Biophysical library screening using a Thermo-FMN assay to identify and characterize Clostridioides difficile FabK inhibitors.

Clostridioides difficile, a gram-positive anaerobic bacterium, is one of the most frequent causes of nosocomial infections. C. difficile infection (CDI) results in almost a half a million infections and approximately 30,000 deaths in the U.S. each year. Broad-spectrum antibacterial use is a strong risk factor for development of recurring CDI. There is a critical need for narrow-spectrum antibacterials with activity limited to C. difficile. The C. difficile enoyl-acyl carrier protein (ACP) reductase II enzyme (CdFabK), an essential and rate-limiting enzyme in the organism's fatty acid biosynthesis pathway (FAS-2), is an attractive target for narrow-spectrum CDI therapeutics as it is not present in many of the non-pathogenic gut organisms. We have previously characterized inhibitors of the CdFabK enzyme with narrow-spectrum anti-difficile activity and favorable in vivo efficacy, ADME, and low dysbiosis. To expand our knowledge of the structural requirements for CdFabK inhibition, we seek to identify new inhibitors with novel chemical scaffolds. Herein we present the optimization of a thermo-FMN biophysical assay based on the principles of differential scanning fluorimetry, or thermal shift, which leverages the fluorescence signal of the FabK enzyme's FMN prosthetic group. The optimized assay was validated by pilot testing a 10K diversity-based chemical library and novel scaffold hit compounds were identified and biochemically characterized. Additionally, we show that the thermo-FMN assay can be used to determine the thermodynamic dissociation constant, Kd, of CdFabK inhibitors.

Identification of Clostridioides difficile mutants with increased daptomycin resistance.

Daptomycin is a cyclic lipopeptide antibiotic used to treat infections caused by some Gram-positive bacteria. Daptomycin disrupts synthesis of the peptidoglycan (PG) cell wall by inserting into the cytoplasmic membrane and binding multiple forms of the undecaprenyl carrier lipid required for PG synthesis. Membrane insertion requires phosphatidylglycerol, so studies of daptomycin can provide insight into assembly and maintenance of the cytoplasmic membrane. Here, we studied the effects of daptomycin on Clostridioides difficile, the leading cause of healthcare-associated diarrhea. We observed that growth of C. difficile strain R20291 in the presence of sub-MIC levels of daptomycin resulted in a chaining phenotype, minicell formation, and lysis-phenotypes broadly consistent with perturbation of membranes and PG synthesis. We also selected for and characterized eight mutants with elevated daptomycin resistance. The mutations in these mutants were mapped to four genes: cdsA (cdr20291_2041), ftsH2 (cdr20291_3396), esrR (cdr20291_1187), and draS (cdr20291_2456). Of these four genes, only draS has been characterized previously. Follow-up studies indicate these mutations confer daptomycin resistance by two general mechanisms: reducing the amount of phosphatidylglycerol in the cytoplasmic membrane (cdsA) or altering the regulation of membrane processes (ftsH2, esrR, and draS). Thus, the mutants described here provide insights into phospholipid synthesis and identify signal transduction systems involved in cell envelope biogenesis and stress response in C. difficile. IMPORTANCE: C. difficile is the leading cause of healthcare-associated diarrhea and is a threat to public health due to the risk of recurrent infections. Understanding biosynthesis of the atypical cell envelope of C. difficile may provide insight into novel drug targets to selectively inhibit C. difficile. Here, we identified mutations that increased daptomycin resistance and allowed us to better understand phospholipid synthesis, cell envelope biogenesis, and stress response in C. difficile.

Phase Variation of Flagella and Toxins in Clostridioides difficile is Mediated by Selective Rho-dependent Termination.

