Proline-Dependent Regulation of Clostridium difficile Stickland Metabolism

Bouillaut, Laurent; Self, William T.; Sonenshein, Abraham L.

doi:10.1128/jb.01492-12

Cited by 150 publications

(224 citation statements)

References 57 publications

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“…The sigD mutant was generated using a group II intron retargeting approach with a modified set of template and delivery plasmids (45,47,48). The intron was retargeted to insert within sigD (fliA, CD0266) after position 228, an insertion site previously used to inactivate this gene (5).…”

Section: Methodsmentioning

confidence: 99%

Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

et al. 2015

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Clostridium difficile is an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration in C. difficile was recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes, pilA1 (CD3513) and pilB1 (CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions. pilA1 is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-round in vitro transcription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation in C. difficile. The Gram-positive spore-forming bacterium Clostridium difficile is the leading cause of nosocomial diarrhea and antibioticassociated colitis in hospital settings (1). Illness caused by C. difficile infections (CDI) may range from mild diarrhea to lifethreatening, fulminant colitis. Throughout its life cycle, C. difficile has to cope with multiple changing environments. In the early steps of CDI, in order to colonize the colon and produce toxins, C. difficile spores first germinate in the intestine in response to stimuli such as the presence of bile salts (2, 3). Vegetative cells need to reach the colon, likely attaching to and colonizing the gut mucosa, where they proliferate and produce toxins. Hence, C. difficile arguably needs to sense and integrate multiple environmental stimuli in order to coordinate the expression of its colonization and virulence factors during its journey through the gastrointestinal tract. The mechanisms allowing this adaptability in C. difficile have been the focus of many recent studies, including those on the agr quorum-sensing system, alternative sigma factors, two-component systems, the global transcription regulator CodY, and the sigma factor TcdR, which promotes TcdA and TcdB toxin expression (4-9).One way by which bacteria sense and respond to changes in their environment is through signal transduction involving secondary messenger molecules. The bacterial second messenger 3=-5= cyclic diguanosine monophosphate (c-di-GMP) plays multiple key roles in bacterial physiology and virulence (10-12). c-di-GMP has been shown to antagonistically control the...

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Section: Methodsmentioning

confidence: 99%

Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

et al. 2015

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“…To generate a C. difficile tcdR mutant, the Targetron method was used (75,76) but with a modified set of template and delivery plasmids (77). The Targetron was targeted to a sequence within tcdR, AAAAAAGCG ATG.…”

Section: Methodsmentioning

confidence: 99%

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

et al. 2013

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The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; 28 ) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.

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“…The resulting PCR product was digested with HindIII and BsrG1 and then ligated into pCE240 (18) that had been digested with the same enzymes, resulting in pDSW1215. pDSW1215 was then digested with SphI and SfoI to obtain a TargeTron fragment, which was cloned into pTHE1037 that had been digested with EcoRI, made blunt-ended using a Klenow fragment, and A plasmid that targets the group II intron to nucleotide 153 of mldB was constructed similarly except as follows: the primer set was CDE914, RP114, RP115, and RP116, and the PCR product was cloned onto HindIII-BsrG1-digested pBL100 (19), resulting in pRAN243. Plasmids pRAN101 (mldA 248 ) and pRAN243 (mldB 153 ) were transformed into the E. coli HB101/pRK24 conjugation donor strain (20).…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of a Gene Cluster Required for Proper Rod Shape, Cell Division, and Pathogenesis in Clostridium difficile

et al. 2014

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Little is known about cell division in Clostridium difficile, a strict anaerobe that causes serious diarrheal diseases in people whose normal intestinal microbiome has been perturbed by treatment with broad-spectrum antibiotics. Here we identify and characterize a gene cluster encoding three cell division proteins found only in C. difficile and a small number of closely related bacteria. These proteins were named MldA, MldB, and MldC, for midcell localizing division proteins. MldA is predicted to be a membrane protein with coiled-coil domains and a peptidoglycan-binding SPOR domain. MldB and MldC are predicted to be cytoplasmic proteins; MldB has two predicted coiled-coil domains, but MldC lacks obvious conserved domains or sequence motifs. Mutants of mldA or mldB had morphological defects, including loss of rod shape (a curved cell phenotype) and inefficient separation of daughter cells (a chaining phenotype). Fusions of cyan fluorescent protein (CFP) to MldA, MldB, and MldC revealed that all three proteins localize sharply to the division site. This application of CFP was possible because we discovered that O 2 -dependent fluorescent proteins produced anaerobically can acquire fluorescence after cells are fixed with cross-linkers to preserve native patterns of protein localization. Mutants lacking the Mld proteins are severely attenuated for pathogenesis in a hamster model of C. difficile infection. Because all three Mld proteins are essentially unique to C. difficile, they might be exploited as targets for antibiotics that combat C. difficile without disrupting the intestinal microbiome.C lostridium difficile is a strictly anaerobic, Gram-positive, spore-forming bacterium that has become the leading cause of hospital-acquired diarrhea in developed countries. The annual impact of C. difficile infections in the United States has been estimated at 14,000 deaths and over $1 billion in excess medical costs (1). Both the severity and the frequency of C. difficile infections are increasing (2), and a recent report on the impact of antibiotic resistance classified the organism as an "urgent threat," the highest threat level (1).C. difficile infections typically occur in people who have been treated with antibiotics that disrupt the flora of the gastrointestinal tract (3, 4). Although C. difficile is resistant to many antibiotics, the infection usually resolves upon treatment with metronidazole or oral vancomycin (5). Unfortunately, disease recurs in ϳ20% of patients, and the prognosis for this cohort is poor (4, 6, 7). The high rate of recurrence has been attributed to germination of C. difficile spores after antibiotic therapy is ended but before restoration of the normal flora (2, 8). For this reason, there is interest in developing antibiotics that target C. difficile selectively and in treatments such as fecal transplants which work by restoring a healthy microbiome (4, 7, 9, 10).Here we describe a cluster of three genes found in C. difficile that is important for morphogenesis, cell division, and pathogenesis. W...

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Proline-Dependent Regulation of Clostridium difficile Stickland Metabolism

Cited by 150 publications

References 57 publications

Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

Identification and Characterization of a Gene Cluster Required for Proper Rod Shape, Cell Division, and Pathogenesis in Clostridium difficile

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