antibiotics Flashcards
Scientific Perspective on Antibiotics
Antibiotics originally used by microorganisms for chemical warfare to outcompete other microbes for resources.
Examples:
Fungi producing penicillin to kill bacteria.
We have modified natural antibiotics to create newer drugs through chemistry.
Penicillin Discovery
Discoverer: Alexander Fleming (1928), although earlier discovered by Ernest Duchesne (work was not recognized).
Serendipitous Discovery:
Penicillium fungi contaminated bacterial plates in Fleming’s lab.
Fleming observed bacteria-free zones around the fungi and investigated further.
Experimental Design with Penicillin
Aim: Demonstrate effectiveness in a living organism.
Steps:
Determine appropriate dose of bacteria.
Set up two groups of mice:
Control: Bacteria only, no penicillin.
Experimental: Bacteria + penicillin.
Observations:
Control group: Mice died.
Experimental group: Mice recovered and were indistinguishable from healthy mice.
Penicillin was found to effectively treat bacterial infections, leading to its widespread use, especially in WWII.
Types of Antibiotics
Bactericidal: Kill bacteria (e.g., penicillin).
Bacteriostatic: Inhibit bacterial growth.
Summary of Antibiotic Function
Antibiotics were not invented by humans but are derived from natural warfare between microorganisms.
Modern antibiotics are modified versions of natural compounds to improve efficacy.
Use of Penicillin in World War II and Experimental Results Summary
Penicillin was purified and used to treat bacterial infections during World War II.
It was effective for soldiers and civilians suffering from bacterial infections, leading to many lives saved.
Experiment Setup:
Two Groups:
Control Group: Given a dose of bacteria but not treated with penicillin.
Treated Group: Given the same dose of bacteria and treated with penicillin for four days.
Observations were recorded at intervals: 6 hours, 12 hours, 24 hours, up to 10 days.
Results:
Control Group:
By day 2, all mice had died due to the bacterial infection.
Treated Group:
First few days: A few mice passed away, but the majority survived.
By day 10, 21 mice were still alive, demonstrating the effectiveness of penicillin in treating the bacterial infection.
Significance:
The survival rate of the treated group was unprecedented.
This success led to widespread use of penicillin in treating bacterial infections during the war and beyond, ultimately saving numerous lives.
mechanism of antibiotic action by targeting specific bacterial pathways or structures
Major Clinically Validated Antibacterial Targets/Pathways:
Inhibition of Cell Wall Synthesis (a):
Antibiotics in this class disrupt peptidoglycan synthesis, which is crucial for bacterial cell wall integrity.
Examples: Penicillins, cephalosporins.
Effect: Weakens the bacterial cell wall, leading to cell lysis and death.
Inhibition of Protein Synthesis (b):
Targets bacterial ribosomes (specifically the 30S or 50S subunits), inhibiting protein synthesis.
Examples: Tetracyclines, macrolides.
Effect: Prevents bacteria from producing essential proteins, leading to growth inhibition or death.
Inhibition of DNA or RNA Synthesis (c):
Interferes with bacterial DNA gyrase or RNA polymerase, preventing replication and transcription.
Examples: Quinolones (target DNA gyrase), rifampicin (targets RNA polymerase).
Effect: Disrupts genetic processes, preventing cell division and growth.
Inhibition of Folate Synthesis (d):
Blocks bacterial folate pathway, which is essential for nucleotide synthesis.
Examples: Sulfonamides, trimethoprim.
Effect: Prevents the synthesis of nucleotides, inhibiting DNA and RNA formation.
Membrane Disruption (e):
Disrupts the bacterial cell membrane, causing leakage of cellular contents.
Examples: Polymyxins.
Effect: Leads to loss of membrane integrity and cell death.
Bacterial Classification: Gram-Positive vs. Gram-Negative
The diagram shows Gram-positive and Gram-negative bacterial structures, highlighting differences in their cell wall composition.
Gram-positive bacteria have a thicker peptidoglycan layer.
Gram-negative bacteria have an outer membrane, which can affect antibiotic permeability.
Penicillin: An Accidental Discovery that Changed Medicine
Penicillin: An Accidental Discovery that Changed Medicine
Discovery:
Penicillium notatum was isolated by Alexander Fleming.
Origin of the Name:
The name Penicillium is derived from the resemblance of the fungus’ spore-forming structure to a paintbrush.
Penicillus is the Latin word for paintbrush.
World War II Impact (1943):
Penicillin was hugely beneficial during WWII for treating wound infections caused by pathogens like Staphylococcus.
Post-War Impact:
After WWII, penicillin was particularly effective in treating rheumatic fever and syphilis.
Other Uses of Penicillium:
Several species of the Penicillium genus are used in food production.
For example, Penicillium roqueforti is used in the production of blue cheeses.
Mechanism of Antibiotic Action
Mechanism of Antibiotic Action: Know Your Bacteria!
Differences between Gram-Negative and Gram-Positive Bacteria are important for understanding how antibiotics work and which antibiotics are effective.
Gram-Negative Bacteria
Cell Wall Structure:
Outer Membrane: Present, containing lipopolysaccharides (LPS), which contribute to the outer structure.
Thin Peptidoglycan Layer: Located in the periplasmic space between the outer membrane and the cytoplasmic membrane.
Porins: Channels present in the outer membrane allowing passage of small molecules.
Key Features:
The outer membrane makes Gram-negative bacteria generally more resistant to certain antibiotics.
Antibiotics often need to penetrate both the outer membrane and the peptidoglycan layer.
Gram-Positive Bacteria
Cell Wall Structure:
No Outer Membrane.
Thick Peptidoglycan Layer: This is much thicker compared to Gram-negative bacteria.
Teichoic Acids: Present within the peptidoglycan layer, contributing to rigidity.
Key Features:
The thick peptidoglycan layer makes Gram-positive bacteria more susceptible to antibiotics like penicillin, which target cell wall synthesis.
Staining and Identification:
Gram Stain:
Gram-Positive: Stains purple due to the thick peptidoglycan layer retaining the crystal violet stain.
Gram-Negative: Stains pink due to the thin peptidoglycan layer and the presence of the outer membrane, which doesn’t retain the crystal violet.
Summary
Gram-Negative Bacteria have an outer membrane and a thin peptidoglycan layer, making them often more resistant to certain antibiotics.
Gram-Positive Bacteria have a thick peptidoglycan layer but no outer membrane, making them more vulnerable to antibiotics targeting cell wall synthesis.
Understanding these differences helps in selecting appropriate antibiotics for effective treatment.
Penicillin – Mechanism of Action
Bacterial Cell Wall:
Bacteria are surrounded by a protective cell wall that provides rigidity and support.
One of the primary components of the bacterial cell wall is peptidoglycan, a structural macromolecule with a net-like composition that maintains the integrity of the bacterial cell.
Peptidoglycan Structure:
Peptidoglycan is a polymer consisting of:
Short chain amino acids (the peptido portion).
A carbohydrate backbone (the glycan portion).
It forms a strong mesh-like structure that gives the cell wall its strength.
Formation of the Cell Wall:
Peptidoglycan chains are cross-linked to form a strong network.
The enzyme DD-transpeptidase, also known as penicillin-binding protein (PBP), is responsible for cross-linking these peptidoglycan chains.
The cross-linking occurs between the carbohydrate chains (N-acetylmuramic acid, NAM) and short peptides, leading to a rigid cell wall structure.
Penicillin’s Action:
Penicillin inhibits the action of penicillin-binding proteins (PBPs).
By binding to PBPs, penicillin blocks the cross-linking of peptidoglycan chains.
Result: The bacterial cell wall becomes weak, ultimately causing cell lysis due to the high internal osmotic pressure, which cannot be contained.
Summary
Penicillin targets the enzymatic cross-linking of peptidoglycan, which is critical for bacterial cell wall integrity.
Without proper cross-linking, the bacterial cell wall cannot maintain its strength, leading to cell death.
penicillin reaction scheme
enicillin Reaction Scheme
Target Enzyme: Transpeptidase (also called penicillin-binding protein (PBP)) is responsible for cross-linking peptidoglycan chains, which is essential for bacterial cell wall integrity.
Reaction Mechanism:
Normal Cross-Linking Process:
Peptidoglycan Chains:
Chain I and Chain II have terminal D-Alanine (D-Ala) residues.
