Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the ...disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR', coding for SinR (112 aa) and SinR' (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus's role in C. difficile physiology. Transcriptome analysis of the sinRR' mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR' acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis.
Clostridium difficile is the principal cause of antibiotic-associated diarrhea. Major metabolic requirements for colonization and expansion of C. difficile after microbiota disturbance have not been ...fully determined. In this study, we show that glutamate utilization is important for C. difficile to establish itself in the animal gut. When the gluD gene, which codes for glutamate dehydrogenase (GDH), was disrupted, the mutant C. difficile was unable to colonize and cause disease in a hamster model. Further, from the complementation experiment it appears that extracellular GDH may be playing a role in promoting C. difficile colonization and disease progression. Quantification of free amino acids in the hamster gut during C. difficile infection showed that glutamate is among preferred amino acids utilized by C. difficile during its expansion. This study provides evidence of the importance of glutamate metabolism for C. difficile pathogenesis.
Toxigenic Clostridium sordellii causes uncommon but highly lethal infections in humans and animals. Recently, an increased incidence of C. sordellii infections has been reported in women undergoing ...obstetric interventions. Pathogenic strains of C. sordellii produce numerous virulence factors, including sordellilysin, phospholipase, neuraminidase, and two large clostridial glucosylating toxins, TcsL and TcsH. Recent studies have demonstrated that TcsL toxin is an essential virulence factor for the pathogenicity of C. sordellii. In this study, we identified and characterized TcsR as the toxin gene (tcsL) regulator in C. sordellii. High-throughput sequencing of two C. sordellii strains revealed that tcsR lies within a genomic region that encodes TcsL, TcsH, and TcsE, a putative holin. By using ClosTron technology, we inactivated the tcsR gene in strain ATCC 9714. Toxin production and tcsL transcription were decreased in the tcsR mutant strain. However, the complemented tcsR mutant produced large amounts of toxins, similar to the parental strain. Expression of the Clostridium difficile toxin gene regulator tcdR also restored toxin production to the C. sordellii tcsR mutant, showing that these sigma factors are functionally interchangeable.
Clostridioides difficile is a Gram-positive, anaerobic bacterium. It is known that C. difficile is one of the major causes of antibiotic associated diarrhea. The enhanced antibiotic resistance ...observed in C. difficile is the result of highly resistant spores produced by the bacterium. In Bacillus subtilis, the sin operon is involved in sporulation inhibition. Two proteins coded within this operon, SinR and SinI, have an antagonistic relationship; SinR acts as an inhibitor to sporulation whereas SinI represses the activity of SinR, thus allowing the bacterium to sporulate. In a previous study, we examined the sin locus in C. difficile and named the two genes associated with this operon sinR and sinR′, analogous to sinR and sinI in B. subtilis, respectively. We have shown that SinR and SinR′ have pleiotropic roles in pathogenesis pathways and interact antagonistically with each other. Unlike B. subtilis SinI, SinR′ in C. difficile carries two domains: the HTH domain and the Multimerization Domain (MD). In this study, we first performed a GST Pull-down experiment to determine the domain within SinR′ that interacts with SinR. Second, the effect of these two domains on three phenotypes; sporulation, motility, and toxin production was examined. The findings of this study confirmed the prediction that the Multimerization Domain (MD) of SinR′ is responsible for the interaction between SinR and SinR’. It was also discovered that SinR’ regulates sporulation, toxin production and motility primarily by inhibiting SinR activity through the Multimerization Domain (MD).
•Clostridioides difficile pathogenicity is controlled by the sporulation inhibition (sin) locus.•The pleiotropic regulator SinR acts as a regulator for sporulation, toxin production, and motility pathways.•Regulation of SinR is achieved by SinR’. The antagonistic relationship between SinR and SinR’ causes inactivation of SinR.•SinR’ encodes an HTH DNA binding domain (1-63 aa) and Multimerization Domain (76-110 aa).•SinR’ directly regulates SinR primarily through a protein-protein interaction with the Multimerization Domain.
