Characterization of a Heme-Regulated Non-Coding RNA Encoded by the prrF Locus of Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen, requires iron for virulence and can obtain this nutrient via the acquisition of heme, an abundant source of iron in the human body. A surplus of either iron or heme can lead to oxidative stress; thus, the Fur (ferric uptake regulator) protein blocks expression of genes required for iron and heme uptake in iron-replete environments. Fur also represses expression of two nearly identical genes encoding the 116- and 114-nucleotide (nt) long PrrF1 and PrrF2 RNAs, respectively. While other Pseudomonads encode for the two PrrF RNAs at separate genomic loci, PrrF1 and PrrF2 are encoded in tandem in all sequenced strains of P. aeruginosa. In this report we characterize a third longer transcript encoded by the prrF locus, PrrH, which is repressed by heme as well as iron. We mapped the PrrH RNA in PA01 using 5′ rapid amplification of cDNA ends (RACE) and northern analysis, demonstrating the PrrH RNA is 325 nt in length. Accordingly, transcription of PrrH initiates at the 5′ end of prrF1, proceeds through the prrF1 terminator and prrF1-prrF2 intergenic sequence (95 nt), and terminates at the 3′ end of the prrF2 gene. We also present evidence that repression of PrrH by heme causes increased expression of previously identified PrrF-regulated genes, as well as newly identified iron- and heme-activated genes. Thus, the PrrH RNA appears to impart a novel heme regulatory mechanism to P. aeruginosa

Dual-Seq Transcriptomics Reveals The Battle For Iron During Pseudomonas Aeruginosa Acute Murine Pneumonia

Determining bacterial gene expression during infection is fundamental to understand pathogenesis. In this study, we used dual RNA-seq to simultaneously measure P. aeruginosa and the murine host’s gene expression and response to respiratory infection. Bacterial genes encoding products involved in metabolism and virulence were differentially expressed during infection and the type III and VI secretion systems were highly expressed in vivo. Strikingly, heme acquisition, ferric-enterobactin transport, and pyoverdine biosynthesis genes were found to be significantly up-regulated during infection. In the mouse, we profiled the acute immune response to P. aeruginosa and identified the pro-inflammatory cytokines involved in acute response to the bacterium in the lung. Additionally, we also identified numerous host iron sequestration systems upregulated during infection. Overall, this work sheds light on how P. aeruginosa triggers a pro-inflammatory response and competes for iron with the host during infection, as iron is one of the central elements for which both pathogen and host fight during acute pneumonia

Regulation of Pseudomonas aeruginosa Virulence by Distinct Iron Sources

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and versatile opportunistic pathogen. Like most other organisms, P. aeruginosa requires iron for survival, yet iron rapidly reacts with oxygen and water to form stable ferric (FeIII) oxides and hydroxides, limiting its availability to living organisms. During infection, iron is also sequestered by the host innate immune system, further limiting its availability. P. aeruginosa’s capacity to cause disease in diverse host environments is due to its ability to scavenge iron from a variety of host iron sources. Work over the past two decades has further shown that different iron sources can affect the expression of distinct virulence traits. This review discusses how the individual components of P. aeruginosa’s iron regulatory network allow this opportunist to adapt to a multitude of host environments during infection

PhuR and HasR are important for heme regulation of PrrF target mRNAs.

<p>Heme utilization mutants lacking one or both outer membrane heme receptors or the HemO heme oxygenase were grown for 18 hours in CM9 +1% glycerol with no added iron (white bars) or 40 µM hemin (hatched bars). RNA was then isolated and analyzed by qRT-PCR for expression of (A) <i>acnB</i>-PA1787, (B) m-<i>acnA</i>-PA0794, and (C) <i>sdhD</i>-PA1582, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009930#s4" target="_blank">Materials and Methods</a>. Error bars show the standard deviation of at least three independent experiments.</p

Effect of heme on the expression of PrrF-regulated genes.

