Production and Uptake of Distinct Endogenous Catecholate-Type Siderophores Are Required for Iron Acquisition and Virulence in Chromobacterium violaceum

Abstract

Bacteria use siderophores to scavenge iron from environmental or host sources. The iron acquisition systems of <i>Chromobacterium violaceum</i>, a ubiquitous environmental bacterium that can cause infections in humans, are still unknown. In this work, we demonstrated that <i>C. violaceum</i> produces putative distinct endogenous siderophores, here named chromobactin and viobactin, and showed that they are each required for iron uptake and virulence. An <i>in silico</i> analysis in the genome of <i>C. violaceum</i> revealed that genes related to synthesis and uptake of chromobactin (<i>cba</i>) and viobactin (<i>vba</i>) are located within two secondary-metabolite biosynthetic gene clusters. Using a combination of gene deletions and siderophore detection assays, we revealed that chromobactin and viobactin are catecholate siderophores synthesized from the common precursor 2,3-dihydroxybenzoate (2,3-DHB) on two nonribosomal peptide synthetase (NRPS) enzymes (CbaF and VbaF) and taken up by two TonB-dependent receptors (CbuA and VbuA). Infection assays in mice revealed that both the synthesis and the uptake of chromobactin or viobactin are required for the virulence of <i>C. violaceum</i>, since only the mutant strains that do not produce any siderophores or are unable to take up both of them were attenuated for virulence. In addition, the mutant strain unable to take up both siderophores showed a pronounced attenuation of virulence <i>in vivo</i> and reduced neutrophil extracellular trap (NET) formation in <i>in vitro</i> assays, suggesting that extracellularly accumulated siderophores modulate the host immune response. Overall, our results revealed that <i>C. violaceum</i> uses distinct endogenous siderophores for iron uptake and its establishment in the host.