1. Bio 319: Antibiotics
Lecture Six
Topic:
•Biotechnology production
•Engineering polyketide antibiotics
•Bioterrorism/Stockpiling
Dr. G. Kattam Maiyoh
GKM/bio 319/antibiotics/2013
Wednesday, April 3, 2013 1
2. DNA Technology and Antibiotics
Production
• Worldwide over 100,000 tons per
year
• Sales ~ Ksh. 400b
• Annually 100-200 antibiotics are
discovered through labor intensive
laboratory research.
• Involves screening of different
organisms for unique antibiotics.
• This is a very costly process yet
only 1-2% of antibiotics so
discovered adds to the disease
fighting arsenal.
• Recombinant DNA technology can
improve this situation.
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3. Advantages
• DNA tec. Can be used to produce new
structurally unique antibiotics with;
– Increased activities against selected targets
– Decreased side effects
– Increased yields
– Decreased cost of production
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4. Organism of Choice
• Streptomyces
• Unlike E-coli not exist as
individual cells but as extended
aggregates of mycelial filaments
• Must remove cell wall to release
individual cells – to allow
distinction between transformed
cells and non transfomed cells.
• Cells are transformed with
different genes based on the
desired antibiotic to be
produced.
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5. Isolation of antibiotic biosynthesis genes:
by complementation
• Mutant cells (not able to produce
antibiotics) are transformed with DNA from
a clone bank constructed from wild-type
chromosomal DNA
• Transformants are then screened for ability
to produce antibiotics
• e.g. for the antibiotic undecylprodigiosin,
this involves color change to red due to
antibiotic presence
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6. cntd
•Plasmid DNA from the clone that supplied a functional
gene and gene product (i.e. complements the mutant) is
used as a hybridization probe to screen another clone bank
of wild-type chromosomal DNA
•Isolate clones with regions that overlap the probe
sequence.
•If antibiotic biosynthetic genes are clustered, then genes
adjacent to complementing gene are likely to be involved
in the biosynthesis of target antibiotic.
•If scattered, more than one mutant are required to
identify the rest of the genes.
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7. Engineering Polyketide Antibiotics (PA)
• Antibiotics synthesized through succesive
enzymatic condensation of small carboxylic
acids e.g. acetate, propionate and butyrate
• Some PA are produce by plants and fungi
• Most are produced by actenomycetes as sec.
metabolites
• To create new PA;
I. study the functions of the enzymes involved in
the biosynthetic pathway
II. Manipulate the genes that encode this enzymes
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8. FA biosynthesis, quick reminder:
Fatty acid biosynthesis is a stepwise assembly
Fatty acid biosynthesis is a stepwise assembly
of acetyl-CoA units (mostly as malonyl-CoA)
of acetyl-CoA units (mostly as malonyl-CoA)
ending with palmitate (C16 saturated)
ending with palmitate (C16 saturated)
3 Phases
Activation
Elongation
Termination
9. Initiation Overall Reaction
Malonyl-CoA + ACP
CH3C~SCo -
OOC-CH2C~S- ACP + HS-CoA
A O O Acyl Carrier
CO2 HS-CoA Protein
CH3C- CH2C~S- ACP
O O
NOTE:
Malonyl-CoA carbons become new COOH end
Nascent chain remains tethered to ACP
CO2, HS-CoA are released at each condensation
10. β-Carbon Elongation
CH3C- CH2C~S- ACP
O O Reduction
NADPH
β-Ketoacyl-ACP reductase
D isomer H
CH3C- CH2C~S- ACP
HO O Dehydration
-H2O β -Hydroxyacyl-ACP dehydrase
H
CH3C- = C- C~S- ACP
NADPH H Reduction
O
Enoyl-ACP reductase
CH3CH2CH2C~S- ACP
O
11. Biosynthesis PA
• Analogous to synthesis of long-chain FA
• Each condensation cycle results in the
formation of , on the growing chain, of a
β-keto group.
