Integrated Genomic and Proteomic Analyses of High-level Chloramphenicol Resistance in Campylobacter jejuni

Abstract

Campylobacter jejuni is a major zoonotic pathogen, and its resistance to antibiotics is of great concern for public health. However, few studies have investigated the global changes of the entire organism with respect to antibiotic resistance. Here, we provide mechanistic insights into high-level resistance to chloramphenicol in C. jejuni, using integrated genomic and proteomic analyses. We identified 27 single nucleotide polymorphisms (SNPs) as well as an efflux pump cmeB mutation that conferred modest resistance. We determined two radical S-adenosylmethionine (SAM) enzymes, one each from an SNP gene and a differentially expressed protein. Validation of major metabolic pathways demonstrated alterations in oxidative phosphorylation and ABC transporters, suggesting energy accumulation and increase in methionine import. Collectively, our data revealed a novel rRNA methylation mechanism by a radical SAM superfamily enzyme, indicating that two resistance mechanisms existed in Campylobacter. This work provided a systems biology perspective on understanding the antibiotic resistance mechanisms in bacteria.

Figure 1

Complete genomes and comparative genomic analysis of parental strain and CAP-resistant strain. Circular representation of chromosome from C. jejuni ATCC 33560 (a) and CAP-resistant strain (b). Circles (from the center toward periphery) indicate the following: first, GC skew; second, G + C content; third, ORFs predicted on the minus strand; fourth, putative gene, rRNA and tRNA in the minus strand; fifth, putative gene, rRNA and tRNA in the sense strand; sixth, COG annotations of genes in the sense strand. (c) Co-linear analysis of genome between C. jejuni ATCC 33560 and C. jejuni NCTC 11168 using MUMmer. The green connecting bars indicated high sequence identity and the red bars a reverse orientation. (d) Number of mutant loci and categories of mutations in CAP-resistant strain.

Figure 2

Gene ontology enrichment analysis of up-regulated and down-regulated DEPs. GO terms of up-regulated and down-regulated DEPs were categorized into biological process (a,d), molecular function (b,e), and cellular component (c,f) using Blast2GO software.

Figure 3

Quantification results from SWATH and SRM analyses.

Figure 4

Schematic representation of disturbed metabolic pathways in CAP-resistant strains including ABC transporter, oxidative phosphorylation, ribosomal assembly, and two-component system. Disturbed metabolic pathway were indicated in RED.