NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target

Abstract

The opportunistic pathogen <i>Pseudomonas aeruginosa</i> produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat <i>P. aeruginosa</i> infections. To identify <i>P. aeruginosa</i> genes required for virulence in multiple hosts, a random library of Tn<i>5</i> mutants in strain PAO1-L was previously screened <i>in vitro</i> for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn<i>5</i> mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits <i>in vitro</i> Construction of an isogenic mutant in this gene presented results similar to those for the Tn<i>5</i> mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of <i>Drosophila melanogaster</i> and <i>Caenorhabditis elegans</i> as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA.<b>IMPORTANCE</b> The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of <i>P. aeruginosa</i> virulence adds complexity to the discovery of these types of targets. Using a sequence of <i>in vitro</i> assays and phylogenetically diverse <i>in vivo</i> disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of <i>nirA</i> mutants in different clinical isolates, high level conservation of its gene product in <i>P. aeruginosa</i> genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.