Purification of the T4SS VirB3/VirB4/VirB6/VirB8 complex of pCF10 and 89K plasmids from the gram positives bacterias Enterococcus faecalis and Streptococcus suis

• Grant number: 14/15508-2
• Support Opportunities: Scholarships abroad - Research
• Start date: April 01, 2015
• End date: March 31, 2016
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Cristiane Rodrigues Guzzo Carvalho
• Grantee: Cristiane Rodrigues Guzzo Carvalho
• Host Investigator: Gabriel Waksman
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: University of London, England

Abstract

Bacteria and some eukaryotic organelles have nanomachines formed by large protein complexes that act in the process of secretion, in most of the cases, of macromolecules to the extracellular medium or directly into the host cell. These systems are called secretion systems and until now, were identified seven general classes of secretion systems and the chaperona/Usher system. There is a great diversity of mechanism of action and function between these secretion systems, but in most of the cases the function is related to the cell-cell interactions that occur within an ecosystem. These associations include biotic associations, pathogenic and associations of mutualism. The Secretion system plays an important role in the process of modulating the interactions of bacteria with the extracellular environment. The aim of this project is to study some membrane integral proteins membrane belonging to the type IV secretion system (T4SS). The T4SS is used by bacteria to inject virulence factors within eukaryotic cells or prokaryotic cells. The T4SS is also involved in injection of DNA from plasmids within the receptor cell, a process called conjugation. The VirB/D4 T4SS, of gram-negative bacteria, is composed by 12 proteins, VirB1 to VirB11 and VirD4. This system can be shared into three main parts, the ATPases (VirD4, VirB11 and VirB4), the components that build the translocation channel (VirB3, VirB6, VirB7, VirB8, VirB9, VirB10) and the pilus proteins (VirB2 e VirB5). We intend to purify the VirB3-VirB4-VirB6-VirB8 complex from T4SS from pCF10 and 89K plasmids of the Gram-positive bacterias Enterococcus faecalis and Streptococcus suis, respectively. This work will be carried out in the laboratory of Professor Dr. Gabriel Waksman in the University of London at Birkbeck College. Prof. Gabriel is one of the few researchers in the world able to do the purification and structural determination by crystallography or by crio-electron microscopy of integral proteins. For this reason, I want to have the opportunity to be in the Gabriel Waksman laboratory to learn how purify membrane integral proteins. This experience will be of immense importance to my scientific career, as far as my scientific maturity and the impact of my scientific search. (AU)