Several other published genomes (Chu or (Horn and orthologues evolved from a common ancestral gene. infectious, medically significant potential human pathogen classified as a category B bioterrorism agent by the Centers for Disease Control and Prevention (www.bt.cdc.gov/agent/agentlist-category.asp). Inhalation via aerosols can cause life-threatening pneumonia (Smith to be significantly more infectious and pathogenic than in humans are not comprehended. All species are obligate intracellular pathogens with a unique developmental life cycle involving two cellular forms. After entering the host cell via endocytosis, metabolically dormant chlamydiaetermed elementary body (EBs)differentiate into larger, actively replicating reticulate body (RBs) within a membrane-bound vacuole termed the inclusion. RBs differentiate back into EBs asynchronously, so the chlamydial inclusion includes both forms (RBs and EBs) at late stages of contamination. After completing development, EBs exit upon lysis of the host cell or nonexocytic extrusion of whole or part of the inclusion (Hybiske and Stephens, 2007 ) Levamlodipine besylate and then either disseminate or infect neighboring cells. All species encode a complete type III secretion (T3S) system that enables the direct translocation of effector proteins across both the bacterial envelope and host plasma membraneCderived inclusion membrane into the host cytosol, where they target specific host proteins and pathways to promote and maintain contamination (Peters inclusion surface (Derre YopN (Fields and Hackstadt, 2000 ), but also modulates the host cytoskeleton (Archuleta (Wang and the impracticality of clonal isolation. Strategies that have been successful include identification based on homology to effectors from other bacterial genera (Hsia (Fields and Hackstadt, 2000 ; Subtil as a surrogate to test putative chlamydial T3S-dependent secreted proteins predicted by the protein homology-based algorithm SIEVE (Samudrala (NCBI G5Q_0070) of strain CAL10 as a putative effector (Hovis protein (SINC), based on its novel localization at the nuclear envelope (NE) of infected and neighboring uninfected cells and association with nuclear membrane proteins. RESULTS is usually syntenic and encodes a poor orthologue of CT694 The putative effector gene was chosen for further investigation because Levamlodipine besylate it posed a paradox: is usually syntenic with of each downstream of the phosphoglycerate kinase gene, (Supplemental Physique S1A); however, the encoded CT694 and SINC proteins are only 12.5% identical, compared with 74% identical phosphoglycerate Mouse monoclonal to NR3C1 kinase proteins. Residual identity to CT694 is usually scattered throughout SINC (e.g., residues 1C11, 151C161, and 458C466), suggesting divergence from a common ancestral gene. Low sequence identity suggested that SINC and CT694 were functionally distinct and might therefore be expressed Levamlodipine besylate at different stages of development in or CAL10 revealed low or background levels of transcripts from 6 to 24 h postinfection (hpi), peaking at 30C42 hpi and decreasing sharply by 42 hpi, with a strong pattern toward statistical significance (= 13.675, = 0.057; Supplemental Physique S1B), much like and and their gene products were expressed at similar occasions during development (Belland CAL10Cinfected HeLa cells fixed with methanol at 24 hpi and stained using antibodies specific for SINC (-SINC) and for elongation factor Tu (CEF-Tu). DNA was DAPI stained; epifluorescence images were obtained on a Zeiss Axio Imager Z.1 (40objective). Bar, 10 m. (B, C) IEM images of CAL10Cinfected HeLa cells fixed with PFA at 24 hpi using colloidal goldCconjugated antibodies specific for SINC (-SINC). (C) Black and white arrowheads identify SINC signals at the NE and a putative nucleoplasmic track consistent with pore-linked filaments, respectively. Bars, 500 nm (white), 100 nm (black). Open in a separate window Physique 2: SINC is usually secreted by chlamydiae and targets the nuclear envelope of infected and uninfected neighboring cells late in development. Immunofluorescence images of CAL10Cinfected HeLa cells fixed with methanol at 36 hpi and stained using antibodies specific for SINC (-SINC) and for elongation factor Tu (CEF-Tu; A) or for SINC alone (B). DNA was stained with DAPI. (A) Confocal images (Zeiss LSM Levamlodipine besylate 510 Meta Confocal Microscope) indicate SINC transmission at the NE. (B) Epifluorescence images (Zeiss Axio Imager Z.1 with ApoTome.2 module) indicate SINC signal at the NE of the infected cell and that of neighboring uninfected cells. Bars, 10 m. At 36 hpi, nearly all chlamydiae within the inclusion were SINC positive as visualized by confocal microscopy (Physique 2A). We also detected strong SINC-specific fluorescence at the host.