title: Blocking phosphatidylcholine utilization in Pseudomonas aeruginosa, via mutagenesis of fatty acid, glycerol and choline degradation pathways, confirms the importance of this nutrient source in vivo.
abstract: Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.
authors: Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors: Zhenxin
authors: Z
authors: Sun
authors: Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors: Yun
authors: Y
authors: Kang
authors: Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors: Michael H
authors: MH
authors: Norris
authors: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors: Ryan M
authors: RM
authors: Troyer
authors: Department of Biological Sciences, Plymouth State University, Plymouth, New Hampshire, United States of America.
authors: Mike S
authors: MS
authors: Son
authors: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors: Herbert P
authors: HP
authors: Schweizer
authors: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors: Steven W
authors: SW
authors: Dow
authors: Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America; Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors: Tung T
authors: TT
authors: Hoang
mesh_terms: D063988:3-Hydroxyacyl-CoA Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002794:Choline / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism / Q000382:microbiology; D005227:Fatty Acids / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D008168:Lung / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009876:Operon; D010713:Phosphatidylcholines / Q000378:metabolism*; D011552:Pseudomonas Infections / Q000378:metabolism / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D010713:Phosphatidylcholines; D063988:3-Hydroxyacyl-CoA Dehydrogenase; D002794:Choline; D005990:Glycerol