Secretome of Transmissible <i>Pseudomonas aeruginosa</i> AES-1R Grown in a Cystic Fibrosis Lung-Like Environment

<i>Pseudomonas aeruginosa</i> is the predominant cause of mortality in patients with cystic fibrosis (CF). We examined the secretome of an acute, transmissible CF <i>P. aeruginosa</i> (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted PAO1. Culture supernatant proteins from rich (LB) and minimal (M9) media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated abundance of PasP protease and absence of AprA protease in AES-1R. CF lung-like artificial sputum medium (ASMDM) contains serum and mucin that generally preclude proteomics of secreted proteins. ASMDM culture supernatants were subjected to 2DLC-MS/MS, which allowed the identification of 57 <i>P. aeruginosa</i> proteins, and qualitative spectral counting was used to estimate relative abundance. AES-1R-specific AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants, while AprA could only be identified in PAO1. Relative quantitation was performed using selected reaction monitoring. Significantly elevated levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated <i>pasP</i> in AES-1R during early (18 h) ASMDM growth, while no evidence of <i>aprA</i> expression could be observed. Genomic screening of CF isolates revealed <i>aes_7139</i> was present in all AES-1 and one pair of sequential nonepidemic isolates. Secreted proteins may be crucial in aiding CF-associated <i>P. aeruginosa</i> to establish infection and for adaptation to the CF lung

Secretome of Transmissible <i>Pseudomonas aeruginosa</i> AES-1R Grown in a Cystic Fibrosis Lung-Like Environment

<i>Pseudomonas aeruginosa</i> is the predominant cause of mortality in patients with cystic fibrosis (CF). We examined the secretome of an acute, transmissible CF <i>P. aeruginosa</i> (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted PAO1. Culture supernatant proteins from rich (LB) and minimal (M9) media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated abundance of PasP protease and absence of AprA protease in AES-1R. CF lung-like artificial sputum medium (ASMDM) contains serum and mucin that generally preclude proteomics of secreted proteins. ASMDM culture supernatants were subjected to 2DLC-MS/MS, which allowed the identification of 57 <i>P. aeruginosa</i> proteins, and qualitative spectral counting was used to estimate relative abundance. AES-1R-specific AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants, while AprA could only be identified in PAO1. Relative quantitation was performed using selected reaction monitoring. Significantly elevated levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated <i>pasP</i> in AES-1R during early (18 h) ASMDM growth, while no evidence of <i>aprA</i> expression could be observed. Genomic screening of CF isolates revealed <i>aes_7139</i> was present in all AES-1 and one pair of sequential nonepidemic isolates. Secreted proteins may be crucial in aiding CF-associated <i>P. aeruginosa</i> to establish infection and for adaptation to the CF lung

Proteomics of <i>Pseudomonas aeruginosa</i> Australian Epidemic Strain 1 (AES-1) Cultured under Conditions Mimicking the Cystic Fibrosis Lung Reveals Increased Iron Acquisition via the Siderophore Pyochelin

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire <i>P. aeruginosa</i> from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and <i>S</i>-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (<i>pchDFG</i> and <i>fptA</i>) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R

Proteomics of <i>Pseudomonas aeruginosa</i> Australian Epidemic Strain 1 (AES-1) Cultured under Conditions Mimicking the Cystic Fibrosis Lung Reveals Increased Iron Acquisition via the Siderophore Pyochelin

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire <i>P. aeruginosa</i> from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and <i>S</i>-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (<i>pchDFG</i> and <i>fptA</i>) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R

<i>C</i>. <i>elegans</i> virulence assay.

<p>Percent survival of <i>C</i>. <i>elegans</i> (n = 50) when grown on a lawn of <i>P</i>. <i>aeruginosa</i> AES-1R, AES-1M, PA14 and PA14Δ<i>hmgA</i>. The dotted line indicates the LT₅₀ (mean time at which 50% of the worms were killed by ingesting <i>P</i>. <i>aeruginosa</i>). The individual points are the means of six biological replicates for AES-1 stains and three biological replicates for PA14 strains. The error bars represent the standard error of the means.</p

Oligonucleotide primers used in this study.

<p>^a Sourced from Choi and Schweizer, 2005.</p><p>^b<i>gene</i>-UpF: Regular 18–20bp forward primer upstream of the target gene</p><p>^c<i>gene</i>-UpR-Gm: 18–20bp reverse primer upstream of the target gene incorporating a 20–25bp overlap homologous to the 5’ end of the gentamicin resistance cassette in pPS856</p><p>^d<i>gene</i>-DnF-Gm: 18–20bp forward primer downstream of the target gene incorporating a 20–25bp overlap homologous to the 3’ end of the gentamicin resistance cassette in pPS856</p><p>^e<i>gene</i>-DnR: Regular 18–20bp forward primer downstream of the target gene.</p><p>Oligonucleotide primers used in this study.</p

Differential expression of <i>hmgA</i> by <i>P</i>. <i>aeruginosa</i> AES-1 by microarray analyses (all experiments conducted in biological duplicate).

<p>*Statistically significant.</p><p>Differential expression of <i>hmgA</i> by <i>P</i>. <i>aeruginosa</i> AES-1 by microarray analyses (all experiments conducted in biological duplicate).</p

Persistence of <i>P</i>. <i>aeruginosa</i> AES-1R, AES-1M and PA14, PA14<i>hmgA</i>, in a C57BL/6 mouse model.

<p><b>A</b>. Mice were infected with 10⁶ CFU of AES-1R and AES-1M and lungs examined at 3-hrs and 24-hrs and 72hrs post-infection for bacterial load (latter not shown). <b>B.</b> Mice were infected with 10⁶ CFU wildtype PA14 and PA14<i>hmgA</i> and lungs were examined at 3-hrs, 24-hrs, and 72-hrs post-infection for bacterial load. AES-1M was recovered at significantly higher level compared to AES-1R at 24-hr. PA14Δ<i>hmgA</i> was recovered at a significantly higher level compared to the wild type PA14 at 24- and 72-hrs post-infection. The significances of differences between strains were determined by ANOVA. 4A: *p<0.001vs AES-1R; 4B* p<0.0001 vs. PA14 WT.</p