Dataset Information

High sporulation and overexpression of virulence factors in biofilms and reduced susceptibility to vancomycin and linezolid in recurrent Clostridium [Clostridioides] difficile infection isolates.

ABSTRACT: Clostridium [Clostridioides] difficile infection (CDI) is one of the leading causes of diarrhea associated with medical care worldwide, and up to 60% of patients with CDI can develop a recurrent infection (R-CDI). A multi-species microbiota biofilm model of C. difficile was designed to evaluate the differences in the production of biofilms, sporulation, susceptibility to drugs, expression of sporulating (sigH, spo0A), quorum sensing (agrD1, and luxS), and adhesion-associated (slpA and cwp84) pathway genes between selected C. difficile isolates from R-CDI and non-recurrent patients (NR-CDI). We obtained 102 C. difficile isolates from 254 patients with confirmed CDI (66 from NR-CDI and 36 from R-CDI). Most of the isolates were biofilm producers, and most of the strains were ribotype 027 (81.374%, 83/102). Most C. difficile isolates were producers of biofilm (100/102), and most were strongly adherent. Sporulation was higher in the R-CDI than in the NR-CDI isolates (p = 0.015). The isolates from R-CDI patients more frequently demonstrated reduced susceptibility to vancomycin than isolates of NR-CDI patients (27.78% [10/36] and 9.09% [6/66], respectively, p = 0.013). The minimum inhibitory concentrations for vancomycin and linezolid against biofilms (BMIC) were up to 100 times and 20 times higher, respectively, than the corresponding planktonic MICs. Expression of sigH, spo0A, cwp84, and agrD1 was higher in R-CDI than in NR-CDI isolates. Most of the C. difficile isolates were producers of biofilms with no correlation with the ribotype. Sporulation was greater in R-CDI than in NR-CDI isolates in the biofilm model of C. difficile. The R-CDI isolates more frequently demonstrated reduced susceptibility to vancomycin and linezolid than the NR-CDI isolates in both planktonic cells and biofilm isolates. A higher expression of sporulating pathway (sigH, spo0A), quorum sensing (agrD1), and adhesion-associated (cwp84) genes was found in R-CDI than in NR-CDI isolates. All of these factors can have effect on the recurrence of the infection.

SUBMITTER: Tijerina-Rodriguez L

PROVIDER: S-EPMC6668830 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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High sporulation and overexpression of virulence factors in biofilms and reduced susceptibility to vancomycin and linezolid in recurrent Clostridium [Clostridioides] difficile infection isolates.

Tijerina-Rodríguez Laura L Villarreal-Treviño Licet L Baines Simon D SD Morfín-Otero Rayo R Camacho-Ortíz Adrián A Flores-Treviño Samantha S Maldonado-Garza Héctor H Rodríguez-Noriega Eduardo E Garza-González Elvira E

PloS one 20190731 7

Clostridium [Clostridioides] difficile infection (CDI) is one of the leading causes of diarrhea associated with medical care worldwide, and up to 60% of patients with CDI can develop a recurrent infection (R-CDI). A multi-species microbiota biofilm model of C. difficile was designed to evaluate the differences in the production of biofilms, sporulation, susceptibility to drugs, expression of sporulating (sigH, spo0A), quorum sensing (agrD1, and luxS), and adhesion-associated (slpA and cwp84) pat ...[more]

PMID: 31365590

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Project description:The ability of the anaerobic gastrointestinal pathogen Clostridioides difficile to survive outside the host relies on the formation of dormant endospores. Spore formation is contingent on the activation of a conserved transcription factor, Spo0A, by phosphorylation. Multiple kinases and phosphatases regulate Spo0A activity in other spore-forming organisms; however, these factors are not well conserved in C. difficile. Previously, we discovered that deletion of a predicted histidine kinase, CD1492, increases sporulation, indicating that CD1492 inhibits C. difficile spore formation. In this study, we investigate the functions of additional predicted orphan histidine kinases CD2492, CD1579, and CD1949, which are hypothesized to regulate Spo0A phosphorylation. Disruption of CD2492 also increased sporulation frequency, similarly to the CD1492 mutant and in contrast to a previous study. A CD1492 CD2492 mutant phenocopied the sporulation and gene expression patterns of the single mutants, suggesting that these proteins function in the same genetic pathway to repress sporulation. Deletion of CD1579 variably increased sporulation frequency; however, knockdown of CD1949 expression did not influence sporulation. We provide evidence that CD1492, CD2492, and CD1579 function as phosphatases, as mutation of the conserved histidine residue for phosphate transfer abolished CD2492 function, and expression of the CD1492 or CD2492 histidine site-directed mutants or the wild-type CD1579 allele in a parent strain resulted in a dominant-negative hypersporulation phenotype. Altogether, at least three predicted histidine kinases, CD1492, CD2492, and CD1579 (herein, PtpA, PtpB and PtpC), repress C. difficile sporulation initiation by regulating activity of Spo0A. IMPORTANCE The formation of inactive spores is critical for the long-term survival of the gastrointestinal pathogen Clostridioides difficile. The onset of sporulation is controlled by the master regulator of sporulation, Spo0A, which is activated by phosphorylation. Multiple kinases and phosphatases control Spo0A phosphorylation; however, this regulatory pathway is not defined in C. difficile. We show that two predicted histidine kinase proteins, CD1492 (PtpA) and CD2492 (PtpB), function in the same regulatory pathway to repress sporulation by preventing Spo0A phosphorylation. We show that another predicted histidine kinase protein, CD1579 (PtpC), also represses sporulation and present evidence that a fourth predicted histidine kinase protein, CD1949, does not impact sporulation. These results support the idea that C. difficile inhibits sporulation initiation through multiple phosphatases.

