Antimicrobial Guanidinylate Polycarbonates Show Oral In Vivo Efficacy Against Clostridioides Difficile.

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.

Inhibition of Hypoxia-Inducible Transcription Factor (HIF-1α) Signaling with Sulfonyl-γ-AApeptide Helices.

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 vanRCd Mutation 343A>G, Resulting in a Thr115Ala Substitution, Is Associated with an Elevated Minimum Inhibitory Concentration (MIC) of Vancomycin in Clostridioides difficile Clinical Isolates from Florida.

Clostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut microbiota and increases the risk of C. difficile infection (CDI), antibiotics are also the primary treatment for CDI. However, only a few antibiotics, including vancomycin, fidaxomicin, and rifaximin, are effective against CDI, and resistance to these antibiotics has emerged recently. In this study, we report the identification of two RT027 C. difficile clinical isolates (TGH35 and TGH64) obtained from symptomatic CDI-diagnosed patients in Tampa, Florida in 2016. These two strains showed an elevated minimum inhibitory concentration (MIC) of vancomycin (MIC = 4 µg/mL, compared to the EUCAST breakpoint of 2 µg/mL) and contained a vanRCd 343A>G mutation resulting in a Thr115Ala substitution in the VanRCd response regulator. This mutation was absent in the vancomycin-sensitive control epidemic strain RT027/R20291. TGH64 was also resistant to rifaximin (MIC ≥ 128 µg/mL) and carried the previously reported Arg505Lys and Ile548Met mutations in RpoB. Furthermore, we report on the antimicrobial resistance (AMR) and genomic characterization of additional C. difficile isolates, including RT106/TGH120, RT017/TGH33, and RT017/TGH51, obtained from the same patient sample cohort representing the highly prevalent and regionally distributed C. difficile ribotypes worldwide. Considering that the VanRCd Thr115Ala mutation was also independently reported in seven C. difficile clinical isolates from Texas and Israel in 2019, we recommend epidemiological surveillance to better understand the impact of this mutation on vancomycin resistance. IMPORTANCE 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. Further, the novel resistance-determining factors can be transferred between different strains and species of bacteria and cause the spread of AMR in clinical, environmental, and community settings. In this study, we have identified a mutation (vanRCd 343A>G) that causes a Thr115Ala substitution and is linked to an increased MIC of vancomycin in clinical isolates of C. difficile obtained from Florida in 2016. Understanding the mechanisms of AMR, especially those of newly evolving strains, is essential to effectively guide antibiotic stewardship policies to combat antibiotic resistance as well as to discover novel therapeutic targets.

Mucosal Vaccination Strategies against Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) presents a major public health threat by causing frequently recurrent, life-threatening cases of diarrhea and intestinal inflammation. The ability of C. difficile to express antibiotic resistance and to form long-lasting spores makes the pathogen particularly challenging to eradicate from healthcare settings, raising the need for preventative measures to curb the spread of CDI. Since C. difficile utilizes the fecal-oral route of transmission, a mucosal vaccine could be a particularly promising strategy by generating strong IgA and IgG responses that prevent colonization and disease. This mini-review summarizes the progress toward mucosal vaccines against C. difficile toxins, cell-surface components, and spore proteins. By assessing the strengths and weaknesses of particular antigens, as well as methods for delivering these antigens to mucosal sites, we hope to guide future research toward an effective mucosal vaccine against CDI.

An HR2-Mimicking Sulfonyl-γ-AApeptide Is a Potent Pan-coronavirus Fusion Inhibitor with Strong Blood-Brain Barrier Permeability, Long Half-Life, and Promising Oral Bioavailability.

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.

Short, Lipidated Dendrimeric γ-AApeptides as New Antimicrobial Peptidomimetics.

Antibiotic resistance is one of the most significant issues encountered in global health. There is an urgent demand for the development of a new generation of antibiotic agents combating the emergence of drug resistance. In this article, we reported the design of lipidated dendrimeric γ-AApeptides as a new class of antimicrobial agents. These AApeptides showed excellent potency and broad-spectrum activity against both Gram-positive bacteria and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The mechanistic studies revealed that the dendrimeric AApeptides could kill bacteria rapidly through the permeabilization of bacterial membranes, analogous to host-defense peptides (HDPs). These dendrimers also did not induce antibiotic resistance readily. The easy access to the synthesis, together with their potent and broad-spectrum activity, make these lipidated dendrimeric γ-AApeptides a new generation of antibacterial agents.

