Functional inhibition of the virulence factor ProA from Legionella pneumophila by novel zinc-binding compounds

The M4 zinc metalloprotease ProA is an important effector protein of the human lung pathogen Legionella pneumophila, which is known as the causative agent of Legionnaires' disease. Due to its versatile spectrum of substrates, ProA contributes to bacterial proliferation, immune evasion, and serious tissue destruction. Successful treatment of the atypical pneumonia is based on antibiotic therapies but cannot always prevent severe symptoms and irreversible lung damage. Additional application of specific inhibitors against virulence factors like ProA might be able to reduce life-threatening complications in patients. In order to identify promising candidates, inhibitory effects of ten novel-designed zinc-binding substances were initially evaluated in azocasein assays. The three best-performing compounds were chosen for subsequent analyses regarding versatile physiological functions of ProA during infection. It was demonstrated that a 20-fold molar excess compared to the enzyme leads to successful inhibition of human collagen IV degradation, cleavage of bacterial flagellin, immune evasion from TLR5-mediated immunity, and PMN inflammation in human lung tissue explants. Interestingly, biochemical assays revealed different tendencies in terms of inhibitor efficacy than studies in a host background. The present results not only indicate great potential of the selected zinc-binding compounds for ProA research but also demonstrate methods which are suitable to test potential drug compounds on different and important organizational levels during infection.