Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part of P. aeruginosa's iron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ⌬prrF1,2 mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identified phuS, encoding a heme binding protein involved in heme acquisition, and vreR, encoding a previously identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-mediated heme regulation. Finally, we showed that the prrF locus encoding the PrrF and PrrH sRNAs is required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ⌬prrF1,2 deletion mutant protects against future challenge with wild-type P. aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P. aeruginosa virulence. Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium and versatile opportunistic pathogen. Iron is required for P. aeruginosa virulence (1-6) and is obtained through several mechanisms. In anaerobic environments, iron in its ferrous form is freely diffusible through the outer membrane (OM) and transported into the cytoplasm by the Feo inner membrane transport system (7,8). However, the insolubility of ferric iron in aerobic environments limits accessibility to this nutrient. Moreover, the sequestration of iron by host proteins creates a substantial barrier to infection (9, 10). To overcome this barrier, P. aeruginosa synthesizes and secretes two siderophores, pyoverdine and pyochelin, which scavenge ferric iron (1-4). P. aeruginosa can also acquire heme, an abundant source of iron in the human host (11). Once internalized, heme is sequestered by the cytosolic PhuS heme chaperone (12). PhuS transfers heme to the iron-regulated HemO heme oxygenase, which degrades heme to biliverdin, releasing carbon monoxide and iron (13,14). Several studies have shown that iron acquisition is essential for P. aeruginosa virulence (1-5) and biofilm formation (15-17), demonstrating the central role of this element in P. aeruginosa pathogenesis.Despite its essentiality, iron and heme can be toxic due to their ability to catalyze the formation of reactive oxygen species. Thus, to maintain iron homeostasis, P. aeruginosa must be able to not only acquire iron but also regulate uptake of iron and heme from the environment, as well as iron use and storage. Expression of genes encoding iron and heme uptake systems in P. aeruginosa is ...

Acinetobacter baumannii is responsible for 10% of all nosocomial infections and has >50% mortality rates when causing ventilator-associated pneumonia. In this proof-of-concept study, we evaluated SPR741, an antibiotic adjuvant that permeabilizes the Gram-negative membrane, in combination with rifampin against AB5075, an extensively drug-resistant (XDR) A. baumannii strain. In standard in vitro assays and in a murine pulmonary model, we found that this drug combination can significantly reduce bacterial burden and promote animal survival despite an aggressive infection.

Pseudomonas aeruginosa 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 Pseudomonas quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the prrF genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the prrF1-prrF2 intergenic (IG) sequence (the PrrH IG sequence). Previous studies showed that the prrF 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 [prrF(ΔH IG )]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by P. aeruginosa. Moreover, we show that the virulence defect of the ΔprrF1-prrF2 mutant is due to decreased bacterial burden during acute lung infection. In vivo analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the ΔprrF1-prrF2 mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by prrF deletion in vivo. Combined, these studies demonstrate that regulation of iron utilization plays a critical role in P. aeruginosa's ability to survive during infection.KEYWORDS Pseudomonas aeruginosa, sRNA, PrrF, PrrH, iron regulation, PQS, small RNA P seudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in a variety of patient populations, including acute blood and lung infections in hospitalized patients and chronic lung infections in individuals with cystic fibrosis (CF) (1-4). Iron is an essential nutrient for virulence in P. aeruginosa (5-9), but it is sequestered by mammalian host proteins such as lactoferrin and transferrin (10). To overcome this barrier to infection, P. aeruginosa secretes two siderophores, pyoverdine and pyochelin, which scavenge ferric iron (Fe 3ϩ ) from host proteins and are required for acute infections (5,(7)(8)(9). P. aeruginosa also obtains iron from host heme using outer membrane heme transporters and a cytosolic HemO heme oxygenase that degrades heme (11,12). Ferrous iron (Fe 2ϩ ) can also be obtained through the Feo system in microaerobic environments, such as those found within biofilms and the CF lung (13-15).

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.

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 ΔalgR 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.