http://iai.asm.org/content/early/2016/09/13/IAI.00735-16 The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile Childress KO, Edwards AN, Nawrocki KL, Woods EC, Anderson SE, McBride SM The formation of spores is critical for survival of Clostridium difficile outside of the host gastrointestinal tract. Persistence of C. difficile spores …
C diff Dlt pathway is controlled by the ECF sigma factor, σV, in response to lysozyme http://iai.asm.org/cgi/content/long/IAI.00207-16v1
http://www.ncbi.nlm.nih.gov/pubmed/26915493?dopt=Abstract A Novel Regulator Controls Clostridium difficile Sporulation, Motility and Toxin Production
Effects of surotomycin on Clostridium difficile viability and toxin production in vitro.
Antimicrob Agents Chemother. 2015 Jul;59(7):4199-205
Authors: Bouillaut L, McBride S, Sorg JA, Schmidt DJ, Suarez JM, Tzipori S, Mascio C, Chesnel L, Sonenshein AL
The increasing incidence and severity of infection by Clostridium difficile have stimulated attempts to develop new antimicrobial therapies. We report here the relative abilities of two antibiotics (metronidazole and vancomycin) in current use for treating C. difficile infection and of a third antimicrobial, surotomycin, to kill C. difficile cells at various stages of development and to inhibit the production of the toxin proteins that are the major virulence factors. The results indicate that none of the drugs affects the viability of spores at 8× MIC or 80× MIC and that all of the drugs kill exponential-phase cells when provided at 8× MIC. In contrast, none of the drugs killed stationary-phase cells or inhibited toxin production when provided at 8× MIC and neither vancomycin nor metronidazole killed stationary-phase cells when provided at 80× MIC. Surotomycin, on the other hand, did kill stationary-phase cells when provided at 80× MIC but did so without inducing lysis.
PMID: 25941230 [PubMed – in process]
An alkaline phosphatase reporter for use in Clostridium difficile.
Anaerobe. 2015 Apr;32:98-104
Authors: Edwards AN, Pascual RA, Childress KO, Nawrocki KL, Woods EC, McBride SM
Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.
PLoS One. 2014;9(11):e112660
Authors: Hudson LE, Fasken MB, McDermott CD, McBride SM, …
Synthetic polymers active against Clostridium difficile vegetative cell growth and spore outgrowth.
J Am Chem Soc. 2014 Oct 15;136(41):14498-504
Authors: Liu R, Suárez JM, Weisblum B, Gellman SH, McBride SM
Nylon-3 polymers (poly-β-peptides) have been investigated as synthetic mimics of host-defense peptides in recent years. These polymers are attractive because they are much easier to synthesize than are the peptides themselves, and the polymers resist proteolysis. Here we describe in vitro analysis of selected nylon-3 copolymers against Clostridium difficile, an important nosocomial pathogen that causes highly infectious diarrheal disease. The best polymers match the human host-defense peptide LL-37 in blocking vegetative cell growth and inhibiting spore outgrowth. The polymers and LL-37 were effective against both the epidemic 027 ribotype and the 012 ribotype. In contrast, neither vancomycin nor nisin inhibited outgrowth for the 012 ribotype. The best polymer was less hemolytic than LL-37. Overall, these findings suggest that nylon-3 copolymers may be useful for combatting C. difficle.
PMID: 25279431 [PubMed – indexed for MEDLINE]
Conserved oligopeptide permeases modulate sporulation initiation in Clostridium difficile.
Infect Immun. 2014 Oct;82(10):4276-91
Authors: Edwards AN, Nawrocki KL, McBride SM
Antimicrobial Peptide Resistance Mechanisms of Gram-Positive Bacteria.
Antibiotics (Basel). 2014 Oct 13;3(4):461-492
Authors: Nawrocki KL, Crispell EK, McBride SM
Initiation of sporulation in Clostridium difficile: a twist on the classic model.
