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

Abstract

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: PQS; PrrF; PrrH; Pseudomonas aeruginosa; iron regulation; sRNA; small RNA.

FIG 1

PrrF expression is maintained in the absence of the PrrH_IG sequence. (A) Diagram of the prrF locus indicating the gene sequence transcribed into the PrrF1 (116 nt), PrrF2 (114 nt), and PrrH (325 nt) sRNAs. The sequence included in each of the prrF complementation constructs is shown below the diagram. Putative and confirmed Rho-independent terminators are indicated by a stem loop. The Fur-regulated promoters of each prrF gene (P_Fur) are indicated by a gray box. The prrF1-prrF2 intergenic (H_IG) region is 92 nt long, the first 40 nt of which were deleted in the prrF(ΔH) complementation construct. The locations of primers and probes used for RT-PCR of the PrrF and PrrH sRNAs throughout this study are indicated above the diagram and described in more detail in the text. (B) Summary of RT-PCR results of the ΔprrF1-prrF2 mutant complemented with the prrF complementation constructs. Detailed RT-PCR data are shown in Fig. S2. (C and D) PAO1 and the ΔprrF1-prrF2 (ΔprrF) strain, transformed with either pUCP18, pUCP-prrF-235 (ΔprrF/WT strain), or pUCP-prrFΔH-235 (ΔprrF/ΔH strain), were grown overnight in DTSB to deplete intracellular iron stores, diluted into M9 medium with or without supplementation of FeCl₃ (100 μM) or heme (5 μM) to an absorbance at 600 nm of 0.08, and then grown for an additional 8 h. RNA was extracted from cultures and analyzed by RT-PCR as described in Materials and Methods. Error bars represent the standard deviations from six independent experiments. The following P values were determined by a two-tailed Student's t test comparing expression levels under low-iron conditions to levels under iron-replete or heme-supplemented conditions: *, P < 0.05; **, P < 0.01; ND, not detected.

FIG 2

PrrF regulates expression of the sodB and antR target mRNAs. PAO1 and the ΔprrF1-prrF2 (ΔprrF) strain, transformed with either pUCP18, pUCP-prrF-235 (ΔprrF/WT strain), or pUCP-prrFΔH-235 (ΔprrF/ΔH strain), were grown overnight in DTSB to deplete intracellular iron stores, subcultured into M9 medium to a final an optical density (600 nm) of 0.08, and then grown for an additional 8 h. RNA was extracted from aerobic and microaerobic cultures and analyzed for expression of sodB (A) and antR (B) expression as described in Materials and Methods. Complementarity that was previously identified (21) between the PrrF sRNAs and the sodB and antR mRNAs is shown above each graph, and the Shine-Dalgarno and/or translational start sites of the mRNAs are underlined. Error bars indicate the standard deviations from six biological replicates. Significance was determined by a two-tailed Student's t test comparing results for each strain to the result with the wild-type vector control (**, P < 0.001).

FIG 3

PrrF mediates iron homeostasis under iron-depleted conditions. (A.) PAO1 and the ΔprrF1-prrF2 (ΔprrF) strain, transformed with either pUCP18, pUCP-prrF-235 (ΔprrF/WT strain), or pUCP-prrFΔH-235 (ΔprrF/ΔH strain), were grown in DTSB overnight and then subcultured into M9 minimal medium to a final optical density at 600 nm (OD₆₀₀) of 0.08 for 24 h in a BioScreen multiwell plate reader. Error bars indicate the standard deviations from three independent experiments. (B to D) Strains were grown in DTSB to deplete intracellular iron stores, diluted to an OD of 0.08 in M9 medium, and then grown for an additional 8 h. Cultures were then analyzed for iron content, pyoverdine production (supernatant fluorescence at 410 nm normalized to culture absorbance at 600 nm), and total siderophore activity (CAS reactivity measured at 410 nm normalized to culture absorbance at 600 nm) as described in Materials and Methods. Error bars indicate the standard deviations of six independent experiments. Significance (*, P < 0.05) was determined by a two-tailed, paired Student's t test comparing results for each strain to those for the wild type.

FIG 4

PrrF promotes the production of numerous AQ metabolites. (A) Model for PrrF regulation of alkylquinolone production. The PrrF sRNAs are predicted to interact with the antR mRNA, which encodes an LysR transcriptional activator of the antABC and catBCA operons. The enzyme complexes encoded by these two operons mediate the degradation of anthranilate for use as a carbon source. Anthranilate can alternatively serve as a biosynthetic precursor to the Pseudomonas quinolone signal (PQS) and other alkylquinolones (AQs). Thus, PrrF regulation of antR provides a molecular switch to promote AQ production under low-iron conditions. (B to H) PAO1 and the ΔprrF1-prrF2 (ΔprrF) strain, transformed with either pUCP18, pUCP-prrF-235 (ΔprrF/WT strain), or pUCP-prrFΔH-235 (ΔprrF/ΔH strain), were grown in DTSB for 16 h, and alkylquinolones were extracted from culture supernatants and analyzed by thin-layer chromatography (TLC) as described in Materials and Methods. (B) A representative image of TLC analysis of PQS and C9-PQS is shown, and densitometry results of each spot normalized to culture density (OD₆₀₀) from three independent experiments are shown below the image (B). Relative intensity of the indicated alkylquinolones extracted from culture supernatants was determined by LC-M/MS as described in Materials and Methods and normalized to culture density (optical density at 600 nm) (C to H). Error bars indicate the standard deviations of three independent experiments. Significance was determined by a two-tailed Student's t test comparing results for the ΔprrF1-prrF2 mutants to those with the wild type: *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

FIG 5

PrrF regulation of phuS is independent of the PrrH_IG. PAO1 and the ΔprrF1-prrF2 (ΔprrF) strain, transformed with either pUCP18, pUCP-prrF-235 (ΔprrF/WT strain), or pUCP-prrFΔH-235 (ΔprrF/ΔH strain), were grown overnight in DTSB to deplete intracellular iron stores, subcultured into M9 medium to a final optical density (600 nm) of 0.08, and then grown for an additional 8 h. RNA was extracted from cultures and analyzed for expression of vreR (A) and phuS (B) as described in Materials and Methods. Complementarity that was previously identified (22, 35) between the PrrH_IG region and the phuS and vreR mRNAs is shown above each graph, and the Shine-Dalgarno and/or translational start sites of the mRNAs are underlined. Error bars indicate the standard deviations from six biological replicates. Significance was determined by a two-tailed Student's t test comparing results for each strain to those with the wild-type vector control. *, P < 0.05.

FIG 6

PrrF is required for growth during acute murine lung infection. Six CD-1 mice were inoculated intranasally with 10⁸ CFU of PAO1 or the ΔprrF1-prrF2 (ΔprrF) strain, transformed with either pUCP18, pUCP-prrF-235 (ΔprrF/WT strain), or pUCP-prrFΔH-235 (ΔprrF/ΔH strain), as described in Materials and Methods. At 16 h postinfection, mice were euthanized, and serial dilutions of nasal washes (A) and lung homogenates (B) were plated on PIA and incubated for 24 h. Significance was determined by a two-tailed Student's t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.