Clostridioides difficile is an intestinal pathogen that exhibits phase variation of flagella and toxins through inversion of the flagellar (flg) switch controlling flagellar and toxin gene expression. The transcription termination factor Rho preferentially inhibits swimming motility of bacteria with the 'flg-OFF' switch sequence. How C. difficile Rho mediates this selectivity was unknown. C. difficile Rho contains an N-terminal insertion domain (NID) which is found in a subset of Rho orthologues and confers diverse functions. Here we determined how Rho distinguishes between flg-ON and -OFF mRNAs and the roles of the NID and other domains of C. difficile Rho. Using in vitro ATPase assays, we determined that Rho specifically binds a region containing the left inverted repeat of the flg switch, but only of flg-OFF mRNA, indicating that differential termination is mediated by selective Rho binding. Using a suite of in vivo and in vitro assays in C. difficile, we determined that the NID is essential for Rho termination of flg-OFF mRNA, likely by influencing the ability to form stable hexamers, and the RNA binding domain is critical for flg-OFF specific termination. This work gives insight into the novel mechanism by which Rho interacts with flg mRNA to mediate phase variation of flagella and toxins in C. difficile and broadens our understanding of Rho-mediated termination in an organism with an AT-rich genome.

Core-genome-mediated promising alternative drug and multi-epitope vaccine targets prioritization against infectious Clostridium difficile.

Prevention of Clostridium difficile infection is challenging worldwide owing to its high morbidity and mortality rates. C. difficile is currently being classified as an urgent threat by the CDC. Devising a new therapeutic strategy become indispensable against C. difficile infection due to its high rates of reinfection and increasing antimicrobial resistance. The current study is based on core proteome data of C. difficile to identify promising vaccine and drug candidates. Immunoinformatics and vaccinomics approaches were employed to construct multi-epitope-based chimeric vaccine constructs from top-ranked T- and B-cell epitopes. The efficacy of the designed vaccine was assessed by immunological analysis, immune receptor binding potential and immune simulation analyses. Additionally, subtractive proteomics and druggability analyses prioritized several promising and alternative drug targets against C. difficile. These include FMN-dependent nitroreductase which was prioritized for pharmacophore-based virtual screening of druggable molecule databases to predict potent inhibitors. A MolPort-001-785-965 druggable molecule was found to exhibit significant binding affinity with the conserved residues of FMN-dependent nitroreductase. The experimental validation of the therapeutic targets prioritized in the current study may worthy to identify new strategies to combat the drug-resistant C. difficile infection.

The microbial metabolite urolithin A reduces Clostridioides difficile toxin expression and toxin-induced epithelial damage.

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacterium responsible for antibiotic-associated pseudomembranous colitis. Clostridioides difficile infection (CDI) symptoms can range from diarrhea to life-threatening colon damage. Toxins produced by C. difficile (TcdA and TcdB) cause intestinal epithelial injury and lead to severe gut barrier dysfunction, stem cell damage, and impaired regeneration of the gut epithelium. Current treatment options for intestinal repair are limited. In this study, we demonstrate that treatment with the microbial metabolite urolithin A (UroA) attenuates CDI-induced adverse effects on the colon epithelium in a preclinical model of CDI-induced colitis. Moreover, our analysis suggests that UroA treatment protects against C. difficile-induced inflammation, disruption of gut barrier integrity, and intestinal tight junction proteins in the colon of CDI mice. Importantly, UroA treatment significantly reduced the expression and release of toxins from C. difficile without inducing bacterial cell death. These results indicate the direct regulatory effects of UroA on bacterial gene regulation. Overall, our findings reveal a novel aspect of UroA activity, as it appears to act at both the bacterial and host levels to protect against CDI-induced colitis pathogenesis. This research sheds light on a promising avenue for the development of novel treatments for C. difficile infection.IMPORTANCETherapy for Clostridioides difficile infections includes the use of antibiotics, immunosuppressors, and fecal microbiota transplantation. However, these treatments have several drawbacks, including the loss of colonization resistance, the promotion of autoimmune disorders, and the potential for unknown pathogens in donor samples. To date, the potential benefits of microbial metabolites in CDI-induced colitis have not been fully investigated. Here, we report for the first time that the microbial metabolite urolithin A has the potential to block toxin production from C. difficile and enhance gut barrier function to mitigate CDI-induced colitis.

Phage transcriptional regulator X (PtrX)-mediated augmentation of toxin production and virulence in Clostridioides difficile strain R20291.

