Evaluation of biological organic peroxides as inhibitors of the AhpC enzyme and their effects on the survival of Pseudomonas aeruginosa

• Grant number: 23/14764-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2024
• End date: December 31, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Marcos Antonio de Oliveira
• Grantee: Laura Fernandes da Silva
• Host Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil

Abstract

The increase in Multiple Drug-Resistant (MDR) bacterial strains is a concern for global public health given the high costs of treatment and the high lethality caused by these pathogens. The antibacterial drugs used to combat pathogenic bacteria interfere with specific biological processes, however, studies show that different antibiotics classes possess a common mechanism that combat pathogens, represented by the generation of reactive oxygen species (ROS). It is important to note that higher eukaryotes defense cells also produce high amounts of ROS as well reactive nitrogen species (RNS) as one of the main fronts of defense against pathogenic organisms. Together, ROS and RNS are so called RONS. Among the different species formed, peroxides represent an important class, because although they have low reactivity, they can cross biological membranes and react with other RONS types, generating very toxic species able to cause damage to all classes of biomolecules. In this context, antioxidant enzymes, especially peroxidases, play an important role in the survival of pathogens and could be a very important target for the search for inhibitors to combat these microorganisms, since the inhibition of their activity has pronounced effects on their mortality. The bacterial peroxiredoxin (Prx) AhpC (Alkyl hydroperoxide reductase C) stands out for its wide distribution in intra- and extracellular environments, abundance and high variety of substrates (hydrogen peroxide, peroxynitrite and alkyl hydroperoxides - organic peroxides), whose decomposition rates can reach constants of 107-8M-1s-1. AhpCs are capable of decomposing peroxides using a reactive cysteine, known as peroxidatic cysteine (CP). It has already been shown that under high concentrations of hydrogen peroxide AhpCs lose their peroxidase activity due to CP hyperoxidation and the enzyme is degraded. Recently, our research group demonstrated that AhpC from Pseudomonas aeruginosa (PaAhpC), a Gram-negative bacterium with MDR strains involved in nosocomial infections with a high mortality rate, has an extraordinary affinity for long-chain lipid peroxides. In addition, results show that these peroxides are capable of oxidizing and promoting hyperoxidation of CP at low concentrations, with second-order constants reaching rates of 106M-1s-1. These results seem important since AhpC has been shown to be a virulence factor in this species. It should also be noted that these lipids, normally found in hosts, are toxic to bacterial cells and a recent explanation is their propensity to form lipid peroxides, which would be the toxicity factors. In plants, lipid peroxides (especially endoperoxides) are found to be involved in cell signaling processes and defense against pathogens, but their biological targets have not yet been fully elucidated. This project aims to evaluate the effect of long-chain lipid peroxides formed in the host or by pathogens (oleic acid peroxide (Ol-OOH), arachidonic acid peroxide (15-HpETE) and the endoperoxides DBEGH(2) 3.5 and DBEGH 3.6 isolated from Drymis brasiliensis, a plant from São Paulo, over the peroxidase activity/inactivation of the P. aeruginosa AhpC enzyme by means of biochemical analyses, as well as investigating in vivo the effect of lipid peroxides and endoperoxides separately or in conjunction with antibiotics normally used to combat P. aeruginosa infections (gentamicin and ciprofloxacin), using wild-type strains, delta pa_ahpc mutants and mutants containing the gene complementation.