title: The Signal Sequence of the Abundant Extracellular Metalloprotease PPEP-1 Can Be Used to Secrete Synthetic Reporter Proteins in Clostridium difficile.
abstract: 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, AmyEoptand sLucoptfor gene expression analysis in C. difficile. AmyEoptextracellular activity results in starch degradation and can be exploited to demonstrate promoter activity in liquid or plate-based assays. sLucoptactivity 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.
authors.affiliation: Department of Medical Microbiology, Section Experimental Microbiology, Leiden University Medical Center , 2333 ZA Leiden, The Netherlands.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Oliveira Paiva
authors.affiliation: Department of Medical Microbiology, Section Experimental Microbiology, Leiden University Medical Center , 2333 ZA Leiden, The Netherlands.
authors.forename: Annemieke H
authors.initials: AH
authors.lastname: Friggen
authors.affiliation: Department of Medical Microbiology, Section Experimental Microbiology, Leiden University Medical Center , 2333 ZA Leiden, The Netherlands.
authors.forename: Shabnam
authors.initials: S
authors.lastname: Hossein-Javaheri
authors.affiliation: Department of Medical Microbiology, Section Experimental Microbiology, Leiden University Medical Center , 2333 ZA Leiden, The Netherlands.
authors.forename: Wiep Klaas
authors.initials: WK
authors.lastname: Smits
mesh_terms: D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001692:Biological Transport; D016360:Clostridioides difficile / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D054884:Host-Pathogen Interactions; D045726:Metalloproteases / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D017422:Sequence Analysis, DNA; D058615:Synthetic Biology
chemical_list: D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D045726:Metalloproteases
keywords: PPEP-1; amylase; luciferase; secretion