The enzyme transpeptidase (E) binds to the peptidoglycan substrate to facilitate cross-linking between the peptidoglycan chains.
Formation of Active Complex:
The enzyme forms a transient enzyme-substrate complex.
A cross-link is formed between the peptidoglycan chains (I and II), strengthening the bacterial cell wall.
Penicillin Action:
Beta-Lactam Ring:
Penicillin has a beta-lactam ring, which is crucial for its function.
The beta-lactam ring structure resembles the D-Ala-D-Ala structure of the peptidoglycan substrate.
Inhibition of Transpeptidase:
Penicillin binds to the active site of the transpeptidase enzyme (E), forming an enzyme-penicillin complex.
This complex is inactive, which means that the enzyme can no longer cross-link peptidoglycan chains.
Irreversible Inhibition:
The binding of penicillin is irreversible. This effectively inhibits the transpeptidase activity, preventing peptidoglycan cross-linking.
Result:
The bacterial cell wall cannot form proper cross-links, leading to a weak cell wall.
Eventually, the bacteria undergo lysis due to the inability to maintain internal pressure.
Key Features:
Beta-Lactam Ring: The beta-lactam ring in penicillin is key to its ability to inhibit transpeptidase.
Competitive Inhibition: Penicillin acts as a mimic of the natural substrate (D-Ala-D-Ala) and binds to the enzyme’s active site, preventing normal function.
Summary
Penicillin inhibits bacterial cell wall synthesis by binding to transpeptidase, preventing the formation of cross-links in the peptidoglycan structure.
This inhibition leads to a weakened bacterial cell wall, ultimately causing bacterial cell death.
Other Cell Wall Inhibitors: Mechanisms
Diversity of Inhibition:
There is more than one way to inhibit a biological process such as bacterial cell wall synthesis.
Vancomycin Mechanism:
Vancomycin is another cell wall inhibitor distinct from beta-lactam antibiotics like penicillin.
It targets the peptidoglycan synthesis process, but its mechanism is different.
Binding to D-Ala-D-Ala:
Vancomycin binds directly to the terminal D-Ala-D-Ala dipeptide on the pentapeptide chain of peptidoglycan subunits.
This binding prevents the cross-linking of peptidoglycan chains by blocking access for transpeptidase enzymes.
Inhibition of Cross-Linking:
By binding to the pentapeptide, vancomycin prevents the necessary cross-linking step needed for a stable cell wall.
The cell wall becomes weak, leading to bacterial cell lysis.
Peptidoglycan Structure:
The diagram illustrates the interaction between vancomycin and the peptidoglycan units:
GlcNAc-MurNAc disaccharide units form the backbone.
The pentapeptide chains are shown with green circles.
Vancomycin (purple) binds to the terminal D-Ala-D-Ala, highlighted in the diagram, to inhibit cell wall formation.
Summary
Vancomycin inhibits cell wall synthesis by binding to D-Ala-D-Ala, preventing cross-linking.
This action differs from penicillin, which inhibits the transpeptidase enzyme directly.
Both lead to a weakened cell wall, resulting in bacterial death, but via different mechanisms.
Mechanism of Antibiotic Action: Inhibition of Protein Synthesis
B) Inhibition of Protein Synthesis
Key Antibiotics:
Aminoglycosides and chloramphenicol are examples of antibiotics that inhibit protein synthesis in bacteria.
Mechanism of Action:
Aminoglycosides bind to bacterial ribosomal RNA.
They bind specifically to the 30S subunit of the ribosome.
This binding leads to disruption of ribosomal structure, causing misreading of mRNA.
This results in the production of mistranslated proteins, which are often misfolded.
The incorporation of misfolded proteins into the bacterial cell membrane causes further damage, leading to increased uptake of aminoglycosides, escalating their effect.
Cellular Outcome:
Misfolded proteins can damage the integrity of the bacterial cell envelope.
This leads to further leakage of cellular contents and ultimately cell death.
Diagram Summary:
The diagram illustrates aminoglycoside binding to the ribosome, resulting in misfolded proteins.
Misfolded proteins insert into the cell membrane, increasing drug uptake and leading to cell death.
Importance:
This mechanism highlights how antibiotics can exploit bacterial protein synthesis machinery to disrupt cell function and cause bacterial death.
Mechanism of Antibiotic Action: Inhibition of DNA or RNA Synthesis
C) Inhibition of DNA or RNA Synthesis
Key Antibiotics:
Rifamycin class of antibiotics, such as rifampicin.
Mechanism of Action:
These antibiotics bind to the RNA polymerase enzyme that is actively involved in transcription.
By binding to RNA polymerase, these antibiotics prevent the transcription process, thereby stopping mRNA synthesis.
Impact on Bacteria:
Inhibition of RNA synthesis leads to a failure in protein production, ultimately causing bacterial cell death.
Example:
Rifampicin is often used to treat infections such as tuberculosis, where it is particularly effective against actively dividing bacteria by preventing RNA synthesis
Mechanism of Antibiotic Action: Inhibition of Folate Synthesis
(Antimetabolites)
D) Inhibition of Folate Synthesis
Key Antibiotics:
Sulfonamides are a class of antibiotics that inhibit a bacteria-specific reaction.
Mechanism of Action:
Sulfonamides act as competitive inhibitors of the enzyme dihydropteroate synthetase, which is involved in folate synthesis.
Dihydropteroate synthetase converts p-aminobenzoic acid (PABA) into folic acid.
By inhibiting this enzyme, sulfonamides prevent the synthesis of folic acid, a key precursor required for the synthesis of nucleotides.
Impact on Bacteria:
Folic acid is crucial for nucleotide production, which is needed for DNA replication.
Inhibition of folate synthesis leads to a deficiency in nucleotide synthesis, which hinders bacterial cell growth and division.
Example:
Sulfanilamide is a typical sulfonamide antibiotic used for its inhibitory action on bacterial folate synthesis.
Mechanism of Antibiotic Action: Membrane Disruption (E)
E) Membrane Disruption
This is the most recent target of widespread clinical utility, often considered a “last resort” treatment.
Two Types of Membrane-Disrupting Antibiotics:
Lipopeptide Antibiotics (e.g., Daptomycin)
Broad Spectrum: Effective against gram-positive bacteria, including MRSA.
Mechanism: Inserts into bacterial membranes and aggregates, forming holes that lead to ion leakage and loss of membrane potential.
Polymyxin
Known for causing severe side effects.
Activity: More active against gram-negative bacteria.
Mechanism: Binds to lipopolysaccharide (LPS) on the outer membrane of gram-negative bacteria and disrupts both outer and inner membranes, leading to cell death.
Membrane Disruption Mechanism: Lipopeptide Antibiotics (e.g., Daptomycin)
Lipopeptide Antibiotics - Daptomycin:
Effective against gram-positive bacteria, including MRSA.
Mechanism of Action:
Step 1: Binding and insertion into the bacterial membrane. Ca²⁺ ions facilitate this process.
Step 2: Oligomerization and channel formation in the membrane.
Step 3: The channels formed lead to depolarization and ion efflux, specifically K⁺, resulting in the loss of membrane potential and eventually cell death.
Structure: The chemical structure of Daptomycin is shown, highlighting its ability to interact with bacterial membranes.
Membrane Disruption Mechanism: Polymyxin
Polymyxin
Characteristics:
Causes severe side effects.
More effective against gram-negative bacteria.
Mechanism of Action:
Binding: Polymyxin binds to lipopolysaccharides (LPS) on the outer membrane of gram-negative bacteria.
Disruption: It disrupts both the outer and inner membranes, leading to the leakage of cellular contents and bacterial cell death.
Modification:
Removal of the hydrophobic tail of polymyxin B creates polymyxin nonapeptide, which can still bind to LPS but loses the ability to kill bacterial cells.
Illustration:
The provided diagram shows polymyxin interacting with Lipid A and phospholipids in the bacterial membrane, eventually causing disruption.
Recap of previous lecture
Antibiotics Recap:
Antibiotics: Compounds that either inhibit bacterial growth (bacteriostatic) or kill bacteria (bactericidal).
Produced naturally by certain fungi and bacteria as a form of chemical warfare to outcompete other microorganisms for resources.
Examples include penicillin, discovered by Fleming, and further purified and tested by Florey and Chain.
Antibiotic Mechanisms:
Five main mechanisms through which antibiotics work, targeting essential bacterial functions:
Inhibition of Protein Synthesis: Antibiotics like chloramphenicol and tetracyclines bind to ribosomes and disrupt protein production.
