Alterations of Metabolic and Lipid Profiles in Polymyxin-Resistant Pseudomonas aeruginosa

Abstract

Multidrug-resistant Pseudomonas aeruginosa presents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance in P. aeruginosa has been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistant P. aeruginosa strains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-type P. aeruginosa strain K ([PAK] polymyxin B MIC, 1 mg/liter) and its paired pmrB mutant strains, PAKpmrB6 and PAKpmrB12 (polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6 and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) in speE (encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6 compared to that in PAKpmrB12 Our results indicate that spermidine may play an important role in high-level polymyxin resistance in P. aeruginosa Interestingly, both pmrB mutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12 mutant exhibited much lower levels of phospholipids than the PAKpmrB6 mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance in P. aeruginosa and highlights its impacts on bacterial metabolism.

Keywords: Pseudomonas aeruginosa; glycerophospholipids; l-Ara4N biosynthesis; lipid A modification; metabolomics; methionine salvage cycle; polymyxin resistance.

FIG 1

Mass characterization of lipid A profiles in polymyxin-susceptible and -resistant P. aeruginosa strains. Wild-type PAK (A), PAKpmrB6 (B), and PAKpmrB12 (C) mutants and the predominant structures of lipid A from the wild-type PAK and/or both pmrB mutants (D). The lipid A samples were analyzed by LC-MS/MS in negative ion mode, which obtained most lipid A peaks with the loss of one hydrogen ([M-H]⁻). R-3-hydroxydecanoate is shown in blue and l-Ara4N is shown in red.

FIG 2

Method comparison in the extraction of lipidomes from P. aeruginosa strain K (PAK). (A) PCA score plot shows that metabolites are grouped due to different extraction methods: red, CHCl₃/MeOH/H₂O ([CMW] 1:1:0.9 [vol/vol], double-phase Bligh-Dyer); green, CMW (1:2:0.8 [vol/vol], single-phase Bligh-Dyer); and blue, MTBE/MeOH/H₂O ([MMW] 10:3:2.5 [vol/vol], MTBE extraction). The colored dots on the PCA score plot represent five biological replicates. (B) The heat map illustrates the relative peak intensities of lipids with three major lipid classes extracted by three different solvents, CMW (left, 1:2:0.8 [vol/vol]), CMW (middle, 1:1:0.9 [vol/vol]), and MMW (right, 10:3:2.5 [vol/vol]). Five independent biological replicates of each extraction method are shown. Colors indicate relative abundance of lipids in each sample based on the relative peak intensity for each lipid: red, high; yellow, mean; blue, low or undetectable. (C) The bar charts show the relative abundance (log scale) of each sample between different extraction methods as well as the quality control (QC) samples. Box plots show the upper and lower quartiles (tops and bottoms of boxes), medians (lines within the boxes), and the spread of data that are not outliers (whiskers).

FIG 3

Lipidomic perturbations between pmrB mutants and the wild-type PAK. (A) Fold changes of GPLs in PAKpmrB6 (blue) and PAKpmrB12 (red) compared to PAK collected from the reversed-phase liquid chromatography (RPLC) method (P < 0.05, FDR < 0.05, one-way ANOVA). (B) Fold changes indicate the decreased levels of fatty acids and acyl-CoA in PAKpmrB6 (blue) and PAKpmrB12 (red) compared to PAK analyzed by the hydrophilic interaction chromatography (HILIC) method (P < 0.05, FDR < 0.05, one-way ANOVA).

FIG 4

Multivariate and univariate statistical analyses of metabolic perturbations between wild-type PAK and the pmrB mutants, PAKpmrB6 and PAKpmrB12. (A) PCA score plots of the two principle components for metabolite levels from the three strains: red, PAK; blue, PAKpmrB6; green, PAKpmrB12. (B) Volcano plots show the fold change and significance of metabolites in both pmrB mutants compared to the wild-type strain. Red plots represent metabolites having a fold change of >2 and a P value of <0.05 (Student's t test), while gray plots represent metabolites that are not significantly changed. Fold changes relative to the untreated control are based upon mean values from five biological replicates in all three strains.

FIG 5

Perturbations of the intermediates in l-Ara4N synthesis pathway. UDP-glucuronate, UDP-l-Ara4N, and UDP-l-Ara4FN were detected using the HILIC method, whereas undecaprenyl phosphate-l-Ara4N was detected using the RPLC method. The green box indicates the metabolite that was significantly increased by >2-fold, while the red boxes show metabolites that were exclusively detected in the pmrB mutants compared to that in the wild-type PAK. Metabolites in gray boxes were not detected through either the HILIC or RPLC method. **, P < 0.01; ***, P < 0.001 (Student's t test).

FIG 6

Metabolic perturbations in methionine salvage cycle and spermidine synthesis pathway. S-Adenosylmethioninamine, putrescine, and spermidine were analyzed using the RPLC method, while the other metabolites were analyzed by the HILIC method. Metabolites in red boxes indicate increased abundance in PAKpmrB6 and/or PAKpmrB12, while blue boxes indicate decreased abundance compared to that in the wild-type PAK. Metabolites in gray boxes were not detected through either the HILIC or RPLC method. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student's t test). Dashed arrow shows the conversion of putrescine to spermidine catalyzed by SpeE in the presence of the cofactor S-adenosylmethioninamine.