NirA is an alternative nitrite reductase from Pseudomonas aeruginosa with potential as an anti-virulence target

Abstract

The opportunistic pathogen Pseudomonas aeruginosa 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, anti-virulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we have identified a Tn5 mutant with an insertion in PA4130 showing reduced levels in a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented similar results as those from the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster, Caenorhabditis elegans as well as decreased toxicity in human cell lines. This mutant also presented an 80% increased survival in murine acute and agar-bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulphite 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 protein is a nitrite reductase operating in a siroheme and 4Fe-4S dependant manner. The preference for nitrite and the production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named as NirA.