RamA is involved in the control of membrane permeability in - - PowerPoint PPT Presentation

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 Marie Curie Meeting 11th April 2008

• Common hospital pathogen from the respiratory

tract in France and other european countries (Belgium, Spain, Austria, Germany…)

• High resistance to a broad spectrum of antibiotics:
• Chromosomally derepressed cephalosporinase
• ESBL
• Alteration of porin content
• Active efflux
• Target mutations

Enterobacter aerogenes in Europe

• Two major clinical clones present in Europe

(one more prevalent ~70%)

• RamA is involved in a MDR phenotype in K.pneumoniae (George et al. 1995)
• RamA in Salmonella Typhimurium (ST) is involved in the response to the
• xydative 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)

• RamA elicits high level of resistance to diverse Antibiotics and is associated with

FQ resistance (Schneiders et al. 2003)

• K. pneumoniae:
• E. aerogenes and E. cloaceae:
• RamA, a transcriptional regulator associated with decreased susceptibility to

tigecycline (Bradford et al. 2006) Salmonella:

• 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)

RamA

Regulation of membrane permeability in gram negative bacteria

Inducers/Stress (Antibiotics)

mar0 marR marA marB

+

acrR acrAB

micF

porins + tolC

? ?ramA

Regulation of permeability and MDR of E. aerogenes

efflux pumps + +

marC marR marA marB

• mpX

mic hns ramR acrR ramA acrA acrB tolC Omp36, 35,37

INFLUX EFFLUX

inducer inducer inducer inducer inducer

? ? ? ? ?

Regulation of permeability and MDR of E. aerogenes

Expression of RamA is responsible for an active efflux of chloramphenicol Measurements of chloramphenicol intracellular accumulation

MIC (µg/ml) ATCC13048 Drugs control + pramA cefepime 0.25 4 Norfloxacin 0.5 2 Chloramphenicol 8 32 Tetracycline 4 16

25 50 75 100 200 400 600 800 T (s) ATCC ATCC,pramA + CCCP ATCC,pramA

Functional characterization Involvement in efflux activation

Porin

kDa

56 37 29

JM109, pRam ATCC13048 JM109 ATCC13048, pRam

Immunodetection of porins by polyclonal antibody prepared against a peptide located within the internal porin L3 loop of enterobacterial porins

Functional characterization Involvement in porin expression

Effect of RamA expression

• n a chromosomal mar::LacZ

RamA is able to modulate the marRAB operon expression

β-galalactosidase assay evaluating activity of the mar promoter by assaying the ß-gal activity of a chromosomal mar::lacZ operon fusion

Functional characterization RamA effect on marRAB operon transcription

β-galactosidase activity (Miller units)

E.coli + RamA E.coli

480 1950 500 1000 1500 2000 2500

• 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

Project

• Focus on E. aerogenes clinical isolates with a MDR

phenotype

• PCR and sequencing
• Different strains :
• 47 clinical isolates with or without MDR

phenotype (efflux and/or impermeability)

48% porin-negative 87% efflux-positive

• 2 laboratory strains (ATCC 13048 and

ATCC 15038)

• Strain CM64 (Efflux) : laboratory mutant
• 9 laboratory strains raised under increasing

Imipenem concentration

Identification of mutations in ramA

• Absent in E. coli
• 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

Characterization of RamA

RamA ----MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIR 56 MarA MMSRRNDNAITIHSILSWIEENLESPLSLEKVSERSGYSKWHLQRMFKKETGHSLGQYIR 60 * * * * ** ** ********* * * *** *** RamA ERKLLLAARDLRDTDQRVYDICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAH--- 113 MarA SRKLTEIAQKLKQSNEPILYLAERYGFESQQTLTRTFKNYFDVPPHKYRITNVPGESRYL 120 *** * * *** **** ** * * ** ** * RamA -------- MarA MPLNNYCC 128

Comparison of RamA

No changes between laboratory strains and clinical isolates

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 ***************************************

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

• f ramA

Comparison of RamA

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%)

marbox

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 Identification of ramR ramR romA ramA 3` 3` 5` 5`

Transcriptional regulators with an HTH binding motif at C-terminal

• Active in homodimer on

DNA

• TetR, QacR, EthR and AcrR

Structure of the TetR repressor

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

*****.. .

RamR of E. aerogenes aligned to putative tetR-family transcriptional regulator [Salmonella enterica subsp. Enterica serovar Typhi str. CT18]

Identities= 123/192 (64%)

Structure of the TetR repressor

13048 VARPKSEDKKQALLEAATAAFAQSGIAASTSAIARSAGVAEGTLFRYFATKDELLNELYLAIKMRLVQTMIAGLNPDEKRPKENARNIWNSYIDWGMRNPMEYRAIRRMALSERITDETR 120 15038 V----------------------------------------------------------------------------------------------------------------------- 120 CM64 V----------------------------------------------------------------------------------------------------------------------- 120 IMP V----------------------------------------------------------------------------------------------------------------------- 120 EA19 V----------------------------------------------------------------------D---------------------------S-------------------- 120 EA7 V----------------------------------------------------------------------D------------------------------------------------ 120 EA103280 V----------------------------------------------------------------------------------------------------------------------- 120 EA112978 V----------------------------------------------------------------------D------------------------------------------------ 120 EA27 V----------------------------------------------------------------------D---------------------------S-------------------- 120 *********************************************************************** *************************** ******************** 13048 IQVKESFPELNEMCQLSVKAVFLSDAYRAFGDALFLSLAETTIEFASHDPQRAREIIALGFEAMWNALHENES 193 15038 S------------------------------------------------------------------------ 193 CM64 --------------------------------- -------------------------------------- 191 IMP ------------------------------------------------------------------------- 193 EA19 S----------------------------------------------------------------ECAA 189 EA7 S------------------------------------------------------------------------ 193 EA103280 ------------------------------------------------------------------------- 193 EA112978 S------------------------------------------------------------------------ 193 EA27 S----------------------------------------------------------------ECAA 189 ******************************** ******************************

