ANTIBIOTICS Lecture 06

Bio 319: AntibioticsLecture Six

Topic: •Biotechnology production

•Engineering polyketide antibiotics•Bioterrorism/Stockpiling

Dr. G. Kattam Maiyoh

Wednesday, April 12, 2023 1GKM/bio 319/antibiotics/2013

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.


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


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.


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

Wednesday, April 12, 2023 GKM/bio 319/antibiotics/2013 5

•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.

cntd


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 enzymesWednesday, April 12, 2023 7GKM/bio 319/antibiotics/2013

FA biosynthesis, quick reminder:

Fatty acid biosynthesis is a stepwise assemblyof acetyl-CoA units (mostly as malonyl-CoA) ending with palmitate (C16 saturated)

Fatty acid biosynthesis is a stepwise assemblyof acetyl-CoA units (mostly as malonyl-CoA) ending with palmitate (C16 saturated)

Activation

Elongation

Termination

3 Phases

Overall Reaction

CH3C~SCoA O

CH3C-

O

CH2C~S-

O

ACP

HS-CoACO2

NOTE:

Malonyl-CoA carbons become new COOH end

Nascent chain remains tethered to ACP

Acyl CarrierProtein

CO2, HS-CoA are released at each condensation

Malonyl-CoA + ACP

-OOC-CH2C~S-

O

ACP + HS-CoA

Initiation

CH3C-

O

CH2C~S-

O

ACP

NADPH

CH3CH2CH2C~S-

O

ACP

CH3C- CH2C~S-

O

ACP

HO

H

CH3C- = C- C~S-

O

ACPH

H

-H2O

NADPH

-Carbon Elongation

D isomer

Reduction

Dehydration

Reduction

-Ketoacyl-ACP reductase

-Hydroxyacyl-ACP dehydrase

Enoyl-ACP reductase

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


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


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 deleted2. Mutation of DNA region encoding enoylreductase activityResults;1. Carbonyl group instead of hydroxyl at C-52. Carbon-carbon double bond at C6 and C7

Wednesday, April 12, 2023 GKM/bio 319/antibiotics/2013 13

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.

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

• 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

BIOLOGICAL AGENTS

• There are several types of agents. They are classified as:– Bacteria– Rickettsia– Viruses– Biotoxins

BACTERIA

• Single celled organisms capable of causing disease. These agents, grown on culture to produce large quantities, can be modified or “weaponized” for greater destruction

• Produces inflamation in tissues and/or toxins

EXAMPLES• ANTHRAX• SMALL POX• PLAGUE• TYPHOID• CHOLERA• TULAREMIA

RICKETTSIA

• Vector borne (ticks, lice, mosquitos) parasitic form of bacteria

• Diseases are difficult to treat

• Variants exist worldwide

EXAMPLES• TYPHUS• ROCKY MT. SPOTTED

FEVER• Q FEVER• INDIA TICK FEVER• MEDITERANEAN TICK

FEVER

VIRUSES

• Smaller than bacteria• RNA or DNA in a protein

coat• Use living cells to

reproduce• Not affected by

antibiotics

EXAMPLES• EBOLA• LASSA FEVER• INFLUENZA• VIRAL HEPATITIS• VIRAL HEMORRHAGIC

FEVERS

BIOTOXINS

• Are poisonous by-products of bacteria, fungi, marine animals or plants

• Do not replicate in the host

• Are not communicable• Highly toxic when

delivered as an aerosol

EXAMPLES

• BOTULINUM• STAPHLOCCOCAL

ENTEROTOXIN B• RICIN

Biological Agents most likely to be used in a terrorist attack

• Bacteria - anthrax, plague, tularemia• Virus - small pox, viral hemorrhagic fever• Biotoxin - botulism

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


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


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%)


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

Rashes

A tentative diagnosis of plague is made

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

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

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

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

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

Emergency Preparedness / Stockpiling

Strategic National Stockpile• Repository of

– Antibiotics– Vaccines– Immunoglobulins– Chemical antidotes– Antitoxins– Life-support medications– IV administration– Airway maintenance

supplies– Medical/surgical items

Containers designed to facilitate transport by roads and railways.

And also by airways.

• 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?

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!

Post-exposure Prophylaxis

Bacteria 1st choice Alternatives

AnthraxCiprofloxacin

Doxycycline

rifampin, penicillin, ampicillin, chloramphenicol, clindamycin, and clarithromycin.

PlagueDoxycycline

CiprofloxacinChloramphenical

TularemiaDoxycycline

Ciprofloxacin

TreatmentBacteria 1st choice Alternatives

AnthraxCiprofloxacin

Doxycycline

rifampin, vancomycin, penicillin, ampicillin, chloramphenicol, imipenem, clindamycin, and clarithromycin.

PlagueGentamicin

Streptomycin

Doxycycline, Ciprofloxacin

Chloramphenical

TularemiaGentamicin

Streptomycin

Doxycycline, Ciprofloxacin, Chloramphenicol

ANTIBIOTICS Lecture 06

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