Weaned piglets are highly susceptible to infections caused by enterotoxigenic Escherichia coli (ETEC), and phytobiotic supplementation has been explored as a potential strategy to prevent or mitigate such infections during the weaning period. However, comprehensive studies on the specific effects of phytobiotics on pig gut health and microbiota composition remains limited. Therefore, this study aimed to investigate the effects of various phytogenic feed additives (PFAs) on intestinal barrier function and gut microbiota composition in weaned piglets challenged with ETEC. A total of 63 weaned piglets ([Yorkshire × Landrace] × Duroc), 28 days old with an initial body weight of 8.03 ± 0.43 kg, were used in a 21-day trial. Piglets were randomly assigned to one of seven treatment groups: NC, negative control (basal diets without E. coli challenge); PC, positive control (basal diets + E. coli challenge); T1, PC + 0.04 % P1; T2, PC + 0.01 % P2; T3, PC + 0.10 % P3; T4, PC + 0.04 % P4; T5, PC + 0.10 % P5. The five phytobiotic materials were as follows: a bitter citrus extract rich in flavonoids (P1), a microencapsulated blend of thymol and carvacrol (P2), a composite of P1 and P2 (P3), a grape polyphenol-based mixture containing green tea and hops (P4), and a fenugreek seed powder rich in saponins (P5). Piglets in challenged groups were orally inoculated with ETEC at a concentration of 1.2 × 10¹⁰ CFU/mL from days 8 to 10. The results showed that PFA supplementation upregulated the expression of tight junction and mucin-related genes including ZO-1 and MUC3. Based on 16S rRNA gene sequencing, distinct microbial shifts were observed depending on the specific PFA composition. The relative abundances of beneficial genera such as Lactobacillus, Olsenella, and Collinsella increased following PFA supplementation. These genera are known to be associated with short-chain fatty acid production, improved gut health, and increased resistance to pathogens. Additionally, increases in Acinetobacter and Medioterraneibacter, which may be involved in the metabolism of plant-derived compounds, were observed. Collectively, our results suggest that PFAs may enhance gut health and contribute to the stabilization of the intestinal microbiota in weaned piglets.

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.

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.

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.

Background/Objectives: Clostridioides difficile is a Gram-positive, spore-forming enteric pathogen that causes intestinal disorders, including inflammation and diarrhea, primarily through toxin production. Standard treatment options for C. difficile infection (CDI) involve a limited selection of antibiotics that are not fully effective, leading to high recurrence rates. Vaccination presents a promising strategy for preventing both CDI and its recurrence. Cell wall protein 2 (Cwp2), a highly immunogenic and abundant surface-exposed C. difficile cell wall protein, plays an important role in the bacterium’s adherence in vitro. In this study, we aimed to analyze the homology and immunogenicity of Cwp2 and its protection efficacy as a vaccine candidate against CDI in mice. Methods: we conducted in silico analyses to assess the homology and immunogenicity of Cwp2, and we evaluated its potential as a vaccine candidate against CDI using a mouse model of immunization and infection. Results: Our in silico analyses predicted the immunogenic region (functional domain) of Cwp2 and revealed its high homology among various toxinotypes and ribotypes (R.T.s) or sequence types (S.T.s). Immunizations of mice with the Cwp2 functional domain (Cwp2_A) induced potent IgG/A antibody responses against Cwp2_A, protected mice from CDI, and reduced C. difficile spore and toxin levels in feces post-infection. Additionally, anti-Cwp2_A sera inhibited the binding of C. difficile vegetative cells to HCT8 cells. Conclusions: Our report demonstrates for the first time the potential of Cwp2_A as an effective vaccine candidate against CDI in mice.