<p>RNA isolated from the indicated strains, grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 µM hemin (hatched bars), or 100 µg/ml FeCl₃ (black bars) was used for qRT-PCR as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009930#s4" target="_blank">Materials and Methods</a>. Error bars represent the standard deviation of expression of (A) <i>acnB</i>-PA1787, (B) m-<i>acnA</i>-PA0794, and (C) <i>sdhD</i>-PA1582 from at least three independent experiments.</p

Quantification of PrrF and PrrH expression.

<p>A. Map of the PrrF-PrrH coding region showing the location of the primers and probes used for qRT-PCR. B-E. RNA was isolated from the indicated strains grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 µM hemin (hatched bars), or 100 µg/ml FeCl₃ (black bars) and used for qRT-PCR analysis of the PrrH (B, D) and PrrF (C, E) RNAs as described in the Materials and Method. Error bars represent the standard deviation of three independent experiments. Asterisks (*) indicate expression was below detection levels.</p

PhuR and HasR are important for heme regulation of PrrH.

<p>Heme utilization mutants lacking one or both outer membrane heme receptors were grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 µM hemin (hatched bars), or 100 µg/ml FeCl₃ (black bars). RNA was then isolated and analyzed by qRT-PCR, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009930#s4" target="_blank">Materials and Methods</a>, for the mutants' ability to mediate heme and iron regulation of the PrrH (A) and PrrF (B) RNAs. Error bars show the standard deviation of at least three independent experiments.</p

Genetic organization of <i>prrF</i> regions from different Pseudomonads.

<p>The tandem gene organization of <i>prrF1</i> and <i>prrF2</i> is restricted to <i>P. aeruginosa</i> strains. Block arrows indicate directionality of the open reading frame, and orthologous genes are represented by the color and pattern of the arrow. Map not drawn to scale.</p

Expression of <i>nirL</i> is activated by heme in a PrrH-dependent manner.

<p>A. Complementarity between the PrrH unique sequence, derived from the <i>prrF1-prrF2</i> intergenic region, and the <i>nirL</i> mRNA. The underlined sequence indicates the start codon for <i>nirL</i> translation. B-C. RNA isolated from wild type PA01 and the (B) <i>prrF</i>, (C) heme receptor, and (D) heme oxygenase mutants, grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 µM hemin (hatched bars), or 100 µg/ml FeCl₃ (black bars), was used for qRT-PCR as described in the Materials and Method. Error bars represent the standard deviation of expression from three independent experiments.</p

The human innate immune protein calprotectin induces iron starvation responses in Pseudomonas aeruginosa

© 2019 Zygiel et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Most microbial pathogens have a metabolic iron requirement, necessitating the acquisition of this nutrient in the host. In response to pathogen invasion, the human host limits iron availability. Although canonical examples of nutritional immunity are host strategies that limit pathogen access to Fe(III), little is known about how the host restricts access to another biologically relevant oxidation state of this metal, Fe(II). This redox species is prevalent at certain infection sites and is utilized by bacteria during chronic infection, suggesting that Fe(II) withholding by the host may be an effective but unrecognized form of nutritional immunity. Here, we report that human calprotectin (CP; S100A8/S100A9 or MRP8/MRP14 heterooligomer) inhibits iron uptake and induces an iron starvation response in Pseudomonas aeruginosa cells by sequestering Fe(II) at its unusual His6 site. Moreover, under aerobic conditions in which the Fe(III) oxidation state is favored, Fe(II) withholding by CP was enabled by (i) its ability to stabilize this redox state in solution and (ii) the production and secretion of redox-active, P. aeruginosa–produced phenazines, which reduce Fe(III) to Fe(II). Analyses of the interplay between P. aeruginosa secondary metabolites and CP indicated that Fe(II) withholding alters P. aeruginosa physiology and expression of virulence traits. Lastly, examination of the effect of CP on cell-associated metal levels in diverse human pathogens revealed that CP inhibits iron uptake by several bacterial species under aerobic conditions. This work implicates CP-mediated Fe(II) sequestration as a component of nutritional immunity in both aerobic and anaerobic milieus during P. aeruginosa infection