Clostridioides difficile is a Gram-positive, anaerobic, and spore-forming bacterial member of the human gut microbiome. The primary virulence factors of C. difficile are toxin A and toxin B. These toxins damage the cell cytoskeleton and cause various diseases, from diarrhea to severe pseudomembranous colitis. Evidence suggests that bacteriophages can regulate the expression of the pathogenicity locus (PaLoc) genes of C. difficile. We previously demonstrated that the genome of the C. difficile RT027 strain NCKUH-21 contains a prophage-like DNA sequence, which was found to be markedly similar to that of the φCD38-2 phage. In the present study, we investigated the mechanisms underlying the φNCKUH-21-mediated regulation of the pathogenicity and the PaLoc genes expression in the lysogenized C. difficile strain R20291. The carriage of φNCKUH-21 in R20291 cells substantially enhanced toxin production, bacterial motility, biofilm formation, and spore germination in vitro. Subsequent mouse studies revealed that the lysogenized R20291 strain caused a more severe infection than the wild-type strain. We screened three φNCKUH-21 genes encoding DNA-binding proteins to check their effects on PaLoc genes expression. The overexpression of NCKUH-21_03890, annotated as a transcriptional regulator (phage transcriptional regulator X, PtrX), considerably enhanced toxin production, biofilm formation, and bacterial motility of R20291. Transcriptome analysis further confirmed that the overexpression of ptrX led to the upregulation of the expression of toxin genes, flagellar genes, and csrA. In the ptrX-overexpressing R20291 strain, PtrX influenced the expression of flagellar genes and the sigma factor gene sigD, possibly through an increased flagellar phase ON configuration ratio.

Elucidation of bezlotoxumab binding specificity to toxin B in Clostridioides difficile.

C. difficile or Clostridioides difficile infection (CDI) is currently one of the major causes of epidemics worldwide. Toxin B from Clostridioides difficile toxin B (TcdB) infection is the main target protein inhibiting CDI recurrence. Clinical research suggested that bezlotoxumab's (Bez) efficiency is significantly reduced in neutralizing the B2 strain compared to the B1 strain. The monoclonal antibody (mAb) functions by binding to the epitope 1 and 2 regions in the combined repetitive oligopeptide (CROP) domain. Some binding residues are distinctively different between B1 and B2 strains. In this work, we aimed to elucidate and compare insights into the interaction of toxins B1 and B2 in complex with Bez by using all-atom molecular dynamics (MD) simulations and binding free energy calculations. The predicted ΔGbinding values suggested that the antibody (Ab) could bind to toxin B1 significantly better than B2, supported by higher salt bridge and hydrogen bonding (H-bonding) interactions, as well as the number of contact residues between the two focused proteins. The toxin B1 residues important for binding with Bez were E1878, T1901, E1902, F1905, N1941, V1946, N2031, T2032, E2033, V2076, V2077, and E2092. The lower susceptibility of Bez towards toxin B2 was primarily due to a change of residue E2033 from glutamate to alanine (A2033) and the loss of E1878 and E1902 contributions, as determined by the intermolecular interaction changes from the dynamic residue interaction network (dRIN) analysis. The obtained data strengthen our understanding of Bez/toxin B binding.

Diversification of division mechanisms in endospore-forming bacteria revealed by analyses of peptidoglycan synthesis in Clostridioides difficile.

The bacterial enzymes FtsW and FtsI, encoded in the highly conserved dcw gene cluster, are considered to be universally essential for the synthesis of septal peptidoglycan (PG) during cell division. Here, we show that the pathogen Clostridioides difficile lacks a canonical FtsW/FtsI pair, and its dcw-encoded PG synthases have undergone a specialization to fulfill sporulation-specific roles, including synthesizing septal PG during the sporulation-specific mode of cell division. Although these enzymes are directly regulated by canonical divisome components during this process, dcw-encoded PG synthases and their divisome regulators are dispensable for cell division during normal growth. Instead, C. difficile uses a bifunctional class A penicillin-binding protein as the core divisome PG synthase, revealing a previously unreported role for this class of enzymes. Our findings support that the emergence of endosporulation in the Firmicutes phylum facilitated the functional repurposing of cell division factors. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a distinct divisome that therefore may represent a unique target for therapeutic interventions.

