Clostridium difficile Extracytoplasmic Function σ Factor σ
            <sup>V</sup>
            Regulates Lysozyme Resistance and Is Necessary for Pathogenesis in the Hamster Model of Infection

Ho, Theresa D.; Williams, Kyle; Chen, Yan; Helm, Richard F.; Popham, David L.; Ellermeier, Craig D.

doi:10.1128/iai.01483-13

Cited by 51 publications

(126 citation statements)

References 51 publications

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“…The attenuated growth of the sigV mutant in lysozyme was complemented by expression of sigV from a plasmid, similar to previous studies (see Fig. S1 in the supplemental material) (21). The sigV mutant did not, however, have a growth defect in polymyxin B (Fig.…”

Section: Impact Of Sigd Sigt and Sigv Disruption On Camp Resistancesupporting

confidence: 70%

“…1A), we hypothesized that these mutants would be less fit in vivo. In a previous study, Ho et al demonstrated that a sigV mutant is significantly attenuated in a hamster model of infection (21). However, that study was performed in the JIR8094 strain, which is attenuated for virulence in vivo (46,50).…”

Section: Impact Of Sigd Sigt and Sigv Disruption On Camp Resistancementioning

confidence: 94%

“…C. difficile encodes orthologs of Spo0A, V (also known as csfV or sigV) and D . Moreover, V is necessary for lysozyme resistance in C. difficile (21). In fact, the C. difficile V anti-sigma factor RsiV binds lysozyme and may serve as a direct lysozyme receptor, as it does in B. subtilis (22).…”

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confidence: 99%

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The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ ^V in Response to Lysozyme

et al. 2016

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Clostridium difficile (also known as Peptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibioticassociated diarrhea throughout the world. Colonization of the intestinal tract is necessary for C. difficile to cause disease. Hostproduced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yet C. difficile is able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition of D-alanine to teichoic acids, is important for C. difficile resistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that a dlt null mutant is severely attenuated for growth in lysozyme and that expression of the dltDABC operon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor V does not induce dlt expression in response to lysozyme, indicating that V is required for regulation of lysozyme-dependent D-alanylation of the cell wall. Using reporter gene fusions and 5= RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independent dlt expression. In addition, we observed that both a sigV mutant and a dlt mutant are more virulent in a hamster model of infection. These findings demonstrate that cell wall D-alanylation in C. difficile is induced by lysozyme in a V -dependent manner and that this pathway impacts virulence in vivo.C lostridium difficile (Peptoclostridium difficile) causes nearly half a million infections in the United States each year, representing a significant public health threat (1). In order to cause infection, C. difficile must colonize the colon. As an important interface between the host and microbiota, the colon is an environment rich in host innate immune molecules and bacteriumderived antimicrobials made by the indigenous microbiota (2-6). These innate immune molecules and bacterially produced antimicrobials include a variety of cationic antimicrobial peptides (CAMPs), such as lysozyme, defensins, and bacteriocins (2,4,[7][8][9]. Understanding how C. difficile is able to resist killing in this antimicrobial-laden environment could better our understanding of the factors that contribute to the progression of C. difficile infections.A common mechanism of resistance to CAMPs in many bacteria is the alteration of the cell surface charge (10-12). One mechanism for increasing the surface charge is through the addition of D-alanine (D-Ala) to teichoic acids in the cell wall (10,12,13). The addition of D-Ala is mediated by four proteins, DltA, DltB, DltC, and DltD, encoded by the dlt operon (13). The Dlt pathway confers lysozyme resistance to Bacillus subtilis and Enterococcus faecalis (14,15). Previously, we demonstrated that the D-alanylation of the cell wall via ...

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Section: Impact Of Sigd Sigt and Sigv Disruption On Camp Resistancesupporting

confidence: 70%

Section: Impact Of Sigd Sigt and Sigv Disruption On Camp Resistancementioning

confidence: 94%

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The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ ^V in Response to Lysozyme

et al. 2016

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“…The cells were fixed with an acetone-ethanol mixture (at a 1:1 ratio). RNA was isolated from these fixed cells using Trizol reagent (Invitrogen) as previously described (16,20). The resulting RNA was treated with DNase I (Turbo DNA-free kit; Ambion) and purified with RNeasy spin columns (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

