Section 5: Nutrition and Antibiotics Flashcards
Vitamins are a…
Micronutrient
Micronutrients - role
Play a vital role in human metabolism since they’re involved in almost every known biochemical reaction and pathway
Synthesising vitamins - animals
Higher animals have lost the capacity to synthesise vitamins during the course of evolution
Vitamins: Biosynthetic pathways - complexity
Can be complex, leading to the suggestion that it’s biologically more efficient to ingest vitamins than to synthesise the enzymes required to construct them from simple molecules
This efficiency comes at a cost of dependence on other organisms for chemicals essential for life
Macronutrients
Carbs, fats, proteins
In humans, the catabolism of macronutrients to supply energy is an important aspect of nutrition
Micronutrients
Vitamins and minerals
Either our bodies can’t synthesise them or they can’t synthesise them in amounts sufficient for our needs –> must obtain vitamins from dietary sources
Vitamins are necessary for…
Metabolic processes
Vitamins - amount
Required in small amounts, i.e. µg to mg
Vitamins are the building blocks for…
Larger molecules
Vitamins - energy yield
Don’t yield energy when degraded
Humans require at least __ vitamins in their diet
12
By contrast, E. Coli only require glucose and organic salts, and make everything else they need
Vitamins - groups
Water-soluble:
Vitamin B group
Vitamin C
Fat-soluble: Vitamin A Vitamin D Vitamin E Vitamin K
Water soluble vs fat soluble vitamins - structure
Water soluble: highly variable in structure
Fat soluble: structurally similar - all isoprenoid compounds
Water-soluble vitamins
Structural variation Functional uniformity Require modification for function - precursor molecules (except vitamin C) Carry mobile metabolic groups; - activated carriers - function as coenzymes (vit B) Readily excreted Easily degraded - don't tend to build up easily in the cell
Fat soluble vitamins
Structurally more similar
Functionally diverse - vit A and D more like hormones
Not easily absorbed from food sources - more difficult to get in sufficient quantity
Generally not activated carriers / coenzymes
Can be toxic in excess (vit A)
Almost all activated carriers are derived from _______
Vitamins
Activated carriers: ATP - group carried and vitamin precursor
Group carried: Phosphoryl
Vitamin precursor: not a vitamin precursor
Activated carriers: NADH and NADPH - group carried and vitamin precursor
Group carried: e-
Vitamin precursor: Nicotinate (niacin) - vitamin B3
Activated carriers: FADH2 - group carried and vitamin precursor
Group carried: e-
Vitamin precursor: Riboflavin - vitamin B2
Activated carriers: Coenzyme A - group carried and vitamin precursor
Group carried: acyl
Vitamin precursor: Pantothenate - vitamin B5
Activated carriers: Tetrahydrofolate - group carried and vitamin precursor
Group carried: 1C units
Vitamin precursor: Folate - vitamin B9
Many of the B vitamins are _________
Activated carriers
What is an activated carrier
A molecule that carries a group that is then transferred to other molecules/groups
B vitamins: Riboflavin (B2) - coenzyme, typical reaction type, consequences of deficiency
Coenzyme: FAD
Reaction: ox-red
Consequences: cheilosis and angular stomatitis (lesions of mouth), dermatitis
B vitamins: Nicotinic acid (niacin) - coenzyme, typical reaction type, consequences of deficiency
Coenzyme: NAD+
Reaction: ox-red
Consequences: pellagra (dermatitis, depression, diarrhea)
B vitamins: Folic acid - coenzyme, typical reaction type, consequences of deficiency
Coenzyme: tetrahydrofolate
Reaction: transfer of 1C components; thymine synthesis
Consequences: anemia, neural-tube defects in development
Deficient in vitamin B2 generally results in…
Inflammatory conditions
Non-coenzyme vitamins: Vitamin C - function and deficiency
Function: antioxidant
Deficiency: scurvy (swollen and bleeding gums, subdermal haemorrhaging)
Vitamin C AKA…
Ascorbic acid
Non-coenzyme vitamins: Vitamin A - function and deficiency
Function: vision, growth, reproduction
Deficiency: night blindness, cornea damage, damage to respiratory and GI tract
Non-coenzyme vitamins: Vitamin D - function and deficiency
Function: regulates calcium and phosphate metabolism
Deficiency: rickets (children); skeletal deformities, impaired growth
osteomalacia (adults); soft, bendy bones
Vitamin C - antioxidant
Reducing agent
Itself is oxidised
Which vitamins function like hormones
A and D
Forms of ascorbic acid
Ascorbate: the ionised form of ascorbic acid
Dehydroascorbic acid: the oxidised form of ascorbate
Humans can’t synthesise vitamin C
Human cells can’t perform the crucial last step of vit C biosynthesis; the conversion of L-gulono-γ-lactone into ascorbic acid, which is catalysed by gulunolactone oxidase
Humans can’t synthesise vitamin C - gulonolactone oxidase