Draft Genome Sequences and Genome Characterization of Three Toxigenic and Two Nontoxigenic Clostridioides difficile Clinical Isolates from Florida, USA.

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.

Recombinant Fusion Protein Vaccine Containing Clostridioides difficile FliC and FliD Protects Mice against C. difficile Infection.

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 immunization of mice with FliCD induced potent IgG and IgA antibody responses against FliCD, protected mice against C. difficile infection (CDI), and decreased the C. difficile spore and toxin levels in the feces after infection. Additionally, the anti-FliCD serum inhibited the binding of C. difficile vegetative cells to HCT8 cells. These results suggest that FliCD may represent an effective vaccine candidate against CDI.

Host Immune Responses to Surface S-Layer Proteins (SLPs) of Clostridioides difficile.

Clostridioides difficile, a nosocomial pathogen, is an emerging gut pathobiont causing antibiotic-associated diarrhea. C. difficile infection involves gut colonization and disruption of the gut epithelial barrier, leading to the induction of inflammatory/immune responses. The expression of two major exotoxins, TcdA and TcdB is the major cause of C. difficile pathogenicity. Attachment of bacterial abundant cell wall proteins or surface S-layer proteins (SLPs) such as SlpA with host epithelial cells is critical for virulence. In addition to being toxins, these surface components have been shown to be highly immunogenic. Recent studies indicate that C. difficile SLPs play important roles in the adhesion of the bacteria to the intestinal epithelial cells, disruption of tight junctions, and modulation of the immune response of the host cells. These proteins might serve as new targets for vaccines and new therapeutic agents. This review summarizes our current understanding of the immunological role of SLPs in inducing host immunity and their use in the development of vaccines and novel therapeutics to combat C. difficile infection.

Regulatory transcription factors of Clostridioides difficile pathogenesis with a focus on toxin regulation.

Clostridioides difficile (CD), a nosocomial gut pathogen, produces two major exotoxins, TcdA and TcdB, which disrupt the gut epithelial barrier and induce inflammatory/immune responses, leading to symptoms ranging from mild diarrhoea to pseudomembranous colitis and potentially to death. The expression of toxins is regulated by various transcription factors (TFs) which are induced in response to CD physiological life stages, nutritional availability, and host environment. This review summarises our current understanding on the regulation of toxin expression by TFs that interconnect with pathways of flagellar synthesis, quorum sensing, motility, biofilm formation, sporulation, and phase variation. The pleiotropic roles of some key TFs suggest that toxin production is tightly linked to other cellular processes of the CD physiology.

This review summarises the current knowledge of the transcription factors involved in regulation of toxin production, which is affected by C. difficile physiological life stages, nutritional availability, and host environment in the gut.

A novel cyclic γ-AApeptide-based long-acting pan-coronavirus fusion inhibitor with potential oral bioavailability by targeting two sites in spike protein.

The receptor-binding domain (RBD) in S1 subunit and heptad repeat 1 (HR1) domain in S2 subunit of SARS-CoV-2 spike (S) protein are the targets of neutralizing antibodies (nAbs) and pan-coronavirus (CoV) fusion inhibitory peptides, respectively. However, neither nAb- nor peptide-based drugs can be used orally. In this study, we screened a one-bead-two-compound (OBTC) cyclic γ-AApeptide library against SARS-CoV-2 S protein and identified a hit: S-20 with potent membrane fusion inhibitory activity, but moderate selectivity index (SI). After modification, one derivative, S-20-1, exhibited improved fusion inhibitory activity and SI (>1000). S-20-1 could effectively inhibit infection by pseudotyped and authentic SARS-CoV-2 and pseudotyped variants of concern (VOCs), including B.1.617.2 (Delta) and B.1.1.529 (Omicron), as well as MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, and HCoV-NL63. It could also inhibit infection of a pseudotyped SARS-related coronavirus WIV1 (SARSr-CoV-WIV1) from bats. Intranasal application of S-20-1 to mice before or after challenge with HCoV-OC43 or SARS-CoV-2 provided significant protection from infection. Importantly, S-20-1 was highly resistant to proteolytic degradation, had long half-life, and possessed favorable oral bioavailability. Mechanistic studies suggest that S-20-1 binds with high affinity to RBD in S1 and HR1 domain in S2 of SARS-CoV-2 S protein. Thus, with its pan-CoV fusion and entry inhibitory activity by targeting two sites in S protein, desirable half-life, and promising oral bioavailability, S-20-1 is a potential candidate for further development as a novel therapeutic and prophylactic drug against infection by SARS-CoV-2 and its variants, as well as future emerging and reemerging CoVs.

