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Clostridioides difficile is a pathogen whose transmission relies on the formation of dormant endospores. Spores are highly resilient forms of bacteria that resist environmental and chemical insults. In recent work, we found that C. difficile SspA and SspB, two small acid-soluble proteins (SASPs), protect spores from UV damage and, interestingly, are necessary for the formation of mature spores. Here, we build upon this finding and show that C. difficile sspA and sspB are required for the formation of the spore cortex layer. Moreover, using an EMS mutagenesis selection strategy, we identified mutations that suppressed the defect in sporulation of C. difficile SASP mutants. Many of these strains contained mutations in CDR20291_0714 (spoIVB2) revealing a connection between the SpoIVB2 protease and the SASPs in the sporulation pathway. This work builds upon the hypothesis that the small acid-soluble proteins can regulate gene expression. Author summary: In prior work, we found that the Clostridioides difficile small acid-soluble proteins, SspA and SspB, are important for the UV resistance that is characteristic of dormant spores (as found in many other spore-forming bacteria). Surprisingly, we also found that the combinatorial deletion of sspA and sspB led to a block in the ability of C. difficile to form dormant spores, a phenotype not observed in other sporulating bacteria. Here, we build upon this work and find that this block can be overcome by mutations in spoIVB2 –σF-regulated protease. Our data suggest that the σG-regulated SspA and SspB directly or indirectly activate spoIVB2 expression during late stages of sporulation and that the identified spoIVB2 alleles overcome the absence of sspA and sspB by increasing spoIVB2 translation efficiency and, thus, abundance during late stages of sporulation. Our data build upon the hypothesis that small acid-soluble proteins can regulate gene expression and indicate that other spore-forming bacteria may have unidentified targets that these proteins may regulate during the sporulation process. [ABSTRACT FROM AUTHOR]

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro‐protein, pro‐σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro‐sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in‐frame deletion mutant fails to produce heat‐resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To cause disease, Clostridium difficile spores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis to C. difficile spore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7[alpha]-dehydroxylation by Clostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment. doi: 10.1128/JB.00610-10

Yersinia type III machines secrete protein substrates across the bacterial envelope and, following assembly of their secretion needles, transport effector Yops into host cells. According to their destination during type III secretion, early, middle, and late secretion substrates can be distinguished; however, the signals and mechanisms whereby these proteins are recognized and transported by the secretion machine are not understood. Here, we examine several hybrids between secretion substrates and the impassable reporter protein glutathione S-transferase (GST). YscP-GST and YopR-GST blocked type III secretion; however, YscF-, YopD-, YopN-, and LcrV-GST did not. Unlike YopR-GST, which can block type III machines only during their assembly, expression of YscP-GST led to an immediate and complete block of all secretion. The secretion signal of YscP was mapped to its first 10 codons or amino acids; however, [YSCP.sub.[DELTA]2-15]-GST, lacking this secretion signal, imposed a partial blockade. YscP-GST copurified with the type III ATPase complex (YscN, YscL, and YscQ) and with YscO, suggesting that the association of specific machine components with the impassable substrate may cause the block in type III secretion.

Spore formation by Ciostridium difficile is a significant obstacle to overcoming hospital-acquired C. difficile-associated disease. Spores are resistant to heat, radiation, chemicals, and antibiotics, making a contaminated environment difficult to clean. To cause disease, however, spores must germinate and grow out as vegetative cells. The germination of C. difficile spores has not been examined in detail. In an effort to understand the germination of C. difficile spores, we characterized the response of C. difficile spores to bile. We found that cholate derivatives and the amino acid glycine act as cogerminants. Deoxycholate, a metabolite of cholate produced by the normal intestinal flora, also induced germination of C. difficile spores but prevented the growth of vegetative C. difficile. A model of resistance to C. difficile colonization mediated by the normal bacterial flora is proposed.

Type III machines of pathogenic Yersinia spp. transport Yop proteins across the bacterial envelope into host cells. Translational fusions of yopE to the dihydrofolate reductase gene (dhfr) or the [beta]-galactosidase gene (lacZ) generate hybrid proteins that block type III injection of Yop proteins into host cells, consistent with the canonical view that impassable DHFR and LacZ hybrids jam secretion machines. Mutations in repressors of posttranscriptional gene regulation, Yersinia enterocolitica yscM1 and yscM2 as well as Yersinia pestis lcrQ, relieve the YopE-DHFR-imposed blockade and restore type III injection into host cells. Genetic suppression of the type III blockade does not, however, promote YopE-DHFR secretion. A model is proposed whereby rejection of YopE-DHFR from the secretion pathway inhibits type III gene expression.

The type III secretion signal of Yersinia enterocolitica YopN was mapped using a gene fusion approach, yopN codons 1 to 12 were identified as critical for signal function. Several synonymous mutations that abolish secretion of hybrid proteins without altering the codon specificity of yopN mRNA were identified.

Yersinia enterocolitica transports YscMl and YscM2 via the type III pathway, a mechanism that is required for the establishment of bacterial infections. Prior to host cell contact, YscM1 and YscM2 exert posttranscriptional regulation to inhibit expression of effector yop genes, which encode virulence factors that travel the type III pathway into the cytoplasm of macrophages. Relief from repression has been predicted to occur via the type Ill secretion of YscMl and YscM2 into the extracellular medium, resulting in the depletion of regulatory molecules from the bacterial cytoplasm. Using digitonin fractionation and fluorescence microscopy of FlAsH-labeled polypeptides in Yersinia-infected cells, we have localized YscMl and YscM2 within the host cell cytoplasm. Type III injection of YscMl and YscM2 required the SycH chaperone. Expression of C-terminal fusions of YscMl and YscM2 to the neomycin phosphotransferase reporter revealed sequences required for regulatory activity and for secretion in the absence of SycH. Coexpression of SycH and glutathione S-transferase (GST)-YscM1 or GST-YscM2, hybrid GST variants that cannot be transported by the type III apparatus, also relieved repression of Yop synthesis. GST-SycH bound to YscM1 and YscM2 and activated effector yop expression without initiation of the bound regulatory molecules into the type III pathway. Further, regulation of yop expression by YscM1, YscM2, and SycH is shown to act independently of factors that regulate secretion, and gel filtration chromotography revealed populations of YscM1 and YscM2 that are not bound to SycH under conditions where Yop synthesis is repressed. Taken together, these results suggest that YscM1-and YscM2-mediated repression may be relieved through binding to the cytoplasmic chaperone SycH prior to their type III injection into host cells.

Some cholate derivatives that are normal components of bile can act with glycine to induce the germination of Clostridium difficile spores, but at least one bile component, chenodeoxycholate, does not induce germination. Here we show that chenodeoxycholate inhibits the germination of C. difficile spores in response to cholate and taurocholate.