Gene that codes for gulonolactone oxidase is present in human genome, but is inactive due to accumulation of several mutations that have turned it into a non-functional pseudogene
Possible (evolutionary) advantages of being unable to synthesise vitamin C
Reaction catalysed by gulono oxidase also produces H2O2
Levels of vit C regulates a key stress-induced transcription factor HIF1 α
Pseudogenes can have a significant role in epigenetic regulation of gene expression
Possible (evolutionary) advantages of being unable to synthesise vitamin C - H2O2
Highly chemically reactive
Can cause damage to cells
Loss of vitamin C is balanced with not making such a reactive species
Possible (evolutionary) advantages of being unable to synthesise vitamin C - HIF1 α
Hypoxia inducible factor α
Activated by low O2 or limited vit C –> indicates nutritionally deficient –> turns on HIF1 α transcription gene –> invokes stress response
Fine tuning based on nutritional status
Possible (evolutionary) advantages of being unable to synthesise vitamin C - pseudogenes
Some pseudogenes can have roles affecting gene expression of other genes
Major causes of nutritional disease
Famine - leads to raft of diff nutritional deficiencies
Vit C deficiency - 2nd most common
Nutritional disease: Age of sail vs today
2 million sailors died of vit C deficiency
Today ~1/100,000 people
Nutritional disease: who is more prone
Elderly
Mentally ill patents
Alcoholics (decrease absorption and storage)
Scurvy - symptoms
Swollen, bleeding gums (gum disease) Poor wound healing Bleeding under skin Bruising Changes to hair Lethargy
Scurvy - experiment
12 sailors with scurvy, divided into groups of 2, were kept on same diet and salted meals, and given 6 diff supplements;
- cider
- elixir of vitriol
- vinegar
- seawater
- lemons and oranges
- an electuary (medicinal paste)
Scurvy - experiment results
The sailors receiving the lemons and oranges quickly improved while the cider appeared to offer modest benefit and the rest had no relief
But, it didn’t prove what it was in the lemon and oranges that helped prevent scurvy
Vitamin C and wound healing
After 6 months of no vit C diet, there’s complete lack of healing; large space occupied by an organised blood clot
After 10 days of intravenous vit C, complete healing of both original wound and that of the biopsy
Vitamin C - electrons
Electron donor (reducing agent / antioxidant) Probs all of its biochemical and molecule roles can be accounted for by this functionality
Ascorbates interacts with enzymes having either…
Monooxygenase or dioxygenase activity
What does ascorbic acid accelerate
Hydroxylation in numerous biosynthetic pathways
Ascorbic acid - acts as an e- donor for…
8 enzymes in humans
3 participate in hydroxylation required for collagen synthesis
Structure of collagen
Amino acid sequence is part of a collagen chain
Every 3rd residue is Gly
Proline and hydroxyproline are also abundant - gly-pro-hyp is a frequent tripeptide
Structure of extracellular collagen
Contains 3 helical peptide chains, each nearly 1000 residues long
Stabilisation of this required hydroxyproline (which requires vit C for synthesis)
- required for inter-strand H bond formation - stabilisation
What is the most abundant protein in mammals
Collagen
Collagen is the main fibrous component of…
Skin, bone, cartilage, teeth
Proline hydroxylase
A dioxygenase enzyme
Takes part in addition of oxygen to 2 diff reactions; one is conversion of αKG to succinate, and second is conversion of proline to hydroxyproline
But first needs activation of oxygen
Hydroxylation of proline in collagen proteins - activation of oxygen
Requires Fe2+
Hydroxylation of proline in collagen proteins - conversion of αKG to succinate
In this process, Fe2+ is oxidised to Fe3+
Hydroxylation of proline in collagen proteins - Fe3+
Inhibits functioning of proline hydroxylase, so needs to be converted back to Fe2+
Vit C does this by donating e- to the Fe3+ –> Fe2+ so proline hydroxylase becomes active again –> can hydroxylate proline to make hydroxyproline
Hydroxylation of proline in collagen proteins - in this process, vitamin C itself is oxidised to…
Dehydroascorbate
Hydroxylation of proline in collagen proteins - if vitamin C isn’t present…
Proline hydroxylase will be inhibited by Fe3+, then proline won’t be converted to hydroxyproline –> collagen won’t have H bonds that stabilise its structure
Macrocytic anaemia: Megaloblastic anaemia - cause
One cause is lack of folate
What is folate required for
Synthesis of precursor molecules of DNA synthesis
Wills factor
A nutritional factor in yeast that prevents and cures macrocytic anaemia
Folic acid / folates (B9): Major structural components
- Bicyclic, heterocyclic, pteridine ring
- p-amino benzoic acid (PABA)
- Glutamic acid
Folic acid / folates (B9): Major structural components - pteridine ring
2 parts:
Pyrimidine
Pyrazine - modifications occur here
Do humans produce folic acid
No - we take it in by diet
Folic acid - active?
