TLR2 and TLR4 was further evaluated in a mouse model. Both Tlr2-/- and Tlr4-/- mice showed more severe CDI disease than wild-type mice in terms of body weight change and fecal content five days after oral challenge with C. difficile. Furthermore, Tlr2-/- mice suffered from more severe disease than Tlr4-/- mice, as evidenced by stool consistency, cecum weight, and survival rate. Conclusion: The TLR2 rs3804099 polymorphism is marginally associated with the development of CDI, and the pathogenic role of TLR2 is further supported by a mouse model.

Clostridioides difficile spores stimulate inflammatory cytokine responses and induce cytotoxicity in macrophages.

Clostridioides difficile is a gram-positive, spore-forming anaerobic bacterium, and the leading cause of antibiotic-associated diarrhea worldwide. During C. difficile infection, spores germinate in the presence of bile acids into vegetative cells that subsequently colonize the large intestine and produce toxins. In this study, we demonstrated that C. difficile spores can universally adhere to, and be phagocytosed by, murine macrophages. Only spores from toxigenic strains were able to significantly stimulate the production of inflammatory cytokines by macrophages and subsequently induce significant cytotoxicity. Spores from the isogenic TcdA and TcdB double mutant induced significantly lower inflammatory cytokines and cytotoxicity in macrophages, and these activities were restored by pre-exposure of the spores to either toxins. These findings suggest that during sporulation, spores might be coated with C. difficile toxins from the environment, which could affect C. difficile pathogenesis in vivo.

Major genetic discontinuity and novel toxigenic species in Clostridioides difficile taxonomy.

Clostridioides difficile infection (CDI) remains an urgent global One Health threat. The genetic heterogeneity seen across C. difficile underscores its wide ecological versatility and has driven the significant changes in CDI epidemiology seen in the last 20 years. We analysed an international collection of over 12,000 C. difficile genomes spanning the eight currently defined phylogenetic clades. Through whole-genome average nucleotide identity, and pangenomic and Bayesian analyses, we identified major taxonomic incoherence with clear species boundaries for each of the recently described cryptic clades CI-III. The emergence of these three novel genomospecies predates clades C1-5 by millions of years, rewriting the global population structure of C. difficile specifically and taxonomy of the Peptostreptococcaceae in general. These genomospecies all show unique and highly divergent toxin gene architecture, advancing our understanding of the evolution of C. difficile and close relatives. Beyond the taxonomic ramifications, this work may impact the diagnosis of CDI.

Fecal Microbiota Transplantation for Recurrent Clostridioides difficile Infection Associates With Functional Alterations in Circulating microRNAs.

BACKGROUND AND AIMS: The molecular mechanisms underlying successful fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection (rCDI) remain poorly understood. The primary objective of this study was to characterize alterations in microRNAs (miRs) following FMT for rCDI. METHODS: Sera from 2 prospective multicenter randomized controlled trials were analyzed for miRNA levels with the use of the Nanostring nCounter platform and quantitative reverse-transcription (RT) polymerase chain reaction (PCR). In addition, rCDI-FMT and toxin-treated animals and ex vivo human colonoids were used to compare intestinal tissue and circulating miRs. miR inflammatory gene targets in colonic epithelial and peripheral blood mononuclear cells were evaluated by quantitative PCR (qPCR) and 3'UTR reporter assays. Colonic epithelial cells were used for mechanistic, cytoskeleton, cell growth, and apoptosis studies. RESULTS: miRNA profiling revealed up-regulation of 64 circulating miRs 4 and 12 weeks after FMT compared with screening, of which the top 6 were validated in the discovery cohort by means of RT-qPCR. In a murine model of relapsing-CDI, RT-qPCR analyses of sera and cecal RNA extracts demonstrated suppression of these miRs, an effect reversed by FMT. In mouse colon and human colonoids, C difficile toxin B (TcdB) mediated the suppressive effects of CDI on miRs. CDI dysregulated DROSHA, an effect reversed by FMT. Correlation analyses, qPCR ,and 3'UTR reporter assays revealed that miR-23a, miR-150, miR-26b, and miR-28 target directly the 3'UTRs of IL12B, IL18, FGF21, and TNFRSF9, respectively. miR-23a and miR-150 demonstrated cytoprotective effects against TcdB. CONCLUSIONS: These results provide novel and provocative evidence that modulation of the gut microbiome via FMT induces alterations in circulating and intestinal tissue miRs. These findings contribute to a greater understanding of the molecular mechanisms underlying FMT and identify new potential targets for therapeutic intervention in rCDI.