Chemistry and Bioactivity of the Deep-Water Antarctic Octocoral Alcyonium sp.

Chemical investigation of an Antarctic deep-water octocoral has led to the isolation of four new compounds, including three illudalane sesquiterpenoids (1-3) related to the alcyopterosins, a highly oxidized steroid, alcyosterone (5), and five known alcyopterosins (4, 6-9). The structures were established by extensive 1D and 2D NMR analyses, while 9 was verified by XRD. Alcyopterosins are unusual for their nitrate ester functionalization and have been characterized with cytotoxicity related to their DNA binding properties. Alcyopterosins V (3) and E (4) demonstrated single-digit micromolar activity against Clostridium difficile, an intestinal bacterium capable of causing severe diarrhea that is increasingly associated with drug resistance. Alcyosterone (5) and several alcyopterosins were similarly potent against the protist Leishmania donovani, the causative agent of leishmaniasis, a disfiguring disease that can be fatal if not treated. While the alcyopterosin family of sesquiterpenes is known for mild cytotoxicity, the observed activity against C. difficile and L. donovani is selective for the infectious agents.

A unique class of Zn2+-binding serine-based PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile.

Treatment with ß-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C. difficile has four different PBPs (PBP1-3 and SpoVD) with various roles in growth and spore formation, and their specific links to ß-lactam resistance in this pathogen are underexplored. Here, we show that PBP2 (known to be essential for vegetative growth) is the primary bactericidal target for ß-lactams in C. difficile. PBP2 is insensitive to cephalosporin inhibition, and this appears to be the main basis for cephalosporin resistance in this organism. We determine crystal structures of C. difficile PBP2, alone and in complex with ß-lactams, revealing unique features including ligand-induced conformational changes and an active site Zn2+-binding motif that influences ß-lactam binding and protein stability. The Zn2+-binding motif is also present in C. difficile PBP3 and SpoVD (which are known to be essential for sporulation), as well as in other bacterial taxa including species living in extreme environments and the human gut. We speculate that this thiol-containing motif and its cognate Zn2+ might function as a redox sensor to regulate cell wall synthesis for survival in adverse or anaerobic environments.

Development of an Effective Nontoxigenic Clostridioides difficile-Based Oral Vaccine against C. difficile Infection.

The symptoms of Clostridioides difficile infection (CDI) are largely attributed to two C. difficile toxins, TcdA and TcdB. Significant efforts have been devoted to developing vaccines targeting both toxins through parenteral immunization routes. Recently, we generated a novel chimeric protein (designated Tcd169), comprised of the glucosyltransferase domain (GT), the cysteine protease domain (CPD), and the receptor binding domain (RBD) of TcdB, and the RBD of TcdA. Parenteral immunizations with Tcd169 provide mice effective protection against infection with a ribotype (RT) 027 C. difficile strain. In this study, we expressed Tcd169 in a nontoxigenic C. difficile CCUG37785 strain (designated NTCD), resulting in strain NTCD_Tcd169 to develop an oral vaccine that can target both C. difficile toxins and colonization/adhesion factors. Oral immunizations with NTCD_Tcd169 spores induced systematic and mucosal antibody responses against, not only both toxins, but also C. difficile flagellins (FliC/FliD). Intriguingly yet importantly, anti-Tcd169 sera raised against Tcd169 protein were significantly cross-reactive with FliC/FliD and two surface layer proteins (SlpA and Cwp2). Oral immunizations with NTCD_Tcd169 spores provided mice effective protection against infection with a hypervirulent RT027 C. difficile strain R20291and significantly reduced R20291spore numbers in feces compared with NTCD or PBS immunized mice. These results imply that the genetically modified, nontoxigenic C. difficile strain expressing Tcd169 may represent a novel mucosal vaccine candidate against CDI. IMPORTANCE Clostridioides difficile is an enteric pathogen, and symptoms of C. difficile infection (CDI) are mainly by two exotoxins TcdA and TcdB. Active vaccination is cost-effective approach to prevent CDI and high rates of recurrence. Ideally, vaccines should target both C. difficile toxins and cell/spore colonization. In this study, we expressed immunodominant fragments of TcdA and TcdB (i.e., Tcd169) in a nontoxigenic C. difficile CCUG37785 strain, generating a promising oral/mucosal vaccine candidate against CDI, by targeting both toxins and colonization of pathogenic C. difficile strains. Importantly, anti-Tcd169 sera raised against Tcd169 protein were significantly cross-reactive with FliC/FliD and two surface layer proteins (SlpA and Cwp2), and all of which are involved in C. difficile adhesion/colonization in vitro and in vivo.

