Regulation studies of signalling transducers PA0847 and HsbD from Pseudomonas aeruginosa and GGDEF and EAL systems in tandem

Author(s)

Éverton Edésio Dinis Silva

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2018-11-21

Examining board members

Marcos Vicente de Albuquerque Salles Navarro; Andre Luis Berteli Ambrosio; Lucas Bleicher; Tie Koide; José Luiz de Souza Lopes

• Advisor: Marcos Vicente de Albuquerque Salles Navarro

Abstract

Membrane signaling protein are widely used by bacteria to adequate their metabolism of environmental cues. They present a great potential of applicability, such as techniques to control bacterial infections and development of biotecnological devices, for example in biosensors. However, little is known regarding the mechanisms of signal transduction in bacteria. In particular, about receptors that posses GGDEF and/or EAL domains, performing synthesis, degradation or recognition of c-di-GMP, which is one of the major bacteria metabolism regulators. For this reason, the present study aimed characterize two important transmembrane signaling proteins from Pseudomonas aeruginosa, coded in genes PA0847 and PA3343 (HsbD) and performed bioinformatic analysis of proteins with GGDEF and EAL in tandem, usually associated with membrane. Previous studies indicated that PA0847 and HsbD are both diguanylate cyclases (DGCs) that significantly impair phenotypes of P. aeruginosa, which is a model organism and common cause of infection in immunocompromised patients. In the present study we have shown that this interaction associated with the presence of a wellconserved pattern from HsbD-Nt, suggests the presence a system used for specific downregulation of c-di-GMP. Furthermore, using isolated domains of PA0847 in thermophoresis, pull down and two-hybrid confirmed the interaction and showed that the full PA0847 cytoplasmic is necessary to interaction with HsbD. Additional amalysis of molecular mass and enzyme kinetic showed that HAMP increases DGC activity, alters the co-purification patern of c-di-GMP and promotes tetramers formation in PA0847 soluble constructs. The presence of sulfate transporter in a common operon with PA0847, associated with computation model of periplasmatic region of PA0847 supports the relation of this protein in sulfate homeostasis. We also identified using new residues groups copled that are potential play roles in GGDEF-EAL proteins. We found by stuctural analisys alternative mechanisms of dimerization in EAL domais that do not posses dimerization site conserved. These results have not been presented elsewhere and provide insights into the bacterial signaling of c-di-GMP pathways. (AU)

• FAPESP's process: 12/25276-6 - Structural, biochemical and phenotypic studies of CHASE receptors associated with bacterial domains GGDEF / EAL
• Grantee: Everton Edesio Dinis Silva
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)