rama is involved in the control of membrane permeability in multidrugresistant (mdr) ·...

RamA is involved in the control of membranepermeability in multidrugresistant (MDR)

E. aerogenes isolatesA. Molitor, J-M. Pagès and A. Davin-Regli

UMR-MD1, Université de la MéditerranéeMarseille, France

Marie Curie Meeting 11th April 2008

Common hospital pathogen from the respiratorytract 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 theoxydative 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 inS.enterica (Ricci et al. 2006)

• RamA elicits high level of resistance to diverse Antibiotics and is associated withFQ resistance (Schneiders et al. 2003)

K. pneumoniae:

E. aerogenes and E. cloaceae:

• RamA, a transcriptional regulator associated with decreased susceptibility totigecycline (Bradford et al. 2006)

Salmonella:

• The overexpression of RamA induces a MDR phenotype associated to a decreasein porin production and an increase in components of the AcrAB-TolC effluxpump in E. aerogenes. Moreover RamA is a transcriptional activator of the marregulon.(Chollet et al. 2004)

• RamA, a transcriptional regulator associated with decreased susceptibility totigecycline (Bradford et al. 2007)

RamA

Regulation of membrane permeability in gram negativebacteria

Inducers/Stress(Antibiotics)

mar0 marR marA marB +

acrR acrABmicF

porins

+

tolC

? ?ramA

Regulation of permeability and MDR of E. aerogenes

effluxpumps

+ +

marC marR marA marB

ompX

mic

hns

ramR

acrR

ramA

acrA acrB tolCOmp36, 35,37

INFLUXEFFLUX

inducerinducer

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

0

25

50

75

100

0 200 400 600 800

T (s)

ATCC

ATCC,pramA + CCCP

ATCC,pramA

Functional characterization Involvement in efflux activation

Porin

kDa

56

37

29

JM10

9, p

RamATCC13

048

JM10

9

ATCC1304

8, p

Ram

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

Functional characterization Involvement in porin expression

Effect of RamA expressionon a chromosomal mar::LacZ

RamA is able to modulate themarRAB operon expression

β-galalactosidase assay evaluating activity of the mar promoter by assaying the ß-galactivity 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

0

500

1000

1500

2000

2500

spc105 spc105,RamA

Role of ramA

Regulation of ramA

Relationship between ramA and progression tohigh-level MDR, persistence in environmentand 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 MDRphenotype

PCR and sequencing

Different strains :

• 47 clinical isolates with or without MDRphenotype (efflux and/or impermeability)

48% porin-negative 87% efflux-positive

• 2 laboratory strains (ATCC 13048 andATCC 15038)

• Strain CM64 (Efflux) : laboratory mutant

• 9 laboratory strains raised under increasingImipenem 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-Helixbinding motifs with conserved motifs necessary to the regulatoryfunction

Characterization of RamA

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

Comparison of RamA

No changes between laboratory strains and clinicalisolates

13048 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 7515038 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75IMP MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75CM64 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75EA7 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75EA19 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75103280 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75112978 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75EA27 MNISAQVIDTIVEWIDDNLHQPLRIDDIARHAGYSKWHLQRLFLQYKGESLGRYIRERKLLLAARDLRDTDQRVY 75 *************************************************************************** 13048 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 11415038 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114IMP DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114CM64 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114EA7 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114EA19 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114103280 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114112978 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114EA27 DICLKYGFDSQQTFTRIFTRTFNQPPGAYRKENHSRAHX 114 ***************************************

Comparison of RamA

« The transcriptional control of the marRAB operon may be affectedby the “marbox” sequence, a cis-acting element within marOidentified as a MarA binding site. »(Alekshun, Levy 1997)

« As MarA is able to autoregulate its expression, we looked for thepresence of a “marbox” in the promoter region of ramA. From theconsensus sequence of the E. coli marbox and the putative MarAbinding site of the E. aerogenes mar-operon, we identified a putativemarbox in the ramA promoter region. »(Chollet et al. 2004)

