A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance

Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for flavodoxin from P . aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H2O2 toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H2O2-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments.
Author Summary Coping with toxic reactive oxygen species (ROS) generated as by-products of aerobic metabolism is a major challenge for O2-thriving organisms, which deploy multilevel responses to prevent ROS-triggered damage, including membrane modifications, induction of antioxidant and repair systems and/or replacement of ROS-sensitive targets by resistant isofunctional versions, among others. The opportunistic pathogen Pseudomonas aeruginosa is frequently exposed to ROS in the environment as well as within the host, and we describe herein a new response by which this microorganism can deal with oxidative stress. This pathway depends on a previously uncharacterized gene that we named fldP (for flavodoxin from P . aeruginosa), which encodes a flavoprotein that belongs to the family of long-chain flavodoxins. FldP exhibited a protective role against ROS-dependent physiological and mutational damage, and contributed to the survival of P. aeruginosa during in vivo infection of flies as well as within mammalian macrophagic cells. Thus, fldP increases the adaptive repertoire of P. aeruginosa to face oxidative stress.