• The repeated steps include;
– Ketoreduction
– Dehydration
– Enoylreduction of the β-keto group of
the growing polyketide chain
Wednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 11
12. Classes of polyketide biosynthetic
enzymes
i. Those involve in the synthesis of aromatic
polyketides (aromatic polyketide syntheses) - have
active sites on same polypeptide
ii. Those with the active sites on separate domains
• In either case, the alteration of a catalytic domain
whose function is known allows for predictable
changes on the structure of the antibiotic
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13. Example: Engineering of Erythromycin
production
• Is synthesized by saccharopolyspora erythraea
• The entire 56kb DNA that contains the ery gene cluster has
been sequenced.
• The erythromycin polyketide synthase gene altered as
follows;
1. DNA encoding beta reductase activity deleted
2. Mutation of DNA region encoding enoylreductase activity
Results;
1. Carbonyl group instead of hydroxyl at C-5
2. Carbon-carbon double bond at C6 and C7
Wednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 13
14. Terrorism
• Terrorism is defined as the unlawful use of force or
violence against persons or property to intimidate or
coerce a government or civilian population in the
furtherance of political or social objectives.
Bioterrorism
• The use, or threatened use, of a micro-organism or the
product of a micro-organism in order to generate fear,
morbidity or mortality in a population.
15. Delivery Mechanisms
• Aerosol route
– Easiest to disperse
– Highest number of people exposed
– Most infectious
– Undetectable to humans
• Food / Waterborne less likely
– Larger volumes required
– More technically difficult
16. • Biologic agents are likely to be used
by terrorists as weapons because:
– They are capable of damaging populations,
economies, and food supplies
– Certain agents are inexpensive to make
– They can be directed at a small group of
people or an entire population
– They can be used to attack people,
economies and food supplies
– They cause fear, panic and social disruption
17. BIOLOGICAL AGENTS
• There are several types of agents. They are
classified as:
– Bacteria
– Rickettsia
– Viruses
– Biotoxins
18. BACTERIA
• Single celled organisms EXAMPLES
capable of causing disease.
• ANTHRAX
These agents, grown on
culture to produce large • SMALL POX
quantities, can be modified • PLAGUE
or “weaponized” for greater
destruction • TYPHOID
• Produces inflamation in • CHOLERA
tissues and/or toxins • TULAREMIA
19. RICKETTSIA
• Vector borne (ticks, lice, EXAMPLES
mosquitos) parasitic • TYPHUS
form of bacteria • ROCKY MT. SPOTTED
• Diseases are difficult to FEVER
treat • Q FEVER
• Variants exist • INDIA TICK FEVER
worldwide
• MEDITERANEAN TICK
FEVER
20. VIRUSES
• Smaller than bacteria
EXAMPLES
• RNA or DNA in a protein
• EBOLA
coat
• LASSA FEVER
• Use living cells to
reproduce • INFLUENZA
• Not affected by • VIRAL HEPATITIS
antibiotics • VIRAL HEMORRHAGIC
FEVERS
21. BIOTOXINS
• Are poisonous by-
EXAMPLES
products of bacteria,
fungi, marine animals
or plants • BOTULINUM
• Do not replicate in the • STAPHLOCCOCAL
host ENTEROTOXIN B
• Are not communicable
• RICIN
• Highly toxic when
delivered as an
aerosol
22. Biological Agents most likely to be
used in a terrorist attack
• Bacteria - anthrax, plague, tularemia
• Virus - small pox, viral hemorrhagic fever
• Biotoxin - botulism
23. Brief history
• Caused by Bacillus anthracis
• Human zoonotic disease
– Spores found in soil worldwide
– Primarily disease of herbivorous animals
• Sheep, goats, cattle
– Occasional human disease
• Epidemics have occurred but uncommon
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24. Bioweapon Potential
• Many countries have weaponized
anthrax
– Former bioweapon programs
• U.S.S.R.,U.S.,U.K., and Japan
– Recent bioweapon programs
• Iraq
– Attempted uses as bioterrorism agent
• WW I: Germans inoculated livestock
• WW II: Alleged Japanese use on prisoners
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25. Features of anthrax suitable as BT
agent
– Fairly easy to obtain, produce and store
– Spores easily dispersed as aerosol
– Moderately infectious
– High mortality for inhalational (86-100%)
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26. PLAGUE
• In nature, fleas living on rodents spread
infection to humans. As a bioterrorist weapon –
inhalation of aerosol leads to pneumonia, sepsis
and infections of bodily organs
• Infectious agent: Yersinia pestis – a gram neg.,
