Optimization and elucidation of cationic peptides antibacterial activity in multidrug-resistant pathogens.

Author(s)

Gabriela Marinho Righetto

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2022-05-09

Examining board members

Ilana Lopes Baratella da Cunha Camargo; Nelma Regina Segnini Bossolan; Eduardo Maffud Cilli; José Luiz de Souza Lopes; Luciene Andrade da Rocha Minarini

• Advisor: Ilana Lopes Baratella da Cunha Camargo

Abstract

Despite advances in the treatment of infectious diseases, pathogenic microorganisms are still a threat to public health. The scarcity of therapeutic options leads to interest in new compounds, so antimicrobial peptides (AMPs) such as Bothropstoxin-I and Plantaricin 149 have been revisited in order to improve and characterize their antimicrobial activity. The minimum inhibitory concentration (MIC) for pathogenic bacteria and the hemolysis of human erythrocytes were indicators used to evaluate the optimization of the activities of the synthesized analogs. The peptides chosen were NA1897, (KKWRWHLKPW)2K and Pep20 (Fmoc-KAVKKLFKKWG), for Bothropstoxin-I and Plantaricin 149, respectively. The MICs of Pep20 and NA1897 were determined for 60 bacteria of different species, strains, and resistance profiles, indicating a broad spectrum of action for both. The killing kinetics of Staphylococcus aureus and Acinetobacter baumannii were determined for both peptides, resulting in quick bacterial death, with a total reduction of survivors in a maximum of two hours. This result and the post-antibiotic effect suggest a concentration-dependent action. We studied the modes of action of these molecules by performing directed evolution in vitro through treatments at subinhibitory concentrations. We evaluated the metabolism of selected strains by phenotypic microarrays and genetic mutations by genome sequencing. Results indicate that these AMPs act on the cytoplasmic membrane and that resistance mechanisms involve a decrease in the anionic charge of the membrane. Membrane depolarization and transmission electron microscopy assays support these hypotheses, although a secondary mechanism of action is possible, mainly for NA1897. The molecules studied here represent antimicrobials of interest, but they are still in early stages of development due to the cytotoxicity presented, with future perspectives of optimization from the biological targets found. (AU)

• FAPESP's process: 18/15887-4 - Optimization and elucidation of cationic peptides antibacterial activity in multidrug-resistant pathogens
• Grantee: Gabriela Marinho Righetto
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)