Transversal gene expression panel to evaluate intestinal health in broiler chickens in different challenging conditions.

There is a high interest on gut health in poultry with special focus on consequences of the intestinal diseases, such as coccidiosis and C. perfringens-induced necrotic enteritis (NE). We developed a custom gene expression panel, which could provide a snapshot of gene expression variation under challenging conditions. Ileum gene expression studies were performed through high throughput reverse transcription quantitative real-time polymerase chain reaction. A deep review on the bibliography was done and genes related to intestinal health were selected for barrier function, immune response, oxidation, digestive hormones, nutrient transport, and metabolism. The panel was firstly tested by using a nutritional/Clostridium perfringens model of intestinal barrier failure (induced using commercial reused litter and wheat-based diets without exogenous supplementation of enzymes) and the consistency of results was evaluated by another experiment under a coccidiosis challenge (orally gavaged with a commercial coccidiosis vaccine, 90× vaccine dose). Growth traits and intestinal morphological analysis were performed to check the gut barrier failure occurrence. Results of ileum gene expression showed a higher expression in genes involved in barrier function and nutrient transport in chickens raised in healthy conditions, while genes involved in immune response presented higher expression in C.perfringens-challenged birds. On the other hand, the Eimeria challenge also altered the expression of genes related to barrier function and metabolism, and increased the expression of genes related to immune response and oxidative stress. The panel developed in the current study gives us an overview of genes and pathways involved in broiler response to pathogen challenge. It also allows us to deep into the study of differences in gene expression pattern and magnitude of responses under either a coccidial vaccine or a NE.

Functional analyses of epidemic Clostridioides difficile toxin B variants reveal their divergence in utilizing receptors and inducing pathology.

Clostridioides difficile toxin B (TcdB) is a key virulence factor that causes C. difficile associated diseases (CDAD) including diarrhea and pseudomembranous colitis. TcdB can be divided into multiple subtypes/variants based on their sequence variations, of which four (TcdB1-4) are dominant types found in major epidemic isolates. Here, we find that these variants are highly diverse in their receptor preference: TcdB1 uses two known receptors CSPG4 and Frizzled (FZD) proteins, TcdB2 selectively uses CSPG4, TcdB3 prefers to use FZDs, whereas TcdB4 uses neither CSPG4 nor FZDs. By creating chimeric toxins and systematically switching residues between TcdB1 and TcdB3, we determine that regions in the N-terminal cysteine protease domain (CPD) are involved in CSPG4-recognition. We further evaluate the pathological effects induced by TcdB1-4 with a mouse intrarectal installation model. TcdB1 leads to the most severe overall symptoms, followed by TcdB2 and TcdB3. When comparing the TcdB2 and TcdB3, TcdB2 causes stronger oedema while TcdB3 induces severer inflammatory cell infiltration. These findings together demonstrate divergence in the receptor preference and further lead to colonic pathology for predominant TcdB subtypes.

Extraintestinal Clostridioides difficile infection: Septic arthritis 12 months after colitis.

A 79-year-old male was diagnosed with a total knee prosthetic infection. He had a history of an episode of Clostridioides difficile-associated diarrhoea a year ago. Clostridioides difficile was isolated from the joint fluid and intraoperative tissue samples, and ribotyping demonstrated that the strain was the same that had caused the diarrheic episode, an infrequent ribotype (RT534). The genetic relatedness suggests that the initial infection occurred during that hospitalization, resulting in a chronic prosthetic infection. We have carried out an extensive literature review of all the cases reported in scientific databases of Clostridioides difficile infections with extraintestinal presentation and their relationship with previous colitis.

Complete genomic sequence and analysis of ß2 toxin gene mapping of Clostridium perfringens JXJA17 isolated from piglets in China.

Clostridium perfringens (Cp) is a ubiquitous opportunistic pathogen of humans and animals in the natural environment and animal intestines. The pathogenicity of Cp depends on the production of toxins encoded by genes on the chromosomes or plasmids. In contemporary literature, there is no clear consensus about the pathogenicity of CpA ß2 toxin. To analyze the homology of the genome of piglet source CpA and its ß2 toxin, we sequenced the whole genome of strain JXJA17 isolated from diarrhea piglets using the Illumina Miseq and Pacbio Sequel platforms. The genome was composed of a circular chromosome with 3,324,072 bp (G + C content: 28.51%) and nine plasmids. Genome and 16S rDNA homology analysis revealed a close relation of the JXJA17 strain with the JGS1495, Cp-06, Cp-16, and FORC_003 strains. These strains were isolated from different samples and belonged to different toxin-types. JXJA17 strain was found to carry two toxin genes (plc and cpb2). In contrast to other Cp strains, the cpb2 of JXJA17 was located on a large plasmid (58 kb) with no co-localization of other toxin genes or antibiotic resistance genes. Analysis of JXJA17 genome homology and its cpb2 would facilitate our further study the relationship between ß2 toxin and piglet diarrhea.