non-motile bacillus
• May be bubonic ( infection of lymph nodes) or
pneumonic (infection of lungs)or septicemic
• Symptoms: cough with bloody sputum, fever,
chill, shortness of breath
28. PLAGUE
• Transmission: may occur person to person by
respiratory droplet inhalation
• Incubation period: 2 to 3 days
• Mortality: 50 to 60%
• Treatment: antibiotics
• Prevention: vaccine ineffective against aerosol
exposure
29. TULAREMIA
• A zoonotic, bacterial infection caused by
Francisella tularensis, a gram negative
coccobacillus
• In nature, bacteria is commonly found in ticks
living on rabbits and transmitted by handling
the animal or by tick bite. Inhalation of
aerosol leads to pneumonia and sepsis
30. TULAREMIA
• Symptoms:sudden and influenza-like with
fever,chills, headache and nausea
• Transmission: not usually person to person
• Incubation period:3 to 5 days(range 1 to 14)
• Mortality:low unless untreated
• Treatment:antibiotics if early, vaccine available
• Prevention: in nature, avoid tick bites and using
gloves when handling infected animals
31. BOTULISM
• Infectious agent: Clostridium botulinum – a
spore forming, anaerobic bacillus
• In nature, may be food borne, wound, or
intestinal. As a bioterrorist weapon, ingestion
or inhalation leads to production of the
neurotoxin and resulting flaccid paralysis
32. BOTULISM
• Symptom: fatigue, weakness, blurred vision, difficulty
in swallowing and speaking, descending muscle
paralysis and respiratory failure
• Transmission: none person to person
• Incubation period: 12 to 72 hours
• Mortality: most lethal compound per weight
• Treatment: antitoxins, respiratory support
• Prevention: vaccine available for types A and B
36. • The Problem with Stockpiling
– Antidotes and treatments are expensive
– Have limited shelf-lives
– Unlikely to be used in large quantities
• 350,000 for prophylaxis
• 2 doses daily for > 7 days of Cipro or Doxycycline
• 4.9 million doses
• Clearly exceeds local supply
• What plans currently exist for such a disaster?
37. Antibiotics to
Counteract Biologic Weapons
• Often older agents are still the
most effective.
• Dosage regimens vary
depending on
– Bacterial agent being
treated
– Treatment vs. prophylaxis
• Most expensive drug is not
necessarily the better drug!
Release of biological weapons could be accomplished in a number of ways. However, it is expected that an aerosol release is the most likely to be encountered in a large-scale bioterrorism event if the goal is to sicken or kill large numbers of people. This is because aerosols are odorless, colorless and relatively easy to disperse over vast areas, and for most of the diseases, it is the route that is most contagious and causes the most severe disease. Many of these agents could theoretically be used to target food or water supplies, however, the psychological impact would likely be more of a concern than the clinical effect because of water purification processes, dilution of infectious material into large volumes, stability of the organisms and the logistical difficulties of dispersion [2].
Bacteria are single-celled organisms capable of causing a variety of diseases in animals, plants and humans. They may also produce potent toxins inside the human body. These single-celled organisms may also be cultured in nutrient media and be used as a bioterrorist agent.
A parasitic form of bacteria. They are smaller than typical bacteria, but larger than viruses. They are gram stain negative and can only multiply inside living cells.
They are smaller than bacteria, and unlike bacteria and rickettsia, they are totally dependent on the host’s living cells for reproduction
A National Repository of Life-Saving Pharmaceuticals and Medical Material The SNS is a national repository of antibiotics, chemical antidotes, vaccines, immunogloblins, antitoxins, life-support medications, IV administration, airway maintenance supplies, and medical/surgical items. The SNS is designed to supplement and re-supply state and local public health agencies in the event of a national emergency anywhere and at anytime within the U.S. or its territories.
Temporary stockpiles have been assembled infrequently in the past. They have typically been assembled for important events that represented a potential terrorist target, such as the 1996 Olympic Games. Factors mitigating against stockpiles have been cost, outdating, and low probability of actual use.
For example: Cipro 500 mg cost $2.25 each Doxycycline 100mg costs $0.08 each
