Bioprospecting by Phage Display of Mimetic Peptides of Chlamydia trachomatis for Use in Laboratory Diagnosis

Abstract

Background: Chlamydia trachomatis infection is a major public health problem and the most common sexually transmitted infection in the world. Although highly prevalent, 70% to 80% of cases are asymptomatic and undiagnosed.

Purpose: To overcome some limitations in terms of rapid diagnosis, phage display technology was used to bioprospect peptide mimetics of C. trachomatis immunoreactive and immunogenic antigens to be selected for the production of synthetic peptides.

Methods: Initially, IgG from 22 individuals with C. trachomatis and 30 negative controls was coupled to G protein magnetic beads. The phage display technique consisted of biopanning, genetic sequencing, bioinformatics analysis and phage ELISA.

Results: Clones G1, H5, C6 and H7 were selected for testing with individual samples positive and negative for C. trachomatis. Reactions were statistically significant (p < 0.05), with a sensitivity of 90.91, a specificity of 54.55, and AUC values >0.8. One-dimensional analysis with C. trachomatis components indicated that the G1 clone aligned with cell wall-associated hydrolase domain-containing protein, the H5 clone aligned with glycerol-3-phosphate acyltransferase PlsX protein, the C6 clone aligned with a transposase and inactivated derivatives, and the H7 clone aligned with GTP-binding protein. Molecular modeling and three-dimensional analysis indicated the best fit of the four clones with a protein known as chlamydial protease/proteasome-like activity factor (CPAF), an important virulence factor of the bacterium.

Conclusion: The peptides produced by phage display are related to the metabolic pathways of C. trachomatis, indicating that they can be used to understand the pathogenesis of the infection. Because of their high sensitivity and AUC values, the peptides present considerable potential for use in platforms for screening C. trachomatis infections.

Keywords: C. trachomatis; CPAF; mimetic peptides; phage display; screening tests.

Figure 1

Comparison of the reactivity of clones identified by phage display between the sera of patients who were positive and negative for C. trachomatis. (A) Phage ELISA with the clones selected by biopanning and sequenced. The clones marked with “*” were selected for use in the ELISA optimization test. Evaluation of reactivity by phage ELISA of individual samples with clones G1 (B), H5 (C), H7 (D) and C6 (E).

Figure 2

Conformational alignment of peptides from clones G1, H5, C6 and H7 in CPAF of C. trachomatis.

Figure 3

Statistical parameters of the mimetic peptides. Sensitivity, specificity, likelihood ratio (LR) and area under the curve (AUC) and ROC curve.