Structural dynamics of the CROPs domain control stability and toxicity of Paeniclostridium sordellii lethal toxin.

Paeniclostridium sordellii lethal toxin (TcsL) is a potent exotoxin that causes lethal toxic shock syndrome associated with fulminant bacterial infections. TcsL belongs to the large clostridial toxin (LCT) family. Here, we report that TcsL with varied lengths of combined repetitive oligopeptides (CROPs) deleted show increased autoproteolysis as well as higher cytotoxicity. We next present cryo-EM structures of full-length TcsL, at neutral (pH 7.4) and acidic (pH 5.0) conditions. The TcsL at neutral pH exhibits in the open conformation, which resembles reported TcdB structures. Low pH induces the conformational change of partial TcsL to the closed form. Two intracellular interfaces are observed in the closed conformation, which possibly locks the cysteine protease domain and hinders the binding of the host receptor. Our findings provide insights into the structure and function of TcsL and reveal mechanisms for CROPs-mediated modulation of autoproteolysis and cytotoxicity, which could be common across the LCT family.

Clostridioides difficile ferrosome organelles combat nutritional immunity.

Iron is indispensable for almost all forms of life but toxic at elevated levels1-4. To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis1,5,6. Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules7,8. However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA9. Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection.

Revised Model for the Type A Glycan Biosynthetic Pathway in Clostridioides difficile Strain 630Δerm Based on Quantitative Proteomics of cd0241-cd0244 Mutant Strains.

The bacterial flagellum is involved in a variety of processes including motility, adherence, and immunomodulation. In the Clostridioides difficile strain 630Δerm, the main filamentous component, FliC, is post-translationally modified with an O-linked Type A glycan structure. This modification is essential for flagellar function, since motility is seriously impaired in gene mutants with improper biosynthesis of the Type A glycan. The cd0240-cd0244 gene cluster encodes the Type A biosynthetic proteins, but the role of each gene, and the corresponding enzymatic activity, have not been fully elucidated. Using quantitative mass spectrometry-based proteomics analyses, we determined the relative abundance of the observed glycan variations of the Type A structure in cd0241, cd0242, cd0243, and cd0244 mutant strains. Our data not only confirm the importance of CD0241, CD0242, and CD0243 but, in contrast to previous data, also show that CD0244 is essential for the biosynthesis of the Type A modification. Combined with additional bioinformatic analyses, we propose a revised model for Type A glycan biosynthesis.

Clostridioides difficile utilizes siderophores as an iron source and FhuDBGC contributes to ferrichrome uptake.

IMPORTANCE: This study is the first example of C. difficile growing with siderophores as the sole iron source and describes the characterization of the ferric hydroxamate uptake ABC transporter (FhuDBGC). This transporter shows specificity to the siderophore ferrichrome. While not required for pathogenesis, this transporter highlights the redundancy in iron acquisition mechanisms that C. difficile uses to compete for iron during an infection.

A sporulation signature protease is required for assembly of the spore surface layers, germination and host colonization in Clostridioides difficile.

A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile, YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σE and σK and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B. subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabGC207A, both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σK-dependent genes, cotA, coding for a coat protein, and cdeM, coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B. subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance.

Targeted inhibition of Wnt signaling with a Clostridioides difficile toxin B fragment suppresses breast cancer tumor growth.

Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of C. difficile toxin B (TcdBFBD), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdBFBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7-positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.

A Colonic Organoid Model Challenged with the Large Toxins of Clostridioides difficile TcdA and TcdB Exhibit Deregulated Tight Junction Proteins.