The REarranged during Transfection (RET) kinase is an oncoprotein and a therapeutic target for RET alteration-positive cancer. Two RET-selective protein tyrosine kinase inhibitors, selpercatinib (LOXO-292) and pralsetinib (BLU-667), have been approved for treating RET-alteration-positive cancer. However, achieving a complete response with a RET kinase inhibitor remains a challenge in most patients. In cell cultures, these inhibitors increase the expression of CCDC6-RET fusion protein, which may affect their effectiveness at low concentrations. To tackle this problem, we synthesized and characterized a panel of RET PROTACs for the dual action of inhibiting RET kinase activity and degrading the oncogenic protein. A lead compound, YW-N-7, was selected based on in vitro PROTAC analyses and in vivo PK data. YW-N-7 exhibited the dual action of specifically inhibiting and degrading RET protein. Proteomic analysis identified RET as the only significantly down-regulated protein in the entire proteome by YW-N-7 compared with the RET kinase inhibitor. In the BaF3/KIF5B-RET tumor model, YW-N-7 inhibited and degraded KIF5B-RET oncoprotein in xenograft tumors in animals, and significantly inhibited tumor growth. This work illustrates the potential of developing a RET PROTAC for simultaneously inhibiting and degrading oncogenic RET kinase for cancer therapy. Shriya Pandey, Yafeng Wang, Xueqing Hu, Ujjwol Khatri, Tao Shen, Vivek Subbiah, Blaine H. Mooers, Ting Chao, Shaohui Wang, Huaxuan Yu, Xingmin Sun, Jianfeng Cai, Jie Wu. Proteolysis targeting chimeras (PROTACs) of oncogenic RET protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7211.

Introduction:A balanced gut microbiota is essential for maintaining digestive, immune, and metabolic health. Kefir, a fermented milk beverage, influences gut microbiota through its probiotic composition and bioactive compounds, exhibiting various health-promoting effects. However, evidence on the effects of kefir on gut microbiota, particularly in healthy populations, is still limited. This study aimed to elucidate the effects of kefir on gut microbiota composition in healthy young adults under a controlled dietary environment. Methods:In this randomized, controlled, parallel-group trial, 28 healthy participants aged 18-30 years were assigned to one of three groups: kefir (n = 13), unfermented milk (n = 9), and yogurt (n = 6). Participants consumed 150 mL of their assigned beverage daily for 2 weeks. Stool samples were collected before and after the intervention to analyze gut microbiota composition using 16S rRNA sequencing. Results:Kefir consumption increased the relative abundance of lactate-producing bacteria, including Bifidobacterium breve, Ruthenibacterium lactatiformans, Weissella koreensis, and Leuconostoc mesenteroides. The genus Blautia also increased, with significant changes observed in Blautia luti and Blautia wexlerae. These shifts in species abundance were associated with increases in the short-chain fatty acid (SCFA) production pathway. Discussion:In summary, this study highlights kefir's potential to modulate gut microbiota composition in healthy individuals, emphasizing its role in supporting gut health.

Clostridioides difficile, as the leading cause of antibiotic-associated diarrhea, contributes to nosocomial and community-acquired infections. Chronic inflammation is a well-known stimulator of epithelial-mesenchymal transition (EMT) pathway, which can subsequently induce carcinogenesis in host tissues. Here, we examined the ability of membrane vesicles (MVs) derived from two clinical C. difficile ribotypes (RT001 and RT084), and the non-toxigenic strain ATCC 700057 to induce EMT process using in vitro and in vivo experiments. The proliferation and viability of HT-29 and SW-480 cells exposed to different concentrations of C. difficile MVs were assessed by trypan blue exclusion test and MTT assay, respectively. The expression level of EMT markers was determined using RT-qPCR and Western blotting. EMT induction was further investigated in 8-week-old male wild-type C57BL/6 mice orally gavaged with MVs for up to 6 weeks. C. difficile MVs did not significantly affect the viability and proliferation of both cell lines. We found upregulation of β-catenin, vimentin, snail, and reduction of E-cadherin genes in colon cancer cells and tissues of mice treated with MVs. Our results demonstrated that RT084 MVs had a stronger impact on EMT induction. Additionally, Western blot analysis of proteins of SW-480 cells corroborated the enhancement of EMT by RT084 MVs. Our findings suggest that C. difficile MVs, especially RT084 MVs, can induce EMT in mice colonic tissues. These findings provide novel mechanistic insights into how C. difficile MVs may promote pro-tumorigenic changes and highlight their potential as platforms for vaccine design, host-pathogen interaction studies, and targeted delivery systems. Further studies are needed to precisely corroborate the role of C. difficile MVs and their content on EMT induction and progression in CDI.

