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Drastically alter Bacterial environment so that multiple systems become inoperative. Therefore, many genes would have to mutate to cause resistance.
Bleach (NaOCl) – Oxidize multiple targets in bacteria
Detergents/soap/alcohol – disrupt membrane
Heat/pH extremes - denature proteins
UV irradiation – grossly damage DNA
Antimicrobial Peptides (AMPs) – lyse membranes
Bacteria chromosome
plasmids
Plasmids in bacteria often contain genes critical for …..antibiotic resistance, toxins, natural product metabolismF factor plasmid (for sexual transmission of plasmids)
Bacteria can transfer antibiotic resistance plasmids between species
Practices that Foster Resistance1. taking antibiotics for non-bacterial illness
2. not taking all of antibiotic
3. non-human use of antibiotics antibiotics as growth promoters in animals
Resistant Bacteria ― strategies
1. mutated target enzyme – evasion strategy
2. enzyme to destroy antibiotic – attack strategy
3. efflux channel – bailout strategy
Fighting Back at Resistant Bacteria
3. Find new targets for Drugs
4. Find new classes of drugs
2. Develop ‘co’-drugs
1. Develop new drugs for same targets
Sulfthiazole resistance ― case study
1985 – 5 isolates of resistant Streptoccoccus Pyogenes saved from patients in Sweden Hospital
1990’s – Genomes from normal and resistant isolates compared – highly mutated genes cloned & expressed in E. coli.
DHPS gene found to be mutated. (evasion strategy)
Pathway genes: folC - folE - folP - folQ - folK
folE = GTP cyclohydrolasefolQ = dihydroneopterin aldolasefolK = hydroxymethydihydropterin pyrophosphatase converts GTP into dihydropteridin unit
folP = DHPS (dihydropteroate synthetase) adds PABA unit
folC = dihydrofolate synthetase adds glutamate unit
H2N- -COOH
H2N-
N
NN
HN
O OHN- -C- NH-CH-COO-
CH2
CH2
COO-
Dihydrofolate Biosynthesis
Pathway genes: folC - folE - folP - folQ - folK
folE = GTP cyclohydrolasefolQ = dihydroneopterin aldolasefolK = hydroxymethydihydropterin pyrophosphatase converts GTP into dihydropteridin unit
folC = dihydrofolate synthetase adds glutamate unit
NH2
O=S=O | NH2
sulfanilamide
sulfathiazole
NH2
N-H
N S
O=S=O
E. Coli - DHPS
sulfonamide
E. Coli - DHPS
KM(inhib) = KM (1 + [I]/Ki)
KM Ki G1 (suscep) 0.7mM 0.2mM G56 (res) 2.5mM 27.4mMDifference 3.6x 137x
DHPS Kinetics
They are analogs of the peptide component of the bacterial cell wall
penicillins and cephalosporins are antibiotic classes that possess lactam ring
b-lactamases of varying specificities are often found in ‘R’ plasmids of resistant bacteria.
penicllin and b-lactams inhibit the cell wall synthesis in bacteria
Lactams contain a 4-membered ring with an amide nitrogen and a keto group.
b-lactamases destroy b-lactams by cleaving (O=C ― N) in lactam structure. Attack strategy destroys antibiotic before it can kill bacteria .
Penicillin inhibits last connection in making bacterial cell wall … Glycopeptide Transpeptidase
Glycopeptide transpeptidase
b-lactam antibiotic
Glycopeptide transpeptidase
b-lactam antibiotic
Polysaccharide
X-X-X-A-A
X-X-X-A-A
G-G-G-G-G
G-G-G-G-G
X-X-X-A-A
X-X-X-A-A
G-G-G-G-G
G-G-G-G-G
Peptidoglycan
Bacterial Cell Wall Completion
X-X-X-A
X-X-X-A
G-G-G-G-G
G-G-G-G-G
G-G-G-G-G
G-G-G-G-G
X-X-X-A-A
X-X-X-A-A
R C = O H - N S CH3
C - C C - CH3
C - N C O COO-
CH3 CH3
- N - C - C - N - C O COO-
penicillin
-D-Ala-D-Ala
mimics AAseq of peptidelinker
b-lactamase
R C = O H - N S CH3
C - C C - CH3
C - N C O COO-
penicillin
O
CH3
C - C C - CH3
C - N C O COO-
O
clavulanate
given along with penicillin it will inhibit penicillinase
OH
OO
Cl
Cl
NNCO
OHOH
OH
N
O
HOOC
O
N
O
OH
N
ON-CH3
O
NH2
HO
Vancomycin binds to D-Ala – D-Ala peptide unit
Resistance due to target mutation in peptidoglycan – D Ala to D – lactate giving 3x less drug affinity due to missing H-bond. replacing C=O with CH2 produces 100x activity to mutant retains only 3% activity to sensitive bacteria. C&E News Feb 13, 2006
Vancomycin (blue)
D-Ala – D-Ala
Efflux Pumps ― bailout strategy
Many efflux pumps expel a broad range of compounds – may have normal anti-toxin function.
efflux pump inhibitors, like b-lactamase inhibitors, could well be analogs of the original antibiotic and have mild antibiotic activity as well.
E. Coli ACRB
Multi-drug efflux transporter
Efflux Pumps
antibiotic
bacteria cell membrane
antibiotic target
effluxpump
EP inhibitor
Pdb – 2f2m EmrEtetraphenylphosphonium
Cl
Cl
ClO
O
Triclosan
inhibits enoyl reductase
Fatty Acid Synthesis
acetylCoA + HCO3- + ATP malonyl CoA +ADP
acetylCoA + ACP acetyl-ACP + CoA
malonylCoA + ACP malonyl-ACP + CoA
acetyl-ACP +malonyl-ACP acetoacetyl-ACP + CO2 + ACP