Biofilm formation is the preferred mode of growth lifestyle for many microorganisms, including bacterial and fungal human pathogens. Biofilm is a strong and dynamic structure that confers a broad range of advantages to its members, such as adhesion/cohesion capabilities, mechanical properties, nutritional sources, metabolite exchange platform, cellular communication, protection and resistance to drugs (e.g., antimicrobials, antiseptics, and disinfectants), environmental stresses (e.g., dehydration and ultraviolet light), host immune attacks (e.g., antibodies, complement system, antimicrobial peptides, and phagocytes), and shear forces. Microbial biofilms cause problems in the hospital environment, generating high healthcare costs and prolonged patient stay, which can result in further secondary microbial infections and various health complications. Consequently, both public and private investments must be made to ensure better patient management, as well as to find novel therapeutic strategies to circumvent the resistance and resilience profiles arising from biofilm-associated microbial infections. In this work, we present a general overview of microbial biofilm formation and its relevance within the biomedical context.

Pseudomonas aeruginosa is a ubiquitous and opportunistic human pathogen that represents a critical problem to the clinician due to the increased number of resistant strains isolated from hospital settings. In addition, there is a great variety of pathologies associated with this versatile Gram-negative bacterium. P. aeruginosa cells are able to produce an incredible arsenal of virulence factors, especially secreted molecules that act singly or together to ensure the establishment, maintenance, and persistence of a successful infection in susceptible hosts. In this context, pseudomonal proteases roles are highlighted due to their ability to cleave key host proteinaceous substrates as well as to modulate several biological processes, for example, escaping and modulating the host immune responses in the bacterial own favor. Proteases secreted by P. aeruginosa include elastase A (LasA), elastase B (LasB), alkaline protease (AP), protease IV (PIV), Pseudomonas small protease (PASP), large protease A (LepA), MucD, and P. aeruginosa aminopeptidase (PAAP). In the present review, we discuss the role of each of these relevant proteases produced by

Coordination of phendione to Ag(+) and Cu(2+) represents a new promising group of anti-infective agents, which revealed a potent anti-P. aeruginosa action against both planktonic- and biofilm-growing cells.

No abstract

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 ]ClO 4 (Ag-phendione) and [Cu(phendione) 3 ](ClO 4 ) 2 .4H 2 O (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 Cu 2+ 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 .