On the Potential Significance of the Intrinsically Disordered Regions in the Clostridiodes difficile Toxins A and B.

BACKGROUND: Clostridiodes (or Clostridium) difficile is a spore-forming, Gram-positive anaerobic bacterium that may cause symptoms ranging from diarrhea to pseudomembranous colitis. During the C. difficile infection (CDI), the two primary bacterial toxins, toxin A (TcdA) or toxin B (TcdB), disrupt host cell function mainly through the inactivation of small GTPases that regulate the actin cytoskeleton. Both toxins have complex structural organization containing several functional domains. METHODS: Analytical bioinformatics tools are used to compare the extent of disorder within TcdA and TcdB proteins, and to see if the existence of structural disorder can be used to explain the difference in the functionality of these toxins. RESULTS: This paper's aim is to offer an overall review of the structural and functional differences between TcdA and TcdB. CONCLUSION: Results of our multifactorial bioinformatics analysis revealed that intrinsic disorder may play a role in the multifunctionality of C. difficile major toxins TcdA and TcdB, suggesting that intrinsic disorder may be related to their pathogenic mechanisms.

Genomic and Phenotypic Characterization of the Nontoxigenic Clostridioides difficile Strain CCUG37785 and Demonstration of Its Therapeutic Potential for the Prevention of C. difficile Infection.

Symptoms of Clostridioides difficile infection (CDI) are attributed largely to two toxins, TcdA and TcdB. About 17-23% of C. difficile isolates produce binary toxin, which enhances C. difficile pathogenesis. Previously, we engineered the nontoxigenic C. difficile strain CCUG37785 (designated as CCUG37785) to express immunogenic fragments of TcdA and TcdB as an oral mucosal CDI vaccine candidate. In this study, we performed genomic and phenotypic analyses of CCUG37785 and evaluated its potential use for preventing and treating CDI. Whole genome sequencing showed that CCUG37785 is ribotype ST3 and lacks toxin genes. Comparative analyses of PaLoc and CdtLoc loci of CCUG37785 revealed 115-bp and 68-bp conserved fragments in these regions, respectively. Phenotypic comparisons between CCUG37785 and C. difficile R20291 (an epidemic hypervirulent BI/NAPI/027 strain, designated as R20291) found that CCUG37785 exhibited significantly higher adhesion and sporulation, significantly lower spore germination and biofilm formation, and comparable motility to R20291. We also showed that oral inoculation of CCUG37785 spores prior to infection with R20291 spores provided mice almost full protection against developing CDI. However, oral inoculation of CCUG37785 spores after infection with R20291 spores only provided minor protection against CDI. Further analysis showed that mice pretreated with CCUG37785 spores secreted significantly less R20291 spores, while mice treated with CCUG37785 spores after infection with R20291 secreted a comparable amount of R20291 spores to mice infected with R20291 spores only. Our data both highlight the potential use of CCUG37785 for the prevention of primary and recurrent CDI in humans and support its use as an oral mucosal vaccine carrier against CDI. IMPORTANCE Clostridioides difficile infection (CDI) symptoms range from diarrhea to intestinal inflammation/lesion and death and are mainly caused by two exotoxins, TcdA and TcdB. Active vaccination provides the attractive opportunity to prevent CDI and recurrence. No vaccine against CDI is currently licensed. Tremendous efforts have been devoted to developing vaccines targeting both toxins. However, ideally, vaccines should target both toxins and C. difficile cells/spores that transmit the disease and cause recurrence. Furthermore, C. difficile is an enteric pathogen, and mucosal/oral immunization would be particularly useful to protect the host against CDI considering that the gut is the main site of disease onset and progression. Data in our current study not only highlight the potential use of CCUG37785 to prevent primary and recurrent CDI in humans but also further support its use as an oral mucosal vaccine carrier against CDI.

