title: Application of β-lactamase reporter fusions as an indicator of effector protein secretion during infections with the obligate intracellular pathogen Chlamydia trachomatis.
abstract: Chlamydia spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform Chlamydia and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 β-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings provide definitive evidence that C. trachomatis CT695 is secreted during infection. Follow-up indirect immunofluorescence studies confirmed CT695 secretion and indicate that this effector can be secreted at multiple points during the chlamydial developmental cycle. Our results indicate that the BlaM-fusion reporter assay will allow efficacious identification of novel secreted proteins. Moreover, this approach can easily be adapted to enable more sophisticated studies of the secretion process in Chlamydia.
authors.affiliation: Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY, United States of America.
authors.forename: Konrad E
authors.initials: KE
authors.lastname: Mueller
authors.affiliation: Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY, United States of America.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Fields
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial; D000069296:Type III Secretion Systems / Q000235:genetics* / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000069296:Type III Secretion Systems; D001618:beta-Lactamases; D000097573:Red Fluorescent Protein; C034413:beta-lactamase TEM-1