Protein structural biology applied in the study of c-di-GMP signaling and bacterial secretion systems

• Grant number: 25/02664-0
• Support Opportunities: Regular Research Grants
• Start date: November 01, 2025
• End date: October 31, 2028
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Cristiane Rodrigues Guzzo Carvalho
• Grantee: Cristiane Rodrigues Guzzo Carvalho
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated researchers: Anacleto Silva de Souza ; Gabriel Guarany de Araujo ; Robson Francisco de Souza

Abstract

This project aims to uncover molecular mechanisms that regulate motility, secretion, and cell-to-cell communication in bacteria, integrating structural, biochemical, and functional approaches. The proposal is organized into two main research axes, interconnected through the use of structural biology techniques - notably cryo-electron microscopy (cryo-EM) and X-ray crystallography - and focused on key proteins from two bacterial models: Leptospira interrogans (Lic) and Xanthomonas citri (Xac).In the first axis, we will continue investigating the intracellular signaling pathway mediated by cyclic di-GMP (c-di-GMP) in L. interrogans, with particular emphasis on the protein YcgR (LIC_11920) and the diguanylate cyclase LIC_11128. Our research group, in collaboration with Prof. Robson de Souza, has recently published in ACS Omega (2025) that YcgR is a chimera containing a PilZN domain and a PilZ domain, with a GAF domain inserted after the first ¿-strand of the PilZ domain. This insertion does not impair LIC_11920's ability to bind c-di-GMP. Our current goal is to characterize the functional role of LIC_11920, one of six YcgR paralogs found in the L. interrogans genome. Initially, we will employ bacterial two-hybrid assays to map its interactions with other cellular proteins. We hypothesize that at least one of these paralogs interacts with components of the flagellar stator, such as MotA and FliG, thereby modulating the torque of the flagellar motor and, consequently, bacterial motility - a mechanism already described in other bacteria.To investigate this mechanism at the structural level, we are building a bacterial two-hybrid library and plan to determine the structure of the MotA¿MotB¿FliL complex using cryo-EM. The heterologous expression and purification of this transmembrane complex represent one of the main technical challenges of the project. In addition, the functional characterization of LIC_11920's interaction partners is complicated by system redundancy: unlike many bacteria that possess a single copy of YcgR and the stator components, L. interrogans encodes at least six YcgR paralogs and two copies of the stator components. This redundancy, combined with the difficulties in genetically manipulating this species, represents an additional barrier that this project seeks to overcome.Another protein of interest in this project is LIC_11128, which contains a sensory PAS domain followed by a GGDEF catalytic domain, characteristic of diguanylate cyclases. The three-dimensional structure of its PAS domain was determined by X-ray crystallography in collaboration with Prof. Armin Wagner's group (UK) and published in 2023. However, its biological function remains unclear, and this will be addressed in the present project.The second research axis focuses on the study of the Type IV pilus (T4P) system of X. citri. In our previous project, we made significant progress in determining the structure of the T4P filament, the PilQ/TsaP secretin complex, and the PilP protein. However, functional data are still lacking to fully understand the roles of these structures in the assembly and function of the secretion system. Therefore, in this project, we aim to investigate the architecture of the PilQ/TsaP/PilP complex in detail, with particular attention to the role of TsaP in T4P biogenesis.In parallel, we characterized a novel D18-symmetry protein structure identified during the purification of bacteriophage ¿Xacm4-11, which infects X. citri via its T4P filament. This shell-like structure is composed of 36 copies of YfdQ. Bioinformatic analyses revealed that the yfdQ gene is frequently located near the yfdP gene in the genome, suggesting a possible functional relationship. Despite these structural advances, the biological role of YfdQ and YfdP in phage physiology remains unknown and is the subject of further investigation in this proposal.We also aim to determine the chemical structure of DSF present in OMVs of Xac. (AU)