BACKGROUND: Clostridioides difficile toxins TcdA and TcdB are responsible for diarrhea and colitis. Lack of functional studies in organoid models of the gut prompted us to elucidate the toxin's effects on epithelial barrier function and the molecular mechanisms for diarrhea and inflammation. METHODS: Human adult colon organoids were cultured on membrane inserts. Tight junction (TJ) proteins and actin cytoskeleton were analyzed for expression via Western blotting and via confocal laser-scanning microscopy for subcellular localization. RESULTS: Polarized intestinal organoid monolayers were established from stem cell-containing colon organoids to apply toxins from the apical side and to perform functional measurements in the organoid model. The toxins caused a reduction in transepithelial electrical resistance in human colonic organoid monolayers with sublethal concentrations. Concomitantly, we detected increased paracellular permeability fluorescein and FITC-dextran-4000. Human colonic organoid monolayers exposed to the toxins exhibited redistribution of barrier-forming TJ proteins claudin-1, -4 and tricellulin, whereas channel-forming claudin-2 expression was increased. Perijunctional F-actin cytoskeleton organization was affected. CONCLUSIONS: Adult stem cell-derived human colonic organoid monolayers were applicable as a colon infection model for electrophysiological measurements. The TJ changes noted can explain the epithelial barrier dysfunction and diarrhea in patients, as well as increased entry of luminal antigens triggering inflammation.

Protective Effects of Alginate and Chitosan Oligosaccharides against Clostridioides difficile Bacteria and Toxin.

Clostridioides difficile infection is expected to become the most common healthcare-associated infection worldwide. C. difficile-induced pathogenicity is significantly attributed to its enterotoxin, TcdA, which primarily targets Rho-GTPases involved in regulating cytoskeletal and tight junction (TJ) dynamics, thus leading to cytoskeleton breakdown and ultimately increased intestinal permeability. This study investigated whether two non-digestible oligosaccharides (NDOs), alginate (AOS) and chitosan (COS) oligosaccharides, possess antipathogenic and barrier-protective properties against C. difficile bacteria and TcdA toxin, respectively. Both NDOs significantly reduced C. difficile growth, while cell cytotoxicity assays demonstrated that neither COS nor AOS significantly attenuated the TcdA-induced cell death 24 h post-exposure. The challenge of Caco-2 monolayers with increasing TcdA concentrations increased paracellular permeability, as measured by TEER and LY flux assays. In this experimental setup, COS completely abolished, and AOS mitigated, the deleterious effects of TcdA on the monolayer's integrity. These events were not accompanied by alterations in ZO-1 and occludin protein levels; however, immunofluorescence microscopy revealed that both AOS and COS prevented the TcdA-induced occludin mislocalization. Finally, both NDOs accelerated TJ reassembly upon a calcium-switch assay. Overall, this study established the antipathogenic and barrier-protective capacity of AOS and COS against C. difficile and its toxin, TcdA, while revealing their ability to promote TJ reassembly in Caco-2 cells.

Nanobodies against C. difficile TcdA and TcdB reveal unexpected neutralizing epitopes and provide a toolkit for toxin quantitation in vivo.

Clostridioides difficile is a leading cause of antibiotic-associated diarrhea and nosocomial infection in the United States. The symptoms of C. difficile infection (CDI) are associated with the production of two homologous protein toxins, TcdA and TcdB. The toxins are considered bona fide targets for clinical diagnosis as well as the development of novel prevention and therapeutic strategies. While there are extensive studies that document these efforts, there are several gaps in knowledge that could benefit from the creation of new research tools. First, we now appreciate that while TcdA sequences are conserved, TcdB sequences can vary across the span of circulating clinical isolates. An understanding of the TcdA and TcdB epitopes that drive broadly neutralizing antibody responses could advance the effort to identify safe and effective toxin-protein chimeras and fragments for vaccine development. Further, an understanding of TcdA and TcdB concentration changes in vivo can guide research into how host and microbiome-focused interventions affect the virulence potential of C. difficile. We have developed a panel of alpaca-derived nanobodies that bind specific structural and functional domains of TcdA and TcdB. We note that many of the potent neutralizers of TcdA bind epitopes within the delivery domain, a finding that could reflect roles of the delivery domain in receptor binding and/or the conserved role of pore-formation in the delivery of the toxin enzyme domains to the cytosol. In contrast, neutralizing epitopes for TcdB were found in multiple domains. The nanobodies were also used for the creation of sandwich ELISA assays that allow for quantitation of TcdA and/or TcdB in vitro and in the cecal and fecal contents of infected mice. We anticipate these reagents and assays will allow researchers to monitor the dynamics of TcdA and TcdB production over time, and the impact of various experimental interventions on toxin production in vivo.

