Effect of phenylalanine arginyl ß-naphthylamide on the imipenem resistance, elastase production, and the expression of quorum sensing and virulence factor genes in Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa is one of the most important nosocomial pathogens that possess the ability to produce multiple antibiotic resistance and virulence factors. Elastase B (LasB) is the major factor implicated in tissue invasion and damage during P. aeruginosa infections, whose synthesis is regulated by the quorum sensing (QS) system. Anti-virulence approach is now considered as potential therapeutic alternative and/or adjuvant to current antibiotics' failure. The aim of this study is primarily to find out the impact of the efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN) on the production of elastase B and the gene expression of lasI quorum sensing and lasB virulence factor in clinical isolates of P. aeruginosa. Five P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Antimicrobial susceptibility of isolates was performed by the disk agar diffusion method. Effect of the PAßN on imipenem susceptibility, bacterial viability, and elastase production was evaluated. The expression of lasB and lasI genes was measured by quantitative real-time PCR in the presence of PAßN. All isolates were identified as multidrug-resistant (MDR) and showed resistance to carbapenem (MIC = 64-256 µg/mL). Susceptibility of isolates to imipenem was highly increased in the presence of efflux inhibitor. PAßN significantly reduced elastase activity in three isolates tested without affecting bacterial growth. In addition, the relative expression of both lasB and lasI genes was diminished in all isolates in the presence of inhibitor. Efflux inhibition by using the EPI PAßN could be a potential target for controlling the P. aeruginosa virulence and pathogenesis. Furthermore, impairment of drug efflux by PAßN indicates its capability to be used as antimicrobial adjuvant that can decrease the resistance and lower the effective doses of current drugs.

Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target.

Elastase B (lasB) is a multifunctional metalloenzyme secreted by the gram-negative pathogen Pseudomonas aeruginosa, and this enzyme orchestrates several physiopathological events during bacteria-host interplays. LasB is considered to be a potential target for the development of an innovative chemotherapeutic approach, especially against multidrug-resistant strains. Recently, our group showed that 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)2]ClO4 (Ag-phendione) and [Cu(phendione)3](ClO4)2.4H2O (Cu-phendione) had anti-P. aeruginosa action against both planktonic- and biofilm-growing cells. In the present work, we have evaluated the effects of these compounds on the (i) interaction with the lasB active site using in silico approaches, (ii) lasB proteolytic activity by using a specific fluorogenic peptide substrate, (iii) lasB gene expression by real time-polymerase chain reaction, (iv) lasB protein secretion by immunoblotting, (v) ability to block the damages induced by lasB on a monolayer of lung epithelial cells, and (vi) survivability of Galleria mellonella larvae after being challenged with purified lasB and lasB-rich bacterial secretions. Molecular docking analyses revealed that phendione and its Ag+ and Cu2+ complexes were able to interact with the amino acids forming the active site of lasB, particularly Cu-phendione which exhibited the most favorable interaction energy parameters. Additionally, the test compounds were effective inhibitors of lasB activity, blocking the in vitro cleavage of the peptide substrate, aminobenzyl-Ala-Gly-Leu-Ala-p-nitrobenzylamide, with Cu-phendione having the best inhibitory action (K i = 90 nM). Treating living bacteria with a sub-inhibitory concentration (½ × MIC value) of the test compounds caused a significant reduction in the expression of the lasB gene as well as its mature protein production/secretion. Further, Ag-phendione and Cu-phendione offered protective action for lung epithelial cells, reducing the A549 monolayer damage by approximately 32 and 42%, respectively. Interestingly, Cu-phendione mitigated the toxic effect of both purified lasB molecules and lasB-containing bacterial secretions in the in vivo model, increasing the survival time of G. mellonella larvae. Collectively, these data reinforce the concept of lasB being a veritable therapeutic target and phendione-based compounds (mainly Cu-phendione) being prospective anti-virulence drugs against P. aeruginosa.