Comparative Proteomics Demonstrates Altered Metabolism Pathways in Cotrimoxazole- Resistant and Amikacin-Resistant Klebsiella pneumoniae Isolates

Abstract

Antibiotic resistance (AMR) has always been a hot topic all over the world and its mechanisms are varied and complicated. Previous evidence revealed the metabolic slowdown in resistant bacteria, suggesting the important role of metabolism in antibiotic resistance. However, the molecular mechanism of reduced metabolism remains poorly understood, which inspires us to explore the global proteome change during antibiotic resistance. Here, the sensitive, cotrimoxazole-resistant, amikacin-resistant, and amikacin/cotrimoxazole -both-resistant KPN clinical isolates were collected and subjected to proteome analysis through liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A deep coverage of 2,266 proteins were successfully identified and quantified in total, representing the most comprehensive protein quantification data by now. Further bioinformatic analysis showed down-regulation of tricarboxylic acid cycle (TCA) pathway and up-regulation of alcohol metabolic or glutathione metabolism processes, which may contribute to ROS clearance and cell survival, in drug-resistant isolates. These results indicated that metabolic pathway alteration was directly correlated with antibiotic resistance, which could promote the development of antibacterial drugs from "target" to "network." Moreover, combined with minimum inhibitory concentration (MIC) of cotrimoxazole and amikacin on different KPN isolates, we identified nine proteins, including garK, uxaC, exuT, hpaB, fhuA, KPN_01492, fumA, hisC, and aroE, which might contribute mostly to the survival of KPN under drug pressure. In sum, our findings provided novel, non-antibiotic-based therapeutics against resistant KPN.

Keywords: Klebsiella pneumoniae (K. pneumoniae); antibiotic resistance; bioinformatics; comparative proteomics; metabolism.

FIGURE 1

Label-Free Quantitative Proteome identify the pattern characteristics of hospital-derived KPN strains. (A) The experimental design and workflow. Klebsiella pneumoniae clinical isolates, including SEN, AMI-resistant, CTX-resistant, and ACB-resistant isolates, and ATCC strains were collected. The cells were subjected to LC-MS/MS analysis for label-free proteome following protein extraction and FASP digestion. Protein quantification were finished using Maxquant software with iBAQ algorithm. (B) The number of proteins detected in each isolate. (C,D) Principal component analysis (C) and Unsupervised hierarchical clustering (D) of the protein profiling of 15 KPN strains.

FIGURE 2

Comparative Proteomics Analyses Revealed the metabolism alteration of CTX-resistant isolates comparing with SEN isolates. (A) Expression profiles (up) and representative function enrichment analysis (down) of differentially expressed genes between CTX-resistant isolates and SEN isolates. Each line represents one protein. Functional terms were labeled and color-coded with p-value (Fisher’s exact test) according to the legend. (B) Expression patterns of proteins participating in the indicated cellular functions/pathways across CTX-resistant or SEN isolates. Values for each protein in all groups are color-coded based on the z-scored protein abundance per cell.

FIGURE 3

Comparative Proteomics Analyses Revealed the metabolism alteration of AMI-resistant isolates comparing with SEN isolates. (A) Expression profiles (up) and representative function enrichment analysis (down) of differentially expressed genes between AMI-resistant isolates and SEN isolates. Each line represents one protein. Functional terms were labeled and color-coded with p-value (Fisher’s exact test) according to the legend. (B) Expression patterns of proteins participating in the indicated cellular functions/pathways across AMI-resistant or SEN isolates. Values for each protein in all groups are color-coded based on the z-scored protein abundance per cell.

FIGURE 4

The expression levels of nine metabolism-associated proteins were highly correlated with the degree of drug-resistance of KPN. (A) Venn diagram summary of the number of up-regulated (up) or down-regulated (down) genes in AMI-resistant (purple), ACB-resistant (pink), and CTX-resistant (blue) isolates, comparing with SEN isolates. The genes only differentially expressed in one of AMI-resistant or CTX-resistant isolates were labeled with dark purple or dark blue, respectively. (B,C) The hist of Pearson correlation coefficients between expression patterns of genes. (D,E) Identification information of metabolic proteins whose expression patterns were significantly correlated with MIC of strains against CTX and AMI.