FEMS Microbiol Lett. 2014 Sep;358(2):110-8
Authors: Edwards AN, McBride SM
The formation of dormant endospores is a complex morphological process that permits long-term survival in inhospitable environments for many Gram-positive bacteria. Sporulation for the anaerobic gastrointestinal pathogen Clostridium difficile is necessary for survival outside of the gastrointestinal tract of its host. While the developmental stages of spore formation are largely conserved among endospore-forming bacteria, the genus Clostridium appears to be missing a number of conserved regulators required for efficient sporulation in other spore-forming bacteria. Several recent studies have discovered novel mechanisms and distinct regulatory pathways that control the initiation of sporulation and early-sporulation-specific gene expression. These differences in regulating the decision to undergo sporulation reflects the unique ecological niche and environmental conditions that C. difficile inhabits and encounters within the mammalian host.
PMID: 24910370 [PubMed – indexed for MEDLINE]
Culturing and maintaining Clostridium difficile in an anaerobic environment.
J Vis Exp. 2013;(79):e50787
Authors: Edwards AN, Suárez JM, McBride SM
Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.
PMID: 24084491 [PubMed – indexed for MEDLINE]
The Clostridium difficile cpr locus is regulated by a noncontiguous two-component system in response to type A and B lantibiotics.
J Bacteriol. 2013 Jun;195(11):2621-31
Authors: Suárez JM, Edwards AN, McBride SM
The intestinal pathogen Clostridium difficile is known to grow only within the intestines of mammals, yet little is known about how the bacterium subsists in this environment. In the intestine, C. difficile must contend with innate defenses within the host, such as cationic antimicrobial peptides (CAMPs) produced by the host and the indigenous microbiota. In this study, we investigated the mechanism of activation and regulation of the CprABC transporter system, which provides resistance to multiple CAMPs and shows homology to the immunity systems of bacterial antimicrobial peptide producers. The CprABC system proved to be controlled by a noncontiguous two-component system consisting of the CprK sensor kinase and an orphan response regulator (CD3320; CprR). The CprK-CprR regulators were shown to activate cprABCK transcription in a manner similar to that by lantibiotic regulatory systems. Unlike lantibiotic producer regulation, regulation by CprK-CprR was activated by multiple lantibiotics produced by diverse Gram-positive bacteria. We identified a motif within these lantibiotics that is likely required for activation of cpr. Based on the similarities between the Cpr system and lantibiotic systems, we propose that the CprABC transporter and its regulators are relatives of lantibiotic systems that evolved to recognize multiple substrates to defend against toxins made by the intestinal microbiota.
PMID: 23543720 [PubMed – indexed for MEDLINE]
Identification of CodY targets in Bacillus anthracis by genome-wide in vitro binding analysis.
J Bacteriol. 2013 Mar;195(6):1204-13
Authors: Château A, van Schaik W, Joseph P, Handke LD, McBride SM, Smeets FM, Sonenshein AL, Fouet A
In Gram-positive bacteria, CodY is an important regulator of genes whose expression changes under conditions of nutrient limitation. Bacillus anthracis CodY represses or activates directly or indirectly approximately 500 genes. Affinity purification of CodY-DNA complexes was used to identify the direct targets of CodY. Of the 389 DNA binding sites that were copurified with CodY, 132 sites were in or near the regulatory regions governing the expression of 197 CodY-controlled genes, indicating that CodY controls many other genes indirectly. CodY-binding specificity was verified using electrophoretic mobility shift and DNase I footprinting assays for three CodY targets. Analysis of the bound sequences led to the identification of a B. anthracis CodY-binding consensus motif that was found in 366 of the 389 affinity-purified DNA regions. Regulation of the expression of the two genes directly controlled by CodY, sap and eag, encoding the two surface layer (S-layer) proteins, was analyzed further by monitoring the expression of transcriptional lacZ reporter fusions in parental and codY mutant strains. CodY proved to be a direct repressor of both sap and eag expression. Since the expression of the S-layer genes is under the control of both CodY and PagR (a regulator that responds to bicarbonate), their expression levels respond to both metabolic and environmental cues.
PMID: 23292769 [PubMed – indexed for MEDLINE]
Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile.
J Bacteriol. 2012 Jul;194(13):3307-16
Authors: Purcell EB, McKee RW, McBride SM, Waters CM, Tamayo R
The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile.
Microbiology. 2011 May;157(Pt 5):1457-65
Authors: McBride SM, Sonenshein AL
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