Responses of carbapenemase-producing and non-producing carbapenem-resistant Pseudomonas aeruginosa strains to meropenem revealed by quantitative tandem mass spectrometry proteomics

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of natural and acquired resistance mechanisms that are enhanced by its large regulatory network. This study describes the proteomic responses of two carbapenem-resistant P. aeruginosa strains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially regulated proteins and pathways. Strain CCUG 51971 carries a VIM-4 metallo-β-lactamase or 'classical' carbapenemase; strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits 'non-classical' carbapenem-resistance. Strains were cultivated with different sub-MICs of meropenem and analyzed, using quantitative shotgun proteomics based on tandem mass tag (TMT) isobaric labeling, nano-liquid chromatography tandem-mass spectrometry and complete genome sequences. Exposure of strains to sub-MICs of meropenem resulted in hundreds of differentially regulated proteins, including β-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic β-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic β-lactamases, efflux pumps, penicillin-binding proteins and downregulation of porins. All components of the H1 type VI secretion system were upregulated in strain CCUG 51971. Multiple metabolic pathways were affected in both strains. Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains of P. aeruginosa exhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility of P. aeruginosa to meropenem.

Keywords: Pseudomonas aeruginosa; carbapenem-resistance; extensively-drug resistant; high-risk clones; multidrug resistance; nano-LC–MS/MS; quantitative shotgun proteomics; tandem mass tag.

Figure 1

Experimental setup of the study. Each strain was cultivated with three different sub-MICs of meropenem and without antibiotic. Subsequently, proteins were extracted, reduced, alkylated and digested with trypsin. Peptides were labeled, using two TMT sets, pooled and analyzed, using nano-LC–MS/MS for protein detection and relative quantitation.

Figure 2

(A) Bar graph showing the number of proteins of Pseudomonas aeruginosa CCUG 51971 upregulated or downregulated in each condition. (B) Bar graph showing the number of proteins of P. aeruginosa CCUG 70744 upregulated or downregulated in each condition. (C) Venn diagram showing the number of upregulated or downregulated proteins overlapping between conditions in strain CCUG 51971. (D) Venn diagram showing the number of upregulated or downregulated proteins overlapping between conditions in strain CCUG 70744.

Figure 3

Genetic map of the H1-T6SS of Pseudomonas aeruginosa CCUG 51971 and relative abundances of its protein products at 256 μg/ml (½ MIC) compared to 0 μg/ml. Annotations from RefSeq, SecReT6 and Pseudomonas Genome Database are indicated, if available.

Figure 4

Functional classification (into COG categories) of the proteins that were upregulated or downregulated in the samples cultivated with the highest sub-MIC of Pseudomonas aeruginosa CCUG 51971 (256 μg/ml) and CCUG 70744 (8 μg/ml). Significantly upregulated or downregulated COG categories are marked as follows: * if p-value < 0.05; ** if p-value < 0.005; *** if p-value < 0.001; **** if p-value < 0.0001.

Figure 5

GO terms that were significantly enriched when the strains were cultivated with the two highest sub-MICs of meropenem. Pseudomonas aeruginosa CCUG 51971 was cultivated with 256 μg/ml (A) and 128 μg/ml (B). P. aeruginosa CCUG 70744 was cultivated with 8 μg/ml (C) and 4 μg/ml (D). In red: GO terms enriched in both strains. In bold: GO terms enriched in both conditions of one strain. BP, biological process; MF, molecular function; CC, cellular component; FDR, false discovery rate; NES, normalized enrichment score.

Figure 6

KEGG pathways that were significantly enriched when the strains were cultivated with the two highest sub-MICs of meropenem. Pseudomonas aeruginosa CCUG 51971 was cultivated with 256 μg/ml (A) and 128 μg/ml (B). P. aeruginosa CCUG 70744 was cultivated with 8 μg/ml (C) and 4 μg/ml (D). The row below each pathway indicates its class. In red: pathways enriched in both strains. In bold, pathways enriched in both conditions of one strain. FDR, false discovery rate; NES, normalized enrichment score.