Project description:Our study sought to compare the strain types of Clostridium difficile causing initial and recurrent episodes of C. difficile infection (CDI) in adult patients with a first episode of CDI or 1 prior episode of CDI within the previous 90 days. Strains originated from patients who had been entered into two phase 3 randomized clinical trials of fidaxomicin versus vancomycin. Isolates of C. difficile from the initial and recurrent episodes within 28 (± 2) days of cure of CDI were compared using restriction endonuclease analysis (REA) typing. Paired isolates were available from 90 of 194 (46%) patients with recurrent CDI. Patients with isolates available were significantly younger (P = .008) and more likely to be from Canadian sites (P = .0001), compared with patients without isolates. In 75 of 90 subjects (83.3%), the identical REA type strain was identified at recurrence and the initial episode (putative relapse). Early recurrences (0-14 days after treatment completion) were relapses in 86.7% and a new strain (reinfection) in 13.3%. Later recurrences (15-31 days after treatment) were relapses in 76.7% and reinfections in 23.3%. Mean time (± standard deviation) to recurrence was 12.2 (± 6.4) days for relapses and 14.7 (± 6.8) days for reinfections (P = .177). The most common BI/NAP1/027 group and the previous US epidemic REA group J/NAP2/001 had a significantly higher combined rate of recurrence with the same strain (relapse), compared with the other REA groups (39 of 42 [93%] vs 36 of 48 [75%], respectively; P = .023). We found a higher than historic rate of recurrent CDI caused by the same isolate as the original episode, a finding that may be related to the relatively short observation period in this study and the high frequency of isolation of epidemic strains, such as groups BI and J, for which relapse rates may be higher than for other REA groups. Caution in generalizing these observations is required, because the patients studied were younger and more likely to be from Canadian sites than were patients with recurrence who did not provide isolates.

Project description:The formation of dormant spores is essential for the anaerobic pathogen Clostridioides difficile to survive outside the host gastrointestinal tract. The regulatory pathways and environmental signals that initiate C. difficile spore formation within the host are not well understood. One second-messenger signaling molecule, cyclic diguanylate (c-di-GMP), modulates several physiological processes important for C. difficile pathogenesis and colonization, but the impact of c-di-GMP on sporulation is unknown. In this study, we investigated the contribution of c-di-GMP to C. difficile sporulation. The overexpression of a gene encoding a diguanylate cyclase, dccA, decreased the sporulation frequency and early sporulation gene transcription in both the epidemic R20291 and historical 630Δerm strains. The expression of a dccA allele encoding a catalytically inactive DccA that is unable to synthesize c-di-GMP no longer inhibited sporulation, indicating that the accumulation of intracellular c-di-GMP reduces C. difficile sporulation. A null mutation in dccA slightly increased sporulation in R20291 and slightly decreased sporulation in 630Δerm, suggesting that DccA contributes to the intracellular pool of c-di-GMP in a strain-dependent manner. However, these data were highly variable, underscoring the complex regulation involved in modulating intracellular c-di-GMP concentrations. Finally, the overexpression of dccA in known sporulation mutants revealed that c-di-GMP is likely signaling through an unidentified regulatory pathway to control early sporulation events in C. difficile. c-di-GMP-dependent regulation of C. difficile sporulation may represent an unexplored avenue of potential environmental and intracellular signaling that contributes to the complex regulation of sporulation initiation. IMPORTANCE Many bacterial organisms utilize the small signaling molecule cyclic diguanylate (c-di-GMP) to regulate important physiological processes, including motility, toxin production, biofilm formation, and colonization. c-di-GMP inhibits motility and toxin production and promotes biofilm formation and colonization in the anaerobic, gastrointestinal pathogen Clostridioides difficile. However, the impact of c-di-GMP on C. difficile spore formation, a critical step in this pathogen's life cycle, is unknown. Here, we demonstrate that c-di-GMP negatively impacts sporulation in two clinically relevant C. difficile strains, the epidemic strain R20291 and the historical strain 630Δerm. The pathway through which c-di-GMP controls sporulation was investigated, and our results suggest that c-di-GMP is likely signaling through an unidentified regulatory pathway to control C. difficile sporulation. This work implicates c-di-GMP metabolism as a mechanism to integrate environmental and intracellular cues through c-di-GMP levels to influence C. difficile sporulation.

Project description:The nosocomial pathogen Clostridioides difficile is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein SpoIVA and the clostridial-organism-specific spore protein SipL, which directly interact. Mutations that disrupt their interaction cause the coat to mislocalize and impair spore formation. In Bacillus subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in the C. difficile SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104-fold less severe than the equivalent mutations in B. subtilis Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased the SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced the SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele specific, our data imply that SipL recognizes the ATP-bound form of SpoIVA and highlight the importance of this interaction for functional C. difficile spore formation.IMPORTANCE The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

Dataset Information

High sporulation and overexpression of virulence factors in biofilms and reduced susceptibility to vancomycin and linezolid in recurrent Clostridium [Clostridioides] difficile infection isolates.

Publications

High sporulation and overexpression of virulence factors in biofilms and reduced susceptibility to vancomycin and linezolid in recurrent Clostridium [Clostridioides] difficile infection isolates.

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