The heme-responsive PrrH sRNA regulates Pseudomonas aeruginosa pyochelin gene expression

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron for growth and virulence, yet this nutrient is sequestered by the innate immune system during infection. When iron is limiting, P. aeruginosa expresses the PrrF1 and PrrF2 small RNAs (sRNAs), which post-transcriptionally repress expression of nonessential iron-containing proteins, thus sparing this nutrient for more critical processes. The genes for the PrrF1 and PrrF2 sRNAs are arranged in tandem on the chromosome, allowing for the transcription of a longer heme-responsive sRNA, termed PrrH. While the functions of PrrF1 and PrrF2 have been extensively studied, the role of PrrH in P. aeruginosa physiology and virulence is not well understood. In this study, we performed transcriptomic and proteomic studies to identify the PrrH regulon. In shaking cultures, the pyochelin synthesis proteins were increased in two distinct prrH mutants compared to the wild type, while the mRNAs for these proteins were not affected by the prrH mutation. We identified complementarity between the PrrH sRNA and the sequence upstream of the pchE mRNA, suggesting the potential for PrrH to directly regulate the expression of genes for pyochelin synthesis. We further showed that pchE mRNA levels were increased in the prrH mutants when grown in static but not shaking conditions. Moreover, we discovered that controlling for the presence of light was critical for examining the impact of PrrH on pchE expression. As such, our study reports on the first likely target of the PrrH sRNA and highlights key environmental variables that will allow for future characterization of PrrH function. IMPORTANCE In the human host, iron is predominantly in the form of heme, which Pseudomonas aeruginosa can acquire as an iron source during infection. We previously showed that the iron-responsive PrrF small RNAs (sRNAs) are critical for mediating iron homeostasis during P. aeruginosa infection; however, the function of the heme-responsive PrrH sRNA remains unclear. In this study, we identified genes for pyochelin siderophore biosynthesis, which mediates uptake of inorganic iron, as a novel target of PrrH regulation. This study therefore highlights a novel relationship between heme availability and siderophore biosynthesis in P. aeruginosa.

Keywords: PrrF; PrrH; Pseudomonas aeruginosa; heme; iron; pyochelin.

FIG 1

Characterization of transcripts produced by the ∆prrH-IG mutant. Organization of the prrF locus and design of complement plasmids (A). The WT comp includes the entire prrF locus plus 235 bp upstream of the prrF1 promoter to ensure regulation of the locus is uninhibited. The ∆H-IG comp also includes the 235 bp region upstream of the prrF1 promoter but has the first 40 bp of the prrF1-prrF2 intergenic region, starting immediately after the Rho-independent terminator, removed. The short comp contains the entire prrF locus but only includes 184 bp upstream of the prrF1 promoter. The locations of qRT-PCR primers and probes are indicated in black. The PrrF primers and probes do not distinguish between PrrF1 and PrrF2. Northern blot probes are labeled in green. For qRT-PCR (B and C), PrrF and PrrH transcription are shown as an average of three biological replicates, relative to WT PAO1 in low iron. The northern blot (D) is a representative of multiple experiments using radiolabeled DNA probes specific to each transcript.

FIG 2

The short prrF complement has a rearrangement in prrF2. qRT-PCR analyses of PrrF (A) and PrrH (B) transcription are shown as an average of three biological replicates, relative to WT PAO1 in low iron. The northern blot (C) is a representative from multiple experiments using radiolabeled DNA probes specific to each transcript (PrrF1, PrrF2, and PrrH). RNAs were isolated from samples collected after 8 h of aerobic growth in M9 media supplemented with 50 nM FeCl₃ (−Fe, low iron) or 100 µM FeCl₃ (+Fe, high iron) at 37°C. (D) The re-arrangement that occurred in the short comp. The locations of the northern blot probes used in C are shown in blue (PrrH) and orange (PrrF2). The qRT-PCR primers/probe for PrrH are shown in red. Sequencing was performed by Eurofins Genomics. Sequencing results were aligned and analyzed using MacVector software.

FIG 3

Short complement and ∆H-IG complement substantially alter the proteome. Proteomics results when comparing protein samples collected after 8 h of aerobic growth in M9 media supplemented with 50 nM FeCl₃ at 37°C. (A) Heatmap of a select group of iron-, heme-, and PrrF-regulated proteins shown as the log₂ fold change of the abundance ratio between high iron and low iron. Undetected proteins are colored in black (ND). (B–D) Volcano plots comparing the protein abundance of the ∆H-IG comp versus WT comp (B), short comp versus WT comp (C), and ∆H-IG comp versus short comp (D). The log₂ fold change is shown on the x-axes, and the −log of the FDR P-value is on the y-axes. Horizontal dashed lines indicate FDR P = 0.05, and vertical dashed lines indicate LFC = ±1. (E and F) Comparisons of the dysregulated proteins in the ∆H-IG comp from experiments 1 and 2 are shown as Venn diagrams. (E) The overlap of the dysregulated proteins in the ∆H-IG comp and short comp, each compared to the WT comp, from experiment 2. (F) The overlap of the dysregulated proteins in the ∆H-IG comp compared to WT between experiments 1 and 2. To be included in the Venn diagram analysis, changes in protein levels must have demonstrated an FDR P-value <0.05 and −0.5 ≤ LFC ≥ 0.5.

FIG 4

Proteins involved in alkylquinolone biosynthesis and pyochelin biosynthesis are dysregulated in prrH mutants. Expression data from proteomics and RNAseq are presented as heat maps, where upregulated proteins are indicated in red, and those downregulated are in blue. Undetected proteins are colored in black (ND). **, P < 0.005; *, P < 0.05.

FIG 5

Static growth and controlling for light promote consistent PrrH repression of the pchEF mRNA. (A) CopraRNA identified sequences with complementarity to PrrH upstream of pchF and within pchI. (B–E) qRT-PCR analysis of the PrrH sRNA (B and D) and pchE mRNA (C and E) levels relative to WT-comp is shown as an average of three or four biological replicates. RNA was isolated from cultures grown in M9 media supplemented with 50 nM FeCl₃, grown in static conditions at 37°C for 8 h, and exposed to either ambient or infrared (IR) light as described in the Materials and Methods. Expression is calculated relative to WT-comp in static, light conditions. Significance was calculated using a two-tailed Student’s t-test with asterisks indicating the following P-values: *, P < 0.05; **, P < 0.005; and ***, P < 0.0005.

FIG 6

Heme negatively affects pchE expression. Relative PrrH (A), pchE (B), and PrrF (C) transcript levels relative to WT PAO1 when grown in the presence or absence of infrared (IR) light. Data are an average of four biological replicates in M9 media supplemented with 50 nM FeCl₃ or 5 µM heme grown statically at 37°C for 8 h. Significance was calculated using an unpaired t-test with two-tailed P values where * indicates P < 0.05, ** indicates P < 0.005, and *** indicates P < 0.0005.