Inhibition of DNA/RNA Synthesis: Rifampicin inhibits RNA polymerase, preventing the synthesis of RNA.
Inhibition of Folate Synthesis: Antibiotics inhibit folate synthesis, critical for amino acid and nucleotide production. This will be discussed in greater detail in the next lecture.
Membrane Disruption:
Daptomycin inserts into bacterial membranes, forming pores, causing cellular contents to leak and eventually leading to cell death.
Polymyxin binds to lipopolysaccharides (LPS) and disrupts bacterial membranes, leading to instability.
Inhibition of Cell Wall Synthesis:
Penicillin binds to DD-transpeptidase via its beta-lactam ring, preventing cross-linking of peptidoglycan and leading to cell wall weakening.
Vancomycin works differently by binding directly to D-alanyl-D-alanine, acting as a physical barrier to prevent cell wall formation.
Kirby-Bauer Technique
Lab Experiment: Testing bacterial resistance to antibiotics.
Agar plates are used as a medium for bacterial growth.
Antibiotic disks (e.g., ampicillin and chloramphenicol) are placed on agar plates.
Zone of inhibition: Area where bacteria do not grow due to the presence of the antibiotic, indicating susceptibility.
No zone of inhibition: Indicates resistance to the antibiotic.
Zone of Inhibition:
This is the clear area around an antibiotic disk where no bacterial growth is visible.
The presence of this clear zone means that the antibiotic is effective at preventing the growth of bacteria. In other words, the bacteria are susceptible to the antibiotic.
The size of the zone of inhibition can indicate how effective the antibiotic is—larger zones mean more susceptibility.
No Zone of Inhibition:
If there is no clear area around the antibiotic disk and the bacteria grow right up to the edge of the disk, it means that the antibiotic did not inhibit bacterial growth.
This indicates that the bacteria are resistant to the antibiotic, meaning they can survive and continue growing even in its presence.
Experiment
This slide presents an example of a “superbug” infection, which is a strain of Klebsiella pneumoniae that was resistant to 26 different antibiotics, leading to the unfortunate death of a 70-year-old woman in Nevada in 2016.
Let’s break down the key elements of the image:
Agar Plates with Disks:
In the two agar plates shown, there are several disks soaked in different antibiotics placed on the agar surface.
The growth of bacteria is represented by the opaque areas, whereas the clear areas around the disks are the zones of inhibition.
Zone of Inhibition:
On these plates, you can see some disks have no clear zones around them, indicating that the bacteria are resistant to those antibiotics.
The absence of a zone of inhibition shows that the antibiotic was ineffective at preventing bacterial growth, which implies antibiotic resistance.
In this case, most disks show little to no inhibition, which aligns with the statement that this bacterium was resistant to all available antibiotics.
Context of the Case:
The infection was likely acquired in India following a femur fracture, and despite treatment attempts in the U.S., no antibiotic was effective in combating the infection.
The bacteria were resistant to carbapenem, a last-resort antibiotic, highlighting the severity of the resistance problem.
This type of experiment helps in determining which antibiotics are effective against a specific bacterial strain and highlights the growing issue of antibiotic resistance, which is a major concern in medicine today. This specific example illustrates a worst-case scenario where no antibiotics were effective, emphasizing the need for new drugs and strategies to combat resistant bacteria.
Examples of antibiotic (AB) resistant bacteria
Drug-resistant Gram-negative bacteria:
Gram-negative bacteria are particularly challenging to treat because they have an outer membrane that makes them resistant to many antibiotics.
These resistant strains have decreased the pool of antibiotics available, which was already limited for Gram-negative bacteria.
MRSA (Methicillin-Resistant Staphylococcus aureus):
MRSA is a strain of Staphylococcus aureus that has become resistant to methicillin and other beta-lactam antibiotics, including penicillin.
The note mentions that 80% of Staphylococcus aureus strains are penicillin-resistant, highlighting the prevalence of resistance in this common pathogen.
MDR-TB (Multi-Drug Resistant Tuberculosis):
MDR-TB refers to tuberculosis that is resistant to at least two of the most potent TB drugs (usually isoniazid and rifampicin), making it much more difficult and costly to treat.
Reasons behind the increase in antibiotic resistance
Increased Misuse of Antibiotics:
The misuse and overuse of antibiotics (AB) are significant contributors to the growing problem of resistance.
When antibiotics are used inappropriately, resistant bacteria can develop and spread.
These resistant bacteria can enter the environment, further contributing to the problem.
Use of Antibiotics in Humans:
In the UK, approximately 214 tons of antibiotics are used annually for human medicine.
There is a concern when antibiotics are inappropriately prescribed, which can lead to increased resistance.
Use of Antibiotics in Food Animals:
Antibiotics are also heavily used in food animals, which contributes to resistance that can transfer to humans.
In the UK, 214 tons of antibiotics are used annually for food animals.
In the USA, the use is much higher, with 6002 tons of antibiotics used each year in food animals.
This slide explains the two main types of antibiotic resistance:
Inherent/Natural Resistance:
This resistance occurs due to natural or physical characteristics of the bacteria.
Some bacteria are naturally resistant to certain antibiotics without any previous exposure or genetic changes.
For example, Gram-negative bacteria have an outer membrane that makes it more difficult for certain antibiotics to penetrate, naturally making them resistant.
Acquired Resistance:
This type of resistance develops when bacteria that were previously susceptible to an antibiotic become resistant.
This can occur through mutation or acquiring resistance genes from other bacteria (e.g., through plasmid transfer).
Acquired resistance often develops within specific sub-populations or strains of bacteria, and it is a major concern because it can spread among bacteria and reduce the effectiveness of antibiotics over time.
Differences between Gram-negative and Gram-positive bacteria
Gram-negative vs. Gram-positive Bacteria:
Gram-negative bacteria have an additional outer membrane that makes them more resistant to certain antibiotics. This outer membrane acts as a barrier, preventing many antibiotics from penetrating.
Gram-positive bacteria have a thicker peptidoglycan layer but lack the outer membrane that Gram-negative bacteria have. This structure makes them more susceptible to certain antibiotics that target the cell wall.
Antibiotics and their Effectiveness:
Broad-spectrum antibiotics (e.g., chloramphenicol, tetracyclines, streptomycin, sulphonamides) are capable of affecting both Gram-positive and Gram-negative bacteria because they target processes common to both.
Vancomycin is unable to penetrate the outer membrane of Gram-negative bacteria, making it ineffective against these bacteria.
Daptomycin also cannot cross the outer membrane, limiting its ability to target Gram-negative bacteria.
The image on the right shows how different antibiotics interact with the bacterial cell structures. Chloramphenicol, for example, is small enough to pass through the porins in the outer membrane of Gram-negative bacteria, while larger molecules like vancomycin cannot pass through, limiting their effectiveness.
How bacteria acquire resistance to antibiotics, specifically through vertical gene transfer and horizontal gene transfer
- Vertical Gene Transfer:
This type of gene transfer occurs when resistance genes are passed from parent bacteria to offspring during DNA replication.
Mutation plays a role here. Although mutations are rare, bacteria reproduce very rapidly, leading to large populations. Even with low mutation rates, the absolute number of cells increases the likelihood of resistance mutations developing.
The mutation frequency for antibiotic resistance is approximately 10⁻⁸ to 10⁻⁹, meaning in every 10⁸ to 10⁹ bacterial cells, one will develop resistance through mutation.
This process is an example of Darwinian evolution driven by natural selection:
In an environment where an antibiotic is present, susceptible bacteria die, while those with resistance survive and reproduce. This leads to an increase in the population of resistant bacteria.
How bacteria acquire resistance to antibiotics, specifically through vertical gene transfer and horizontal gene transfer
Vertical Gene Transfer:
This type of gene transfer occurs when resistance genes are passed from parent bacteria to offspring during DNA replication.
Mutation plays a role here. Although mutations are rare, bacteria reproduce very rapidly, leading to large populations. Even with low mutation rates, the absolute number of cells increases the likelihood of resistance mutations developing.
The mutation frequency for antibiotic resistance is approximately 10⁻⁸ to 10⁻⁹, meaning in every 10⁸ to 10⁹ bacterial cells, one will develop resistance through mutation.
