Association of tubal factor infertility with elevated antibodies to Chlamydia trachomatis caseinolytic protease P

Abstract

Objective: The objective of the study was to assess antibodies against Chlamydia trachomatis heat shock proteins (HSP) in patients with tubal factor infertility (TFI), infertility controls (IFC), and fertile controls (FC). HSPs assist organisms in surviving caustic environments such as heat.

Study design: Twenty-one TFI, 15 IFC, and 29 FC patients were enrolled after laparoscopic tubal assessment. The titers of antibodies against C trachomatis organisms and 14 chlamydial HSPs were compared among the 3 groups.

Results: TFI patients developed significantly higher levels of antibodies against C trachomatis and specifically recognizing chlamydial HSP60 and caseinolytic protease (Clp) P, a subunit of the ATP-dependent Clp protease complex involved in the degradation of abnormal proteins.

Conclusion: In addition to confirming high titers of antibodies against C trachomatis organisms and HSP60 in TFI patients, we identified a novel link of TFI with anti-ClpP antibodies. These findings may provide useful information for developing a noninvasive screening test for TFI and constructing subunit anti-C trachomatis vaccines.

FIGURE 1. Reactivity of human antibodies with chlamydial fusion proteins arrayed to microplate wells

The bacterial lysates containing individual chlamydial GST fusion proteins or GST alone (listed along the X-axis) were directly added to glutathione-coated microplates. Human antisera from 3 groups of patients (listed along the Y-axis) were first preabsorbed with bacterial lysates containing GST alone and then reacted with the plate-immobilized chlamydial fusion proteins. The human antibody binding was detected with a goat antihuman IgG conjugated with HRP plus the soluble substrate ABTS (Sigma, St Louis, MO) and measured in OD values at 405 nm. A reaction with an OD value of 2 SD about the mean was considered positive as indicated with horizontal bars. The number of positive individuals from different groups of patients was compared with Pearson’s χ² test. The number of sera that positively recognized HSP60 (P = .001) or ClpP (P = .03) was significantly higher in the TFI group when compared with either the IFC or the FC groups. ClpP, caseinolytic protease P; FC, fertile controls; GST, glutathione S-transferase; HSP, heat shock proteins; IFC, infertility controls; OD, optical density; SD, standard deviation; TFI, tubal factor infertility.

FIGURE 2. Absorption of human sera with endogenous C trachomatis antigens blocks the binding of human antibodies to chlamydial fusion proteins

The bacterial lysates containing individual chlamydial GST fusion proteins or GST alone (as listed along the left side of the figure) were allowed to bind to microplates, and the ELISA was carried out as described in the legend for Figure 1. The 4 human antisera from the TFI group as listed on top of the figure were preabsorbed with bacterial lysates containing GST alone and then further absorbed with either HeLa alone or chlamydia-infected HeLa lysates prior to reacting with the chlamydial fusion proteins on the microplate. Please note that none of the 4 sera bound to the other subunit of the ClpP complex (GST-CT706) and the binding of the 4 sera to both GST-CT431 and GST-CT858 was completely blocked by absorption with the chlamydia-infected but not HeLa alone lysates. ClpP, caseinolytic protease P; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; TFI, tubal factor infertility.

FIGURE 3. Expression of CT110 (HSP60) and CT431 (ClpP) during C trachomatis infection

HeLa cells grown on coverslips were infected with C trachomatis serovar D organisms, and at various times after infection as listed on the top of the figure, the infected cultures were processed for immunofluorescence labeling with mouse antibodies against HSP60 or ClpP (red). The samples were colabeled with an anti-MOMP antibody (green) and a DNA dye (blue). Please note that HSP60 was detected as early as 12 hours (yellow = red overlapping with green), whereas ClpP was detected only by 24 hours (white arrows). ClpP, caseinolytic protease P; HSP, heat shock proteins; MOMP, major outer membrane protein.