Identification of Proteus and

Providencia porins

RTN - MTR meeting

Que-Tien Tran, Marseille, April 11th 2008

Dealing with infectious diseases

• Emergence of nosocomial infections

• Decreasing antibiotic susceptibility among Gram-negativebacteria (GNB) in clinic – Multidrug resistant (MDR) isolates frequently observed

• Inefficacity of conventional antibacterial agents - Intrinsicresistance

• Requirement of new alternative efficacious molecules for therapeutic

• Full understanding about resistance mechanisms and theirregulation is difficult but urgently necessary

The bacterial outer membranes

• First barrier of cell defense

• Molecular filter for hydrophilic molecules or permeability barrierfor hydrophobic substances (especially in enteric bacteria).

• Modification of the permeability may concern several antibioticclasses

• Intrinsic resistance of bacteria

Porins

• Pore-forming proteins in outer membrane of GNB, mitochondria, plastids

• Uptake pathway of small molecules (≤600 Da) includingantibiotics: β-lactams, quinolones…

• Modified porin expression (decreasing or mutation) → change of membrane permeability

• Most studied porins: E. coli OmpF, OmpC & PhoE

• Porins classification:

– General (OmpF, OmpC , PhoE) or specific (LamB, ScrY…)

– Trimer (OmpF, OmpC, PhoE) or monomer (OmpG…)

Internal loop3

A) Trimer viewed from the top. B) Monomer viewed from the side.

C) Residues at the constriction zone of the monomer viewed from

the top (Cowan et al., 1992)

E. coli OmpF

Constriction domain of OmpF porin

It is now required to extend our expertise concerning the E. coli porins to other less-studied Gram-negative pathogens that cause emerging infectious diseases due to their

high resistance level.

Proteus sp. and Providencia sp.

• GNB, Enterobacteriaceae, tribe Proteeae (Proteus, Providencia, Morganella)

• Straight rods, motile, facultative anaerobic

• Environment, gastrointestinal flora

• Opportunistic pathogens, urinary tract infection (bladder stone) → Risk group 2 (lab accessibility in Marseille)

• Hospital-acquired infection

• They are closed in term of DNA relatedness but distant from E. coli

not yetP. mirabilis by Sanger InstituteGenomic

annotation

Aminoglycosides,

fluoroquinolone, C3G,

carbapenem

Aminoglycosides, quinolone,

C3G-C4G, carbapenem,

azthreonam, ticarcillin-

clavulanic acid, trimethoprime-

sulfamethoxazole

Antibiotic

susceptibility

Providencia stuartii

P. heimbachae

P. rettgeri

P. rustigianii

P. alcalifaciens

Proteus vulgaris

P. mirabilis

P. myxofaciens

Species

Providencia sp.Proteus sp.

Proteus sp. and Providencia sp.

Providencia stuartii

• Cutaneous colonisation

• Species with isolates the most resistant towards antibiotics of the family

• Susceptibility : C3G

Carbapenem

Aminoglycoside

Fluoroquinolone

• Antibiotic resistance

– Cephalosporinase (AmpC)

– ESBL: TEM, SHV, CTX-M, VEB…

– Permeability: LPS modification

Outer membrane proteins ?!

ND1621640.125≤0.06≤0.061P. vulgaris 5860

ND644>2>220.125≤0.062P. mirabilis 99594

ND3240.250.250.25≤0.06≤0.062P. mirabilis ATCC 29906 (PR14)

12851264>512>256>2560,514P. stuartii 2636

ND3264>512>256>256114P. stuartii 99645

3225664>512>256>2561-0.548-4P. stuartii NEA16

1282563225664128≤0.060.252P. stuartii 65237

ND3216410.50.250.252P. stuartii 19539

≤0.06322≤0.06≤0.06≤0.06≤0.06≤0.062P. stuartii ATCC 29914

SFXCMFOXCAZCPOFEPMPMEPMIPM

Abbreviations: IPM (imipenem), EPM (ertapenem), MPM (meropenem), FEP (cefepime), CPO (cefpirome),

CAZ (ceftazidime), FOX (cefoxitin), CM (chloramphenicol) , SFX (sparfloxacine), ND (not determined).