This process is an example of Darwinian evolution driven by natural selection:
In an environment where an antibiotic is present, susceptible bacteria die, while those with resistance survive and reproduce. This leads to an increase in the population of resistant bacteria.
what does it mean by inherent or acquired
Inherent (Natural) Resistance
This type of resistance is due to natural or physical characteristics of the bacterium.
It is something that a bacterium naturally possesses because of its structural or genetic traits.
For example, Gram-negative bacteria have an additional outer membrane that prevents many antibiotics from entering, providing them inherent resistance to certain antibiotics.
This type of resistance is always present and is passed on as part of the bacterium’s standard genetic characteristics.
2. Acquired Resistance
Acquired resistance happens when bacteria that were previously susceptible to an antibiotic develop resistance.
This resistance is not naturally present but develops over time due to genetic changes.
Bacteria can acquire resistance either through:
Mutations in their existing genes (vertical gene transfer), which are passed to their offspring.
Horizontal gene transfer (HGT), where they gain resistance genes from other bacteria (through conjugation, transduction, or transformation).
Acquired resistance is a major concern because it means that bacteria that were once easily treated can evolve and become resistant to antibiotics due to overuse or misuse of these drugs.
Horizontal Gene Transfer (HGT)
Horizontal Gene Transfer (HGT)
HGT involves the transfer of genetic material between individual bacteria, which can occur not only between the same species but also between different bacterial species.
Unlike vertical gene transfer, which passes genes from parent to offspring, HGT allows bacteria to acquire resistance genes from neighboring bacteria, which helps spread resistance more rapidly.
Mechanisms of HGT
There are three main mechanisms of horizontal gene transfer that bacteria use to exchange genetic material:
Conjugation: Direct transfer of DNA between two bacterial cells that are temporarily joined by a structure called a pilus. It often involves the transfer of plasmids, which can carry resistance genes.
Transduction: Transfer of bacterial DNA through bacteriophages (viruses that infect bacteria). Bacteriophages can accidentally pick up bacterial DNA, including resistance genes, and transfer it to another bacterium.
Transformation: Uptake of naked DNA from the environment by bacteria. This DNA may come from dead bacteria that have released their genetic material.
These mechanisms allow bacteria to share genetic material, which contributes to the rapid spread of antibiotic resistance across different bacterial populations and species. This adaptability makes HGT a major concern for the development and spread of antibiotic-resistant pathogens.
Conjugation: one of the main mechanisms of horizontal gene transfer (HGT) in bacteria
Conjugation
Conjugation is considered the main mechanism of horizontal gene transfer.
It involves the direct contact between two bacteria through a structure called a pilus.
Plasmids, which are small circular DNA molecules, are key players in this process. They contain genes that can include antibiotic resistance.
During conjugation, a pilus forms between the donor cell and the recipient cell, allowing the plasmid (containing resistance genes) to be transferred from one bacterium to another.
This mechanism allows antibiotic resistance to spread quickly through a bacterial population, much faster than what could occur through random mutations or vertical gene transfer (where resistance is inherited by offspring).
Bacterial transduction
a mechanism of horizontal gene transfer that contributes to antibiotic resistance among bacteria. Here’s a summary of the steps involved in transduction:
Bacteriophage Infection of Donor Cell:
A bacteriophage, which is a virus that infects bacteria, injects its genetic material into the donor bacterial cell. In this process, it may integrate into the bacterial chromosome and accidentally incorporate antibiotic resistance genes during replication.
Release of Phage Particles:
The newly formed phage particles are released from the infected donor cell, and some may carry fragments of the donor bacterial DNA, which can include antibiotic resistance genes.
Infection of Recipient Cell:
The bacteriophage carrying the donor bacterial DNA infects a recipient bacterial cell. During this process, the transferred DNA from the phage may become integrated into the chromosome of the recipient cell.
bacterial transformation
Release of DNA from Donor Cell:
When a bacterial cell (the donor cell) dies and undergoes lysis, it releases fragments of its DNA into the surrounding environment. This DNA can include antibiotic-resistance genes.
Uptake by Recipient Cell:
A recipient bacterial cell takes up this naked DNA from the environment. This process is called transformation. The DNA fragments are then integrated into the recipient cell’s chromosome or plasmid, allowing the recipient to acquire new traits, such as antibiotic resistance.
bacterial transformation
Release of DNA from Donor Cell:
When a bacterial cell (the donor cell) dies and undergoes lysis, it releases fragments of its DNA into the surrounding environment. This DNA can include antibiotic-resistance genes.
Uptake by Recipient Cell:
A recipient bacterial cell takes up this naked DNA from the environment. This process is called transformation. The DNA fragments are then integrated into the recipient cell’s chromosome or plasmid, allowing the recipient to acquire new traits, such as antibiotic resistance.
efflux pumps
Efflux Pumps:
Bacteria use efflux pump proteins to actively pump antibiotics out of the bacterial cell. This mechanism reduces the intracellular concentration of the antibiotic, keeping it below therapeutic levels, which prevents the antibiotic from effectively targeting and killing the bacteria.
Mechanism of Action:
The efflux pumps are variants of membrane pumps found in all bacterial cells, used to transport various substances in and out of the cell. When antibiotics enter the cell, the efflux pumps actively expel them back into the environment before they can accumulate to effective concentrations.
Impact on Antibiotic Efficacy:
For antibiotics to be effective, they must reach their specific bacterial targets in sufficient concentrations and act within a reasonable timeframe. Efflux pumps interfere with this by maintaining antibiotic levels that are too low to inhibit essential bacterial processes, such as protein synthesis.
An example provided is resistance to tetracyclines (a class of antibiotics including aminoglycosides), where efflux pumps prevent concentrations from reaching levels that would block protein synthesis.
principal resistance mechanism employed by bacteria: modification of antibiotics by enzymes
Antibiotic Modification:
The antibiotic chloramphenicol is depicted in the image. It can be enzymatically inactivated through the addition of specific chemical groups, such as acetyl groups or phosphate groups.
These modifications are performed by specific resistance enzymes, such as:
Acetyl transferases: Enzymes that add acetyl groups to the antibiotic.
Phosphoryl transferases: Enzymes that add phosphate groups.
Impact of Modification:
By modifying the antibiotic, these enzymes effectively “decorate” the periphery of the chloramphenicol molecule. This alteration interferes with the antibiotic’s ability to bind to its target, typically ribosomes, thereby preventing it from inhibiting protein synthesis.
As a result, the antibiotic can no longer exert its intended effect, allowing the bacteria to survive despite the presence of the antibiotic.
Example of Inactivation:
The image shows the structural transformation of chloramphenicol into acetylated chloramphenicol. This modification changes the structure such that the antibiotic is no longer effective in binding to bacterial ribosomes, thus neutralizing its activity.
principal resistance mechanism against antibiotics, specifically how antibiotics are destroyed by enzymes produced by resistant bacteria
Enzymatic Destruction of Antibiotics:
The antibiotic penicillin is shown as an example. The structure of penicillin includes a β-lactam ring, which is crucial for its antibacterial activity.
Resistant bacteria produce an enzyme called β-lactamase (also known as penicillinase), which breaks a bond in the β-lactam ring of penicillin, converting it into an inactive form called penicilloic acid. By destroying this part of the molecule, the antibiotic becomes ineffective.
Mechanism of Action:
β-lactamase is secreted by bacteria into the periplasmic space, where it acts on β-lactam antibiotics like penicillin before they can reach their targets, such as bacterial cell wall enzymes.
The enzyme is extremely effective: a single β-lactamase molecule can hydrolyze 1000 penicillin molecules per second. Given that each resistant bacterial cell can secrete many β-lactamase enzymes, large amounts of penicillin can be inactivated quickly.
Overcoming β-lactamase Resistance:
To overcome β-lactamase-mediated resistance, new antibiotics were developed. For instance, methicillin is a second-generation semi-synthetic derivative of penicillin, designed to be resistant to β-lactamase cleavage. This made methicillin effective against bacteria that produce β-lactamase, although eventually some bacteria also developed resistance to methicillin (leading to MRSA – Methicillin-resistant Staphylococcus aureus).
principal resistance mechanism involving modification of the target site for the antibiotic polymyxin， which results in resistance.
Mcr-1 Gene and Enzyme Production:
The Mcr-1 gene is a mobile gene that can be transferred between bacteria via a plasmid.
Mcr-1 encodes an enzyme that modifies the target site of the antibiotic polymyxin on bacterial cells, specifically on the lipopolysaccharide (LPS) of the bacterial outer membrane.