The human gut microbiome is a complex ecosystem shaped by both intrinsic and extrinsic factors, with external elements such as diet and exercise significantly influencing its diversity and composition. In this study, we evaluated gut microbiome shifts in adolescents participating in a four-week camp with controlled diets, lifestyle, and a healthy living environment. Stool samples were collected before and after the camp period and analyzed through 16S rRNA gene sequencing to assess changes in microbial composition and diversity. Post-intervention, gut microbiome diversity increased significantly, with notable changes in the relative abundance of taxa such as Lachnospira, Alistipes, and Barnesiella, which are associated with enhanced immune function and gut health. Additionally, functional prediction using PICRUSt indicated an increase in genes associated with energy production and metabolism, suggesting a broader functional impact of lifestyle modifications on gut microbial functionalities. These findings revealed the potential causal relationships between lifestyle modifications and gut microbiome shifts, providing valuable insights into the interactions between environment, diet, and the gut microbiota.

Clostridioides difficile (C. difficile) infection (CDI) is a life-threatening healthcare-associated infection occurring worldwide. C. difficile toxins (toxin A and toxin B) are the major virulence factors, causing CDI-related diarrhea and complications. Recent studies have shown that sulfated glycosaminoglcans (GAGs) are involved in mediating the cellular entry of these toxins. Although interactions between GAGs and toxins were reported, their binding kinetics and the structure features of glycans that facilitate toxin interaction have not been thoroughly studied. This research utilized surface plasmon resonance (SPR) to directly measure the kinetics of interactions between heparin and various toxins. Both toxin A and toxin B bind to heparin with high affinity (KD = 3.3 nM and 13.5 nM, respectively). SPR competition assay showed that both toxin A and B prefer binding to longer heparin chains and that all sulfation on the heparin chain is crucial for the heparin-toxin interaction. Finally, an in vitro assay showed that heparin and non-anticoagulant heparin inhibit the cell rounding caused by toxin A in HeLa cells.

Clostridioides difficile strains of sequence type (ST) 37, primarily including PCR ribotype (RT) 017, are prevalent in mainland China. Our study aimed to compare the major virulence factors of an epidemic C. difficile isolate of ST37 type (Xy06) from China with the well-characterized C. difficile reference strains R20291 (RT027) and CD630E (ST54), as well as a Chinese ST54 strain (Xy07) isolated from the same hospital. The Xy06 genome was predicted to harbor two complete prophages and several transposon-like elements. Comparative analysis of PaLoc revealed a truncated tcdA gene, a functional tcdB gene, a functional tcdC gene, and well-conserved tcdR and tcdE genes. Phenotypic comparisons showed that Xy06 was a robust producer of TcdB, readily sporulated and germinated, and strongly bound to human gut epithelial cells. In a mouse model of C. difficile infection, Xy06 was more virulent than strains CD630E and Xy07 and was comparable to strain R20291 in virulence. Our data suggest the potential threat of the epidemic ST37 strains in China.

The rearranged-during-transfection (RET) kinase is a validated target for the treatment of RET-altered cancers. Currently approved RET-selective kinase inhibitors, selpercatinib (LOXO-292) and pralsetinib (BLU-667), increase the oncogenic RET protein level upon treatment, which may affect their efficacy. We seek to reduce the oncogenic RET protein level and RET kinase activity simultaneously. Here, we report the development of proteolysis targeting chimera (PROTAC) degraders of oncogenic RET protein. Compound YW-N-7 exhibited dual action of selectively inhibiting and depleting RET protein both in vitro and in vivo. Proteomic analysis indicated that YW-N-7 is highly specific to RET. In cell cultures, reducing RET fusion protein potentiated the activity of LOXO-292. Furthermore, YW-N-7 showed significant activity in inhibiting KIF5B-RET-driven xenograft tumors in animals. This study exemplifies the feasibility of simultaneously inhibiting and degrading oncogenic RET kinase for cancer therapy.

