Mechanisms of PrrF-Mediated Iron regulation in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a gram-negative bacterium and opportunistic pathogen that infects people with compromised immune systems. P. aeruginosa is highly resistant to multiple antibiotics, in part due to its ability to organize into robust biofilm communities. Iron is required for P. aeruginosa virulence and biofilm formation, but iron can also be toxic to the bacteria. Therefore, iron acquisition and utilization are tightly regulated in response to iron availability. Iron homeostasis is maintained in part by the production of two small RNAs (sRNAs), PrrF1 and PrrF2, which repress the expression of iron containing proteins in iron-depleted conditions. PrrF1 and PrrF2 are encoded in tandem on the P. aeruginosa genome, allowing the expression of a longer heme-regulated sRNA named PrrH. Due to the unique sequence of this sRNA, PrrH is hypothesized to regulate distinct mRNAs involved in heme metabolism. Our lab previously showed that the locus for the PrrF and PrrH sRNAs is required for virulence in an acute murine lung infection model, yet the mechanisms guiding PrrF and PrrH regulation of virulence have not yet been determined. In this work, we identified Hfq, an RNA-binding protein that stabilizes many bacterial sRNAs, as a potential PrrF-and PrrH-binding protein. Using gel-shift assays, I showed that Hfq has a strong binding affinity for PrrF1 and PrrF2. Moreover, I showed that Hfq increases the annealing rate of PrrF with one of its mRNA targets, antR. I identified nucleotides required for PrrF interaction with antR in vitro and in vivo, and I showed that PrrF1 and PrrF2 regulation of antR is redundant. PrrF regulation of antR was previously shown to promote the production of key virulence-related metabolites, including the Pseudomonas quinolone signal (PQS). Accordingly, we showed that the PrrF1 and PrrF2 sRNAs have redundant function in the production of PQS and structurally related metabolites. I further showed that Hfq binds to the PrrH sRNA in vitro, and I performed preliminary in vitro analysis of the PrrF sRNAs with additional mRNA targets involved in iron metabolism and biofilm formation. Lastly, I demonstrated that iron and PrrF allow for increased P. aeruginosa biofilm formation in the presence of certain antibiotics. Altogether, these studies established the mechanisms of PrrF regulation of a key virulence trait, and they provided the basis for future work into the biochemical and genetic basis of PrrF-mediated virulence in P. aeruginosa.