Mechanism of Resistance:
Polymyxin (e.g., colistin) normally binds to LPS molecules on the outer membrane of Gram-negative bacteria through electrostatic interactions with negatively charged groups on the LPS.
The enzyme encoded by Mcr-1 modifies lipid A of the LPS by adding a phosphoethanolamine (pEtN) group, which neutralizes the negative charge of lipid A. This modification reduces the affinity of polymyxin for the LPS, preventing the antibiotic from binding effectively.
Impact on Polymyxin Activity:
In the absence of Mcr-1, polymyxin can bind to LPS and exert its antibacterial effects by disrupting the bacterial outer membrane, leading to cell death.
In the presence of Mcr-1, however, the addition of pEtN to LPS prevents polymyxin from binding effectively, allowing the bacteria to survive and resist the action of polymyxin (as depicted in the image with polymyxin being unable to bind to the modified LPS).
two additional resistance mechanisms used by methicillin-resistant Staphylococcus aureus (MRSA) to evade the effects of antibiotics
Release of Fatty Decoys by MRSA:
One of the limited treatment options for MRSA infections is the antibiotic daptomycin, yet 1 in 3 infections remains resistant to this drug.
MRSA cells produce and release membrane phospholipid decoys. These decoys are fatty molecules that mimic the bacterial cell membrane and trick the antibiotic daptomycin into binding to them rather than the actual bacterial membrane.
Daptomycin is designed to bind to bacterial membranes and disrupt their structure, leading to cell death. By releasing these decoys, MRSA prevents the antibiotic from reaching and damaging its own cell membrane, thereby avoiding destruction.
Modification of Transpeptidase/Penicillin-binding Protein:
MRSA has acquired a gene encoding a modified transpeptidase (penicillin-binding protein) that has lower affinity for β-lactam antibiotics like methicillin.
This modification allows MRSA to survive even in the presence of these antibiotics, which normally inhibit the enzymes responsible for cell wall synthesis.
Additionally, MRSA changes the outer membrane charge, likely to repel positively charged antibiotics, further contributing to resistance.
antibiotic (AB) resistance
Antibiotic Resistance is categorized into two types:
Inherent/Natural Resistance:
This resistance is due to the physical characteristics of the bacteria.
An example mentioned is Gram-negative bacteria, which naturally have some resistance mechanisms. This kind of resistance means there are no major concerns about antibiotics impacting these bacteria (“No worries”).
Acquired Resistance:
This occurs when bacteria that were previously susceptible to antibiotics become resistant. This happens due to the acquisition of resistance genes.
There are two mechanisms of acquiring resistance:
Vertical Transfer:
Resistance genes are passed from bacteria to their offspring.
Mutations occur randomly, and these mutations are passed down through the same species or strain.
Example mentioned: “MEGA plate,” which is a laboratory experiment showing how bacteria adapt to increasing antibiotic concentrations.
Horizontal Transfer:
Involves the transfer of resistance genes between unrelated strains.
Requires close proximity between bacterial cells to facilitate this gene transfer.
Different mechanisms of horizontal gene transfer in bacteria
Conjugation:
This involves a bacterium donating a plasmid to another bacterium.
Plasmids are circular pieces of DNA that often contain resistance genes, which can give bacteria an advantage in antibiotic-rich environments. The transfer occurs through a structure called a pilus, which connects the donor to the recipient.
Transduction:
In this process, a bacterium is infected by a virus (bacteriophage).
The virus acts as a vector to transfer antibiotic resistance genes from one bacterium to another.
Transformation:
This occurs when bacteria take up DNA from their environment, often from dead bacterial cells.
The DNA may contain resistance genes, allowing the receiving bacterium to gain new traits, such as antibiotic resistance.
Overview of the principal resistance mechanisms for bacteria in the context of antibiotics
Drug Efflux (1):
Bacteria use efflux pumps to actively pump antibiotics out of the cell. This reduces the intracellular concentration of the drug below therapeutic levels, rendering the antibiotic ineffective.
The note in the image indicates that these pumps lower the drug concentration “below therapeutic levels,” preventing it from acting on its target.
Enzymatic Modifications of Antibiotics (2):
Bacteria can modify antibiotics chemically, often through the addition of chemical groups like acetyl or phosphate groups.
This prevents the antibiotic from binding to its target site and neutralizes its effects.
Enzymatic Breakdown of Antibiotics (3):
Bacteria can produce enzymes such as β-lactamase, which breaks down β-lactam antibiotics (like penicillins and cephalosporins), rendering them ineffective.
This is a common mechanism where the antibiotic is physically degraded and inactivated.
Modification of Target Site (4):
Bacteria can alter the structure of their target sites (such as proteins or other molecules) to which the antibiotic binds.
This can involve changing the lipopolysaccharide (LPS) membrane, which prevents binding of antibiotics like polymyxins. Additionally, changes in the bacterial chromosome can lead to these modifications.
perspectives on antibiotics access issues, use, and misuse
Why Use Antibiotics?:
An example of their effectiveness is the significant reduction in deaths from pneumococcal pneumonia, with mortality rates decreasing from 20-40% to 5% due to the use of antibiotics.
Antibiotic Resistance:
Antibiotic resistance is widely recognized as a major medical issue. However, it is not the only issue when it comes to antibiotics. Access and appropriate use are also major concerns.
Access to Antibiotics:
Not everyone has access to antibiotics, which can lead to preventable deaths. For example, universal provision of antibiotics could prevent 75% of deaths from pneumonia.
There are alternative strategies, such as increasing vaccination rates (e.g., Haemophilus influenzae type b (Hib) and pneumococcal vaccines), which could help reduce the need for antibiotics by preventing bacterial infections in the first place.
Antibiotics access issues, use, and misuse
Why Use Antibiotics?:
Antibiotics have been proven effective in reducing mortality from certain infections. For example, pneumococcal pneumonia deaths decreased from 20-40% to 5% after antibiotics became widely used.
Antibiotic Resistance:
Antibiotic resistance is widely recognized as a major public health concern, but it is important to remember that it is not the only issue related to antibiotic use. Access and equitable distribution are also significant challenges.
Access Issues:
Not everyone has access to antibiotics. Limited access means that preventable deaths occur, particularly in vulnerable populations. For instance, universal provision of antibiotics could prevent 75% of deaths from pneumonia.
Scaling up vaccinations, such as Haemophilus influenzae type b (Hib) and pneumococcal vaccines, can reduce the burden of bacterial infections and subsequently limit the need for antibiotics.
Innovation gap
Strategies
Current strategies to overcome antibiotic resistance, specifically focused on β-lactamase inhibitors
Role of β-lactamase in Resistance:
β-lactamase is an enzyme produced by some bacteria that can inhibit β-lactam antibiotics (e.g., penicillin, amoxicillin) by hydrolyzing the β-lactam ring, which is crucial for the antibiotic’s activity. This renders the antibiotic ineffective.
Using β-Lactam Antibiotics with Inhibitors:
β-lactam antibiotics can be used in combination with β-lactamase inhibitors like clavulanic acid to counteract resistance.
Clavulanic acid itself lacks antibiotic activity, but it binds to β-lactamase, preventing it from hydrolyzing the β-lactam ring of antibiotics.
This restores the effectiveness of β-lactam antibiotics such as penicillin and amoxicillin.
Clavulanic acid is often used in combination with amoxicillin, resulting in a formulation known as Augmentin.
Other β-Lactamase Inhibitors:
Sulbactam is another example of a β-lactamase inhibitor that functions similarly to clavulanic acid, helping to restore the activity of β-lactam antibiotics.
Ongoing Research:
New β-lactamase inhibitors are being investigated to develop more effective solutions against resistant bacterial strains.
Bacterial Cell Structure
Bacterial Cell Structure:
The bacterial cell membrane is shown, with the cytoplasm beneath it.
Bacteria produce enzymes, such as transpeptidase (also known as penicillin-binding protein), which helps form cross-links in the peptidoglycan cell wall to stabilize its structure.
Penicillin Action:
Penicillin is added to the bacterial environment.
Penicillin binds to the penicillin-binding protein (transpeptidase), inhibiting the formation of cross-links in the bacterial cell wall.
Bacterial Resistance Mechanism:
When bacteria acquire a resistance gene, they can produce beta-lactamase.
