Marie Curie Meeting 11 th April 2008 RamA is involved in the control of membrane permeability in multidrugresistant (MDR) E. aerogenes isolates A. Molitor, J-M. Pagès and A. Davin-Regli UMR-MD1, Université de la Méditerranée Marseille, France
Enterobacter aerogenes in Europe Common hospital pathogen from the respiratory tract in France and other european countries (Belgium, Spain, Austria, Germany…) Two major clinical clones present in Europe (one more prevalent ~70%) High resistance to a broad spectrum of antibiotics : Chromosomally derepressed cephalosporinase ESBL Target mutations Alteration of porin content Active efflux
RamA K. pneumoniae: • RamA is involved in a MDR phenotype in K.pneumoniae ( George et al. 1995) • RamA elicits high level of resistance to diverse Antibiotics and is associated with FQ resistance ( Schneiders et al . 2003) • RamA, a transcriptional regulator associated with decreased susceptibility to tigecycline ( Bradford et al. 2006) Salmonella: • RamA in Salmonella Typhimurium (ST) is involved in the response to the oxydative stress ( Straaten et al . 2003) • Deletion of ramA has no effect on virulence in Salmonella Typhimurium ( Straaten et al . 2003) • ramA is more important than mar and sox in the development of MDR in S .enterica ( Ricci et al. 2006 ) E. aerogenes and E. cloaceae: • The overexpression of RamA induces a MDR phenotype associated to a decrease in porin production and an increase in components of the AcrAB-TolC efflux pump in E. aerogenes . Moreover RamA is a transcriptional activator of the mar regulon.( Chollet et al . 2004) • RamA, a transcriptional regulator associated with decreased susceptibility to tigecycline ( Bradford et al. 2007)
Regulation of membrane permeability in gram negative bacteria
Regulation of permeability and MDR of E. aerogenes Inducers/Stress (Antibiotics) ? ? ramA + mar0 marR marA marB + + + acrR acrAB tolC micF efflux pumps porins
Regulation of permeability and MDR of E. aerogenes inducer ? inducer marC marR marA marB ? ramR ramA inducer ? inducer inducer ? mic Omp36, 35,37 ompX acrR acrA acrB tolC ? hns EFFLUX INFLUX
Functional characterization Involvement in efflux activation Measurements of chloramphenicol intracellular accumulation 100 MIC ( µ g/ml) ATCC ATCC13048 75 Drugs control + p ramA ATCC,p ramA + CCCP 50 cefepime 0.25 4 25 0.5 2 Norfloxacin ATCC,p ramA Chloramphenicol 8 32 0 0 200 400 600 800 Tetracycline 4 16 T (s) Expression of RamA is responsible for an active efflux of chloramphenicol
Functional characterization Involvement in porin expression ATCC13048, pRam JM109, pRam ATCC13048 JM109 kDa 56 Porin 37 29 Immunodetection of porins by polyclonal antibody prepared against a peptide located within the internal porin L3 loop of enterobacterial porins
Functional characterization RamA effect on marRAB operon transcription β -galactosidase activity (Miller units) 2500 1950 2000 Effect of RamA expression on a chromosomal mar :: LacZ 1500 1000 RamA is able to modulate the 480 500 marRAB operon expression 0 E.coli E.coli + RamA β -galalactosidase assay evaluating activity of the mar promoter by assaying the ß-gal activity of a chromosomal mar ::lacZ operon fusion
Project Role of ramA Regulation of ramA Relationship between ramA and progression to high-level MDR, persistence in environment and virulence of nosocomial bacteria Genetic organization of ramA and its flanking regions Special attention: ram -gene only present in E. aerogenes , K. pneumoniae and Salmonella
Identification of mutations in ramA Focus on E. aerogenes clinical isolates with a MDR phenotype PCR and sequencing Different strains : • 2 laboratory strains (ATCC 13048 and ATCC 15038) • 47 clinical isolates with or without MDR phenotype (efflux and/or impermeability) 48% porin-negative 87% efflux-positive • 9 laboratory strains raised under increasing Imipenem concentration • Strain CM64 (Efflux) : laboratory mutant
Characterization of RamA RamA ----MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIR 56 MarA MMSRRNDNAITIHSILSWIEENLESPLSLEKVSERSGYSKWHLQRMFKKETGHSLGQYIR 60 * * * * ** ** ********* * * *** *** RamA ERKLLLAARDLRDTDQRVYDICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAH--- 113 MarA SRKLTEIAQKLKQSNEPILYLAERYGFESQQTLTRTFKNYFDVPPHKYRITNVPGESRYL 120 *** * * *** **** ** * * ** ** * RamA -------- MarA MPLNNYCC 128 A 113aa protein 45% of homology with MarA Belongs to the AraC-XylS family of regulators: 2 Helix-Turn-Helix binding motifs with conserved motifs necessary to the regulatory function Absent in E. coli
