Ahps from Pseudomonas aeruginosa and lipid peroxides: from antioxidant defense to subversion of the hosts imune system

• Grant number: 25/01281-0
• Support Opportunities: Scholarships in Brazil - Master
• Start date: July 01, 2025
• End date: June 30, 2027
• 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
• Associated research grant: 13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

The increase in Multi Drug Resistance bacterial (MDR) is a worldwide problem and highlights the importance of searching for new drugs and biological targets to overcome these pathogens. Studies indicate that antimicrobials have the convergent ability to produce reactive oxygen species (ROS), helping to kill the pathogen. ROS and reactive nitrogen species (RNS) are also produced by the host immune system to fight pathogens. Among the species produced, hydroperoxides represent a major challenge for bacteria since they are able to generate reactive radical species and organic peroxides, such as lipid peroxides in bacterial membranes, which alter their organization and can lead to their rupture, with consequent death of the pathogen. Lipid peroxides are also produced in the host, through enzymes such as lipoxygenases (Lox) and cyclooxygenases (Cox), mostly derived from arachidonic acid (ARA) and linoleic acid (LIA), which, in addition to being toxic to bacteria, play signaling roles in events of infection and inflammation. Interestingly, in Pseudomonas aeruginosa, an etiological agent with MDR strains, these peroxides can also be metabolized by two enzymes, PaLoxA and PaCYP168A1O, resulting in products typically produced by defense cells, which subvert the host's protective function. However, since these peroxides are also toxic, they need to be detoxified. In this context, microorganisms have developed highly efficient enzymes able to decompose peroxides, including Ahps, a group of enzymes that represent important pharmacological targets due to their diversity, distribution and high reactivity on substrates. The expression of these enzymes is controlled by transcription factors such as OxyR, but also by the quorum sensing factor MvfR, which is related to the control of virulence elements and antibiotic resistance. It has already been shown that the AhpC isoform of P. aeruginosa (PaAhpC) is a virulence factor, and its expression is related to resistance to the beta-lactam meropenem and to H2O2, a function shared with a second isoform called AhpB, which has not yet been characterized. However, no work has systematically evaluated the importance of Ahps in the decomposition of lipid-derived peroxides. As part of my scientific initiation, I collaborate in a study which shown that PaAhpC has a high affinity for lipid peroxides derived from oleic and linoleic acid, and that these substrates can hyperoxidize the enzyme (irreversible inhibition). The aim of my CI was to assess whether PaAhpC was capable of breaking down ARA-derived lipid peroxides formed by the host cell, 15-HpETE and PGG2, and we demonstrated that the enzyme has a high affinity for these intermediates, as well is able to decompose endoperoxide function, such as that present in PGG2. This project aims to deepen the understanding of the Ahps importance in host infection, since the metabolization of signaling molecules can have implications for the interaction with defense cells, so we will carry out the functional characterization of the second Ahp peroxidase isoform from P. aeruginosa (PaAhpB), which indicates peculiar characteristics in relation to the PaAhpC isoform. We will also evaluate the sensitivity of wild-type P. aeruginosa cells and delta-ahpc and delta-ahpb mutants to the antibiotic meropenem in combination with lipid peroxides. We also aim to carry out tests on mouse macrophages isolated from bone marrow (BMDM) and epithelial cells isolated from patients with cystic fibrosis (A549) using wild-type, delta-ahpc, delta-ahpb and complemented PA14 cells. The last two procedures are planned to be carried out in the laboratory of Prof. Laurence Rahme at Harvard University. (AU)