Glycine fermentation by C. difficile promotes virulence and spore formation, and is induced by host cathelicidin.

Clostridioides difficile is a leading cause of antibiotic-associated diarrheal disease. C. difficile colonization, growth, and toxin production in the intestine is strongly associated with its ability to use amino acids to generate energy, but little is known about the impact of specific amino acids on C. difficile pathogenesis. The amino acid glycine is enriched in the dysbiotic gut and is suspected to contribute to C. difficile infection. We hypothesized that the use of glycine as an energy source contributes to colonization of the intestine and pathogenesis of C. difficile. To test this hypothesis, we deleted the glycine reductase (GR) genes grdAB, rendering C. difficile unable to ferment glycine, and investigated the impact on growth and pathogenesis. Our data show that the grd pathway promotes growth, toxin production, and sporulation. Glycine fermentation also had a significant impact on toxin production and pathogenesis of C. difficile in the hamster model of disease. Furthermore, we determined that the grd locus is regulated by host cathelicidin (LL-37) and the cathelicidin-responsive regulator, ClnR, indicating that the host peptide signals to control glycine catabolism. The induction of glycine fermentation by LL-37 demonstrates a direct link between the host immune response and the bacterial reactions of toxin production and spore formation.

Mucosal-associated invariant T cells from Clostridioides difficile-infected patients exhibit a distinct proinflammatory phenotype and enhanced cytotoxic activity.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells mainly found in the mucosa and peripheral blood. We have recently demonstrated that Clostridioides difficile activates MAIT cells in vitro. However, their role in the pathogenesis of C. difficile infection (CDI) in human patients remains elusive to date. In this study, we performed comprehensive immunophenotyping of MAIT cells derived from CDI patients and compared their phenotype to that of patients with inflammatory bowel diseases (IBD) and healthy controls. Our study revealed that blood MAIT cells from CDI patients exhibit an interleukin 17a (IL-17a)-dominated proinflammatory phenotype and an increased readiness to synthesize the proinflammatory cytokine interferon γ (IFN-γ) following in vitro re-stimulation. Moreover, the cytotoxic activity of MAIT cells, as measured by surface CD107a and intracellular granzyme B expression, was strongly increased in CDI. Multi epitope ligand cartography (MELC) analysis of intestinal biopsies from CDI patients revealed that MAIT cells exhibit an increased production of granzyme B and increased cytotoxicity compared to the control group. Together with previously published in vitro data from our group, our findings suggest that MAIT cells are functionally involved in the immune response against C. difficile and contribute to the pathogenesis of CDI.

Sequence Similarity among Structural Repeats in the Piezo Family of Mechanosensitive Ion Channels.

Members of the Piezo family of mechanically activated cation channels are involved in multiple physiological processes in higher eukaryotes, including vascular development, cell differentiation, touch perception, hearing, and more, but they are also common in single-celled eukaryotic microorganisms. Mutations in these proteins in humans are associated with a variety of diseases, such as colorectal adenomatous polyposis, dehydrated hereditary stomatocytosis, and hereditary xerocytosis. Available 3D structures for Piezo proteins show nine regions of four transmembrane segments each that have the same fold. Despite the remarkable similarity among the nine characteristic structural repeats in the family, no significant sequence similarity among them has been reported. Using bioinformatics approaches and the Transporter Classification Database (TCDB) as reference, we reliably identified sequence similarity among repeats based on four lines of evidence: (1) hidden Markov model-profile similarities across repeats at the family level, (2) pairwise sequence similarities between different repeats across Piezo homologs, (3) Piezo-specific conserved sequence signatures that consistently identify the same regions across repeats, and (4) conserved residues that maintain the same orientation and location in 3D space.