Clostridium difficile is a gram-positive anaerobic, motile, spore-forming opportunistic bacterium. It is a nosocomial pathogen, and the symptoms of C. difficile infection (CDI) range from mild ...diarrhea to life-threatening pseudomembranous colitis and toxic megacolon. Antibiotic use is the primary risk factor for the development of CDI as it disrupts the healthy protective gut flora which enables C. difficile to colonize and establish in the colon. C. difficiledamages the host tissue by secreting toxins and disseminates in the environment by forming spores. The two-major toxin-encoding genes, tcdA, and tcdB are located within a 19.6 kb pathogenicity locus (PaLoc), which also includes the gene encoding an RNA polymerase sigma factor tcdR, that is essential for toxin gene expression. We created a site-directed mutation in tcdR in the epidemic-type C. difficileR20291 strain and found that disruption of tcdR affected sporulation in addition to toxin production. Spores of the tcdR mutant were more heat- sensitive and required nearly three-fold higher taurocholate to germinate when compared to the wild-type (WT). Transmission Electron Microscopic analysis of the tcdR mutant spores also revealed a weakly assembled exosporium. Consistent with our phenotypic assays, our comparative transcriptome analysis also showed significant downregulation of sporulation genes in the tcdR mutant when compared to the WT strain. Our findings on tcdR suggest that the regulatory networks of toxin production and sporulation in C. difficileR20291 strain are interlinked with each other. Transcriptome analysis revealed the sin operon to be significantly downregulated in the tcdR mutant which made us hypothesize the link between sin operon regulation and sporulation. The sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition in B. subtilis. SinR in B. subtilis mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, and SinI/SinR interaction determines whether or not the SinR can inhibit target gene expression. The C. difficilegenome carries two sinR homologs in the operon, and we named it as sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. To identify the regulation of sin on sporulation, we created a site-directed mutation in the sin locus in two different C. difficilestrains R20291 and JIR8094. Comparative transcriptome analysis of the sinRR' mutants revealed their pleiotropic roles in controlling several essential pathways including sporulation, toxin production, and motility (STM) in C. difficile. We performed several genetic and biochemical experiments, to prove that SinR regulates transcription of crucial regulators in STM pathways, which includes sigD, spo0A, and codY. Unlike B. subtilis, SinR’ acts as an antagonist of SinR and SinR’/SinR determines SinR activity. Our in vivo experiment using hamster model also demonstrated the importance of sin locus for successful C. difficilecolonization. Our findings above reveal that sin locus acts as a central link that regulates essential pathways including sporulation, toxin production, and motility, which are critical for C. difficilepathogenesis. The final section of this dissertation analyzes a variant codY gene in the epidemic C. difficile R20291 strain. In this strain the CodY, a global nutrient sensor-regulator carry a missense mutation where the 146th tyrosine residue is replaced with asparagine (CodYY146N). Our preliminary study with the mutated CodYY146N suggested its differential role in its regulatory activity. Further analysis of CodYY146N might give some possible clues behind the hypervirulent nature of epidemic R20291 strain. Taken together, studies performed on both tcdR and sinR mutants reveal a significant amount of crosstalk occurring between the powerful regulators of STM pathways under the directionality of tcdR and SinR in determining their ultimate cell fate. Our findings on CodYY146N suggest how the bacteria could switch to a hypervirulence mode by manipulating one of its vital regulators like CodY.
Doctor of Philosophy
Genetics Interdepartmental Program
Revathi Govind
Clostridium difficile is a gram-positive anaerobic, motile, spore-forming opportunistic bacterium. It is a nosocomial pathogen, ...and the symptoms of C. difficile infection (CDI) range from mild diarrhea to life-threatening pseudomembranous colitis and toxic megacolon. Antibiotic use is the primary risk factor for the development of CDI as it disrupts the healthy protective gut flora which enables C. difficile to colonize and establish in the colon. ;
C. difficile damages the host tissue by secreting toxins and disseminates in the environment by forming spores. The two-major toxin-encoding genes, tcdA, and tcdB are located within a 19.6 kb pathogenicity locus (PaLoc), which also includes the gene encoding an RNA polymerase sigma factor TcdR, that is essential for toxin gene expression. We created a site-directed mutation in tcdR in the epidemic-type C. difficile R20291 strain and found that disruption of tcdR affected sporulation in addition to toxin production. Spores of the tcdR mutant were more heat- sensitive and required nearly three-fold higher taurocholate to germinate when compared to the wild-type (WT). Transmission Electron Microscopic analysis of the tcdR mutant spores also revealed a weakly assembled exosporium. Consistent with our phenotypic assays, our comparative transcriptome analysis also showed significant downregulation of sporulation genes in the tcdR mutant when compared to the WT strain. Our findings on tcdR suggest that the regulatory networks of toxin production and sporulation in C. difficile R20291 strain are interlinked with each other.;
Transcriptome analysis revealed the sin operon to be significantly downregulated in the tcdR mutant which made us hypothesize the link between sin operon regulation and sporulation. The sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition in B. subtilis. SinR in B. subtilis mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, and SinI/SinR interaction determines whether or not the SinR can inhibit target gene expression. ;
The C. difficile genome carries two sinR homologs in the operon, and we named it as sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. To identify the regulation of sin on sporulation, we created a site-directed mutation in the sin locus in two different C. difficile strains R20291 and JIR8094. Comparative transcriptome analysis of the sinRR’ mutants revealed their pleiotropic roles in controlling several essential pathways including sporulation, toxin production, and motility (STM) in C. difficile. ;
We performed several genetic and biochemical experiments, to prove that SinR regulates transcription of crucial regulators in STM pathways, which includes sigD, spo0A, and codY. Unlike B. subtilis, SinR’ acts as an antagonist of SinR and SinR’/SinR determines SinR activity. Our in vivo experiment using hamster model also demonstrated the importance of sin locus for successful C. difficile colonization. Our findings above reveal that sin locus acts as a central link that regulates essential pathways including sporulation, toxin production, and motility, which are critical for C. difficile pathogenesis. ;
The final section of this dissertation analyzes a variant codY gene in the epidemic C. difficile R20291 strain. In this strain the CodY, a global nutrient sensor-regulator carry a missense mutation where the 146th tyrosine residue is replaced with asparagine (CodYsuperscript Y146N). Our preliminary study with the mutated CodYsuperscript Y146N suggested its differential role in its regulatory activity. Further analysis of CodYsuperscript Y146N might give some possible clues behind the hypervirulent nature of epidemic R20291 strain. ;
Taken together, studies performed on both tcdR and sinR mutants reveal a significant amount of crosstalk occurring between the powerful regulators of STM pathways under the directionality of TcdR and SinR in determining their ultimate cell fate. Our findings on CodYsuperscript Y146N suggest how the bacteria could switch to a hypervirulence mode by manipulating one of its vital regulators like CodY.
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