It’s a precursor molecule, so must be modified to make it into its active form (tetrahydrofolate)
Conversion of folic acid to tetrahydrofolate
2 successive reductions using NADPH –> NADP+
Folate –> dihydrofolate –> tetrahydrofolate
Conversion of folic acid to tetrahydrofolate - NADPH
The electron donor
i.e. reducing agent
Conversion of folic acid to tetrahydrofolate - catalysis
Both reduction reactions are catalysed by the NADPH-specific enzyme; dihydrofolate reductase (DHFR)
Glutamate - forms
Polyglutamate
Monoglutamate
(must be able to recognise!)
Addition of additional glutamic acid residues in liver cells yield a poly-γ-glutamate tail
Why is polyglutamate converted
It can’t be absorbed, so is converted to monoglutamate
Sources of folate
Bacteria, yeast and higher plants
In these sources, folates are polyglutamate form
Sources of folate - humans
Diet
Polyglutamate - intestine
In the intestine, polyglutamate is converted into monoglutamate form
Absorbed by active transport
Conversion of folic acid to tetrahydrofolate - where
Intestinal cells
Tetrahydrofolate - storage
Can be stored in liver (50% of THF in body)
Liver - glutamate form
Converted to polyglutamate
- retains THF in liver cells
- polyglutamate has higher affinity for enzymes
Folic acid: Major functional groups of 1C units
Methyl (CH3)
Methylene (CH2)
Formyl (HCO)
Coenzymes derived from folic acid (THF)
Participate in generation and utilisation of 1C functional groups
Folates are essential for…
Cell growth and tissue development
Folate must come from _______ in mammals
Exogenous sources
Because we can’t synthesise these derivates de novo
Folic acid: Where do the 1C units join
At the nitrogen 5 or nitrogen 10 position
Folic acid: Major sources of 1C units
Amino acids (serine)
Histidine
Glycine
Formate
Folic acid: Major end products of 1C metabolism
Methionine - involved in protein synthesis
dTMP - building block for DNA and RNA
Formyl-methionyl-tRNA - derivative of methionine, used by bacteria and mitochondria
Purines - building block for DNA and RNA
Antimetabolite
Synthetic compound
Usually structurally related to metabolite
Interferes with metabolite to which it’s related
Antimetabolite: Anticancer
Inhibit human DHFR
Antimetabolite: Antibacterial
Inhibit bacterial DHFR
Antimetabolite: Antiparasitic
Inhibit protozoa DHFR
Antimetabolite - cancer - how does it work
Reduce’s cells ability to proliferate by binding to the DHFR and inhibiting production of THF
Division of cancer cells
Divide faster than normal cells
Antimetabolite: Sulfanilamide and its derivatives
Competitively inhibit synthesis of folic acid –> decreases synthesis of nucleotides needed for replication of DNA
Antimetabolite: Methotrexate
Competitively inhibits DHFR
[Folic acid analogue is used to treat psoriasis, rheumatoid arthritis and neoplastic diseases]
Neural tube detects (NTDs) reflect…
A combination of genetic predisposition and environmental influences (folic acid)
Neural tube detects (NTDs) - how does this happen
Normally the CNS begins as a plate of cells which folds on itself to form a tube
Failure of closure results in NTDs
Neural tube detects (NTDs) - types
2 main forms; Anencephaly (main cranial defect) Spina bifida (main caudal defect)
Neural tube detects (NTDs): Anencephaly
Cerebral cortex fails to develop
~1/3 of cases of NTDs
Invariability lethal - death either before or shortly after birth
Neural tube detects (NTDs): Spina bifida
Spinal cord develops abnormally
~2/3 of cases of NTDs
Causes paralysis of lower extremities and impaired bladder and bowel function
Not usually fatal unless accompanied by other conditions
NTDs - prevention
Researchers found that 50-75% of NTDs can be prevented when women supplement their diet with folic acid
NTDs: Methylation hypothesis
Proposed that folate deficiency causes NTDs due to decreased methylation of various molecules that are essential to cellular processes
Vitamin A - synthesis in humans
Humans can’t synthesise vitamin A
Vitamin A (retinol) - sources
Cartenoids, especially β-carotene in (often orange) plants
Esterified retinol from animal sources (originally sourced from carotenoids)
Function of β-carotene in plants
Photosynthesis
Act as an accessory pigment - absorb light which is converted into chemical forms of energy
β-carotene vs chlorophyll
Both undergo photosynthesis, but absorb light in a slightly diff wavelength to chlorophyll
β-carotene: Conversion
