Expression and structural modelling of the components GspC and GspD from the type II secretion system of Leptospira interrogans

• Grant number: 22/07160-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2022
• End date: January 31, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Cristiane Rodrigues Guzzo Carvalho
• Grantee: Gabriel Sánchez Hueck
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Leptospirosis is a zoonosis caused by bacteria of the genus Leptospira, a gram-negative spirochaete of translational motility. L. interrogans is the main cause of leptospirosis in humans, being characterized by varied virulence mechanisms such as tissue invasion, biofilm formation, adhesion to several types of cell lines, and secretion of enzymatic effectors which establish pathogenesis. An important target of investigation in L. interrogans is the Type II Secretion System (T2SS), which may be an important means of transport of bacterial-borne toxic effectors and adhesins, to the extracellular milieu. T2SSs are formed by an intermembrane complex that includes an inner membrane platform, a filamentous pseudopilus, which pushes out folded proteins, as well as an outer membrane secretin channel. Little is known about the T2SS in L. interrogans, other than the fact that its genome conserves almost all components of the complex. The mechanisms of protein secretion and signaling also remain to be understood. The aim of this project is to resolve the outer membrane complex of Leptospira interrogans T2SS, comprised of the secretin GspD, bound to the periplasmatic GspC. We will attempt heterologous co-expression of these components in E. coli, and will carry out overexpression essays, expecting to induce channel formation, which will be inspected in membrane fractions. Once identified, we aim to purify the complex and obtain images through electron microscopy.(AU)