Structure and function of bacterial secretion systems

• Grant number: 17/17303-7
• Support type: Research Projects - Thematic Grants
• Duration: March 01, 2018 - February 28, 2023
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Shaker Chuck Farah
• Grantee: Shaker Chuck Farah
• Home Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Co-Principal Investigators: Cristiane Rodrigues Guzzo Carvalho ; Roberto Kopke Salinas
• Assoc. researchers: Robson Francisco de Souza ; Rodrigo Villares Portugal
• Associated grant(s): 18/26394-9 - Multi-User Equipment approved in grant 2017/17303-7: single tilt cryotransfer holder, AP.EMU; 18/26396-1 - Multi-User Equipment approved in grant 2017/17303-7: vitrification robot, AP.EMU; 18/20391-8 - Implementation of modern multidimensional heteronuclear NMR techniques in the 800 MHz NMR instrument of the analytical instrumentation center of the University of São Paulo, AV.EXT; 18/20526-0 - Multiuser equipment granted in project 2017/17303-7: AKTA liquid cromatography system, AP.EMU; 18/08822-3 - The role of the com system in biofilm development mediated by the type IV pilus in Xanthomonas citri subsp. citri, AV.EXT
• Associated scholarship(s): 19/12234-2 - Isolation and structural studies on the Xanthomonas citri Type IV pilus and T4SS pilus, BP.PD; 19/09482-4 - The R-bodies of Xanthomonas citri, BP.IC; 18/21076-9 - Structure of the Xanthomonas citri Type IV pilus secretin, BP.DR; + associated scholarships 18/21450-8 - Cryo-EM structural studies on the Xanthomonadaceae Type IV secretion systems, BP.PD; 18/09277-9 - Investigation of the structural requirements for recognition of T4SS-related toxins by the coupling protein VirD4, BP.PD; 16/00458-5 - Structural and functional characterization of Xanthomonas citri Type IV Secretion System, BP.PD; 15/18237-2 - Genetic elucidation of the functional and regulatory mechanisms underlying the Type IV Secretion System in Xanthomonas citri and how it mediates contact dependent killing, BP.PD; 14/04294-1 - Structural and functional studies of the Xanthomonas citri Type IV Secretion System, BP.PD - associated scholarships

Abstract

Bacterial macromolecular protein secretion systems are large (multi-mega Dalton) multiprotein complexes that transport effector proteins through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells. These systems also mediate the horizontal transfer of genetic material (conjugation) between bacterial cells and specialized homologous forms of these systems have evolved into bacterial pili and flagella. Therefore, macromolecular secretion systems are important determinants for bacterial adaptation, competition and survival in diverse environmental niches. During the course of the previous thematic project, we demonstrated the importance of type IV pili (T4P) in surface twitching motility and biofilm formation in Xanthomonas citri and characterized key regulators of T4P biogenesis. We also showed that the Xanthomonas citri Type IV secretion system (T4SS) provides these cells the capacity to kill other Gram-negative bacterial species in a contact-dependent manner. This was the first demonstration of the involvement of a T4SS in bacterial killing and points to this special class of T4SS as an important determinant for Xanthomonad survival during competitive encounters with other bacterial species in the environment. In this project, we propose to pursue several lines of research into the molecular structure, molecular function and physiological relevance of these two complex nano-machines in Xanthomonas citri as well as in other members of the Xanthomonadaceae family. Specifically, we plan to pursue two general lines of research. Project 1: studies on the Xanthomonadaceae Type IV secretion systems. We plan to i) determine the structure of individual T4SS components as well as multiprotein complexes; ii) perform structural and ezymological studies on the toxins secreted by this system as well as their cognate inhibitors; iii) study the molecular basis of recognition of toxins by the T4SS apparatus; iv) study the cellular localization and regulation of expression of the T4SS, and iv) extend our studies from the Xanthomonas citri system to homologous T4SSs found in other bacterial species including Stenotrophomonas, Lysobacter and in some species of the betaproteobacteria orders Burkholderiales and Neisseriales. Project 2: studies on the Xanthomonas Type IV pilus. We plan to investigate i) how PilB ATPase activity is regulated by PilZ, FimX and c-diGMP and other inner membrane (platform) components; ii) the structures of the PilB-PilZ and PilB-PilZ-FimX complexes; iii) the roles of the conserved minor pilins in Xanthomonadaceae species; iv) the role of PilU, the third and poorly understood ATPase; v) how specific environmental signals control T4P-dependent functions in Xanthomonas specie; vi) how T4P contribute to Xanthomonas competitiveness in antagonistic interactions with other bacterial species, and vii) the structure of the extracellular pilus (polymer of PilA subunits). Our studies will employ a variety of experimental methods including X-ray crystallography, Nuclear Magnetic Resonance (NMR), cryoelectron microscopy of single particles, Small-Angle X-ray Scattering (SAXS), molecular dynamics simulations, the characterization of gene knockouts, gene expression analysis, fluorescence microscopy, enzymology and bioinformatics. This project involves the training of undergraduate and graduate students, post-doctoral researchers and collaboration with researchers here in Brazil and overseas. (AU)

