Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile Colonization In Vivo

Citation: Girinathan, B. P., Braun, S., Sirigireddy, A. R., Lopez, J. E., & Govind, R. (2016). Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile Colonization In Vivo. Plos One, 11(7), 18. doi:10.1371/journal.pone.0160107Clostridium 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

Integrating sporulation, toxin production and motility by redefining the role of TcdR and characterizing the sin region in Clostridium difficile

Doctor of PhilosophyGenetics Interdepartmental ProgramRevathi GovindClostridium 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 (CodY[superscript Y146N]). Our preliminary study with the mutated CodY[superscript Y146N] suggested its differential role in its regulatory activity. Further analysis of CodY[superscript 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 CodY[superscript Y146N] suggest how the bacteria could switch to a hypervirulence mode by manipulating one of its vital regulators like CodY

Pleiotropic roles of Clostridium difficile sin locus.

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

Characterization of <i>sin</i> locus (<i>sinRR’</i>) mutant in <i>C</i>. <i>difficile</i>.

<p><b>(A)</b> Functional categorization of genes affected by <i>sin</i> locus mutation in R20291 strains based on RNA seq data. <b>(B)</b> Western blot analysis with SinR and SinR’ specific antibodies demonstrating the absence of both SinR and SinR’ in the <i>sinRR’</i> mutants and their presence after the complementation. GDH detection using anti-GDH antibodies was used as loading control. <b>(C)</b> Growth curve of the parent (R20291), <i>sinRR’</i> mutant and the <i>sinRR’</i> mutant complemented strains in TY medium. The data shown are means ± standard errors of three replicates.</p

Under-expressed sporulation genes in R20291::<i>sinRR’</i>.

<p>Under-expressed sporulation genes in R20291::<i>sinRR’</i>.</p

Effect of <i>sinR</i> or <i>sinR’</i> overexpression in the R20291 strain.

<p>The <i>sinR</i> or the <i>sinR’</i> gene was cloned under tetracycline-inducible promoter and the resulting plasmid constructs were introduced into wildtype (WT) R20291 strain for overexpression. <b>(A)</b> Toxin ELISA, <b>(B)</b> Motility assay <b>(C)</b> Sporulation frequency. The data shown are means ± standard errors of three biological replicates. Statistical analysis was performed using one way-ANOVA with Dunnett’s multiple comparisons test comparing values to the average of the parent with vector control (***<0.0005, *< 0.05 <i>p</i>-value).</p

Schematic diagram showing <i>sin</i> locus regulation of genes involved in toxin production, sporulation, and motility in <i>C. difficile</i>.

<p>Known genetic interactions are marked in red and the predicted interactions are marked in blue.</p

CodY controls the <i>sin</i> locus expression.

<p><b>(A)</b> CodY-6His binding to <i>sin</i> locus promoter region. The <i>tcdR</i> upstream and a non-specific DNA probe was as positive and negative controls respectively. <b>(B)</b> Western blot analysis of UK1 and UK1::<i>codY</i> mutants to detect SinR and SinR’ proteins.</p

Mutation in the <i>sin</i> locus affects <i>C</i>. <i>difficile</i> flagellar synthesis.

<p><b>(A)</b> Heat map showing the lower expression of flagellar and motility-related genes in the R2091::<i>sinRR’</i> mutant compared to the parent. Color intensity in each cell represents corresponding Log₂ expression values in the color scale bar. <b>(B)</b> Transmission electron micrographs of negatively stained <i>C</i>. <i>difficile</i> cells. White arrows point to flagella. <b>(C)</b> Motility of R20291, <i>sinRR’</i> mutant and the <i>sinRR’</i> mutant complemented strains in BHIS with 0.3% agar. The <i>sigD</i> mutant and the <i>sinRR’</i> mutant expressing <i>sigD</i> from an inducible promoter were included in this analysis. The swim diameters (mm) was measured every 24 h for a total of 120 h is shown and the data shown are means ± standard errors of three biological replicates. The experiments were repeated at least three times independently (*, <i>p</i>≤0.05 by a two-tailed Student's <i>t</i>-test).</p

Sporulation in <i>sinRR’</i> mutant.

<p><b>(A)</b> Phase contrast microscopy of paraformaldehyde-fixed R20291::<i>sinRR’</i> strains revealed no spores. <b>(B)</b> R20291::<i>sinRR’</i> was asporogenic as shown in representative TEM images in comparison with the parent strain. Black arrows indicate mature spores in parent strains. <b>C.</b> Asporulation phenotype of <i>sinRR’</i> mutant could not be complemented. Sporulation frequency (CFU/ml of ethanol resistant spores) of R20291, <i>sinRR’</i> mutant and mutant complemented with different constructs were determined. The <i>sinRR’</i> mutant strain expressing <i>spo0A</i> from its own promoter was also included in this analysis. Below the sporulation frequency graph is the multiplex-western blot analysis of <i>sinRR’</i> mutant complemented strain proteins using Spo0A and GDH specific antibodies.</p