Recent developments in systems biology and genetic engineering toward design of vaccines for TB.

Tuberculosis (TB) is one of the most prevalent diseases worldwide. The currently available Bacillus Calmette-Guérin vaccine is not sufficient in protecting against pulmonary TB. Although many vaccines have been evaluated in clinical trials, but none of them yet has proven to be more successful. Thus, new strategies are urgently needed to design more effective TB vaccines. The emergence of new technologies will undoubtedly accelerate the process of vaccine development. This review summarizes the potential and validated applications of emerging technologies, including: systems biology (genomics, proteomics, and transcriptomics), genetic engineering, and other computational tools to discover and develop novel vaccines against TB. It also discussed that the significant implementation of these approaches will play crucial roles in the development of novel vaccines to cure and control TB.

Pathobionts: mechanisms of survival, expansion, and interaction with host with a focus on Clostridioides difficile.

Pathobionts are opportunistic microbes that emerge as a result of perturbations in the healthy microbiome due to complex interactions of various genetic, exposomal, microbial, and host factors that lead to their selection and expansion. Their proliferations can aggravate inflammatory manifestations, trigger autoimmune diseases, and lead to severe life-threatening conditions. Current surge in microbiome research is unwinding these complex interplays between disease development and protection against pathobionts. This review summarizes the current knowledge of pathobiont emergence with a focus on Clostridioides difficile and the recent findings on the roles of immune cells such as iTreg cells, Th17 cells, innate lymphoid cells, and cytokines in protection against pathobionts. The review calls for adoption of innovative tools and cutting-edge technologies in clinical diagnostics and therapeutics to provide insights in identification and quantification of pathobionts.

FliW and CsrA Govern Flagellin (FliC) Synthesis and Play Pleiotropic Roles in Virulence and Physiology of Clostridioides difficile R20291.

Clostridioides difficile flagellin FliC is associated with toxin gene expression, bacterial colonization, and virulence, and is also involved in pleiotropic gene regulation during in vivo infection. However, how fliC expression is regulated in C. difficile remains unclear. In Bacillus subtilis, flagellin homeostasis and motility are coregulated by flagellar assembly factor (FliW), flagellin Hag (FliC homolog), and Carbon storage regulator A (CsrA), which is referred to as partner-switching mechanism "FliW-CsrA-Hag." In this study, we characterized FliW and CsrA functions by deleting or overexpressing fliW, csrA, and fliW-csrA in C. difficile R20291. We showed that fliW deletion, csrA overexpression in R20291, and csrA complementation in R20291ΔWA (fliW-csrA codeletion mutant) dramatically decreased FliC production, but not fliC gene transcription. Suppression of fliC translation by csrA overexpression can be relieved mostly when fliW was coexpressed, and no significant difference in FliC production was detected when only fliW was complemented in R20291ΔWA. Further, loss of fliW led to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model of C. difficile infection (CDI), while fliW-csrA codeletion decreased toxin production and mortality in vivo. Our data suggest that CsrA negatively modulates fliC expression and FliW indirectly affects fliC expression through inhibition of CsrA post-transcriptional regulation. In light of "FliW-CsrA-Hag" switch coregulation mechanism reported in B. subtilis, our data also suggest that "FliW-CsrA-fliC/FliC" can regulate many facets of C. difficile R20291 pathogenicity. These findings further aid us in understanding the virulence regulation in C. difficile.