A novel oxidized low-density lipoprotein-binding protein from Pseudomonas aeruginosa

• Published in: Microbiology (Society for General Microbiology) 2008-02, Vol.154 (2), p.654-665
• Main Authors: Rao, Jayasimha, DiGiandomenico, Antonio, Unger, Jason, Bao, Yongde, Polanowska-Grabowska, Renata K, Goldberg, Joanna B
• Format: Article
• Language: English
• Subjects: Aspergillus fumigatus; Bacterial Proteins - analysis; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Cloning, Molecular; Cystic Fibrosis - microbiology; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Humans; Kinetics; Lipoproteins, LDL - metabolism; Lysophosphatidylcholines - metabolism; Microbiology; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Pseudomonas aeruginosa; Pseudomonas aeruginosa - metabolism; Recombinant Proteins - analysis; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.2007/011429-0

1 Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA, USA 2 Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, VA, USA Correspondence Joanna B. Goldberg jbg2b{at}virginia.edu A novel protein, PA0122, has been identified in Pseudomonas aeruginosa and shown to bind to oxidized low-density lipoprotein (Ox-LDL). The PA0122 gene was recognized based on gene expression pattern differences between two strains of P. aeruginosa isolated from the sputum of an individual with cystic fibrosis (CF). There was an approximately eightfold increase in PA0122 expression in the non-mucoid strain 383, compared to that in the mucoid strain 2192. Quantitative real-time RT-PCR (qRT-PCR) supported PA0122 transcript expression differences between strains 383 and 2192 and revealed growth-phase dependence, with the highest level of expression at early stationary phase (OD 600  1.5). PA0122 encodes a 136 aa ‘conserved hypothetical’ protein that has similarity to Aspergillus fumigatus Asp-haemolysin, which is an Ox-LDL-binding protein, and possessed a motif that is homologous to the fungal aegerolysin family of proteins. Antibodies produced to purified recombinant PA0122 recognized a 16 kDa protein band in cell lysates as well as in the supernatant fractions of strain 383. The PA0122 protein expression pattern was growth phase-dependent, with maximal production observed at OD 600  1.5 that was consistent with the PA0122 transcript expression profile. Subcellular fractionation studies revealed differences in the localization of PA0122 between strains 383 and 2192. In 383, PA0122 was observed in the cytoplasm and in membrane fractions. In 2192, PA0122 was found in the cytoplasm but was not detected in membrane fractions. Surface plasmon resonance revealed that recombinant PA0122 binds with high affinity to Ox-LDL and to its major subcomponent, lysophosphatidylcholine, but not to non-oxidized LDL. Abbreviations: CF, cystic fibrosis; ECL, enhanced chemiluminescence; LDL, low-density lipoprotein; lysoPC, lysophosphatidylcholine; nmd, non-mucoid derivative; Ox-LDL, oxidized low-density lipoprotein; Ox-PL, oxidized phospholipid; qRT-PCR, quantitative real-time RT-PCR; QS, quorum sensing; SPR, surface plasmon resonance Two supplementary tables showing genes differentially expressed between the P. aeruginosa strains studied and QS-regulated genes whose expression was upregulated in strai