Retrocyclin inhibits Gardnerella vaginalis biofilm formation and toxin activity

Abstract

Background: Retrocyclins are cyclic antimicrobial peptides that have been shown to be both broadly active and safe in animal models. RC-101, a synthetic retrocyclin, targets important human pathogens and is a candidate vaginal microbicide. Its activity against microbes associated with bacterial vaginosis is unknown.

Methods: We investigated the effect of RC-101 on toxin activity, bacterial growth and biofilm formation of Gardnerella vaginalis in vitro.

Results: RC-101 potently inhibits the cytolytic activity of vaginolysin, the Gardnerella vaginalis toxin, on both erythrocytes and nucleated cells. RC-101 lacks inhibitory activity against planktonic G. vaginalis but markedly decreases biofilm formation.

Conclusions: These dual properties, toxin inhibition and biofilm retardation, justify further exploration of RC-101 as a candidate agent for bacterial vaginosis prevention.

Figure 1.

RC-101 inhibits VLY-mediated cytolysis. (a) VLY (20 ng/mL) and indicated concentrations of RC-101 or vehicle control were added to a 1% solution of human erythrocytes, and haemolysis was measured by haemoglobin release assay. (b) VLY (1 μg/mL) and indicated concentrations of RC-101 or vehicle control were added to HeLa cells and lysis assessed by lactate dehydrogenase release. Percentage lysis is reported relative to cells exposed to 0.05% Triton X-100 (representing 100% lysis). Bars represent standard errors. *P < 0.05; ***P < 0.001.

Figure 2.

RC-101 inhibits G. vaginalis de novo biofilm formation but does not break down established biofilms or inhibit planktonic growth. (a) Bacteria were grown for 24 h in the presence of vehicle control or RC-101 at the indicated concentrations and biofilm formation determined by safranin staining, solubilization and measurement of OD₄₅₀. (b) G. vaginalis biofilms were grown for 24 h without treatment and then exposed to vehicle control or RC-101 at the indicated concentrations for an additional 24 h prior to staining. (c) G. vaginalis was exposed to RC-101 or vehicle control for 1 or 3 h and bacterial concentration determined by quantitative culture. (d) G. vaginalis growth was measured by monitoring OD₅₉₀ in the presence of RC-101 (final concentration 12.5 mg/L) or vehicle control. *P < 0.05; ***P < 0.001.