RamA, which controls expression of the MDR efflux pump AcrAB-TolC, is regulated by the Lon protease

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@article{38ec7d3d9a3847e4a8ce28bf69e2cf3f,
title = "RamA, which controls expression of the MDR efflux pump AcrAB-TolC, is regulated by the Lon protease",
abstract = "OBJECTIVES: RamA regulates the AcrAB-TolC multidrug efflux system. Using Salmonella Typhimurium, we investigated the stability of RamA and its impact on antibiotic resistance.METHODS: To detect RamA, we introduced ramA::3XFLAG::aph into plasmid pACYC184 and transformed this into Salmonella Typhimurium SL1344ramA::cat and lon::aph mutants. An N-terminus-deleted mutant [pACYC184ramA(Δ2-21)::3XFLAG::aph] in which the first 20 amino acids of RamA were deleted was also constructed. To determine the abundance and half-life of FLAG-tagged RamA, we induced RamA with chlorpromazine (50 mg/L) and carried out western blotting using anti-FLAG antibody. Susceptibility to antibiotics and phenotypic characterization of the lon mutant was also carried out.RESULTS: We show that on removal of chlorpromazine, a known inducer of ramA, the abundance of RamA decreased to pre-induced levels. However, in cells lacking functional Lon, we found that the RamA protein was not degraded. We also demonstrated that the 21 amino acid residues of the RamA N-terminus are required for recognition by the Lon protease. Antimicrobial susceptibility and phenotypic tests showed that the lon mutant was more susceptible to fluoroquinolone antibiotics, was filamentous when observed by microscopy and grew poorly, but showed no difference in motility or the ability to form a biofilm. There was also no difference in the ability of the lon mutant to invade human intestinal cells (INT-407).CONCLUSIONS: In summary, we show that the ATP-dependent Lon protease plays an important role in regulating the expression of RamA and therefore multidrug resistance via AcrAB-TolC in Salmonella Typhimurium.",
keywords = "Anti-Bacterial Agents, Bacterial Proteins, Carrier Proteins, Drug Resistance, Bacterial, Gene Expression Regulation, Bacterial, Multidrug Resistance-Associated Proteins, Protease La, Protein Stability, Salmonella typhimurium, Trans-Activators",
author = "Vito Ricci and Blair, {Jessica M A} and Piddock, {Laura J V}",
year = "2014",
month = mar,
doi = "10.1093/jac/dkt432",
language = "English",
volume = "69",
pages = "643--50",
journal = "Journal of Antimicrobial Chemotherapy",
issn = "0305-7453",
publisher = "Oxford University Press",
number = "3",

}

RIS

TY - JOUR

T1 - RamA, which controls expression of the MDR efflux pump AcrAB-TolC, is regulated by the Lon protease

AU - Ricci, Vito

AU - Blair, Jessica M A

AU - Piddock, Laura J V

PY - 2014/3

Y1 - 2014/3

N2 - OBJECTIVES: RamA regulates the AcrAB-TolC multidrug efflux system. Using Salmonella Typhimurium, we investigated the stability of RamA and its impact on antibiotic resistance.METHODS: To detect RamA, we introduced ramA::3XFLAG::aph into plasmid pACYC184 and transformed this into Salmonella Typhimurium SL1344ramA::cat and lon::aph mutants. An N-terminus-deleted mutant [pACYC184ramA(Δ2-21)::3XFLAG::aph] in which the first 20 amino acids of RamA were deleted was also constructed. To determine the abundance and half-life of FLAG-tagged RamA, we induced RamA with chlorpromazine (50 mg/L) and carried out western blotting using anti-FLAG antibody. Susceptibility to antibiotics and phenotypic characterization of the lon mutant was also carried out.RESULTS: We show that on removal of chlorpromazine, a known inducer of ramA, the abundance of RamA decreased to pre-induced levels. However, in cells lacking functional Lon, we found that the RamA protein was not degraded. We also demonstrated that the 21 amino acid residues of the RamA N-terminus are required for recognition by the Lon protease. Antimicrobial susceptibility and phenotypic tests showed that the lon mutant was more susceptible to fluoroquinolone antibiotics, was filamentous when observed by microscopy and grew poorly, but showed no difference in motility or the ability to form a biofilm. There was also no difference in the ability of the lon mutant to invade human intestinal cells (INT-407).CONCLUSIONS: In summary, we show that the ATP-dependent Lon protease plays an important role in regulating the expression of RamA and therefore multidrug resistance via AcrAB-TolC in Salmonella Typhimurium.

AB - OBJECTIVES: RamA regulates the AcrAB-TolC multidrug efflux system. Using Salmonella Typhimurium, we investigated the stability of RamA and its impact on antibiotic resistance.METHODS: To detect RamA, we introduced ramA::3XFLAG::aph into plasmid pACYC184 and transformed this into Salmonella Typhimurium SL1344ramA::cat and lon::aph mutants. An N-terminus-deleted mutant [pACYC184ramA(Δ2-21)::3XFLAG::aph] in which the first 20 amino acids of RamA were deleted was also constructed. To determine the abundance and half-life of FLAG-tagged RamA, we induced RamA with chlorpromazine (50 mg/L) and carried out western blotting using anti-FLAG antibody. Susceptibility to antibiotics and phenotypic characterization of the lon mutant was also carried out.RESULTS: We show that on removal of chlorpromazine, a known inducer of ramA, the abundance of RamA decreased to pre-induced levels. However, in cells lacking functional Lon, we found that the RamA protein was not degraded. We also demonstrated that the 21 amino acid residues of the RamA N-terminus are required for recognition by the Lon protease. Antimicrobial susceptibility and phenotypic tests showed that the lon mutant was more susceptible to fluoroquinolone antibiotics, was filamentous when observed by microscopy and grew poorly, but showed no difference in motility or the ability to form a biofilm. There was also no difference in the ability of the lon mutant to invade human intestinal cells (INT-407).CONCLUSIONS: In summary, we show that the ATP-dependent Lon protease plays an important role in regulating the expression of RamA and therefore multidrug resistance via AcrAB-TolC in Salmonella Typhimurium.

KW - Anti-Bacterial Agents

KW - Bacterial Proteins

KW - Carrier Proteins

KW - Drug Resistance, Bacterial

KW - Gene Expression Regulation, Bacterial

KW - Multidrug Resistance-Associated Proteins

KW - Protease La

KW - Protein Stability

KW - Salmonella typhimurium

KW - Trans-Activators

U2 - 10.1093/jac/dkt432

DO - 10.1093/jac/dkt432

M3 - Article

C2 - 24169580

VL - 69

SP - 643

EP - 650

JO - Journal of Antimicrobial Chemotherapy

JF - Journal of Antimicrobial Chemotherapy

SN - 0305-7453

IS - 3

ER -