“…The resulting RNA was treated with DNase I (Turbo DNA-free kit; Ambion) and purified with RNeasy spin columns (Qiagen). The resulting RNA was processed by the University of Iowa Carver Center for Genomics (Iowa City, IA) with custom C. difficile Roche Nimblegen microarrays (20). RNA from three independent biological replicates grown on different days was used.…”

Section: Methodsmentioning

confidence: 99%

Ferric Uptake Regulator Fur Control of Putative Iron Acquisition Systems in Clostridium difficile

Ellermeier

2015

J Bacteriol

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Clostridium difficile is an anaerobic, Gram-positive, spore-forming opportunistic pathogen and is the most common cause of hospital-acquired infectious diarrhea. Although iron acquisition in the host is a key to survival of bacterial pathogens, high levels of intracellular iron can increase oxidative damage. Therefore, expression of iron acquisition mechanisms is tightly controlled by transcriptional regulators. We identified a C. difficile homologue of the master bacterial iron regulator Fur. Using targetron mutagenesis, we generated a fur insertion mutant of C. difficile. To identify the genes regulated by Fur in C. difficile, we used microarray analysis to compare transcriptional differences between the fur mutant and the wild type when grown in highiron medium. The fur mutant had increased expression of greater than 70 transcriptional units. Using quantitative reverse transcriptase PCR (qRT-PCR), we analyzed several of the Fur-regulated genes identified by the microarray and verified that they are both iron and Fur regulated in C. difficile. Among those Fur-and iron-repressed genes were C. difficile genes encoding 7 putative cation transport systems of different classes. We found that Fur was able to bind the DNA upstream of three Fur-repressed genes in electrophoretic mobility shift assays. We also demonstrate that expression of Fur-regulated putative iron acquisition systems was increased during C. difficile infection using the hamster model. Our data suggest that C. difficile expresses multiple iron transport mechanisms in response iron depletion in vitro and in vivo. IMPORTANCEClostridium difficile is the most common cause of hospital-acquired infectious diarrhea and has been recently classified as an "urgent" antibiotic resistance threat by the CDC. To survive and cause disease, most bacterial pathogens must acquire the essential enzymatic cofactor iron. While import of adequate iron is essential for most bacterial growth, excess intracellular iron can lead to extensive oxidative damage. Thus, bacteria must regulate iron import to maintain iron homeostasis. We demonstrate here that C. difficile regulates expression of several putative iron acquisition systems using the transcriptional regulator Fur. These import mechanisms are induced under iron-limiting conditions in vitro and during C. difficile infection of the host. This suggests that during a C. difficile infection, iron availability is limited in vivo.A lmost all living organisms require iron as a cofactor for essential metabolic chemistry (1). Although iron is one of the most abundant of Earth's elements, ferric iron has very limited solubility in aqueous, nonacidic, or oxygenated environments (1). Ferric iron is most often found as iron oxides or hydroxides, which cannot be used by most organisms. Competition over bioavailable iron is fierce both among bacteria in complex microbial communities and between bacterial pathogens and their eukaryotic hosts (2).Organisms have evolved various mechanisms of iron transport to obtain this essentia...

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Proteolytic Activation of Extra Cytoplasmic Function (ECF) σ Factors

Hastie

Ellermeier

2016

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria

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Clostridium difficile Extracytoplasmic Function σ Factor σ ^V Regulates Lysozyme Resistance and Is Necessary for Pathogenesis in the Hamster Model of Infection

Cited by 51 publications

References 51 publications

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ ^V in Response to Lysozyme

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ ^V in Response to Lysozyme

Ferric Uptake Regulator Fur Control of Putative Iron Acquisition Systems in Clostridium difficile

Proteolytic Activation of Extra Cytoplasmic Function (ECF) σ Factors

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Clostridium difficile Extracytoplasmic Function σ Factor σ V Regulates Lysozyme Resistance and Is Necessary for Pathogenesis in the Hamster Model of Infection

Cited by 51 publications

References 51 publications

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ V in Response to Lysozyme

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ V in Response to Lysozyme

Ferric Uptake Regulator Fur Control of Putative Iron Acquisition Systems in Clostridium difficile

Proteolytic Activation of Extra Cytoplasmic Function (ECF) σ Factors

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Clostridium difficile Extracytoplasmic Function σ Factor σ ^V Regulates Lysozyme Resistance and Is Necessary for Pathogenesis in the Hamster Model of Infection

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ ^V in Response to Lysozyme

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ ^V in Response to Lysozyme