Now: We can be sure, that a marbox is present in frontof ramA

Comparison of RamA

13048 CATTTAACGCCTGGTGGCGC----------------------GGAGAGA------ATG------- 6515038 C-----------------------------------------GGAGAGA---------------- 65IMP C-----------------------------------------GGAGAGA---------------- 65CM64 C-----------------------------------------GGAGAGA---------------- 65EA7 C-----------------------------------------G A---------------- 60EA19 C-----------------------------------------GGAGAGA---------------- 65103280 C-----------------------------------------GGAGAGA---------------- 65112978 C-----------------------------------------GGAGAGA---------------- 65EA27 C-----------------------------------------G A---------------- 60

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

Deletion between Marbox and start codon in 44 of 47clinical isolates (93.6%)

marbox

romA - encodes a putative protein of the outer membranewhich could interfere with porins translation

ramR - encodes a protein of 193aa belonging to the family ofthe TetR repressors as AcrR with an HTH motif

Identification of ramR

ramR

romA ramA 3`

3`

5`

5`

Transcriptional regulators withan HTH binding motif atC-terminal

Active in homodimer onDNA

TetR, QacR, EthR and AcrR

Structure of the TetR repressor

ATCC13048 VARPKSEDKKQALLEAATAAFAQSGIAASTSAIARSAGVAEGTLFRYFATKDELLNELYL 60EA27 V-----------------A-F---------SA---S---V--------------L-E--- 60Salmonella M-----------------Q-I---------AV---N--I---------------I-T--- 60

:***************** *:*********:.***.**:***************:* ***

ATCC13048 AIKMRLVQTMIAGLNPDEKRPKENARNIWNSYIDWGMRNPMEYRAIRRMALSERITDETR 120EA27 AI-MR-V-T--DG-NPDEKRP-ENA-N------D--MRNSMEY----RM-L--R--D--R 120Salmonella HL-QN-C-S--ME-DRSITDA-TMT-F------S--LNHPARH----QL-V--K-—K--E 120

:* .* *:** *: . . .* :* ******.**:.:. .:****::*:**:**.**.

ATCC13048 IQVKESFPELNEMCQLSVKAVFLSDAYRAFGDALFLSLAETTIEFASHDPQRAREIIALG 180EA27 SQVKES----NEM-QL--KA--L--A------A---S-----IE--SH--Q--R-I---- 180Salmonella QRADDM----RDL-HR--LM--S--Y------L---L-----DF--RD--R--E-I---- 180

:..: ****.::*: ** **:** ******.***:*****::**::** ** * ****

ATCC13048 FEAMWNALHENESQ- 194EA27 -----ECAA------ 189Salmonella -----RALTREEQ-- 193

*****.. .

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

Identities= 123/192 (64%)

Structure of the TetR repressor

13048 VARPKSEDKKQALLEAATAAFAQSGIAASTSAIARSAGVAEGTLFRYFATKDELLNELYLAIKMRLVQTMIAGLNPDEKRPKENARNIWNSYIDWGMRNPMEYRAIRRMALSERITDETR 12015038 V----------------------------------------------------------------------------------------------------------------------- 120CM64 V----------------------------------------------------------------------------------------------------------------------- 120IMP V----------------------------------------------------------------------------------------------------------------------- 120EA19 V----------------------------------------------------------------------D---------------------------S-------------------- 120EA7 V----------------------------------------------------------------------D------------------------------------------------ 120EA103280 V----------------------------------------------------------------------------------------------------------------------- 120EA112978 V----------------------------------------------------------------------D------------------------------------------------ 120EA27 V----------------------------------------------------------------------D---------------------------S-------------------- 120 *********************************************************************** *************************** ******************** 13048 IQVKESFPELNEMCQLSVKAVFLSDAYRAFGDALFLSLAETTIEFASHDPQRAREIIALGFEAMWNALHENES 19315038 S------------------------------------------------------------------------ 193CM64 --------------------------------- -------------------------------------- 191IMP ------------------------------------------------------------------------- 193EA19 S----------------------------------------------------------------ECAA 189EA7 S------------------------------------------------------------------------ 193EA103280 ------------------------------------------------------------------------- 193EA112978 S------------------------------------------------------------------------ 193EA27 S----------------------------------------------------------------ECAA 189 ******************************** ******************************

