Nudix-type RNA pyrophosphohydrolase regulates pathogenesis-associated factors in Pseudomonas aeruginosa

The PA0336 protein from Pseudomonas aeruginosa belongs to the family of widely distributed Nudix pyrophosphohydrolases which catalyze the hydrolysis of pyrophosphate bonds in a variety of nucleoside diphosphate derivatives. The amino acid sequence of the PA0336 protein is highly similar to that of the RppH Nudix RNA pyrophosphohydrolase from E. coli which removes pyrophosphate from 5'-end of triphosphorylated RNA transcripts. Trans-complementation experiments showed that the P. aeruginosa enzyme can functionally substitute for RppH in E. coli cells indicating that, similarly to RppH, the Pseudomonas hydrolase mediates RNA turnover in vivo. In order to elucidate the biological significance of the PA0336 protein in Pseudomonas cells, a PA0336 mutant strain was constructed. The mutated strain considerably increased level of the virulence factor pyocyanin compared to wild type, suggesting that PA0336 could be involved in down-regulation of P. aeruginosa pathogenicity. This phenotype was reversed by complementation with the wild type, but not catalytically inactive PA0336, indicating that the catalytic activity was indispensable for its biological function. To study the role of PA0336 further, transcriptomes of the PA0336 mutant and the wild type strain were compared using RNA sequencing. The cellular level of a number of transcripts was affected by the lack of PA0336. We focused our attention on pathogenesis-related genes. Up-regulated in the PA0336 mutant were transcripts coding for, i. a., proteins involved in the regulation and/or production of pyocyanin, biofim-associated alginates and exotoxins. The results from the global analysis were verified by determining the cellular level of chosen transcripts by quantitative RT-PCR method. Pathogenesis tests in Caenorhabditis elegans showed that the PA0336 mutant of P. aeruginosa was significantly more virulent than the parental strain, confirming further that the P. aeruginosa RNA pyrophosphohydrolase PA0336 modulates bacterial pathogenesis by down-regulating production of virulence factors. Overall design: Study comparing RNA expression of P. aeruginosa PA0336 mutant strain with wild type reference, both in biological triplicates, by RNA-seq performed on Ion Torrent Proton platform

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Kujawa M, Lirski M, Ziecina M, Drabinska J, Modzelan M, Kraszewska E