Pseudomonas aeruginosa AlgR Phosphorylation Status Differentially Regulates Pyocyanin and Pyoverdine Production

Field: Immunology and Microbiology (Life Sciences)

Pseudomonas aeruginosa employs numerous, complex regulatory ele- ments to control expression of its many virulence systems. The P. aeruginosa AlgZR two-component regulatory system controls the expression of several crucial viru- lence phenotypes. We recently determined, through transcriptomic profiling of a PAO1 ΔalgR mutant strain compared to wild-type PAO1, that algZR and hemCD are cotranscribed and show differential iron-dependent gene expression. Previous ex- pression profiling was performed in strains without algR and revealed that AlgR acts as either an activator or repressor, depending on the gene. Thus, examination of P. aeruginosa gene expression from cells locked into different AlgR phosphorylation states reveals greater physiological relevance. Therefore, gene expression from strains carrying algR alleles encoding a phosphomimetic (AlgR D54E) or a phosphoablative (AlgR D54N) form were compared by microarray to PAO1. Transcriptome analyses of these strains revealed 25 differentially expressed genes associated with iron siderophore biosynthesis or heme acquisition or production. The PAO1 algR D54N mutant produced lower levels of pyoverdine but increased expression of the small RNAs prrf1 and prrf2compared to PAO1. In contrast, the algR D54N mutant produced more pyocyanin than wild-type PAO1. On the other hand, the PAO1 algR D54E mutant produced higher levels of pyoverdine, likely due to increased expression of an iron-regulated gene encoding the sigma factor pvdS, but it had decreased pyocyanin production. AlgR specifically bound to the prrf2 and pvdS promoters in vitro. AlgR-dependent pyoverdine production was additionally influenced by carbon source rather than the extracellular iron concen- tration per se. AlgR phosphorylation effects were also examined in a Drosophila melano- gaster feeding, murine acute pneumonia, and punch wound infection models. Abroga- tion of AlgR phosphorylation attenuated P. aeruginosa virulence in these infection models. These results show that the AlgR phosphorylation state can directly, as well as indirectly, modulate the expression of iron acquisition genes that may ultimately impact the ability of P. aeruginosa to establish and maintain an infection.\nIMPORTANCE Pyoverdine and pyocyanin production are well-known P. aeruginosa virulence factors that obtain extracellular iron from the environment and from host proteins in different manners. Here, we show that the AlgR phosphorylation state in- versely controls pyoverdine and pyocyanin production and that this control is car- bon source dependent. P. aeruginosa expressing AlgR D54N, mimicking the constitu- tively unphosphorylated state, produced more pyocyanin than cells expressing wild- type AlgR. In contrast, a strain expressing an AlgR phosphomimetic (AlgR D54E) produced higher levels of pyoverdine. Pyoverdine production was directly controlled through the prrf2 small regulatory RNA and the pyoverdine sigma factor, PvdS. Ab- rogating pyoverdine or pyocyanin gene expression has been shown to attenuate vir- ulence in a variety of models. Moreover, the inability to phosphorylate AlgR attenu- ates virulence in three different models, a Drosophila melanogaster feeding model, a murine acute pneumonia model, and a wound infection model. Interestingly, AlgR- dependent pyoverdine production was responsive to carbon source, indicating that this regulation has additional complexities that merit further study.