The Pseudomonas aeruginosa PrrF Small RNAs Regulate Iron Homeostasis during Acute Murine Lung Infection

Abstract

<i>Pseudomonas aeruginosa</i> is a Gram-negative opportunistic pathogen that requires iron for virulence. Iron homeostasis is maintained in part by the PrrF1 and PrrF2 small RNAs (sRNAs), which block the expression of iron-containing proteins under iron-depleted conditions. The PrrF sRNAs also promote the production of the <i>Pseudomonas</i> quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the <i>prrF</i> genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the <i>prrF1-prrF2</i> intergenic (IG) sequence (the PrrH_IG sequence). Previous studies showed that the <i>prrF</i> locus is required for acute lung infection. However, the individual functions of the PrrF and PrrH sRNAs were not determined. Here, we describe a system for differentiating PrrF and PrrH functions by deleting the PrrH_IG sequence [<i>prrF</i>(ΔH_IG)]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by <i>P. aeruginosa</i> Moreover, we show that the virulence defect of the Δ<i>prrF1-prrF2</i> mutant is due to decreased bacterial burden during acute lung infection. <i>In vivo</i> analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the Δ<i>prrF1-prrF2</i> mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by <i>prrF</i> deletion <i>in vivo</i> Combined, these studies demonstrate that regulation of iron utilization plays a critical role in <i>P. aeruginosa</i>'s ability to survive during infection.