Pseudomonas aeruginosa
AlgR Phosphorylation Status Differentially Regulates Pyocyanin and Pyoverdine Production
Authors: Little Alexander S. , Okkotsu Yuta , Reinhart Alexandria A. , Damron F. Heath , Barbier Mariette , Barrett Brandon , Oglesby-Sherrouse Amanda G. , Goldberg Joanna B. , Cody William L. , Schurr Michael J. , Vasil Michael L. , Schurr Michael J. 
Published: March 2018

ABSTRACT

Pseudomonas aeruginosa
employs numerous, complex regulatory elements to control expression of its many virulence systems. The
P. aeruginosa
AlgZR two-component regulatory system controls the expression of several crucial virulence phenotypes. We recently determined, through transcriptomic profiling of a PAO1 Δ
algR
mutant strain compared to wild-type PAO1, that
algZR
and
hemCD
are cotranscribed and show differential iron-dependent gene expression. Previous expression 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
prrf2
compared 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 concentration
per se
. AlgR phosphorylation effects were also examined in a
Drosophila melanogaster
feeding, murine acute pneumonia, and punch wound infection models. Abrogation 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.

IMPORTANCE
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 inversely controls pyoverdine and pyocyanin production and that this control is carbon source dependent.
P. aeruginosa
expressing AlgR D54N, mimicking the constitutively 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. Abrogating pyoverdine or pyocyanin gene expression has been shown to attenuate virulence in a variety of models. Moreover, the inability to phosphorylate AlgR attenuates 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.