In vitro microbicidal activities of cecropin peptides D2A21 and D4E1 and gel formulations containing 0.1 to 2% D2A21 against Chlamydia trachomatis

Abstract

Topically applied microbicides that eradicate pathogens at the time of initial exposure represent a powerful strategy for the prevention of sexually transmitted infections. To aid in the further development of an effective topical microbicide, we assessed the minimum cidal concentration (MCC) of two cecropin peptides, D2A21 and D4E1, and gel formulations containing 0.1 to 2% D2A21 against Chlamydia trachomatis in vitro. The MCC of peptide D2A21 was 5 microM (18.32 microg/ml), and that of peptide D4E1 was 7.5 microM (21.69 microg/ml). The MCC of gel formulations containing 2% D2A21 was 0.2 mM (0.7 mg/ml), and that of gel formulations containing 0.5% D2A21 was 0.2 mM (0.7 mg/ml). There was no significant variation in the results when two different C. trachomatis strains were tested, and the addition of 10% human blood did not significantly alter the MCCs. pH values above and below 7 reduced the activity of the D2A21 peptide alone, but the MCC of the 2% D2A21 gel formulation was only slightly altered at the various pHs tested. Ultrastructural studies indicated that C. trachomatis membranes were disrupted after D2A21 exposure, resulting in leakage of the cytoplasmic contents. These in vitro results suggest that these cecropin peptides may be an effective topical microbicide against C. trachomatis and support the need for further evaluation.

FIG. 1.

The amino acid sequences and structures of the peptides D2A21 and D4E1. (A) The amphipathic nature of the D2A21 peptide is shown with the hydrophobic amino acids on the top of the molecule. The peptide is assumed to be in an alpha-helical conformation when it is near or in the membrane. The hydrogens are not shown. (B) The D4E1 peptide is depicted in an amphipathic beta-sheet conformation. The nonpolar face is on the top, while the polar charged face is below the plane of the molecule.

FIG. 2.

Comparison of the antichlamydial activities of serial dilutions of antimicrobial peptides D2A21 and D4E1. The MCC preinoculation assay was used, and the percent killing of C. trachomatis serovar D after 5 and 120 min of exposure to each peptide dilution or the controls was plotted. The initial concentrations of polymyxin B and penicillin G were 1.4 mM (2 mg/ml) and 5.4 mM (2 mg/ml), respectively. Values were calculated in comparison to that of the organism-only control according to the following formula: (mean IFU of organism control − mean IFU of test)/mean IFU of organism control × 100. The standard deviations from triplicate tests are indicated by error bars. Results for the polymyxin B positive and penicillin G negative controls are also shown.

FIG. 3.

Activities of the gel formulations containing 2% D2A21 against C. trachomatis serovar D in the modified ultrafiltration MCC assay (A), 0.5% D2A21 in the standard MCC assay (B), or 0.1% D2A21 in the standard MCC assay (C). The percent killing of the D2A21-containing gel formulation after 5 and 120 min in the MCC preinoculation assay is plotted, and standard deviations from triplicate tests are indicated by error bars. Values were calculated in comparison to that of an organism-only control according to the following formula: (mean IFU of organism control − mean IFU of test)/mean IFU of organism control × 100. Results for the polymyxin B positive and penicillin G negative controls as well as the placebo control are also shown.

FIG. 4.

Percent killing of C. trachomatis serovar D preexposed to the indicated concentrations of the peptide D2A21 for 5 and 120 min in the presence and absence of 10% whole human blood. Error bars indicate standard deviations of triplicate tests. Values for the organism and for polymyxin B and penicillin G controls were also determined in the MCC assay without the addition of 10% whole human blood.

FIG. 5.

Percent killing of C. trachomatis serovar D preexposed to dilutions of the 2% D2A21 gel formulation after 5 and 120 min in the presence and absence of 10% whole human blood. Error bars indicate standard deviations of triplicate tests. Values for the organism and for polymyxin B and penicillin G controls were also determined in the modified ultrafiltration MCC assay without the addition of 10% whole human blood.

FIG. 6.

D2A21 activity at pH 4, 5, 6, 7, and 8 at 120 min. The percent killing of C. trachomatis serovar D by the peptide D2A21 is shown at each pH value, with standard deviations (from triplicate tests) indicated by error bars for each concentration. Values were calculated in comparison to that of the organism control at the same pH value. The standard MCC assay protocol was followed.

FIG. 7.

Antichlamydial activities of gel formulations containing 2% D2A21 (A), 0.5% D2A21 (B), or 0.1% D2A21 (C) at pH 4, 5, 6, 7, and 8 at 120 min. The percent killing of C. trachomatis serovar D by each of the gel formulations containing D2A21 is shown at each pH value, with standard deviations (from triplicate tests) indicated by error bars for each concentration. Values were calculated in comparison to that of the organism control at the same pH value. Results for the polymyxin B positive and penicillin G negative controls are also shown. The modified ultrafiltration MCC method was used with the 2% gel, and the standard MCC protocol was followed for the 0.5 and 0.1% gels.

FIG. 8.

Transmission electron micrographs of C. trachomatis serovar D exposed to peptide D2A21 for 90 min. (A) Organisms treated with D2A21 compared to the untreated control appear as if the inner membrane had lost its structural integrity (gray arrow) or they are in the process of leaking their cytoplasmic contents (black arrow). (B) Untreated organisms incubated in SPG only and processed for microscopy. It is important to note the intact outer membrane structures and electron-dense cytoplasmic mass. The figures shown are representative of the entire field. Electron micrographs were scanned on an Agfa Arcus II flatbed scanner in Adobe Photoshop, version 5.0. Bar = 0.5 μm.