Beta-lactamase binds to penicillin and breaks apart its bond, preventing penicillin from inhibiting transpeptidase.
This allows the bacteria to continue forming the cell wall and maintain its structure.
Overcoming Resistance:
Clavulanic acid is added along with penicillin.
Clavulanic acid resembles penicillin and binds permanently to beta-lactamase, inhibiting it.
This allows penicillin to remain active, bind to transpeptidase, and inhibit cell wall cross-linking, leading to bacterial cell death.
Combined Therapy:
A combination of an antibiotic (penicillin) and a beta-lactamase inhibitor (clavulanic acid) ensures the effectiveness of the antibiotic.
This strategy is used to overcome resistance and effectively kill bacteria.
Pivampicillin
Pivampicillin (Figure A):
Modification: Pivampicillin is a prodrug of ampicillin. Once administered, it is hydrolyzed into ampicillin and pivalic acid.
Benefit: This modification improves absorption, leading to 2-3 fold increased serum concentrations of ampicillin.
Sultamicillin (Figure B):
Modification: Sultamicillin is a covalent linkage between ampicillin and sulbactam, which is a β-lactamase inhibitor.
Benefit: This covalent linking increases microbial activity 4-32 fold, making the drug more effective in overcoming resistance caused by β-lactamase-producing bacteria.
Why Chemically Cross-Link Molecules?:
The cross-linking ensures simultaneous absorption of both molecules (ampicillin and its inhibitor) in a constant ratio.
These molecules are hydrolyzed to release two active components, allowing for enhanced therapeutic action against resistant bacteria.
overview of modifications in the core structures of different antibiotic classes to improve their effectiveness against pathogens and overcome resistance
Antibiotic Core Modifications
Penicillins:
Generation 1: Penicillin G
Generation 2: Amoxicillin (improved effectiveness against broader spectrum bacteria)
Generation 3: Ticarcillin
Generation 4: Piperacillin
Modifications: Peripheral chemical modifications (colored red) are made to the core scaffold (colored black) to create successive generations with improved properties.
Cephalosporins:
Generation 1: Cefalotin
Generation 2: Cefuroxime
Generation 3: Ceftazidime
Generation 4: Cefepime
Modifications to cephalosporins have allowed enhanced activity and resistance to beta-lactamases.
Quinolones:
Generation 1: Nalidixic acid
Generation 2: Ciprofloxacin
Generation 3: Levofloxacin
Generation 4: Moxifloxacin
The modifications have improved activity against Gram-positive and Gram-negative bacteria, along with pharmacokinetic properties.
Tetracyclines:
Older Generation: Oxytetracycline and Doxycycline
Newer Generation: Tigecycline
Tigecycline, for example, has modifications to combat resistance mechanisms such as efflux pumps and ribosomal protection proteins.
Key Takeaways
Synthetic Tailoring: Chemical modifications (shown in red) of core scaffolds (shown in black) are used to create successive generations of antibiotics, increasing their effectiveness against resistant bacteria.
Challenges: There is an urgent need for new antibiotic core scaffolds due to rising resistance.
Characteristics of New Antibiotics:
Active against both Gram-positive and Gram-negative pathogens.
Lack of cross-resistance to existing drugs.
Ability to be synthetically tailored for further modifications.
efflux pump resistance
Resistance by Antibiotic Efflux
Efflux pumps in bacterial cell membranes actively pump antibiotics out of the cell, reducing the intracellular concentration and rendering the antibiotic ineffective.
Tetracycline is a common target for such efflux pumps, leading to reduced efficacy as it cannot accumulate inside the cell to inhibit protein synthesis.
Strategies to Overcome Resistance
Solution 1: Modify Existing Scaffold
Tigecycline is a modified derivative of tetracycline.
It is designed such that it is no longer a substrate for the efflux pump, allowing it to stay inside the bacterial cell.
Once inside, tigecycline can inhibit protein synthesis effectively, targeting the ribosome and preventing bacterial growth.
Solution 2: Use a New Scaffold
A different approach is to develop a new antibiotic scaffold that bypasses the efflux mechanism entirely.
Retapamulin is an example of such a scaffold. It is not a substrate for the efflux pump and works by binding to a different site on the ribosome, effectively inhibiting protein synthesis.
This provides an alternative way to inhibit bacterial growth without being affected by the efflux resistance mechanism.
Key Takeaway
Efflux Pump Resistance: Bacteria use efflux pumps to resist antibiotics by removing them from the cell. This strategy is especially common against tetracyclines.
Modification Approaches:
Modify Existing Antibiotic Scaffolds: Chemical modification (as with tigecycline) prevents the antibiotic from being recognized by the efflux pump.
Create New Antibiotic Scaffolds: Designing completely new antibiotics like retapamulin that can evade efflux mechanisms and bind to unique ribosomal sites provides another effective solution.
Vancomycin and Its Target
Vancomycin and Its Target
Vancomycin is a glycopeptide antibiotic that targets the peptidoglycan cell wall of bacteria.
It works by binding to the D-Ala-D-Ala terminus of the pentapeptide chain in peptidoglycan, thereby preventing cross-linking of the cell wall, which is crucial for bacterial survival.
Mechanism of Resistance
Some bacteria develop resistance to vancomycin by altering the binding site.
Specifically, they replace the terminal D-Ala-D-Ala sequence with D-Ala-D-lactate (D-Ala-D-Lac).
This modification significantly reduces vancomycin’s ability to bind to the target site due to the altered affinity between vancomycin and the new D-Ala-D-Lac structure.
As a result, vancomycin cannot effectively inhibit cell wall synthesis, allowing resistant bacteria to survive.
Structural Representation
In the diagram:
Vancomycin is represented in purple and binds to the pentapeptide terminus.
The pentapeptide chain is shown with green circles, which connect to the peptidoglycan backbone.
The substitution from D-Ala-D-Ala to D-Ala-D-Lac is highlighted as a key mechanism that prevents vancomycin from binding effectively.
Key Points
Resistance Mechanism: Bacterial resistance to vancomycin occurs due to a chemical modification of the target site, specifically changing D-Ala-D-Ala to D-Ala-D-Lac.
Reduced Binding Affinity: This modification reduces the binding affinity of vancomycin, making it unable to inhibit cell wall synthesis effectively.
Clinical Implications: This resistance mechanism poses a significant challenge in treating infections caused by vancomycin-resistant bacteria, such as vancomycin-resistant Enterococci (VRE).
Vancomycin Resistance and Derivatives
Resistance Mechanism: Bacteria develop resistance to vancomycin by changing the D-Ala-D-Ala binding site to D-Ala-D-Lac, reducing the affinity of vancomycin for the peptidoglycan cell wall.
Rational Design: To counter this resistance, new vancomycin derivatives have been developed by modifying the structure to enhance binding and target additional bacterial processes.
Modifications and Their Effects
Increased Membrane Permeability:
Modifications to the vancomycin structure (e.g., adding a lipophilic group) have made it possible for the antibiotic to induce membrane permeability, making it more potent by facilitating better access to bacterial targets.
Inhibition of Transglycosylation:
The modifications also allow vancomycin to inhibit transglycosylase, an enzyme involved in the synthesis of peptidoglycan, thereby inhibiting cell wall biosynthesis by an additional mechanism.
Dual Binding:
The modified vancomycin can bind to both D-Ala-D-Ala and D-Ala-D-Lac, overcoming the change that normally leads to resistance.
This means that vancomycin can now inhibit transpeptidase effectively, blocking cross-linking and ultimately weakening the bacterial cell wall.
Structural Changes
The diagram shows several chemical modifications to the vancomycin molecule:
New functional groups are added to enhance binding to the modified D-Ala-D-Lac, allowing it to retain its antibacterial activity.
These changes are also intended to target multiple bacterial pathways, reducing the likelihood of resistance development.
Benefits of Modified Vancomycin
Three Mechanisms of Action: The modified vancomycin targets bacteria in three different ways:
Inhibiting transpeptidase activity.
Inhibiting transglycosylase.
Increasing membrane permeability.
Potent and Durable Activity: Having multiple mechanisms of action reduces the chances of bacteria developing resistance because they would need to overcome all three barriers simultaneously.
Reduced Resistance: The rational design of these derivatives has led to lower minimum inhibitory concentrations (MIC) against vancomycin-resistant Enterococci (VRE), indicating increased potency against resistant bacteria.
