title: Identification of New Resistance Mechanisms in Escherichia coli against Apidaecin 1b Using Quantitative Gel- and LC-MS-Based Proteomics.
abstract: Bacteria have acquired resistance mechanisms to overcome antibiotic treatments, triggering major concerns about the return of epidemic infections. Antimicrobial peptides identified in insects, animals, and plants represent a huge pool of promising lead structures that can be further developed for medical applications. Short proline-rich antimicrobial peptides (PrAMPs) have gained much attention due to their clinically interesting activity spectrum, serum protease stability, efficacy in murine infection models, and low adverse effects. Here we induced resistances by incubating Escherichia coli with increasing concentrations of apidaecin 1b, a PrAMP isolated from honeybees, and quantitatively evaluated the proteomes between wild-type and resistant strains. Surprisingly, 2D differential gel electrophoresis did not reveal differences, indicating that the expression levels of dominant proteins were very similar. Reversed-phase chromatography coupled online to a mass spectrometer identified 2131 proteins in the soluble fraction (cytosolic fraction) and 1296 proteins in the nonsolubilized pellet (membrane fraction). Overall 29 proteins showed a statistically significant upregulation in the resistant E. coli strain, whereas 18 proteins were downregulated. Interestingly, periplasmic chaperone FimC, fimbrial protein FimA, and mannose-binding domain protein FimH, which are part of the fimbrial complex, were not detected in the resistant strain that was also unable to form biofilms. Furthermore, the expression of a few other proteins known as virulence factors was downregulated. Additionally, the expression level of isochorismatase hydrolase (YcaC) decreased in the membrane fraction of the resistant strain to 35%, and the corresponding knockout mutant of E. coli BW25113 was eight times less susceptible to apidaecin 1b and the related designer peptide Api88.
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig , 04103 Leipzig, Germany.|Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig , 04103 Leipzig, Germany.
authors.forename: Rico
authors.initials: R
authors.lastname: Schmidt
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig , 04103 Leipzig, Germany.|Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig , 04103 Leipzig, Germany.
authors.forename: Andor
authors.initials: A
authors.lastname: Krizsan
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig , 04103 Leipzig, Germany.|Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig , 04103 Leipzig, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Volke
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig , 04103 Leipzig, Germany.|Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig , 04103 Leipzig, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Knappe
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig , 04103 Leipzig, Germany.|Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig , 04103 Leipzig, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hoffmann
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000302:isolation & purification / Q000494:pharmacology; D001516:Bees / Q000737:chemistry; D002850:Chromatography, Gel; D056148:Chromatography, Reverse-Phase; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D013058:Mass Spectrometry; D020543:Proteome / Q000187:drug effects*; D040901:Proteomics / Q000379:methods*
chemical_list: D023181:Antimicrobial Cationic Peptides; D020543:Proteome; C061361:apidaecin
keywords: Api88; Apidaecin 1b; Escherichia coli; fimbrial complex; isochorismatase hydrolase (YcaC); resistance