Comparison of RamA 13048 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75 15038 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75 IMP MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75 CM64 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75 EA7 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75 EA19 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75 103280 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75 112978 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75 EA27 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75 *************************************************************************** 13048 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114 15038 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114 IMP DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114 CM64 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114 EA7 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114 EA19 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114 103280 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114 112978 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114 EA27 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114 *************************************** No changes between laboratory strains and clinical isolates
Comparison of RamA « The transcriptional control of the marRAB operon may be affected by the “marbox” sequence, a cis -acting element within marO identified as a MarA binding site. » (Alekshun, Levy 1997) « As MarA is able to autoregulate its expression, we looked for the presence of a “marbox” in the promoter region of ramA . From the consensus sequence of the E. coli marbox and the putative MarA binding site of the E. aerogenes mar- operon, we identified a putative marbox in the ramA promoter region. » (Chollet et al. 2004) Now: We can be sure, that a marbox is present in front of ramA
Comparison of RamA marbox 13048 CATTTAACGCCTGGTGGCGC- --------------------- GGAGAGA ----- -ATG- ------ 65 15038 C-------------------- --------------------- GGAGAGA ----- ----- ------ 65 IMP C-------------------- --------------------- GGAGAGA ----- ----- ------ 65 CM64 C-------------------- --------------------- GGAGAGA ----- ----- ------ 65 EA7 C-------------------- --------------------- G A ----- ----- ------ 60 EA19 C-------------------- --------------------- GGAGAGA ----- ----- ------ 65 103280 C-------------------- --------------------- GGAGAGA ----- ----- ------ 65 112978 C-------------------- --------------------- GGAGAGA ----- ----- ------ 65 EA27 C-------------------- --------------------- G A ----- ----- ------ 60 ********************* ********************* * * ***** ***** ****** Deletion between Marbox and start codon in 44 of 47 clinical isolates (93.6%)
Identification of ramR 5` romA ramA 3` 3` ramR 5` romA - encodes a putative protein of the outer membrane which could interfere with porins translation ramR - encodes a protein of 193aa belonging to the family of the TetR repressors as AcrR with an HTH motif
Structure of the TetR repressor Transcriptional regulators with an HTH binding motif at C-terminal TetR, QacR, EthR and AcrR Active in homodimer on DNA
Structure of the TetR repressor RamR of E. aerogenes aligned to putative tetR-family transcriptional regulator [ Salmonella enterica subsp. Enterica serovar Typhi str. CT18] ATCC13048 VARPKSEDKKQALLEAATAAFAQSGIAASTSAIARSAGVAEGTLFRYFATKDELLNELYL 60 EA27 V-----------------A-F---------SA---S---V--------------L-E--- 60 Salmonella M-----------------Q-I---------AV---N--I---------------I-T--- 60 :***************** *:*********:.***.**:***************:* *** ATCC13048 AIKMRLVQTMIAGLNPDEKRPKENARNIWNSYIDWGMRNPMEYRAIRRMALSERITDETR 120 EA27 AI-MR-V-T--DG-NPDEKRP-ENA-N------D--MRNSMEY----RM-L--R--D--R 120 Salmonella HL-QN-C-S--ME-DRSITDA-TMT-F------S--LNHPARH----QL-V--K-—K--E 120 :* .* *:** *: . . .* :* ******.**:.:. .:****::*:**:**.**. ATCC13048 IQVKESFPELNEMCQLSVKAVFLSDAYRAFGDALFLSLAETTIEFASHDPQRAREIIALG 180 EA27 SQVKES----NEM-QL--KA--L--A------A---S-----IE--SH--Q--R-I---- 180 Salmonella QRADDM----RDL-HR--LM--S--Y------L---L-----DF--RD--R--E-I---- 180 :..: ****.::*: ** **:** ******.***:*****::**::** ** * **** ATCC13048 FEAMWNALHENESQ- 194 EA27 -----ECAA------ 189 Salmonella -----RALTREEQ-- 193 *****.. . Identities= 123/192 (64%)
Recommend
More recommend