Cleaved in intestine through middle –> 2x all-trans-retinal –> all trans-retinol –> all-trans-retinoic acid and 11-cis-retinal
What form of vitamin A is transported around the body
All-trans-retinol
What are the more active forms of vitamin A
All-trans-retinoic acid
11-cis-retinal
Vitamin A: β-carotene - 11-cis-retinal
Role in vision
Vitamin A: β-carotene - all-trans-retinoic acid
Hormone-like functions
- binds to retinoic acid receptors (RAR)
- regulates gene expression
- development, immune function, reproduction
Vitamin A: Retinyl ester
Hydrophobic
A form of vitamin A that travels around in chylomicrons and can be taken up by cells
How do we store vitamin A
Retinyl ester is taken up by chylomicrons into the liver cells where it’s stored
What happens to the stored vitamin A when we need it
Retinyl ester is converted into retinol, which is then transported around the body by retinol binding protein (RBP) –> binds to STRA6 and gets internalised
Vitamin A transport: STRA6
A receptor in the cell membrane with a pore-like structure
Vitamin A transport: STRA6 - steps
When RBP comes to a cell that expresses STRA6, it binds to it which transfers the retinol through the STRA6
Retinol then binds to other molecules to transfer through the hydrophilic area of cell
Ways to transport vitamin A around the body
Lipid vesicles and RBP
- storage and release
- regulate peripheral levels of vit A
Why do we need to regulate vitamin A in body
Don’t want to waste vitamin A
Can be toxic in excess
Vitamin A - specificity
Receptor (STRA6) allows tissue specificity - regulates which tissues have access to vit A
General concept - regulating molecules
Regulate in periphery (storage)
Regulate release by signals (change in environment or metabolic state)
Regulate transport of molecules
Regulate tissue specificity (receptors and channels)
Vitamin A: Rhodopsin
11-cis-retinal combines with opsin via a lysine residue to form rhodopsin, which is a protein present in many rod cells within the eye
Vitamin A: Rhodopsin - function
Converts light energy into a visual signal
When does excessive vit A consumption occur
Typically occur when vitamin A is ingested in its preformed state (liver)
Doesn’t occur when ingesting carotenoids including β-carotene
Hypervitaminosis A - symptoms
Acute: abdominal pain, nausea, vomiting, dizziness
Chronic: bone abnormalities, joint pain, visual disturbances, appetite loss, dizziness, peeling, oily/itchy skin, respiratory infection
Vitamin D - dietary sources
Fish liver oils, fortified foods
supplements
Rickets - symptoms
Bone pain
Dental deformities
Decreased muscle strength
Skeletal deformities, e.g. bowlegs, rib-cage
Rickets - type of disease
Multi-factorial disease
Which vitamin is a cholesterol derivative
Vitamin D
How is vitamin D taken into our bodies
Can take in by diet, but most are made within our own cells
Vitamin D - precursor molecule
7-dehydrocholesterol
Synthesising vitamin D - steps
Dehydrocholesterol –UV–> previtamin D3 –> vitamin D3 –> liver: calcidiol –> kidneys: calcitriol
What form of vitamin D is found within our bodies
Vitamin D3
Vitamin D transport - steps
Previtamin D3 is hydrophobic, so is bound to VDBP (vitamin D binding protein) –> transported to liver then to kidneys (active form)
Vitamin D: Active form - functions
2 diff ways; when it gets to cells, it binds to VDR (vitamin D receptor)
- can function by activating pathways within cell (signal transduction)
- more commonly, is involved in upregulation of a no of genes
Vitamin D - functions
Bone: increases bone mineralisation
Intestine: increases absorption of Ca2+ and Pi
Immune cells: induces differentiation of immune cells
Vitamin D: Bone mineralisation
The depositing of Ca2+ and phosphate ions in bones
Vitamin D - excess
Can lead to disease if taken in excess
Where Ca2+ and phosphate become deposited in soft tissues instead of bones
What is excess vitamin D due to
Due to taking too much vitamin D in supplementation
Types of antibiotics (based on mode of action)
Bacteriocidal: Antibiotics that kill bacteria
- penicillin
Bacteriostatic: antibiotics that block growth
- tetracyclines
- sulphonamides
Penicillin - history
First discovered by Ernest Duchesne, but was forgotten until Alexander Fleming rediscovered it
What was the first true antibiotic
Penicillin
What are most antibiotics in human use
Natural products, elaborated by one species of microbe (bacteria or fungi) as chemical weapons, often in times of crowding, to kill off other microbes in the neighbouring microenvironment
