| Congresso Brasileiro de Microbiologia 2023 | Resumo: 126-1 | ||||
Resumo:Infections caused by the Candida genus are emerging, with an alarming increase in occurrences and severity. Although certain Candida species are commensals, imbalances in the host-microorganism interaction can favor infections. Considering that mortality from fungal infections has been rising, and the rate of resistance to classic antifungal drugs has increased as well, the development of new control strategies is highly necessary. In this context, interest in peptides with therapeutic functions has enhanced. Proteus mirabilis urease (PMU) is composed of a trimer of (ABC)3 trimers, denoted as α, β and γ. As seen for other ureases, PMU also induces effects independent of its enzymatic action, including activation of human platelets, antifungal and entomotoxic activities. Notably, studies utilizing isolated domains of PMU indicate that the β domain (PmUreβ) accounts for most of these biological activities. This study explores the effects and mechanism of action of PmUreβ on Candida spp., with potential biotechnological implications as an antimicrobial peptide. For recombinant peptide production, the coding region of the respective gene was cloned and expressed in E. coli, followed by purification using nickel affinity chromatography. Then, we investigated whether the peptide had toxic effects on C. albicans and non-albicans species. PmUreβ concentrations of 2.25 µM, 4.5 µM, and 9 µM were utilized, and cell viability was assessed after 24 hours of yeast cell incubation in the presence or absence of the peptide. Significant reduction in cell viability was observed at 30 °C with 4.5 µM PmUreβ, while the same magnitude of toxic effect was not observed at host temperature (37 °C). To assess potential cellular alterations induced by PmUreβ, microscopic analyses of the cultures were performed. After 3 h of treatment with PmUreβ (9 µM), we observed intense formation of cell clusters in samples exposed to the peptide compared to the control. Scanning Electron Microscopy (SEM) confirmed the presence of potential extracellular matrix fibers surrounding these cell clusters and the possible occurrence of extracellular vesicles. Also, Candida cells appeared to be damaged by PmUreβ, as evidenced by the deformed cell surfaces, suggesting the cell wall as a target. Numerous non-polar bud scars on cells treated with the peptide were observed, suggesting that the normal yeast division process may have been disrupted. In addition, PmUreβ demonstrated the ability to reduce biofilm formation, as confirmed by the XTT assay. Comparative RNA-Seq revealed that genes involved in cell membrane and cell wall integrity are affected by PmUreβ treatment. The Sorbitol Protection Assay demonstrated that PmUreβ can alter cell membrane permeability, and ROS production was increased in the presence of the peptide. As ROS can induce lipid peroxidation, membrane structure alterations leading to increased permeability might be attributed to the peptide's fungitoxic effect. Moreover, the cytotoxic effect of PmUreβ was evaluated on mammalian cells (HEK293). Analyses of cell viability, MTT determination, and trypan blue exclusion assays indicated that PmUreβ has not shown harmful effect on these cells. Collectively, these findings suggest that PmUreβ possesses promising potential as a fungitoxic peptide, thus highlighting its biotechnological interest for further development and application. Palavras-chave: Antifungal activity, Antimicrobial peptide, Candida albicans, Cell wall, Urease Agência de fomento:Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, Brazil). | |||||