Comparison of RamR

Ala72Asp Pro100Ser

Iso121Ser Deletion154/155

C-Terminal

MarA and MarR

No changes in aminoacid sequence between laboratorystrains 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 (Thymineinstead of Cytosine)

No changes in aminoacid sequence between laboratorystrains and clinical isolates

Results

RamADeletion Ala72Asp Pro100Ser Iso121Ser Deletion 154/155 C-Terminal

ATCC13048ATCC15038 X

CM64 XIMP

EA 19 X X X XEA 7 X X X

EA 103280EA 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 startcodon for clinical isolates (exception EA19, EA103280 andEA112978)

Effect on ramA-transcriptionmust be studied

No changes in promoter region of ramA for laboratory strains andimipenem/chloramphenicol treated strains

93.6% of MDR clinicalisolates show deletion

Discussion

RamR:

Several mutations between laboratory strains and clinical isolates

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

93.6%of clinical isolates (except EA7, EA103280 andEA112978)

C-Terminal

just CM64Deletion154/155

97.8% of clinical isolates (except EA103280) andATCC15038

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, responsiblefor DNA-binding

HTH domain DNA-binding


13048 VARPKSEDKKQALLEAATAAFAQSGIAASTSAIARSAGVAEGTLFRYFATKDELLNELYLAIKMRLVQTMIAGLNPDEKRPKENARNIWNSYIDWGMRNPMEYRAIRRMALSERITDETR 12015038 V----------------------------------------------------------------------------------------------------------------------- 120CM64 V----------------------------------------------------------------------------------------------------------------------- 120IMP V----------------------------------------------------------------------------------------------------------------------- 120EA19 V----------------------------------------------------------------------D---------------------------S-------------------- 120EA7 V----------------------------------------------------------------------D------------------------------------------------ 120EA103280 V----------------------------------------------------------------------------------------------------------------------- 120EA112978 V----------------------------------------------------------------------D------------------------------------------------ 120EA27 V----------------------------------------------------------------------D---------------------------S-------------------- 120

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

13048 IQVKESFPELNEMCQLSVKAVFLSDAYRAFGDALFLSLAETTIEFASHDPQRAREIIALGFEAMWNALHENES 19315038 S------------------------------------------------------------------------ 193CM64 --------------------------------- -------------------------------------- 191IMP ------------------------------------------------------------------------- 193EA19 S----------------------------------------------------------------ECAA 189EA7 S------------------------------------------------------------------------ 193EA103280 ------------------------------------------------------------------------- 193EA112978 S------------------------------------------------------------------------ 193EA27 S----------------------------------------------------------------ECAA 189

******************************** ******************************:. *

Effect on ramA-transcriptionmust be studied

Discussion

RamR:

Various changes e.g. neutral -> acidic, nonpolar -> polarC-Terminal

Deletion154/155

Nonpolar -> polar, tinyIso121Ser

Prolin able to induce a bendPro100Ser

Nonpolar, neutral -> polar, acidicAla72Asp

May be important for ligandbinding and/or dimerisation


Outlook

1. Identify possible structural/functional changes of RamR

2. Compare possible mutagen effects of several antibiotics onRamR

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. coliand E. aerogenes bearing ramA and/or ramR

Clone ramR of laboratory strain in clinical isolate

Clone ramR of clinical isolate in laboratory strain

Cloning



Outlook

Gene reporter to check the effect ofantibiotics as effectors of MDR

Tool to measure expression of regulators

β-Gal assay / fused gene probe





Outlook

Measure and compare trancription of ramAand ramR in laboratory strains and clinicalisolates

Realtime PCR





rama is involved in the control of membrane permeability in multidrugresistant (mdr) ·...

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