What have many antibiotics been isolated from
Fungi (e.g. penicillins) and diff strains of filamentous bacterium (streptomyces)
Antibiotics: Semi-synthetic modifications of total synthesis
Both produced newer generations of antibiotics
Semi = purify product from body and change it chemically
Penicillin: Where did the fungus come from
Agar plates were open to environment and one spore of mould was transported from Freeman’s lab to Fleming’s lab
Agar plate left for 9 days, and penicillium excreted substances (‘mould juice’) which inhibited bacteria growth around it
Penicillin: Temperature
Grows in temps below 20 degrees
Staphylococcal: Temperature
Grows in temps around mid-20 degrees
Penicillin can only affect…
New bacterial growth
Who purified penicillin
Florey and Chain
Penicillin: Florey’s breakthrough results - preliminary experiments
Dose of bacteria required to kill a mouse
Penicillin: Florey’s breakthrough results - experiment
Peritoneal injections of bacterial culture into mice
After a period of time, they treated 2 groups of mice differently - untreated control (no penicillin) and treated mice (8 hourly injections for 4 days)
Penicillin: Florey’s breakthrough results - experiment results
Treated mice: First 36 hours, quite sick (a few died)
As time progressed, had vastly improved health
After 48 hours, infected mice were indistinguishable from healthy mice
Penicillin: Florey’s breakthrough results - experiment - control vs treated mice
Control: Within 2 days, all mice (24/24) were dead
Treated: A few died, but 21/24 survived
Penicillin: Florey’s breakthrough results - high dosage
Other experiment showed that penicillin (even at a high dose) led to an advantage for mice (still a few survived)
What does Penicillin come from
Comes from a fungus called penicillium
Penicillium - structure
Looks like a paintbrush
Penicillin: WWII
Hugely beneficial to treat wound pathogens such as Staphylococcus
Penicillin: Following WII
Used in treatment of rheumatic fever and syphilis
Penicillium: Food
Several species of Penicillium play a central role in production of cheese and various meat products
Mechanism of antibiotic action: 5 antibacterial targets/pathways
Inhibition of cell wall synthesis Inhibition of protein synthesis Inhibition of DNA or RNA synthesis Inhibition of folate synthesis Membrane disruption
Gram -ve bacteria
Have an additional outer membrane - important because it can affect how some antibiotics get into the cell
Penicillin: Inhibition of bacterial cell wall synthesis - steps
- Penicillin (or other inhibitor) is added to growth medium with a dividing bacterium
- Cell begins to grow, but is unable to synthesise new cell wall to accommodate the expanding cell
- As cellular growth continues, cytoplasm covered by PM begins to squeeze out through gap(s) in cell wall
- Cell continues to increase in size, but is unable to pinch off extra cytoplasmic material into 2 daughter cells
- Cell wall is shed entirely –> spheroplast, which is extremely vulnerable - prone to lysis
Penicillin: Inhibition of bacterial cell wall synthesis - why is the bacterial cell unable to ‘pinch off’ extra cytoplasmic material into 2 daughter cells
Because formation of a division furrow depends on ability to synthesise new cell wall
Penicillin: Inhibition of bacterial cell wall synthesis - what do you grow the bacteria in
In hypertonic solution - only allows cells that have a membrane around it to survive
In presence of penicillin, bacteria seemed to die unless…
They were growing in a hypertonic solution
Bacteria cells - structure
Surrounded by a protective envelope (cell wall)
Bacteria cells: Cell wall - peptidoglycan
A structural macromolecule with a net-like composition - provides rigidity and support to outer cell wall
A polymer consisting of short chain amino acids the peptido portion carbohydrate backbone (glycan portion)
Bacteria cells: Cell wall - peptidoglycan - how does it form the cell wall
A single peptidoglycan chain is cross-linked to other chains through the action of enzyme DD-transpeptidase (final step)
DD-transpeptidase
An enzyme
AKA penicillin binding protein (PBP)
Bacterial cell synthesis - steps (normal)
Transpeptidase cleaves off D-alanine and facilitates binding of other peptide chain to D-alanine –> cross-linked chains via covalent bonds - strengthens cell wall
