title: Expression and localization of predicted inclusion membrane proteins in Chlamydia trachomatis.
abstract: Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a membrane-bound vacuole termed the inclusion. Early in the infection cycle, the pathogen extensively modifies the inclusion membrane through incorporation of numerous type III secreted effector proteins, called inclusion membrane proteins (Incs). These proteins are characterized by a bilobed hydrophobic domain of 40 amino acids. The presence of this domain has been used to predict up to 59 putative Incs for C. trachomatis; however, localization to the inclusion membrane with specific antibodies has been demonstrated for only about half of them. Here, we employed recently developed genetic tools to verify the localization of predicted Incs that had not been previously localized to the inclusion membrane. Expression of epitope-tagged putative Incs identified 10 that were previously unverified as inclusion membrane localized and thus authentic Incs. One novel Inc and 3 previously described Incs were localized to inclusion membrane microdomains, as evidenced by colocalization with phosphorylated Src (p-Src). Several predicted Incs did not localize to the inclusion membrane but instead remained associated with the bacteria. Using Yersinia as a surrogate host, we demonstrated that many of these are not secreted via type III secretion, further suggesting they may not be true Incs. Collectively, our results highlight the utility of genetic tools for demonstrating secretion from chlamydia. Further mechanistic studies aimed at elucidating effector function will advance our understanding of how the pathogen maintains its unique intracellular niche and mediates interactions with the host.
authors.affiliation: Host Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Mary M
authors.initials: MM
authors.lastname: Weber
authors.affiliation: Host Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Laura D
authors.initials: LD
authors.lastname: Bauler
authors.affiliation: Host Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lam
authors.affiliation: Host Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA ted_hackstadt@nih.gov.
authors.forename: Ted
authors.initials: T
authors.lastname: Hackstadt
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D015964:Gene Expression Regulation, Bacterial; D006367:HeLa Cells; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D058977:Molecular Sequence Annotation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000069296:Type III Secretion Systems / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism; D014709:Vero Cells; D015011:Yersinia pseudotuberculosis / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000069296:Type III Secretion Systems