Cassia Moreira Santos^1,2, Leo Kei Iwai², Angela Silva Barbosa²

Proteases are among the virulence determinants of a number of medically important pathogens. During the infectious process, pathogens produce proteases that may cleave peptide bonds, leading to irreversible structural changes, in addition to causing tissue damage and excessive inflammation. The mechanisms underlying entry, dissemination, persistence and tissue damage resulting from infection by the spirochete bacterium Leptospira - the causative agent of leptospirosis - are still poorly known, but studies carried out by our group are suggestive that proteases secreted by this bacterium contribute to the various stages of host colonization. The biggest bottleneck related to the study of leptospiral proteases has been the limitation with regard to the identification of targeted substrates. In this project, we will apply degradomics to study the effects of proteases secreted by leptospires on human plasma molecules. Initially, Golden Syrian hamsters were intraperitoneally inoculated with Leptospira interrogans serovar Copenhageni strain L1-130 or serovar Manilae strain L495 (10^8 bacteria). Following disease progression, bacteria were isolated from the kidneys and maintained in EMJH medium. The leptospiral secretome was obtained by incubating 10^9 bacteria in 500 uL of PBS at 37 °C for 4 hours. The supernatant was centrifuged, filtered through a 0.22 um membrane, and stored at –80 °C. To assess the proteolytic activity of the secretome, 5 ug was incubated with purified plasma proteins C3b, C5, and fibronectin, followed by evaluation of proteolysis via Western blot. For experiments using human plasma, whole blood samples were collected from volunteers in 3.8% citrate-phosphate tubes, after approval by Plataforma Brasil, and stored at –80 °C. In the initial experiments, 5 ug of leptospiral secretome was incubated with a plasma pool for 1, 6, and 24 hours at 37 °C. Due to the wide range of proteins present in plasma, the most abundant proteins were depleted using perchloric acid prior to proteomic analysis. Samples were reduced, alkylated, and desalted using the Single-Pot, Solid-Phase-enhanced Sample Preparation (SP3) protocol, followed by trypsin digestion and vacuum centrifugation. The peptides were then resuspended in formic acid and analyzed using an Orbitrap Exploris 480 mass spectrometer coupled to a Vanquish Neo nano-LC system. Leptospires proteases were able to fully or partially degrade purified C3b, C5, and plasma fibronectin, corroborating previous findings from our group. Depletion of abundant plasma proteins was effective, as confirmed by the absence of bands on silver-stained acrylamide gels. However, preliminary proteomic analysis identified only 220 shared proteins—a number lower than expected based on previous plasma proteomic studies. Additionally, it remains unclear whether perchloric acid affects protein integrity, a critical issue since a major objective is to identify plasma targets of Leptospira proteases. Therefore, the next steps will involve peptidomic analyses of plasma treated and untreated with perchloric acid to evaluate potential effects on protein structure, as well as N-terminomics of plasma incubated with the secretome. These approaches aim to provide a comprehensive view of proteolysis mediated by proteases secreted by pathogenic Leptospira.

Agradecimentos: grants #2023/11939-8, #2013/07467-1, São Paulo Research Foundation (FAPESP).