Diagnostic Modality of Clostridioides difficile Infection Predicts Treatment Response and Outcomes in Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) patients are at increased risk of developing Clostridioides difficile infection (CDI). Common methods to diagnose CDI involve a combination of tests including the toxin A/B enzyme immunoassay (Toxin) and toxin gene polymerase chain reaction assay (PCR). Disease outcomes in Toxin+ versus Toxin-PCR+ IBD patients remain unclear. AIMS: This study aimed to examine the response to antibiotics and risk of IBD therapy escalation in Toxin+ versus Toxin-PCR+ patients. METHODS: IBD patients at an academic center with CDI diagnosis based on Toxin+ or Toxin-PCR+ from 2012 to 2017 were identified. Comparisons of response to antibiotics within 30 days and escalation of IBD therapy within 90 days of CDI diagnosis between these two groups were analyzed by Chi-square analysis. Multivariable regression analysis examined factors associated with antibiotic response. RESULTS: Among 92 patients included, 61% had Crohn's disease and 39% had ulcerative colitis. 70% tested Toxin-PCR+. 60% received vancomycin or fidaxomicin to treat CDI. 82% of Toxin+ patients responded to antibiotics compared to 25% of Toxin-PCR+ patients (p < 0.001). 21% of Toxin+ patients required IBD therapy escalation compared to 63% of Toxin-PCR+ patients (p < 0.001). When adjusted for the types of antibiotics used, IBD subtypes, and immunosuppression status, positivity to Toxin (OR 14.85, CI 4.62-47.72) was the most significant predictor of response to antibiotics. CONCLUSIONS: Toxin+ compared to Toxin-PCR+ IBD patients had a significantly higher rate of response to antibiotics and lower chances of requiring IBD therapy escalation. Future outcome studies involving CDI in IBD patients should be stratified by modality of diagnosis.

Identification and Characterization of MAPK Signaling Pathway Genes and Associated lncRNAs in the Ileum of Piglets Infected by Clostridium perfringens Type C.

Clostridium perfringens type C (C. perfringens type C) is one of the main microbial pathogens responsible for piglet diarrhea worldwide, causing substantial economic losses for pig-rearing industries. The mitogen-activated protein kinase (MAPK) signaling pathway is a key regulator of inflammatory bowel disease, especially necrotic enteritis. However, whether and how the MAPK signaling pathway is involved in regulating the process of piglet diarrhea when challenged by C. perfringens type C are still unknown. Here, we screened 38 differentially expressed genes (DEGs) in piglets' ileum tissues experimentally infected with C. perfringens type C that were enriched in the Sus scrofa MAPK signaling pathway, based on our previous transcriptome data. Of these DEGs, 12 genes (TRAF2, MAPK8, and GADD45G, among others) were upregulated whereas 26 genes (MAPK1, TP53, and CHUK, among others) were downregulated in the infected group. Our results showed that MAPK1, TP53, MAPK8, MYC, and CHUK were in the core nodes of the PPI network. Additionally, we obtained 35 lncRNAs from the sequencing data, which could be trans-targeted to MAPK signaling pathway genes and were differentially expressed in the ileum tissues infected with C. perfringens. We used qRT-PCR to verify the expression levels of genes and lncRNAs related to the MAPK signaling pathway; their expression patterns were consistent with RNA sequencing data. Our results provide strong support for deeply exploring the role of the MAPK signaling pathway in diarrhea caused by C. perfringens type C.

Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B.

Clostridioides difficile toxins (TcdA and TcdB) are major exotoxins responsible for C. difficile infection (CDI) associated diseases. The previously reported TcdB variants showed distinct biological features, immunoactivities, and potential pathogenicity in disease progression. Here, we performed global comparisons of amino acid sequences of both TcdA and TcdB from 3,269 C. difficile genomes and clustered them according to the evolutionary relatedness. We found that TcdB was much diverse and could be divided into eight subtypes, of which four were first described. Further analysis indicates that the tcdB gene undergoes accelerated evolution to maximize diversity. By tracing TcdB subtypes back to their original isolates, we found that the distribution of TcdB subtypes was not completely aligned with the phylogeny of C. difficile. These findings suggest that the tcdB genes not only frequently mutate, but also continuously transfer and exchange among C. difficile strains.