The emerging antibiotic resistance has been named by the World Health Organization (WHO) as one of the top 10 threats to public health. Notably, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VREF) are designated as serious threats, whereas Clostridioides difficile (C. difficile) is recognized as one of the most urgent threats to human health and unmet medical need. Herein, they report the design and application of novel biodegradable polymers - the lipidated antimicrobial guanidinylate polycarbonates. These polymers showed potent antimicrobial activity against a panel of bacteria with fast-killing kinetics and low resistance development tendency, mainly due to their bacterial membrane disruption mechanism. More importantly, the optimal polymer showed excellent antibacterial activity against C. difficile infection (CDI) in vivo via oral administration. In addition, compared with vancomycin, the polymer demonstrated a much-prolonged therapeutic effect and virtually diminished recurrence rate of CDI. The convenient synthesis, easy scale-up, low cost, as well as biodegradability of this class of polycarbonates, together with their in vitro broad-spectrum antimicrobial activity and orally in vivo efficacy against CDI, suggest the great potential of lipidated guandinylate polycarbonates as a new class of antibacterial biomaterials to treat CDI and combat emerging antibiotic resistance.

EDITORIAL article Front. Microbiol., 23 June 2023Sec. Antimicrobials, Resistance and Chemotherapy Volume 14 - 2023 | https://doi.org/10.3389/fmicb.2023.1221606

Neutralizing antibodies and fusion inhibitory peptides have the potential required to combat the global pandemic caused by SARS-CoV-2 and its variants. However, the lack of oral bioavailability and enzymatic susceptibility limited their application, necessitating the development of novel pan-CoV fusion inhibitors. Herein we report a series of helical peptidomimetics, d-sulfonyl-γ-AApeptides, which effectively mimic the key residues of heptad repeat 2 and interact with heptad repeat 1 in the SARS-CoV-2 S2 subunit, resulting in inhibiting SARS-CoV-2 spike protein-mediated fusion between virus and cell membranes. The leads also displayed broad-spectrum inhibitory activity against a panel of other human CoVs and showed strong potency in vitro and in vivo. Meanwhile, they also demonstrated complete resistance to proteolytic enzymes or human sera and exhibited extremely long half-life in vivo and highly promising oral bioavailability, delineating their potential as pan-CoV fusion inhibitors with the potential to combat SARS-CoV-2 and its variants.

Draft genome sequences of five Clostridioides difficile clinical isolates were obtained in Florida, USA. Three isolates, designated TGH29 (sequence type 1 [ST1]/clade 2), TGH79 (ST11/clade 5), and TGH91 (ST35/clade 1), contained toxin-encoding genes. The two nontoxigenic strains were classified as TGH114 (ST109/clade 4) and TGH132 (ST15/clade 1). Antimicrobial resistance determinants and plasmids were detected and putative prophages predicted in some isolates.

Bacterial flagella are involved in infection through their roles in host cell adhesion, cell invasion, auto-agglutination, colonization, the formation of biofilms, and the regulation and secretion of nonflagellar bacterial proteins that are involved in the virulence process. In this study, we constructed a fusion protein vaccine (FliCD) containing the Clostridioides difficile flagellar proteins FliC and FliD.

The development of inhibitors that selectively block protein-protein interactions (PPIs) is crucial for chemical biology, medicinal chemistry, and biomedical sciences. Herein, we reported the design, synthesis, and investigation of sulfonyl-γ-AApeptide as an alternative strategy of canonical peptide-based inhibitors to disrupt hypoxia-inducible factor 1α (HIF-1α) and p300 PPI by mimicking the helical domain of HIF-1α involved in the binding to p300. The designed molecules recognized the p300 protein with high affinity and potently inhibited the hypoxia-inducible signaling pathway. Gene expression profiling supported the idea that the lead molecules selectively inhibited hypoxia-inducible genes involved in the signaling cascade. Our studies also demonstrated that both helical faces consisting of either chiral side chains or achiral sulfonyl side chains of sulfonyl-γ-AApeptides could be adopted for mimicry of the α-helix engaging in PPIs. Furthermore, these sulfonyl-γ-AApeptides were cell-permeable and exhibited favorable stability and pharmacokinetic profiles. Our results could inspire the design of helical sulfonyl-γ-AApeptides as a general strategy to mimic the protein helical domain and modulate many other PPIs.

The perpetually evolving antimicrobial resistance (AMR) of C. difficile is an important contributor to its epidemiology and is a grave concern to global public health. This exacerbates the challenge of treating the infections caused by this multidrug-resistant causative organism of potentially life-threatening diarrhea.