The Fur family of transcription factors and the response to iron and zinc in Chromobacterium violaceum

Author(s)

Renato Elias Rodrigues de Souza Santos

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2021-10-27

Examining board members

José Freire da Silva Neto; Tie Koide; Nadia Monesi; Aline Maria da Silva

• Advisor: José Freire da Silva Neto

Abstract

The transition metals iron and zinc are micronutrients essential to all forms of life. These metals perform catalytic, structural, and regulatory functions, acting as a cofactor of numerous proteins in bacteria and eukaryotes. In the context of infection, both the vertebrate host and the pathogens compete for these metals. Bacteria use numerous strategies to overcome the mechanisms of host nutritional immunity and to gain access to iron and zinc. In this work, we characterized the transcription factors Fur and Zur regarding their roles in maintaining iron and zinc homeostasis and in the virulence of the environmental bacterium Chromobacterium violaceum, an opportunistic human pathogen. Obtaining null mutant Δfur was only possible under iron limitation, indicating that Fur is conditionally essential in C. violaceum. The spontaneous mutant FurR40S, which has Fur partially functional, showed increased production of siderophores and susceptibility to oxidative stress. The Δfur mutant had pleiotropic phenotypes, such as increased siderophore production, reduced growth and survival in the presence of iron and hydrogen peroxide, and decreased biofilm production and swimming motility. Many of the general fitness problems of the Δfur mutant were reversed in the Δfur T8 strain that has an insertion of the T8 transposon at a CRISPR/Cas locus. Both the Δfur and Δfur T8 strains showed strong attenuation of virulence in mice, indicating the importance of Fur in the pathogenesis of C. violaceum. Finally, we demonstrate that in iron sufficiency Fur acts as a repressor of cbaF and cbuA genes related to iron use via siderophores. Regarding the Zur transcription factor, β-galactosidase assays revealed that this regulator acts as a repressor of an operon that encodes Zur, two hypothetical proteins, and the highaffinity transporter ZnuABC. The promoter of this operon has a Zur-box and was derepressed in zinc limitation. The only phenotype found for the Δzur mutant was an increase in quorum sensing molecules in its culture supernatants. However, in the absence of the ZnuABC transporter, C. violaceum was more susceptible to zinc deficiency induced by treatment with EDTA or the metal-binding protein calprotectin. We demonstrate the importance of zinc and the ZnuABC transporter in violacein production, biofilm formation, bacterial competition, and antibiotic susceptibility. In vitro infection assays revealed that the ΔznuCBA mutant was more susceptible to killing by neutrophils, corroborating collaborative data that this mutant is attenuated for virulence in mice. Together, these characterizations demonstrate the relevance of the Fur and Zur regulators and the ZnuABC transporter in maintaining iron and zinc homeostasis and their importance in the virulence of C. violaceum. (AU)

• FAPESP's process: 17/03342-0 - The Fur family of transcription factors and the response to iron and zinc in Chromobacterium violaceum
• Grantee: Renato Elias Rodrigues de Souza Santos
• Support Opportunities: Scholarships in Brazil - Doctorate