Evolutionary analysis and structure modelling of the Rcs-repressor IgaA unveil a functional role of two cytoplasmic small β-barrel (SBB) domains

Leticia Rodríguez; Marcos Peñalver; Patricia Casino; Francisco García-Del Portillo

doi:10.1016/j.heliyon.2023.e16661

Evolutionary analysis and structure modelling of the Rcs-repressor IgaA unveil a functional role of two cytoplasmic small β-barrel (SBB) domains

Heliyon. 2023 May 26;9(6):e16661. doi: 10.1016/j.heliyon.2023.e16661. eCollection 2023 Jun.

Authors

Leticia Rodríguez¹, Marcos Peñalver^{1

2}, Patricia Casino^{3

4

5}, Francisco García-Del Portillo¹

Affiliations

¹ Laboratory of Intracellular Bacterial Pathogens, National Center for Biotechnology-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.
² Departamento de Biología Molecular, Universidad Autónoma de Madrid (UAM), Madrid, Spain.
³ Departamento de Bioquímica y Biología Molecular, Universitat de València, Burjassot, Spain.
⁴ Instituto Universitario de Biotecnología y Biomedicina BIOTECMED, Universitat de València, Burjassot, Spain.
⁵ CIBER de Enfermedades Raras (CIBERER-ISCIII), Madrid, Spain.

Abstract

The Rcs sensor system, comprising the RcsB/RcsC/RcsD and RcsF proteins, is used by bacteria of the order Enterobacterales to withstand envelope damage. In non-stress conditions, Rcs is repressed by IgaA, a membrane protein with three cytoplasmic regions (cyt-1, cyt-2 and cyt-3). How the Rcs-IgaA axis evolved within Enterobacterales has not been yet explored. Here, we report phylogenetic data supporting co-evolution of IgaA with RcsC/RcsD. Functional exchange assays showed that IgaA from Shigella and Dickeya, but not from Yersinia or the endosymbionts Photorhabdus and Sodalis, repress the Rcs system of Salmonella. IgaA from Dickeya, however, repress only partially the Rcs system despite being produced at high levels in the complementation assay. The modelled structures of these IgaA variants uncovered one periplasmic and two cytoplasmic conserved β-rich architectures forming partially closed small β-barrel (SBB) domains. Conserved residues map in a connector linking cytoplasmic SSB-1 and SBB-2 domains (E180-R265); a region of cyt-1 facing cyt-2 (R188-E194-D309 and T191-H326); and between cyt-2 and cyt-3 (H293-E328-R686). These structures validated early in vivo studies in Salmonella that assigned a role in function to R188, T191 and G262, and in addition revealed a previously unnoticed "hybrid" SBB-2 domain to which cyt-1 and cyt-2 contribute. IgaA variants not functional or partially functional in Salmonella lack H192-P249 and R255-D313 interactions. Among these variants, only IgaA from Dickeya conserves the helix α6 in SSB-1 that is present in IgaA from Salmonella and Shigella. RcsF and RcsD, which interact directly with IgaA, failed to show structural features linked to specific IgaA variants. Altogether, our data provide new insights into IgaA by mapping residues selected differently during evolution and involved in function. Our data also infer contrasting lifestyles of Enterobacterales bacteria as source of variability in the IgaA-RcsD/IgaA-RcsF interactions.

Keywords: Enterobacterales; Evolution; IgaA; Rcs system; SBB domain; Salmonella.