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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
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