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04142022_B.cenocepacia_pigment_specific_metabolism

ABSTRACT: Metabolomic study of B. cenocepacia J2315 (produces pyomelanin) and K56-2 (lacks pyomelanin) WT strains and mutant strains in which the pyomelanin production phenotype are reversed were cultured in synthetic CF sputum media (SCFM2). Metabolites were extracted from cultures via ethyl acetate and C18 SPE extractions, and extracts were pooled before acquiring UPLC/HRMS/MS data to evaluate pyomelanin-specifc metabolism.

INSTRUMENT(S): impact II

ORGANISM(S): Burkholderia Cenocepacia K56-2valvano (ncbitaxon:985076) Burkholderia Cenocepacia J2315 (ncbitaxon:216591)

SUBMITTER: Neha Garg

PROVIDER: MSV000089255 | MassIVE | Wed Apr 13 21:58:00 BST 2022

REPOSITORIES: MassIVE

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Project description:Pseudomonas aeruginosa airway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. In vitro models that closely mimic CF sputum are needed to improve understanding of the pathobiology of P. aeruginosa in the CF airway. We developed an artificial sputum medium (ASMDM) that more closely resembles the composition of CF sputum than current media. In order to validate the utility of ASMDM, we used GeneChip microarrays to compare expression data of P. aeruginosa UCBPP-PA14 (PA14) in ASMDM with published data for this strain grown under the same conditions in an artificial medium containing 10% (v/v) CF sputum. Thirty-seven of 39 nutrition-related genes were differentially expressed in the same manner in both media. However, 24 quorum-sensing (QS) genes, 23 Type III secretion system and several anaerobic respiration genes were more highly expressed in ASMDM than in sputum-containing medium. When grown to stationary phase in ASMDM, PA14 differentially expressed about 50 biologically significant genes compared to stationary phase growth in Luria Broth; genes involved in iron acquisition (pfeA, fepC) and in assimilatory nitrate reduction (nasC, nirD) were upregulated, while 24 QS genes, including the regulator rhlR, lasA, rsaL, aprADEI and phenazine genes phzC2DD2EG2 were downregulated. Downregulation of QS-regulated virulence genes has been noted in chronic P. aeruginosa infection. ASMDM thus appears highly suitable for studies on gene expression of (i) P. aeruginosa strains from acutely and chronically infected CF patients and (ii) established biofilms that are a hallmark of advanced CF lung disease. PA14 was grown in four different ways: 1. Logarithmic growth for early gene expression Cells were grown in MOPS-Glucose and separately in ASMDM. The average of two biological duplicates in each case was compared to the other to determine differential gene expression. 2. Stationary phase growth for gene expression Cells were grown in Luria Broth and separately in ASMDM. The average of two biological duplicates in each case was compared to the other to determine differential gene expression.

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04142022_B.cenocepacia_pigment_specific_metabolism

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