Key Takeaways
The chemical modification of vancomycin derivatives helps to overcome resistance mechanisms by targeting multiple bacterial processes.
Rational drug design allows for the development of antibiotics that bind effectively even when the bacterial target is altered.
The modifications result in enhanced membrane permeability and dual binding ability, making the antibiotic more effective and reducing the potential for resistance.
the search for new antibiotic scaffolds from underexplored ecological niches and the role of genomic analysis in this pursuit
Need for New Antibiotic Scaffolds
There is an urgent need for new antibiotic scaffolds due to the rising threat of bacterial resistance.
Natural Sources:
Over two-thirds of clinically used antibiotics are natural products or semisynthetic derivatives.
To discover new antibiotics, researchers look to nature—exploring diverse and often isolated environments.
Potential Sources for New Antibiotics
Newly Isolated Bacteria:
Hot springs, isolated environments, plants, marine life, and animals are all potential sources of novel antibiotic-producing bacteria.
These environments contain unique microbial communities that might produce novel antibiotics to survive in their competitive habitats.
Soil Screening:
Soil is a highly promising but underexplored source, as very little is actually known about the microbial diversity present in soil samples.
Screening soil for novel bacterial species and their metabolites has the potential to yield new antibiotic compounds.
Genomic Approaches
Genome Sequencing:
Sequencing the genomes of a large number of bacteria and fungi has revealed many silent biosynthetic gene clusters—up to two dozen per organism.
These clusters have the potential to encode natural products, but they are often not expressed under standard laboratory conditions.
Activating Silent Gene Clusters:
A variety of methods are used to activate silent biosynthetic gene clusters to evaluate the novelty and activity of the resulting small molecules.
Turning on these silent genes can help discover new antibiotics with unique properties, providing a way to bypass the limitations of conventional cultivation methods.
Rational Design of Completely New Antibiotics
Linezolid is highlighted as an example of a de novo synthetic drug—meaning that it is a completely new scaffold that does not occur in nature.
The design of Linezolid was based on detailed knowledge of the target, rather than modifying an existing natural product.
Mechanism of Action: Inhibition of Protein Synthesis
Protein synthesis is a vital bacterial process that occurs in three stages:
Initiation: The assembly of the ribosome on the mRNA and the binding of the first tRNA.
Elongation: Successive tRNAs bring amino acids, which are added to the growing peptide chain.
Termination: The completed protein is released once the ribosome reaches a stop codon.
Linezolid’s Unique Target:
Linezolid inhibits the initiation phase of protein synthesis by preventing the assembly of the ribosomal complex.
This mechanism is different from other antibiotics like chloramphenicol, macrolides, and gentamicin, which generally inhibit the elongation phase of protein synthesis.
By targeting a different aspect of protein synthesis (initiation rather than elongation), Linezolid represents a novel approach, making it effective against bacteria that may have resistance to other classes of protein synthesis inhibitors.
Key Insights
Novel Scaffold Design: Linezolid is an entirely new chemical structure, specifically designed based on molecular biology and biochemical insights into how bacterial ribosomes work.
Reduced Resistance Risk: Since Linezolid targets the initiation phase rather than elongation, it can be effective against bacteria that are resistant to other protein synthesis inhibitors. This unique mechanism reduces the risk of cross-resistance.
Summary
The development of new antibiotics, such as Linezolid, through rational drug design and de novo synthesis allows researchers to create antibiotics with novel mechanisms of action.
Targeting Initiation: By targeting different steps in a critical bacterial process, new antibiotics can overcome resistance mechanisms that limit the effectiveness of existing drugs.
Macronutrients and micronutrients
Key points
General info on vitamins
Overview of vitamins and their classification based on solubility
Vitamins are classified into two main categories based on their solubility:
Water-Soluble Vitamins:
Vitamin B Group (B1, B2, B3, B5, B6, B7, B9, B12)
Vitamin C
Fat-Soluble Vitamins:
Vitamin A
Vitamin D
Vitamin E
Vitamin K
Characteristics of Water-Soluble Vitamins
Structurally Diverse, Functionally Similar: Although structurally diverse, they have similar functions related to metabolism (e.g., Vitamin B complex).
Vitamin B Requires Modification: Vitamin B group vitamins often require modification to become biologically active.
Role in Metabolism: Vitamin B group acts as mobile metabolic carriers (e.g., coenzymes in various biochemical reactions).
Readily Absorbed and Excreted: Water-soluble vitamins are easily absorbed and transported in the bloodstream, and they are also readily excreted when consumed in excess, which reduces the risk of toxicity.
Characteristics of Fat-Soluble Vitamins
Structurally Similar, Functionally Diverse: Fat-soluble vitamins have similar structures but diverse functions (e.g., Vitamin A for vision, Vitamin D for calcium regulation, etc.).
Less Readily Absorbed: These vitamins are less readily absorbed compared to water-soluble vitamins and depend on dietary fats for absorption.
Risk of Toxicity: Because they are stored in body fat and liver, fat-soluble vitamins can be toxic if taken in excess.
Transportation in the Body: Since they are fat-soluble, they need specific mechanisms to be transported throughout the body, often involving binding to proteins or incorporation into lipoproteins.
B vitamins, their corresponding coenzymes, their role in metabolic reactions, and the consequences of deficiency
Coenzymes Derived from Vitamins
Coenzymes are small organic molecules essential for enzyme activity, and many are derived from vitamins, particularly the B vitamins.
Breakdown of Selected B Vitamins:
Riboflavin (Vitamin B2)
Coenzyme: Flavin adenine dinucleotide (FAD)
Typical Reaction Type: Oxidation-reduction reactions
Consequence of Deficiency:
Cheilosis (cracking at the corners of the mouth)
Angular stomatitis (lesions around the mouth)
Dermatitis (skin inflammation)
Nicotinic Acid (Niacin, Vitamin B3)
Coenzyme: Nicotinamide adenine dinucleotide (NAD⁺)
Typical Reaction Type: Oxidation-reduction reactions
Consequence of Deficiency:
Pellagra, which includes symptoms of dermatitis, depression, and diarrhea
Folic Acid
Coenzyme: Tetrahydrofolate (THF)
Typical Reaction Type: Transfer of one-carbon components, crucial for thymine synthesis
Consequence of Deficiency:
Anemia (reduced red blood cells)
Neural tube defects in fetal development (e.g., spina bifida)
Other B Vitamins (Not highlighted in red but included for reference):
Thiamine (Vitamin B1):
Coenzyme: Thiamine pyrophosphate
Role: Aldehyde transfer
Deficiency: Beriberi (weight loss, heart problems, neurological dysfunction)
Pyridoxine (Vitamin B6):
Coenzyme: Pyridoxal phosphate
Role: Group transfer to/from amino acids
Deficiency: Depression, confusion, convulsions
Vitamin B12:
Coenzyme: 5’-Deoxyadenosyl cobalamin
Role: Transfer of methyl groups and intramolecular rearrangements
Deficiency: Anemia, pernicious anemia, methylmalonic acidosis
Summary
B Vitamins play critical roles in metabolism by serving as precursors to coenzymes.
These coenzymes participate in key biochemical reactions like oxidation-reduction and carbon transfer.
Deficiency in any of these vitamins can lead to serious health conditions, such as skin disorders, mental health issues, anemia, and developmental defects.
Non-Coenzyme Vitamins and Their Functions
Vitamin C (Ascorbic Acid)
Function: Antioxidant—helps protect cells from oxidative damage by neutralizing free radicals.
Deficiency: Scurvy
Symptoms include swollen and bleeding gums and subdermal hemorrhaging due to weakened connective tissue.
Vitamin A
Function: Plays a role in vision, as well as growth and reproduction.
Deficiency:
Night blindness (inability to see well in low light).
Cornea damage and increased susceptibility to infections affecting the respiratory and gastrointestinal tract.
Vitamin D
Function: Regulates calcium and phosphate metabolism, crucial for bone health.
Deficiency:
Rickets in children, characterized by soft, bendy bones and bowed legs.
Osteomalacia in adults, which involves pain in bones and hips, and increased risk of bone fractures, particularly in the hip.
Vitamin E
Function: Antioxidant—protects cell membranes from oxidative damage.
Deficiency (not highlighted): Can lead to inhibition of sperm production and lesions in muscles and nerves (rare).
Vitamin K
Function: Involved in blood coagulation, essential for proper blood clotting.
