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Chlamydia trachomatis-infected patients display variable antibody profiles against the nine-member polymorphic membrane protein family.

ABSTRACT: Genomic analysis of the Chlamydiaceae has revealed a multigene family encoding large, putatively autotransported polymorphic membrane proteins (Pmps) with nine members in the sexually transmitted pathogen Chlamydia trachomatis. While various pathogenesis-related functions are emerging for the Pmps, observed genotypic and phenotypic variation among several chlamydial Pmps in various Chlamydia species has led us to hypothesize that the pmp gene repertoire is the basis of a previously undetected mechanism of antigenic variation. To test this hypothesis, we chose to examine the serologic response of C. trachomatis-infected patients to each Pmp subtype. Immune serum samples were collected from four populations of patients with confirmed C. trachomatis genital infection: 40 women with pelvic inflammatory disease from Pittsburgh, PA; 27 and 34 adolescent/young females from Oakland, CA, and Little Rock, AR, respectively; and 58 adult male patients from Baltimore, MD. The Pmp-specific antibody response was obtained using immunoblot analysis against each of the nine recombinantly expressed Pmps and quantified by densitometry. Our results show that nearly all C. trachomatis-infected patients mount a strong serologic response against individual or multiple Pmp subtypes and that the antibody specificity profile varies between patients. Moreover, our analysis reveals differences in the strengths and specificities of the Pmp subtype-specific antibody reactivity relating to gender and clinical outcome. Overall, our results indicate that the Pmps elicit various serologic responses in C. trachomatis-infected patients and are consistent with the pmp gene family being the basis of a mechanism of antigenic variation.

SUBMITTER: Tan C

PROVIDER: S-EPMC2715660 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Chlamydia trachomatis-infected patients display variable antibody profiles against the nine-member polymorphic membrane protein family.

Tan Chun C Hsia Ru-ching RC Shou Huizhong H Haggerty Catherine L CL Ness Roberta B RB Gaydos Charlotte A CA Dean Deborah D Scurlock Amy M AM Wilson David P DP Bavoil Patrik M PM

Infection and immunity 20090601 8

Genomic analysis of the Chlamydiaceae has revealed a multigene family encoding large, putatively autotransported polymorphic membrane proteins (Pmps) with nine members in the sexually transmitted pathogen Chlamydia trachomatis. While various pathogenesis-related functions are emerging for the Pmps, observed genotypic and phenotypic variation among several chlamydial Pmps in various Chlamydia species has led us to hypothesize that the pmp gene repertoire is the basis of a previously undetected me ...[more]

PMID: 19487469

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Project description:The sexually transmitted pathogen Chlamydia trachomatis (CT) is able to replicate and survive in human intestinal epithelial cells, being the gastro-intestinal tract a suitable site of residence for this microorganism. In this context, no detailed information about the mechanisms of cell death in intestinal cell lines after a chlamydial infection is available. The aim of this study was to compare the effect of two different CT serovars (D and L2) on the survival/death of different intestinal cell lines (Caco-2 and COLO-205), using endocervical cells (HeLa) as a reference model of genital infection. Seventy two hours after chlamydial infection at different multiplicity of infection (MOI) levels, the viability of HeLa, Caco-2 and COLO 205 cells was evaluated through dose-response experiments by means of a MTS-based assay. To get deeper insights in the mechanisms of cell death induced by CT, cell viability was assessed in presence of different inhibitors (i.e. pan-caspase inhibitor Z-VAD, necroptosis inhibitor Necrostatin-1, hydrogen peroxide scavenger catalase, caspase-1 inhibitor Ac-YVAD-cmk). Moreover, the activation of effector caspases and the presence of cellular apoptotic/necrotic changes were evaluated at different time points after CT infection. Our results demonstrated that, for both chlamydial serovars, intestinal cell lines are more resistant to CT-induced cell death compared to HeLa, thus representing a suitable 'niche' for chlamydial residence and replication. In literature, apoptosis has been widely described to be the main cell death mechanism elicited by chlamydia infection. However, our data demonstrate that necroptosis plays a relevant role, proceeding in parallel with apoptosis. The protective effect of catalase suggests the involvement of oxidative stress in triggering both cell death pathways. Moreover, we demonstrated that caspase-1 is involved in CT-induced cell death, potentially contributing to host inflammatory response and tissue damage. Cells infected by L2 serovar displayed a higher activation of effector caspases compared to cells infected with serovar D, suggesting a serovar-specific activation of apoptotic pathways and potentially explaining the greater virulence of L serovars. Finally, we found that Chlamydia elicits the early externalization of phosphatidylserine on the external leaflet of plasma membrane independently of caspase activation.

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

Dataset Information

Chlamydia trachomatis-infected patients display variable antibody profiles against the nine-member polymorphic membrane protein family.

Publications

Chlamydia trachomatis-infected patients display variable antibody profiles against the nine-member polymorphic membrane protein family.

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