The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the antR mRNA.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions, P. aeruginosa transcribes two highly identical (95%) small regulatory RNAs (sRNAs), PrrF1 and PrrF2, which are required for virulence in acute murine lung infection models. The PrrF sRNAs promote the production of 2-akyl-4(1H)-quinolone metabolites (AQs) that mediate a range of biological activities, including quorum sensing and polymicrobial interactions. Here, we show that the PrrF1 and PrrF2 sRNAs promote AQ production by redundantly inhibiting translation of antR, which encodes a transcriptional activator of the anthranilate degradation genes. A combination of genetic and biophysical analyses was used to define the sequence requirements for PrrF regulation of antR, demonstrating that the PrrF sRNAs interact with the antR 5' untranslated region (UTR) at sequences overlapping the translational start site of this mRNA. The P. aeruginosa Hfq protein interacted with UA-rich sequences in both PrrF sRNAs (Kd [dissociation constant] = 50 nM and 70 nM). Hfq bound with lower affinity to the antR mRNA (0.3 µM), and PrrF was able to bind to antR mRNA in the absence of Hfq. Nevertheless, Hfq increased the rate of PrrF annealing to the antR UTR by 10-fold. These studies provide a mechanistic description of how the PrrF1 and PrrF2 sRNAs mediate virulence traits, such as AQ production, in P. aeruginosaIMPORTANCE The iron-responsive PrrF sRNAs play a central role in regulating P. aeruginosa iron homeostasis and pathogenesis, yet the molecular mechanisms by which PrrF regulates gene expression are largely unknown. In this study, we used genetic and biophysical analyses to define the interactions of the PrrF sRNAs with Hfq, an RNA annealer, and the antR mRNA, which has downstream effects on quorum sensing and virulence factor production. These studies provide a comprehensive mechanistic analysis of how the PrrF sRNAs regulate virulence trait production through a key mRNA target in P. aeruginosa.

Low-Dose Oxygen Enhances Macrophage-Derived Bacterial Clearance following Cigarette Smoke Exposure.

Background. Chronic obstructive pulmonary disease (COPD) is a common, smoking-related lung disease. Patients with COPD frequently suffer disease exacerbations induced by bacterial respiratory infections, suggestive of impaired innate immunity. Low-dose oxygen is a mainstay of therapy during COPD exacerbations; yet we understand little about whether oxygen can modulate the effects of cigarette smoke on lung immunity. Methods. Wild-type mice were exposed to cigarette smoke for 5 weeks, followed by intratracheal instillation of Pseudomonas aeruginosa (PAO1) and 21% or 35-40% oxygen. After two days, lungs were harvested for PAO1 CFUs, and bronchoalveolar fluid was sampled for inflammatory markers. In culture, macrophages were exposed to cigarette smoke and oxygen (40%) for 24 hours and then incubated with PAO1, followed by quantification of bacterial phagocytosis and inflammatory markers. Results. Mice exposed to 35-40% oxygen after cigarette smoke and PAO1 had improved survival and reduced lung CFUs and inflammation. Macrophages from these mice expressed less TNF-α and more scavenger receptors. In culture, macrophages exposed to cigarette smoke and oxygen also demonstrated decreased TNF-α secretion and enhanced phagocytosis of PAO1 bacteria. Conclusions. Our findings demonstrate a novel, protective role for low-dose oxygen following cigarette smoke and bacteria exposure that may be mediated by enhanced macrophage phagocytosis.