Degradomic analysis of human plasma proteins targeted by Leptospira proteases

• Grant number: 23/11939-8
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: July 01, 2024
• End date: April 30, 2027
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Angela Silva Barbosa
• Grantee: Cassia Moreira Santos
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

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 degradamics to study the effects of proteases secreted by leptospires on human plasma molecules. The plasma will be incubated with leptospires or with extracellular proteins secreted by the bacteria. The N-terminal groups of the degradation products will be initially marked using TAILS approach (Terminal Amine Isotopic Labeling of Substrates) with the TMT isobaric marker, reduced with DTT, alkylated with iodoacetamide, and digested with trypsin. Samples will be analyzed in the Orbitrap Exploris 480 mass spectrometer coupled to the Vanquish Neo liquid nano-chromatograph. The application of this proteomic approach based on mass spectrometry to evaluate the Leptospira-host interface may improve our knowledge on the alterations that occur in the plasma involving molecules of innate immunity and on the coagulation cascade as well as other pathways that may open perspectives for the identification of new substrates. In addition, this approach can improve our understanding on the mechanisms used by this bacterium to cause disease in vivo, and may help the development of therapeutic tools to combat the disease.