Scientific publications

(5)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

BAYER-SANTOS, ETHEL; CENENS, WILLIAM; MATSUYAMA, BRUNO YASUI; OKA, GABRIEL UMAJI; DI SESSA, GIANCARLO; MININEL, IZABEL DEL VALLE; ALVES, TIAGO LUBIANA; FARAH, CHUCK SHAKER. The opportunistic pathogen Stenotrophomonas maltophilia utilizes a type IV secretion system for interbacterial killing. PLOS PATHOGENS, v. 15, n. 9 SEP 2019. Web of Science Citations: 0.

ANTUNES, VICTOR U.; LLONTOP, EDGAR E.; VASCONCELOS, FERNANDA N. DA COSTA; DE LOS SANTOS, YOSSEF LOPEZ; OLIVEIRA, RONALDO J.; LINCOPAN, NILTON; FARAH, CHUCK S.; DOUCET, NICOLAS; MITTERMAIER, ANTHONY; FAVARO, DENIZE C. Importance of the beta 5-beta 6 Loop for the Structure, Catalytic Efficiency, and Stability of Carbapenem-Hydrolyzing Class D beta-Lactamase Subfamily OXA-143. BIOCHEMISTRY, v. 58, n. 34, p. 3604-3616, AUG 27 2019. Web of Science Citations: 0.

BAYER-SANTOS, ETHEL; CESETI, LUCAS DE MORAES; FARAH, CHUCK SHAKER; ALVAREZ-MARTINEZ, CRISTINA ELISA. Distribution, Function and Regulation of Type 6 Secretion Systems of Xanthomonadales. FRONTIERS IN MICROBIOLOGY, v. 10, JUL 17 2019. Web of Science Citations: 0.

SGRO, GERMAN G.; OKA, GABRIEL U.; SOUZA, DIORGE P.; CENENS, WILLIAM; BAYER-SANTOS, ETHEL; MATSUYAMA, BRUNO Y.; BUENO, NATALIA F.; DOS SANTOS, THIAGO RODRIGO; ALVAREZ-MARTINEZ, CRISTINA E.; SALINAS, ROBERTO K.; FARAH, CHUCK S. Bacteria-Killing Type IV Secretion Systems. FRONTIERS IN MICROBIOLOGY, v. 10, MAY 21 2019. Web of Science Citations: 2.

SGRO, GERMAN G.; COSTA, TIAGO R. D.; CENENS, WILLIAM; SOUZA, DIORGE P.; CASSAGO, ALEXANDRE; DE OLIVEIRA, LUCIANA COUTINHO; SALINAS, ROBERTO K.; PORTUGAL, V, RODRIGO; FARAH, CHUCK S.; WAKSMAN, GABRIEL. Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri. NATURE MICROBIOLOGY, v. 3, n. 12, p. 1429-1440, DEC 2018. Web of Science Citations: 9.