Susceptibility to antibiotics by minimal inhibitory

concentration (MIC in µg/ml)

Analysis of outer membrane proteins

1. P. stuartii ATCC 29914

2. P. stuartii 65237

3. P. stuartii 19539

4. P. stuartii NEA16

5. P. mirabilis ATCC 29906

6. P. vulgaris 5860

Negative detection of the antigenic site located inside porin

which is reported as loop 3 marker of enterobacterial porins

In vitro resistance selection with cefepime

1) P. stuartii ATCC 29914 (no

antibiotic treatment)

2) FEP 0.06 µg/ml

3) FEP 0.09 µg/ml

4) FEP 0.25 µg/ml

5) FEP 0.5 µg/ml

The abbreviation means that

the variant was selected at the

concentration of cefepime

indicated.

Same response observed with cefoxitin

Resistance selection with imipenem

1) P. stuartii ATCC 29914 (no

antibiotic treatment),

2) IPM 1 µg/ml,

3) IPM 2 µg/ml,

4) IPM 4 µg/ml,

5) IPM 6 µg/ml,

6) IPM 8 µg/ml.

The abbreviation indicates that the

variants were selected at the

concentration of imipenem

mentioned.

Analysis of outer membrane proteins

in resistant variants

• Several proteins involved in response to antibiotic by losing or decreasing the expression

• Cefepime & cefoxitin variants present a decrease of production of immunorelated porins

Resistance by lowering cephalosporin uptake

• No resistant variant selected with imipenem exhibit a modification of immunorelated porins

Porin expression in response to imipenem is different than to cephalosporins (at least with the concentrations tested)

Comparison of predicted a.a sequences OmpPm &

OmpPv with OmpF, OmpC, PhoE

The internal loop 3 domain

In Proteus, L3 loop length is well conserved with important

modification of residues compared to E. coli general porins

This may explain the negative detection of the enterobacterial

loop 3 marker by F4 antibody

• Semi-conservation of amino acid sequences compared

to E. coli general porins

• Conservation of typical porin structure with:16 β-strands

8 periplasmic turns

8 extracellular loops

• Several porin key residues conserved

• Important modification in the internal L3 loop

Proteus porin

Special conformation of the eyelet inside porin ?

Different functional and structural organization of

porin channel ?!

Involvement in antibiotic susceptibility ? Role of L3

loop ?

Discussion on OmpPm

What about Providencia porin?

Southern blotting

Templates: genomic DNA

Positive controls: ompPm amplicon

Different restrictases used,

Positively clear signal obtained with HindIII

Probe: Proteus mirabilis ompPm

Hybridization t°: 50°C

P. mirabilis

ompPm gDNA

P. stuartii gDNA

EcoRI HindIII

Fragment about

3.5 Kb

Southern blotting

Providencia porin amplification by PCR

1 2 3 4 5 6 7

PCR for Providencia porin amplification

The products were cloned into pGem-T-easy vector for sequencing

1 2 3 4 5 6 7

Providencia porin sequence compared

to Proteus homolog

The protein sequence is bout 50% identity compared to

OmpF, OmpC and PhoE porins

ompPst

Adaptor PCR as a technique of choice

to get the whole gene

5’ 3’??

Adaptor PCR

Adaptor PCR in Providencia stuartii

Ta = 50°C Ta = 45°C

Restrictases : NcoI

NcoI_adaptor

Sequencing: in waiting

Future aims

Expression – Regulation of the porins

Physico-chemical characterization

Antibiotic flux through the porins

…

Acknowledgement

Prof. Mathias Winterhalter

Prof. Matthias Ullrich

Dr. Helge Weingart

Dr. Jean-Marie Pagès

Dr. Anne Davin-Régli

Biophysics group

Microbiology group

Laboratory UMR-MD1,

Facultés de Médecine et de

Pharmacie, Marseille

Thank you for your attention!