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.

FIG. 1.

Serum reactivity of C. trachomatis-infected patients against purified EB lysates. Proteins from urographin-purified EBs were separated by SDS-PAGE and analyzed by immunoblot analysis. Lane 1, Coomassie blue-stained EB proteins; lane 2, control serum from C. trachomatis-negative patient; lanes 3 to 10, serum samples from C. trachomatis-infected patients (lanes 3 and 4, PEACH PID group; lanes 5 and 6, Oakland adolescent female group; lanes 7 and 8, Arkansas adolescent female group; lanes 9 and 10, Baltimore male adult group). Molecular mass markers (kDa) are indicated in each row.

FIG. 2.

Serum reactivity of C. trachomatis-infected patients against the Pmp family. Partially purified proteins rPmpA to -I (subtypes are indicated above each lane) were fractionated by SDS-PAGE and Coomassie blue-stained (a) or immunoblotted with anti-His tag antibody (1:1,500) (b), control serum from a healthy individual (1:100) (c), or serum samples (1:100) from patients with C. trachomatis genital infection. Representatives of the PEACH PID group (serum no. 19) (d), Oakland adolescent females (serum no. 19) (e), and Baltimore male adults (serum no. 21) (f) are shown. Stars indicate specific reactivity against full-size proteins rPmpA to -I. Patient serum samples reacting with bands that do not include full-size rPmp were recorded as negative. Molecular mass markers (kDa) are indicated in each row.

FIG. 3.

Profiling of the Pmp-specific antibody responses in C. trachomatis-infected patients. The antibody reactivity against rPmps was quantified and color-coded for each patient as described in Materials and Methods. Reactivity lower than Q1 is coded as light yellow, that between Q1 and the median as gold, that between the median and Q3 as orange, and that above Q3 as dark red. (A) PEACH PID women; (B) Oakland adolescent females; (C) Arkansas adolescent females; and (D) Baltimore male adults. The patient serum numbers are indicated along the side of each panel.

FIG. 4.

Quartile distribution and statistical significance of Pmp-specific relative antibody reactivity in the three patient populations. (A) Antibody reactivity against each rPmp was quantified using ImageQuant 5.2, and the distributions of immunoreactivity were analyzed and compared in the following three patient populations: PEACH PID women, pooled adolescent females, and Baltimore male adults. The box corresponds to reactivity between Q1 and Q3 of the total distribution. The horizontal line inside the box denotes the median, and the upper and lower horizontal lines correspond to the maximum and minimum reactivities of the total distribution, respectively. (B) Statistical analysis was performed to compare the relative antibody reactivities against individual rPmps between different patient populations as described in Materials and Methods. The P values are listed for each comparison, and the asterisks indicate statistically significant difference in two or three group comparisons.