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Chlamydia trachomatis cytotoxicity associated with complete and partial cytotoxin genes.

ABSTRACT: Chlamydia trachomatis is an obligate intracellular human bacterial pathogen that infects epithelial cells of the eye and genital tract. Infection can result in trachoma, the leading cause of preventable blindness worldwide, and sexually transmitted diseases. A common feature of infection is a chronic damaging inflammatory response for which the molecular pathogenesis is not understood. It has been proposed that chlamydiae have a cytotoxic activity that contributes to this pathology, but a toxin has not been identified. The C. trachomatis genome contains genes that encode proteins with significant homology to large clostridial cytotoxins. Here we show that C. trachomatis makes a replication-independent cytotoxic activity that produces morphological and cytoskeletal changes in epithelial cells that are indistinguishable from those mediated by clostridial toxin B. A mouse chlamydial strain that encodes a full-length cytotoxin caused pronounced cytotoxicity, as did a human strain that has a shorter ORF with homology to only the enzymatically active site of clostridial toxin B. Cytotoxin gene transcripts were detected in chlamydiae-infected cells, and a protein with the expected molecular mass was present in lysates of infected epithelial cells. The protein was present transiently in infected cells during the period of cytotoxicity. Together, these data provide compelling evidence for a chlamydial cytotoxin for epithelial cells and imply that the cytotoxin is present in the elementary body and delivered to host cells very early during infection. We hypothesize that the cytotoxin is a virulence factor that contributes to the pathogenesis of C. trachomatis diseases.

SUBMITTER: Belland RJ

PROVIDER: S-EPMC61153 | biostudies-literature | 2001 Nov

REPOSITORIES: biostudies-literature

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Chlamydia trachomatis cytotoxicity associated with complete and partial cytotoxin genes.

Belland R J RJ Scidmore M A MA Crane D D DD Hogan D M DM Whitmire W W McClarty G G Caldwell H D HD

Proceedings of the National Academy of Sciences of the United States of America 20011113 24

Chlamydia trachomatis is an obligate intracellular human bacterial pathogen that infects epithelial cells of the eye and genital tract. Infection can result in trachoma, the leading cause of preventable blindness worldwide, and sexually transmitted diseases. A common feature of infection is a chronic damaging inflammatory response for which the molecular pathogenesis is not understood. It has been proposed that chlamydiae have a cytotoxic activity that contributes to this pathology, but a toxin ...[more]

PMID: 11707582

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Project description:Messenger RNA of C trachomatis infected monocytes of the three healthy donors was compared with the corresponding mock-infected samples. For mock infection, SPG buffer was used instead of the C trachomatis suspension, all other procedures were performed identically. Cells were resuspended in solution D and total RNA was extracted by application of phenol:chloroform 5:1 (pH 4.5, Ambion, Austin, TX) followed by precipitation in isopropanol at -80°C for 1 hr and incubation with RQ1 DNase (Promega, Madison, WI) at 37 °C for 1 hr. The signal intensity of the G3PDH housekeeping gene on the microarray membrane was used as indirect quality marker for the RNA utilized as only experiments were included in the analysis that revealed a G3PDH signal intensity within 1.5 times the standard deviation of all membranes evaluated in our study. The entire microarray procedure and its analysis has recently been validated and reported in detail. Total RNA of all donors was pooled and for each microarray experiment 150 µg were reverse transcribed and amplified by SMART™-PCR (BD Biosciences Clontech, Palo Alto, CA), a technology that allows reverse transcription of small amounts of total RNA and subsequent amplification of the entire cDNA. Probes were labeled with 32P and subsequently over night hybridized to a filter-based nylon membrane containing immobilized cDNA-specific sequences from a total of 1,184 genes (Human Atlas Array 1.2, BD Biosciences Clontech, Palo Alto, CA). For analysis, we focused solely on the 159 cytokines, chemokines and their receptors as given by the manufacturer. Signal intensities were retrieved by a STORM 860 scanner (Molecular Dynamics, Sunnyvale, CA) in combination with the AtlasImage 2.0 software (BD Biosciences Clontech). Data from all signal intensities were then subtracted by the local background intensity measured around each gene. The local background intensities of all individual genes were subsequently averaged, resulting in the mean background intensity of a particular membrane. Gene expression in our experiments was determined by a spot intensity of a single transcript that exceeded twice the mean background. Normalisation to background and to the G3PDH housekeeping gene was achieved by the global (sum) normalization method. Signal intensity data are given as the ratio of C trachomatis infected vs mock infected probes. Keywords: expression analysis

Project description:PurposeChlamydia trachomatis is the leading infectious cause of blindness. The goal of the current study was to search for biomarkers associated with C. trachomatis-induced ocular pathologies.MethodsWe used a whole genome scale proteome array to systematically profile antigen specificities of antibody responses to C. trachomatis infection in individuals from trachoma-endemic communities with or without end-stage trachoma (trichiasis) in The Gambia.ResultsWhen 61 trichiasis patients were compared with their control counterparts for overall antibody reactivity with organisms of different chlamydial species, no statistically significant difference was found. Both groups developed significantly higher titers of antibodies against C. trachomatis ocular serovars A and B than ocular serovar C, genital serovar D, or Chlamydia psittaci, whereas the titers of anti-Chlamydia pneumoniae antibodies were the highest. When antisera from 33 trichiasis and 26 control patients (with relatively high titers of antibodies to C. trachomatis ocular serovars) were reacted with 908 C. trachomatis proteins, 447 antigens were recognized by at least 1 of the 59 antisera, and 10 antigens by 50% or more antisera, the latter being designated as immunodominant antigens. More importantly, four antigens were preferentially recognized by the trichiasis group, with antigens CT414, CT667, and CT706 collectively reacting with 30% of trichiasis antisera but none from the normal group, and antigen CT695 reacting with 61% of trichiasis but only 31% of normal antisera. On the other hand, eight antigens were preferentially recognized by the control group, with antigens CT019, CT117, CT301, CT553, CT556, CT571, and CT709 together reacting with 46% of normal antisera and none from the trichiasis group, whereas antigen CT442 reacted with 35% of normal and 19% of trichiasis antisera respectively.ConclusionsThe current study, by mapping immunodominant C. trachomatis antigens and identifying antigens associated with both ocular pathology and protection, has provided important information for further understanding chlamydial pathogenesis and the development of subunit vaccines.

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Chlamydia trachomatis cytotoxicity associated with complete and partial cytotoxin genes.

Publications

Chlamydia trachomatis cytotoxicity associated with complete and partial cytotoxin genes.

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