Bacterial DiseasesBubonic Plague Tuberculosis Cholera

Sepsis Lyme Disease

Antibiotics

1929 – Penicillin discovered

1933 – Sulfa drugs synthesized

1969 – US surgeon Gen “end infectious diseases???

Today – bacteria with multi-drug resistance. Concern over resistance to ‘last resort’ antibiotics.

1865 – Pasteur - Decay due to living organisms

1867 – Lister – phenol is disinfectant

Enterococcus faecalis

A leading cause of hospital infections

vanomycin = antibiotic of last resort

E faecalis resistant strains for years

can transfer resistance genes to Staphylococcus aureus in lab - MRSA

virulent cause of pneumonia, endocarditis, sepsis etc.

Examples of Antibiotic Targets

Cell Wall Formation - penicillin, cephalosporins, vancomycin

Replication – novobiocin & DNA Gyrase

Transcription – rifampicin & RNA Pol

Translation – puromycin & ribosome ‘A’

Folate biosynthesis – sulfa drugs & DHPS

Fatty Acid synthesis – triclosan & enoyl reductase

Killing Bacteria without Resistance

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

DHPS DHFS

PABA → → dihydrofolate

folP = DHPS (dihydropteroate synthetase) adds PABA unit ― 16% divergence

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

acetoacetyl-ACP + NADPH hydroxybutyryl-ACP + NADP+

hydroxybutyryl-ACP Crotonyl-ACP + H2O

Crotonyl-ACP + NADPH butyryl-ACP + NADP+

Enoyl-ACP reductase

Enoyl reductase (step in fatty acid synthesis)

triclosan

Enoyl reductase (step in fatty acid synthesis)

triclosan

Parikh et. al. (2000) Biochemistry 39, 7645-7650

Triclosan inhibits enoyl-ACP reductase from Mycobacterium Tuberculosis Ki ~ 0.22 mM for crotonyl-ACP & NADH

Y158 F Ki ~ 47 & 36 mM

M161 V triclosan resistant Ki ~ 4.3 mM also less sensitive to isoniazid

triclosan could stimulate TB resistant strains of mycobacterium

New Antibiotics

oxazolidimones (linezolid) – binds 30S subunit of ribosome and prevents mRNA & fMet-tRNA binding.

gemifloxacin – DNA gyrase inhibitor used on respiratory tract infections

daptomycin – blocks peptidoglycan and lipoteichoic acid synthesis (cell wall formation) works on vanomycin resistant enterococci

BPI Protein - (bacterial permeability increasing) naturally found in bactria killing wbc’s – good in combo

Antimicrobial Peptides – defensins & protegrins may function as voltage-gated pores specific for

acidic phospholipids found only in bacteria

New Targets for Antibiotics

sortase – cleaves loosely bound surface proteins in gram (+) bacteria to activate infectivity proteins. (doesn’t kill bacteria)

deformylase – removes formyl group from amino end of bacterial polypeptides – includes actinonen (natural cpd)

Efflux Pump Inhibitors