Mechanisms of antibiotic resistance

Alessio Branchini13 dicembre 2010

Corso di Laurea in Scienze Biomolecolari e Cellulari

Macromolecole Biologiche

Parte degli argomenti trattati si ritrovano su testi quali:

- James D. Watson, “Biologia molecolare del gene” ,

SESTA EDIZIONE, Zanichelli

- David L. Nelson, “I principi di biochimica di

Lehninger” , QUARTA EDIZIONE, Zanichelli

Basic bacterial cell structure

Mechanisms of antibiotic function

Mechanisms of antibiotic resistance• Increased export (efflux pumps)

• Hydrolysis of the drug (β-lactamase)

• Modification of the drug target

• Modification of the antibiotic

• Reduced uptake

• Cell-to-cell transfer

Mechanisms of antibiotic resistance• Increased export (efflux pumps)

• Hydrolysis of the drug (β-lactamase)

• Modification of the drug target

• Modification of the antibiotic

• Reduced uptake

• Cell-to-cell transfer

Efflux pumps are membrane-spanning protein complexes with different substrate specificity

Increased export (efflux pumps)

AcrB = Acriflavine resistance protein B MFP = Membrane Fusion ProteinOMF = Outer Membrane Factor

The genetic elements encoding efflux pumps may be encoded onchromosomes and/or plasmids, thus contributing to both intrinsic(natural) and acquired resistance

The base structure of the HAE1 family efflux pumps

OMF

MFP

RND

HAE1 = Hydrophobe/Amphiphile Efflux-1 family

The RND pump functions asan H+/substrate antiporter

12 transmembrane α-helices

AcrB-AcrA-TolC complex exports drugs by a proton motive force-dependent mechanism in E. coli

(OMF)

(MFP)

(RND)

AcrB is a homotrimer composed by three domains

- Transmembrane domain ���� insertion in the bilayer- Pore domain ���� site of substrate interaction/transport - TolC docking domain ���� interaction with TolC

1 α-helix from each monomer

Top view of pore domain

(~30Å)

(~40Å)

(~50Å)

Drug transport mechanism of AcrB

Yu et al., J Bacteriol, 2003

Drug-binding site of AcrB

Putative residues involved in substrate binding:

Hydrophobicsubstrate-binding

pocket

Leu25Lys29Asp 99Asp101Val105Asn109Gln112Pro116Phe136Gln178

Val382Ala385Phe386Gly387Phe458Phe459Phe610Phe615Phe617Phe628

Hydrophobic interactions have a major role in AcrB-substrate

binding

Cysteine-scanning study on AcrB drug-binding site

“Empty”cells

AcrB-wt plasmid

Murakami et al., J Biol Chem, 2004

A fluorescent compound is used to study the efflux activity(decreased fluorescence) of different Cys-substituted mutants

Each AcrB monomer contains a proton translocationsite reponsible for the creation of the proton motiveforce which allows the antirporter-based extrusion ofsubstrates

Top view of transmembrane domain

Alanine-scanning study for identifying AcrB residues involved in H + transport

wt-AcrB = 100% activity

AcrB alternating site rotation transport mechanismTolC

AcrA

L = Loose

T = Tight

O = Open

Conformational states

Proton translocation site(D407, D408, K940,

R971 and T978)

Seeger et al., Curr. Drug Targets, 2008

AcrB alternating site rotation transport mechanismTolC

AcrA

High

Low

No

Binding affinity

L = Loose

T = Tight

O = Open

Conformational states

Seeger et al., Curr. Drug Targets, 2008

Mechanisms of antibiotic resistance• Increased export (efflux pumps)

• Hydrolysis of the drug (β-lactamase)

• Modification of the drug target

• Modification of the antibiotic

• Reduced uptake

• Cell-to-cell transfer

Target of β-lactam antibiotics: PBP (Penicillin-Binding Protein), an

enzyme involved in the formation of bacterial cell wall

Hydrolysis of the drug by β-lactamases

Modification of the target (PBP)Resistance

Hydrolysis of the drug ( β-lactamases)

Resistance to β-lactam antibiotics

E. cloacae � chromosomal AmpC β-lactamase is expressed atlow levels but is inducible by β-lactams

Peptidoglycan-derivedmuropeptide

Tripeptide releasedby AmpD

Mureinpentapeptideprecursor

Resistance to β-lactam antibiotics

E. cloacae � chromosomal AmpC β-lactamase is expressed atlow levels but is inducible by β-lactams

Peptidoglycan-derivedmuropeptide

Resistance to β-lactam antibiotics

E. cloacae � chromosomal AmpC β-lactamase is expressed atlow levels but is inducible by β-lactams

Peptidoglycan-derivedmuropeptide

ELEMENTI MOBILI –

TRASFERIMENTO RESISTENZA

Mechanisms of antibiotic resistance• Increased export (efflux pumps)

• Hydrolysis of the drug (β-lactamase)

• Modification of the drug target

• Modification of the antibiotic

• Reduced uptake

• Cell-to-cell transfer

Horizontally acquired DNA usually encodes functions that ar eof selective advantage to the organism

R factors � the first example of horizontally transferred antibiotic resistance

The resistance genes are either carried on plasmid-integrated transposons orinserted in an integron

Transposable elements are

involved in the collection

of different resistance

genes, thus leading to

MULTIDRUG RESISTANCE

tnp = transposasetnpR = regulator sequenceres = resolvase binding sitebla = beta-lactamase

Interest in antibiotic resistance and transmissible plasmidsrevealed an important role for mobile elements in dissemination ofresistance genes and in promotion of gene acquisition

Mobile elements encoding a transposaseallowing insertions and excisionsTRANSPOSONS =

Resistance gene capture by integrons

(attC)

cc

cc

intI = Integrase

attI = Recombination site

Pc = Promoter for the integrated gene cassette

Pint = Promoter for the integrase gene

sulI = Gene for sulfonamide resistance

Gene capture systems that utilize site-specificrecombination � insertion of resistance genesdownstream from a strong promoter

INTEGRONS =

Resistance gene capture by integrons

and multidrug resistance

(attC)

c

Up to 8 resistance

genes

Cell-to-cell transfer of resistanceThe “mating-pair”formation complex

TraG

Cell-to-cell transfer of resistanceThe “mating-pair”formation complex

TraG

5’-end