Comparison of RamR Ala72Asp Pro100Ser Iso121Ser Deletion 154/155 C-Terminal

MarA and MarR

• No changes in aminoacid sequence between laboratory

strains and clinical isolates

MarA:

• Clinical isolates show a different nucleotide at position 366 of 387

(Guanine instead of Adenine)

MarR:

• EA103280 shows a different nucleotide at position 328 of 378 (Thymine

instead of Cytosine)

• No changes in aminoacid sequence between laboratory

strains and clinical isolates

Results

RamA Deletion Ala72Asp Pro100Ser Iso121Ser Deletion 154/155 C-Terminal ATCC13048 ATCC15038 X CM64 X IMP EA 19 X X X X EA 7 X X X EA 103280 EA 112978 X X EA 27 X X X X X RamR

Represents 91.5% of clinical strains

Discussion

RamA:

• No changes in RamA itself
• BUT: Deletion of five nucleotides between marbox and start

codon for clinical isolates (exception EA19, EA103280 and EA112978)

Effect on ramA-transcription must be studied

• No changes in promoter region of ramA for laboratory strains and

imipenem/chloramphenicol treated strains

93.6% of MDR clinical isolates show deletion

Discussion

RamR:

Several mutations between laboratory strains and clinical isolates

ALSO: Several MDR E. aerogenes strains, isolated in Nîmes (researches made by Jean-Philippe Lavigne), show same differencies.

93.6%of clinical isolates (except EA7, EA103280 and EA112978) C-Terminal just CM64 Deletion154/155 97.8% of clinical isolates (except EA103280) and ATCC15038 Iso121Ser 93.6% of clinical isolates (except EA7, EA103280, EA112978) Pro100Ser: 97.8% of clinical isolates (except EA103280) Ala72Asp

Discussion

RamR:

BUT: Mutations are not located in helix-turn-helix motive, responsible for DNA-binding

HTH domain DNA-binding Several mutations between laboratory strains and clinical isolates

13048 VARPKSEDKKQALLEAATAAFAQSGIAASTSAIARSAGVAEGTLFRYFATKDELLNELYLAIKMRLVQTMIAGLNPDEKRPKENARNIWNSYIDWGMRNPMEYRAIRRMALSERITDETR 120 15038 V----------------------------------------------------------------------------------------------------------------------- 120 CM64 V----------------------------------------------------------------------------------------------------------------------- 120 IMP V----------------------------------------------------------------------------------------------------------------------- 120 EA19 V----------------------------------------------------------------------D---------------------------S-------------------- 120 EA7 V----------------------------------------------------------------------D------------------------------------------------ 120 EA103280 V----------------------------------------------------------------------------------------------------------------------- 120 EA112978 V----------------------------------------------------------------------D------------------------------------------------ 120 EA27 V----------------------------------------------------------------------D---------------------------S-------------------- 120

*********************************************************************** ***************************.********************

13048 IQVKESFPELNEMCQLSVKAVFLSDAYRAFGDALFLSLAETTIEFASHDPQRAREIIALGFEAMWNALHENES 193 15038 S------------------------------------------------------------------------ 193 CM64

• -------------------------------- -------------------------------------- 191

IMP

• ------------------------------------------------------------------------ 193

EA19 S----------------------------------------------------------------ECAA 189 EA7 S------------------------------------------------------------------------ 193 EA103280

• ------------------------------------------------------------------------ 193

EA112978 S------------------------------------------------------------------------ 193 EA27 S----------------------------------------------------------------ECAA 189 ******************************** ******************************:. *

Effect on ramA-transcription must be studied

Discussion

RamR:

Various changes e.g. neutral -> acidic, nonpolar -> polar C-Terminal Deletion154/155 Nonpolar -> polar, tiny Iso121Ser Prolin able to induce a bend Pro100Ser Nonpolar, neutral -> polar, acidic Ala72Asp

May be important for ligand binding and/or dimerisation

Several mutations between laboratory strains and clinical isolates

Outlook

• 1. Identify possible structural/functional changes of RamR
• 2. Compare possible mutagen effects of several antibiotics on

RamR

ATCC 13048 CM64 ≠ ATCC 13048 Imp = ATCC 13048

Chloramphenicol Imipenem

Outlook

• 3. Measure the transcription of ramA and ramR both

in vitro and in vivo

Attempt is to design a plasmid suitable for E. coli and E. aerogenes bearing ramA and/or ramR Clone ramR of laboratory strain in clinical isolate Clone ramR of clinical isolate in laboratory strain

Cloning

• 1. Identify possible structural/functional changes of RamR
• 2. Compare possible mutagen effects of several antibiotics on

RamR

Outlook

Gene reporter to check the effect of antibiotics as effectors of MDR Tool to measure expression of regulators

β-Gal assay / fused gene probe

• 3. Measure the transcription of ramA and ramR both

in vitro and in vivo

• 1. Identify possible structural/functional changes of RamR
• 2. Compare possible mutagen effects of several antibiotics on

RamR

Outlook

Measure and compare trancription of ramA and ramR in laboratory strains and clinical isolates

Realtime PCR

• 3. Measure the transcription of ramA and ramR both

in vitro and in vivo

• 1. Identify possible structural/functional changes of RamR
• 2. Compare possible mutagen effects of several antibiotics on

RamR