Bacterial cell synthesis - steps (penicillin)
Penicillin and D-Ala peptides have similar structure, so D-D-transpeptidase can’t tell them apart
Transpeptidase binds to penicillin and cleaves β-lactam ring –> forms an enzyme-penicillin complex which irreversibly binds to and inactivates transpeptidase
Mechanism of antibiotic action: Inhibition of protein synthesis - example
Aminoglycosides - inhibit protein synthesis in bacteria
Mechanism of antibiotic action: Inhibition of protein synthesis - how do they work
Bind to bacterial rRNA (30s subunit), disrupt ribosomal structure –> mistranslated proteins that can misfold –> cell death
Mechanism of antibiotic action: Inhibition of protein synthesis - incorporation of misfolded membrane proteins into cell envelope can lead to…
Increased drug uptake by allowing more drugs to enter the membrane
Mechanism of antibiotic action: Inhibition of DNA or RNA synthesis - example
Rifamycin class of antibiotics (e.g. rifampicin)
Mechanism of antibiotic action: Inhibition of DNA or RNA synthesis - how do they work
Bind to actively transcribing RNA polymerase –> inhibits production of RNA
Mechanism of antibiotic action: Inhibition of folate synthesis (antimetabolites) - example
Sulfonamides - antibiotics that inhibit a bacteria-specific reaction
Mechanism of antibiotic action: Inhibition of folate synthesis (antimetabolites) - sulfonamides
Competitively inhibit dihydropteroate synthetase (enzyme involved in synthesis of folic acid)
Peptidoglycan cell walls - humans
Not present in humans
Mechanism of antibiotic action: What’s the most recent target of widespread clinical utility
Membrane disruption
Mechanism of antibiotic action: Membrane disruption - example
Lipopeptide antibiotics, e.g. daptomycin
Mechanism of antibiotic action: Membrane disruption - structure
A peptide sequence to which a fatty acid moiety is covalently attached
Huge lipo group and a peptide group
Mechanism of antibiotic action: Membrane disruption - how does it work
Mechanism of action unclear
Likely to include insertion into membrane –> membrane disruption and loss of MP and lysing of cell
Classes of AB-resistant pathogens that are a medical concern
MRSA - methicillin-resistant staphylococcus aureus
Drug-resistant gram -ve bacteria
Drug-resistant mycobacterium tuberculosis
Classes of AB-resistant pathogens that are a medical concern: MRSA
80% of staph are resistant to penicillin
Methicillin was designed to overcome penicillin
Classes of AB-resistant pathogens that are a medical concern: Drug-resistant gram -ve bacteria
e.g. Kirebsiella pneumoniae
Additional outer membrane - restricts type of antibiotics we can use
Classes of AB-resistant pathogens that are a medical concern: TB
TB can develop many resistances
What’s driving the development of drug resistance
Increased use of antibiotics –> increased level of resistance
Human: 1.4M kg/yr, about 1/2 inappropriately prescribed
Food animals: often fed antibiotics as part of food source; ~14M kg/year
Where does resistance to AB come from
Inherent / natural resistance
Acquired resistance
AB-resistance: Inherent/natural resistance
Natural/physical characteristics
Pre-determined natural things the bacteria already has, e.g. gram -ve bacteria have additional membrane
AB-resistance: Acquired resistance
Bacteria that were previously susceptible are now resistant
Resistance developing in a sub-pop or strains of bacteria
Acquired bacterial resistance - types
Vertical gene transfer
Horizontal gene transfer
Acquired bacterial resistance: Vertical gene transfer
Transfer of spontaneous resistance gene mutations in bacterial chromosome to bacterial progeny during DNA replication
i.e. parent to progeny
Acquired bacterial resistance: Vertical gene transfer - commonality
Mutation is a v rare event, but v fast growth of bacteria and absolute no of cells attained means it doesn’t take long before resistance develop
Spontaneous mutation frequency for antibiotic resistance is ~10^-8 or -9
Acquired bacterial resistance: Vertical gene transfer - Darwinian evolution
Process is driven by natural selection
In the selective environment of the antibiotic, the wild-type are killed and resistant mutant is allows to grow and flourish
Acquired bacterial resistance: Horizontal gene transfer
Genetic material contained in small packets of DNA can be transferred between individual bacteria of same or diff species
Acquired bacterial resistance: Horizontal gene transfer - mechanisms
