The HtrA-Like Protease CD3284 Modulates Virulence of Clostridium difficile

Bakker, Dennis; Buckley, Anthony M.; Jong, Anne de; Winden, Vincent J. C. van; Verhoeks, Joost P. A.; Kuipers, Oscar P.; Douce, Gill; Kuijper, Ed J.; Smits, Wiep Klaas; Corver, Jeroen

doi:10.1128/iai.02336-14

Cited by 24 publications

(35 citation statements)

References 86 publications

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“…In contrast, the toxicity of bacterial supernatants toward Vero cells is generally only detectable from stationary growth phase. 28 Consistent with our findings, we did not detect significant extracellular levels of toxins in the exponential phase (T5), but readily detected toxins from the transition into stationary phase (T8) ( Figure 3B). Intracellularly, toxin A was detectable by immunoblotting during exponential growth ( Figure 3C).…”

supporting

confidence: 94%

The Signal Sequence of the Abundant Extracellular Metalloprotease PPEP-1 Can Be Used to Secrete Synthetic Reporter Proteins in Clostridium difficile

et al. 2016

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Clostridium difficile is an opportunistic pathogen and the main cause of antibiotic-associated diarrhea. Adherence of C. difficile to host cells is modulated by proteins present on the bacterial cell surface or secreted into the environment. Cleavage of collagen-binding proteins is mediated by the zinc metalloprotease PPEP-1, which was identified as one of the most abundant secreted proteins of C. difficile. Here, we exploit the PPEP-1 signal sequence to produce novel secreted enzymes. We have constructed two functional secreted reporters, AmyE and sLuc for gene expression analysis in C. difficile. AmyE extracellular activity results in starch degradation and can be exploited to demonstrate promoter activity in liquid or plate-based assays. sLuc activity could reliably be detected in culture supernatant when produced from an inducible or native promoter. The secreted reporters can be easily assessed under aerobic conditions, without the need of complex sample processing.

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confidence: 94%

The Signal Sequence of the Abundant Extracellular Metalloprotease PPEP-1 Can Be Used to Secrete Synthetic Reporter Proteins in Clostridium difficile

et al. 2016

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“…2C). The actual difference in hamster survival is underestimated as the bacterial knockout strain has greater resistance to clindamycin (as a consequence of the ClosTron insertion containing an ermB gene conferring concomitant resistance to clindamycin) and hence a growth advantage compared to WT cells in vivo following pre-treatment of animals with clindamycin [24]. Pre-treatment of hamsters with the antibiotic clindamycin is essential as it disrupts the intestinal microbiota and thereby incites C. difficile infection.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, Southern blot analysis using an ermB specific probe was used to confirm a specific single insertion in the genome. All C. difficile strains were grown as described previously [24].…”

Section: Generation Of a C Difficile Ppep-1 Knockout Strainmentioning

confidence: 99%

Clostridium difficile secreted Pro‐Pro endopeptidase PPEP‐1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831

et al. 2015

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a b s t r a c tThe Clostridium difficile cd2830 gene product is a secreted metalloprotease, named Pro-Pro endopeptidase (PPEP-1). PPEP-1 cleaves C. difficile cell surface proteins (e.g. CD2831). Here, we confirmed that PPEP-1 has a unique preference for prolines surrounding the scissile bond. Moreover, we show that it exhibits a high preference for an asparagine at the P2 position and hydrophobic residues at the P3 position. Using a PPEP-1 knockout C. difficile strain, we demonstrate that the removal of the collagen binding protein CD2831 is fully attributable to PPEP-1 activity. The PPEP-1 knockout strain demonstrated higher affinity for collagen type I with attenuated virulence in hamsters.

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“…A single intraperitoneal injection of clindamycin (10 g/g body weight) was administered either 24 h prior to infection (single-strain infections) or 48 h prior to infection (competitions). The timing of the clindamycin injection was modified to 48 h prior to inoculation in the competition experiments to ensure that clindamycin concentrations did not favor growth of the mutants, which contain the ermB gene, which provides some resistance to clindamycin (86,87). Mice were inoculated with 10 5 CFU of C. difficile spores (single-strain infections) or 2 ϫ 10 5 total spores (coinfections) by oral gavage.…”

Section: Methodsmentioning

confidence: 99%

Type IV Pili Promote Clostridium difficile Adherence and Persistence in a Mouse Model of Infection

et al. 2018

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Cyclic diguanylate (c-di-GMP) is a second messenger that regulates the transition from motile to sessile lifestyles in numerous bacteria and controls virulence factor production in a variety of pathogens. In , c-di-GMP negatively regulates flagellum biosynthesis and swimming motility and promotes the production of type IV pili (TFP), biofilm formation, and surface motility Flagella have been identified as colonization factors in , but the role of TFP in adherence to host cells and in colonization of the mammalian gut is unknown. Here we show that c-di-GMP promotes adherence to epithelial cells, which can be partly attributed to the loss of flagella. Using TFP-null mutants, we demonstrate that adherence to epithelial cells is partially mediated by TFP and that this TFP-mediated adherence requires c-di-GMP regulation. In a mouse model of colonization, the TFP-null mutants initially colonized the intestine as well as the parental strain but were cleared more quickly. Moreover, compared to the parent strain, strains lacking TFP were particularly deficient in association with the cecal mucosa. Together these data indicate that TFP and their positive regulation by c-di-GMP promote attachment of to the intestinal epithelium and contribute to persistence of in the host intestine.

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The HtrA-Like Protease CD3284 Modulates Virulence of Clostridium difficile

Cited by 24 publications

References 86 publications

The Signal Sequence of the Abundant Extracellular Metalloprotease PPEP-1 Can Be Used to Secrete Synthetic Reporter Proteins in Clostridium difficile

The Signal Sequence of the Abundant Extracellular Metalloprotease PPEP-1 Can Be Used to Secrete Synthetic Reporter Proteins in Clostridium difficile

Clostridium difficile secreted Pro‐Pro endopeptidase PPEP‐1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831

Type IV Pili Promote Clostridium difficile Adherence and Persistence in a Mouse Model of Infection

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