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Although some outer membrane (OM) proteins involved in antibiotic resistance have been previously reported, the OM proteins regulating chlortetracycline (CTC) resistance are largely unknown. In this study, we employed a subproteomics approach to identify altered OM proteins of Escherichia coliin response to CTC exposure. Upregulation of TolC and downregulation of LamB, FadL, OmpC, OmpT, and OmpW were found in E. colistrains exposed to CTC at a high concentration that was increased suddenly and at a half-minimum inhibitory concentration (MIC) that was kept constant in the culture medium. These changes in the level of protein expression were validated using Western blotting. In addition, the possible roles of these altered proteins and their regulation mechanisms in response to CTC exposure were investigated using genetically modified strains with gene deletion of these altered proteins. It was found that deletion of tolC, fadL, ompC, ompT, or ompWresulted in a decrease in the MICs and survival capabilities of the gene-deleted strains, whereas the absence of lamBled to an improvement of the two abilities. The downregulation of LamB expression in the CTC-resistant E. colistrain and the increased antibiotic resistance in its gene-deleted strain suggested a negative regulation mechanism in E. coliin response to CTC exposure. Meanwhile, the direction of the regulation pattern in response to CTC exposure was different from that in E. coliin response to exposure to other antibiotics. These findings uncover a novel antibiotic-resistant mechanism in which bacteria respond to exposure to antibiotics through alteration of the direction of regulation of OM proteins.

Edwardsiella tarda,the causative agent of Edwardsiellosis, imposes medical challenges in both the clinic and aquaculture. The emergence of multidrug resistant strains makes antibiotic treatment impractical. The identification of molecules that facilitate or promote antibiotic efficacy is in high demand. In the present study, we aimed to identify small molecules whose abundance is correlated with kanamycin resistance in E. tardaby gas chromatography–mass spectrometry. We found that the abundance of fructose was greatly suppressed in kanamycin-resistant strains. The incubation of kanamycin-resistant bacteria with exogenous fructose sensitized the bacteria to kanamycin. Moreover, the fructose also functioned in bacteria persisters and biofilm. The synergistic effects of fructose and kanamycin were validated in a mouse model. Furthermore, the mechanism relies on fructose in activating TCA cycle to produce NADH, which generates proton motive force to increase the uptake of the antibiotics. Therefore, we present a novel approach in fighting against multidrug resistant bacteria through exploration of antibiotic-suppressed molecules.

Membrane proteins of Gram-negative bacteria are key molecules that interface the cells with the environment. Despite recent proteomic identification of numerous oligomer proteins in the Escherichia colicell envelope, the protein complex of E. colimembrane proteins and their peripherally associated proteins remain ill-defined. In the current study, we systematically analyze the subproteome of E. colicell envelope enriched in sarcosine-insoluble fraction (SIF) and sarcosine-soluble fraction (SSF) by using proteomic methodologies. One hundred and four proteins out of 184 spots on 2D electrophoresis gels are identified, which includes 31 outer membrane proteins (OMPs). Importantly, our further proteomic studies reveal a number of previously unrecognized membrane-interacting protein complexes, such as the complex consisting of OmpW and fumarate reductase. This established complete proteomic profile of E. colienvelope also sheds new insight into the function(s) of E. coliouter envelope.