Conjugation
Transduction
Transformation
Plasmid
Small circular piece of DNA
Generally confers an advantage to the bacteria
Passed down to their progeny
What is thought to be the main mechanism of horizontal gene transfer
Conjugation
Acquired bacterial resistance: Horizontal gene transfer - conjugation
Transmission of resistance genes following direct contact between 2 bacteria via pilus (bridge)
Plasmids - conjugation
Plasmids are key players in conjugation exchange
Located in cytoplasm of donor and recipient cell, and exchange through the pilus
Acquired bacterial resistance: Horizontal gene transfer - what does conjugation allow
Allows resistance to spread among a pop of bacterial cells much faster than simple mutation and vertical gene transfer would permit
Acquired bacterial resistance: Horizontal gene transfer - transduction
Antibiotic resistance genes are transferred between 2 closely related bacteria by bacteria-specific viruses (bacteriophages)
Resistance genes generally integrated into chromosome of recipient cell
Acquired bacterial resistance: Horizontal gene transfer - what does transduction require
A virus (bacteriophage), which picks up a resistance gene in one bacteria and transfers it into a recipient cell
Acquired bacterial resistance: Horizontal gene transfer - transformation
Occurs when naked DNA is released into the external environment, normally due to death and lysis of an organism and is taken up by another bacterium
Antibiotic-resistance can be integrated into chromosome or plasmid of recipient cell
Principal resistance mechanisms for bacterial survival
Efflux pumps
Enzymatic degradation of antibiotic
Enzymatic modification of antibiotic
Principal resistance mechanisms for bacterial survival: Efflux pump
Pumps antibiotics back out of bacterial cells through efflux pump proteins to keep intracellular drug conc below therapeutic levels
Pumps are variants of membrane pumps possessed by all bacteria to move molecules in and out of cells
For antibiotics to be effective…
They must reach their specific bacterial targets and act in a reasonable timeframe
Principal resistance mechanisms for bacterial survival: Efflux pumps - example
Resistance to tetracyclines (aminoglycosides) - conc too low to block protein synthesis because when they come into the cell, they’re quickly pumped back out
Principal resistance mechanisms for bacterial survival: What do efflux pumps prevent
Prevents antibiotic from interacting with target inside cell
Principal resistance mechanisms for bacterial survival: Enzymatic degradation of antibiotic
Antibiotic is destroyed by chemical modification by enzyme that’s elaborated by resistant bacteria
e.g. penicillin
Principal resistance mechanisms for bacterial survival: Enzymatic degradation of antibiotic - Penicillin
Deactivation of β-lactam in penicillin by expression of β-lactamase by resistant bacteria
Lactamase-producing bacteria secrete the enzyme into the periplasm to destroy β-lactam ring in antibiotics before they can reach their targets
β-lactamase
A hydrolytic enzyme
AKA penicillinase
Expressed by resistant bacteria
Principal resistance mechanisms for bacterial survival: Enzymatic degradation of antibiotic - How many penicillin molecules can be hydrolysed
A single β-lactamase can hydrolyse 1000 penicillin molecules per second - very effective
Why was methicillin designed
It’s a second generation semi-synthetic derivative of penicillin, designed to be resistant to β-lactamase cleavage
Principal resistance mechanisms for bacterial survival: Enzymatic modification of antibiotic
Antibiotic is modified by an enzyme so it’s no longer effective
Principal resistance mechanisms for bacterial survival: Enzymatic modification of antibiotic - resistance enzymes
Acetyl transferases
Phosphoryl transferases
Principal resistance mechanisms for bacterial survival: Enzymatic modification of antibiotic - example
Antibiotic chloramphenicol can be enzymatically inactivated by addition of acetyl or phosphate groups
These modifications decorate the periphery of the antibiotic and interrupt binding to ribosomes - physical barrier
Transferases
Transfer a group onto a molecule
Other resistance mechanisms: MRSA
Release fatty decoys (vesicles)
Daptomycin binds to inserts into these vesicles rather than going into bacterial cell
Decreases amount of daptomycin getting into bacterial cell
