title: Translocation of ExsE into Chinese hamster ovary cells is required for transcriptional induction of the Pseudomonas aeruginosa type III secretion system.
abstract: Transcription of the Pseudomonas aeruginosa type III secretion system (T3SS) is induced under Ca(2+)-limiting growth conditions or following the contact of the bacteria with host cells. The regulatory response to low Ca(2+) levels is initiated by the T3SS-mediated secretion of ExsE, a negative regulatory protein that prevents T3SS gene transcription. In the present study, we demonstrated that ExsE plays an analogous role in transcriptional induction following host cell contact. By using a flow cytometry assay, the host contact-dependent induction of T3SS gene expression was found to be dependent upon the presence of functional type III translocation machinery. Using three independent assays, we demonstrated that ExsE was translocated into Chinese hamster ovary cells in a T3SS-dependent manner. Deletion mapping experiments indicated that the amino terminus of ExsE is required both for secretion under Ca(2+)-limiting growth conditions and for translocation into host cells. A P. aeruginosa mutant expressing an exsE allele lacking codons 3 through 20 was deficient in ExsE secretion and translocation and showed constitutive repression of T3SS gene expression under Ca(2+)-limiting growth conditions. The mutant also failed to induce T3SS gene expression following host cell contact and demonstrated a significant reduction in T3SS-dependent cytotoxicity towards Chinese hamster ovary cells, indicating that the translocation of ExsE is required for the host contact-dependent induction of T3SS gene expression.
authors.affiliation: Department of Microbiology, 540B EMRB, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Urbanowski
authors.forename: Evan D
authors.initials: ED
authors.lastname: Brutinel
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Yahr
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D016466:CHO Cells; D020013:Calcium Signaling / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D021381:Protein Transport / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; C095093:ExsA protein, Pseudomonas aeruginosa; C470740:ExsD protein, Pseudomonas aeruginosa; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators