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Gene expression in PAO1, clonal AES-1 and non-clonal isolates of Pseudomonas aeruginosa

ABSTRACT: Pseudomonas aeruginosa (P. aeruginosa) lung infection is a significant cause of mortality in patients with cystic fibrosis (CF). Most CF patients acquire unique P. aeruginosa strains from the environment; however clonal strains have been identified in CF communities in several countries. Two clonal strains infect 10% to 40% of patients in three CF clinics in mainland eastern Australia. The expression profiles of four planktonically-grown isolates of one Australian clonal strain (AES-1), and four non–clonal CF P. aeruginosa isolates were compared to each other and to the reference strain PAO1 using the Affymetrix P. aeruginosa PAO1 genome array, to gain insight into properties mediating the enhanced infectivity of AES-1. The isolates were subsequently grown as 3-day old biofilms and similarly extracted for RNA and compared as above. Data analysis was carried out using BIOCONDUCTOR software. Keywords: Comparative strain hybridization

ORGANISM(S): Pseudomonas aeruginosa

PROVIDER: GSE6122 | GEO | 2008/12/05

SECONDARY ACCESSION(S): PRJNA97617

REPOSITORIES: GEO

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Project description:Pseudomonas aeruginosa is a Gram-negative, opportunistic bacterium and a major etiological agent in monogenic disease cystic fibrosis (CF). High density colonies of P. aeruginosa are often isolated from hypoxic mucus plugs in respiratory tract of CF patients, indicating high adaptive capacities of the bacterium. Despite the high prevalence and related patient mortality, the protein machinery enabling the bacterium to adapt to this hypoxic environment remains to be fully elucidated. We investigated this by performing both SWATH mass spectrometry and data-dependent SPS-MS3 of TMT labelled peptides to profile the proteomes of two P. aeruginosa CF isolates, PASS2 and PASS3, and a laboratory reference strain, PAO1, grown under hypoxic stress (O2<1%) and aerobic conditions in media that mimics the nutrient components of the CF lung. 3,967 P. aeruginosa proteins were quantitated (FDR <1%) across all three strains, reflecting approximately 71% of predicted ORFs in PAO1 and representing the most comprehensive proteome of clinically relevant P. aeruginosa to date. Comparative analysis revealed 735, 640 and 364 proteins were altered by two-fold or more when comparing low oxygen to aerobic growth in PAO1, PASS2 and PASS3 respectively. Strikingly, under hypoxic stress, all strains showed concurrent increased abundance of proteins required for both aerobic (cbb3-1 and cbb3-2 terminal oxidases) and anaerobic denitrification and arginine fermentation, with the two clinical isolates showing higher relative expression of proteins in these pathways. Additionally, functional annotation revealed that clinical strains portray a unique expression profile of replication, membrane biogenesis and virulence proteins during hypoxia which may endow these bacteria with a survival advantage. These protein profiles illuminate the diversity of P. aeruginosa mechanisms to adapt to low oxygen and shows that CF isolates initiate a robust molecular response to persist under these conditions.

Project description:PAO1 and FRD1 were cultured planktonically and in biofilms with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined for PAO1 cultured planktonically, for FRD1 cultured planktonically, for PAO1 cutured in biofilms, and for FRD1 cultured in biofilms. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.

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Gene expression in PAO1, clonal AES-1 and non-clonal isolates of Pseudomonas aeruginosa

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