title: Effect of transcriptional activators SoxS, RobA, and RamA on expression of multidrug efflux pump AcrAB-TolC in Enterobacter cloacae.
abstract: Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae. However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli, the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae, EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB, thus conferring antimicrobial resistance as well as a stress response in E. cloacae. In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The involvement of these proteins in conferring antimicrobial resistance through upregulation of acrAB was demonstrated in E. cloacae.
authors.affiliation: Servicio de Microbiología-Instituto de Investigación Biomédica, Complexo Hospitalario Universitario A Coruña, A Coruña, Spain.
authors.forename: Astrid
authors.initials: A
authors.lastname: Pérez
authors.forename: Margarita
authors.initials: M
authors.lastname: Poza
authors.forename: Jesús
authors.initials: J
authors.lastname: Aranda
authors.forename: Cristina
authors.initials: C
authors.lastname: Latasa
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Medrano
authors.forename: María
authors.initials: M
authors.lastname: Tomás
authors.forename: Antonio
authors.initials: A
authors.lastname: Romero
authors.forename: Iñigo
authors.initials: I
authors.lastname: Lasa
authors.forename: Germán
authors.initials: G
authors.lastname: Bou
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology*; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D016972:Enterobacter cloacae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D005434:Flow Cytometry; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics* / Q000502:physiology*
chemical_list: C573020:AcrAB-TolC protein, Enterobacter cloacae; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D015534:Trans-Activators