Universal provision of antibiotics could prevent ___ of deaths from pneumonia
75%
Innovation gap
Between 1962 and 2000, no major classes of antibiotics were introduced
Ideal antibiotic - characteristics
Kills/inhibits growth of harmful bacteria but doesn’t affect beneficial bacteria
Able to act regardless of site of infection
Exceptional blood/fluid circulation
Broad spectrum
Large therapeutic window
Modifiable / not able to lead to resistance
Ideal antibiotic: Exceptional blood/fluid circulation
Ability to travel well in blood and fluids of body and able to access all infections
Ideal antibiotic: ADME
Easily Absorbed, Distributed, well Metabolised and easily Excreted
Ideal antibiotic: Broad spectrum
Ideally treat all bacteria at once
Ideal antibiotic: Large therapeutic window
A window over which each drug works where it’s safe dose is effective
Does the ideal antibiotic exist
No - but the ability to modify antibiotics is useful
What is clavulanic acid
β-lactamase inhibitor
Clavulanic acid - how does it work
Lacks antibiotic activity but irreversibly binds to β-lactamase to prevent it from hydrolysing β-lactam antibiotics (e.g. penicillin and amoxicillin)
Clavulanic acid - structure
Looks similar to penicillin
Clavulanic acid - what is it used with
Used in combination with antibiotics - soaks up β-lactamase so the antibiotic can function to inhibit bacterial cell wall synthesis
Strategies to overcome resistance: Approaches to develop new antibiotics
Modification of common core structures of diff antibiotic classes using medicinal chemistry
Identification of new antibiotic scaffolds through searches of underexplored ecological niches and bacterial taxa
Bioinformatic analysis of bacterial genomes
Strategies to overcome resistance: Modification of common core structures
Scaffold alterations; two strategies:
- Tetracycline scaffold can be chemically modified –> tetracycline derivative (e.g. tigecycline) that’s no longer a substrate for the efflux pump
- A new scaffold (e.g. retapamulin) which isn’t a substrate for efflux and binds to a diff site in ribosome can be used instead of tetracycline
Strategies to overcome resistance: Modification of common core structures - synthetic tailoring
Where the new generation only looks slightly diff - just enough for the bacterial resistance gene to no longer recognise it
Strategies to overcome resistance: Modification of common core structures - characteristics of a new antibiotic which would be good for synthetic scaffolds
Active against gram +ve and -ve pathogens
Lack of cross-resistance to existing drugs
Able to be synthetically tailored
Strategies to overcome resistance: Identification of new antibiotic scaffolds
More than 2/3 of clinically used antibiotics are natural products or semisynthetic derivatives
Strategies to overcome resistance: Identification of new antibiotic scaffolds - examples
Komodo dragons live in bacterial-infested waters
Marine life
Plant defenses
Antibiotic-contaminated lake
Strategies to overcome resistance: Bioinformatic analysis of bacterial genomes
Genome sequences of bacteria and fungi have up to 2 dozen silent clusters for natural product biosynthesis
Approaches used to turn on silent biosynthetic gene clusters to evaluate the activity of resultant small molecules
Strategies to overcome resistance: AMPs and lipopeptides
Insert into bacterial membrane –> physically damages bacterial morphology
AMPs
Antimicrobial peptides
Strategies to overcome resistance: AMPs and lipopeptides - disadvantage
Limited medical use - break down easily in body, so it’s hard to get them delivered to target without having them destroyed on the way
Strategies to overcome resistance: AMPs and lipopeptides - nanoparticles
Potentially nanoparticles made of AMP-like peptides could enhance their activity due to multivalent interactions
More stable
Strategies to overcome resistance: AMPs and lipopeptides - SNAPPs
Structurally nanoengineered antimicrobial peptide polymers
Strategies to overcome resistance: AMPs and lipopeptides - SNAPPs - how does it work
Proceeds via a multimodal mechanism of bacterial cell death by:
- outer membrane destabilisation (gram -ve)
- unregulated ion movement across cytoplasmic membrane (change in ion efflux)
- induction of apoptotic-like death pathway
Strategies to overcome resistance: AMPs and lipopeptides - why is multimodal mechanism importan
Bacteria will find it harder to overcome all 3 types of death induction