Deficiency (not highlighted): Can result in subdermal hemorrhaging due to impaired clotting ability.
Summary
Non-Coenzyme Vitamins: These vitamins have diverse functions beyond enzymatic reactions, such as antioxidant protection, vision, bone health, and blood clotting.
Deficiencies in these vitamins can lead to serious health issues, including scurvy, rickets, night blindness, and impaired coagulation.
Why humans cannot synthesize Vitamin C
Inability to Synthesize Vitamin C
Vitamin C Biosynthesis Pathway:
In most mammals, Vitamin C (L-ascorbate) is synthesized from glucose through a series of enzymatic reactions.
The crucial last step in Vitamin C biosynthesis involves the conversion of L-gulono-γ-lactone into ascorbic acid (Vitamin C).
This step is catalyzed by the enzyme gulonolactone oxidase.
Humans Lack Gulonolactone Oxidase:
Human cells lack the ability to complete the conversion of L-gulono-γ-lactone into ascorbic acid because the enzyme gulonolactone oxidase is non-functional.
The gene that codes for gulonolactone oxidase is present in the human genome, but due to multiple mutations, it has become a pseudogene—meaning it no longer produces a functional enzyme.
Evolutionary Loss:
The loss of the functional gulonolactone oxidase enzyme occurred in humans, as well as in guinea pigs, some bats, and primates. These species have also lost the ability to synthesize Vitamin C and must obtain it from their diet.
Summary of Pathway
The biosynthesis of Vitamin C involves multiple steps, starting from simple sugars (e.g., glucose or mannose) through intermediate compounds.
In mammals that can synthesize Vitamin C, the conversion of L-gulono-γ-lactone to L-ascorbate (Vitamin C) is the final step, which humans cannot perform due to a non-functional gulonolactone oxidase enzyme.
Consequences
As a result, humans must obtain Vitamin C from dietary sources, such as citrus fruits, vegetables, and other fresh produce.
Vitamin C deficiency can lead to scurvy, which manifests as symptoms like bleeding gums, weakened connective tissue, and impaired wound healing.
Key Takeaway
Humans and some other animals cannot synthesize Vitamin C due to the loss of function of a crucial enzyme in the biosynthesis pathway. This loss requires dietary intake of Vitamin C to meet physiological needs.
Energy Conservation:
The pathways to produce vitamins are complex and energy-consuming. It may be more “economical” to obtain Vitamin C through diet rather than synthesizing it.
Reduction in Harmful Byproducts:
The enzyme gulono oxidase, which is involved in Vitamin C synthesis, also produces hydrogen peroxide (H₂O₂), a reactive oxygen species.
This can cause cellular damage, so not synthesizing Vitamin C may help reduce the production of such harmful byproducts.
Regulation of Stress Response:
Levels of Vitamin C regulate the hypoxia-inducible factor 1-alpha (HIF1α), a transcription factor activated by low oxygen or limited Vitamin C.
This mechanism allows for fine-tuning of stress responses based on nutritional status.
Role of Pseudogenes:
Pseudogenes, remnants of genes once responsible for Vitamin C synthesis, can play a role in epigenetic regulation of gene expression.
Experiment conducted by Sir James Lind
The slide describes the experiment conducted by Sir James Lind, a ship’s surgeon in the British Navy, on the treatment and prevention of scurvy. In the experiment:
Twelve sailors with scurvy were divided into groups of two.
All sailors were kept on the same basic diet (mainly hardtack and salted meats).
Each group received one of six different supplements: cider, elixir of vitriol, vinegar, seawater, lemons and oranges, or an electuary.
The results showed that:
Sailors who received lemons and oranges improved quickly.
Those given cider showed some improvement, while others showed no significant improvement.
The key question here is: “What did this experiment really prove?”
This experiment proved that citrus fruits (lemons and oranges) were effective in treating scurvy. It suggested a connection between these fruits and the prevention or cure of scurvy, highlighting the importance of vitamin C (though Lind did not understand the specific nutrient at the time). This laid the foundation for later understanding that scurvy is caused by a deficiency of vitamin C, which is abundant in citrus fruits.
Information on ascorbic acid (Vitamin C)
The slide provides information on ascorbic acid (Vitamin C) and its chemistry and biochemistry:
Vitamin C as an Electron Donor: Vitamin C acts as an electron donor (reducing agent or antioxidant) for enzymes that exhibit monooxygenase or dioxygenase activity. This property makes it effective in biochemical redox reactions.
Role in Hydroxylation: Vitamin C accelerates hydroxylation in various biosynthetic pathways, which is crucial for the modification of molecules in the body.
Enzyme Activity: Vitamin C acts as an electron donor for eight enzymes in the human body. These enzymes are involved in several important physiological processes, including:
Collagen Synthesis: Hydroxylation of proline and lysine is critical for stabilizing collagen, making Vitamin C essential for the formation and maintenance of connective tissues.
Tyrosine Metabolism: It is involved in the catabolism and regulation of tyrosine.
Carnitine Biosynthesis: Vitamin C plays a role in the synthesis of carnitine, which is crucial for transporting fatty acids into mitochondria for energy production.
The slide also depicts chemical structures of ascorbic acid, its deprotonated form (ascorbate), and its oxidized form (dehydroascorbic acid).
Structure of collagen
Amino Acid Sequence of Collagen:
Glycine is found at every third position in the amino acid sequence of a collagen chain.
Proline and Hydroxyproline are also abundant in collagen. They form a frequent tripeptide sequence: glycine-proline-hydroxyproline.
Structure of Collagen:
Collagen is made up of three helical peptide chains, each about 1,000 residues long, that form a triple helix.
The figure illustrates the collagen structure, starting from the primary sequence to the complete triple helix.
Role of Hydroxyproline:
The stabilization of the collagen triple helix requires hydroxyproline.
Hydroxyproline formation is dependent on Vitamin C, which is essential for the hydroxylation of proline.
Hydroxyproline is crucial for inter-strand hydrogen bond formation, which stabilizes the collagen structure.
Regulation of HIFs (Hypoxia-Inducible Factors)
Regulation of HIFs (Hypoxia-Inducible Factors)
HIF-1α and HIF-1β are transcription factors involved in the cellular response to oxygen levels.
Under normoxic conditions (normal oxygen levels), HIF-1α undergoes hydroxylation by prolyl hydroxylase domain enzymes (PHD1, PHD2, and PHD3) and factor-inhibiting HIF (FIH).
Hydroxylated HIF-1α is targeted for proteasomal degradation, thus preventing it from activating genes involved in the hypoxic response.
Vitamin C plays a role in the function of PHD enzymes, acting as a cofactor that helps in the hydroxylation of HIF-1α.
Hypoxic Conditions (Low Oxygen Levels)
When oxygen levels are low (hypoxia), hydroxylation by PHD enzymes is inhibited.
HIF-1α accumulates, binds to HIF-1β, and translocates to the nucleus, where it binds to hypoxia response elements (HREs) on DNA.
This results in the activation of genes involved in:
Metabolism (adjusting to low oxygen availability)
Survival (promoting cell survival under low oxygen)
Angiogenesis (formation of new blood vessels to improve oxygen supply)
Role of Vitamin C
Vitamin C, as an electron donor, is crucial for the activity of PHD enzymes, which require it to hydroxylate HIF-1α.
Without adequate Vitamin C, the hydroxylation process is impaired, leading to increased stability of HIF-1α and activation of the hypoxia response, even under normal oxygen conditions.
Hydroxylation of Proline
Hydroxylation of Proline
Proline hydroxylase is the enzyme that catalyzes the conversion of proline to hydroxyproline in collagen.
This reaction requires several cofactors and substrates, including:
α-ketoglutarate as a co-substrate, which is converted to succinate.
Molecular oxygen (O₂) and Fe²⁺ (ferrous iron) are also required for the hydroxylation reaction.
Vitamin C (ascorbic acid) is crucial for maintaining Fe²⁺ in its reduced form.
Role of Vitamin C
Vitamin C functions as a reducing agent that regenerates Fe²⁺ from Fe³⁺ after the oxidation occurs during the hydroxylation reaction.
Without Vitamin C, Fe²⁺ cannot be reduced back, leading to decreased activity of proline hydroxylase, which affects the synthesis of hydroxyproline.
Hydroxyproline is essential for stabilizing the collagen triple helix through hydrogen bonding, which provides structural strength to collagen.
Reaction Products
The hydroxylation of proline produces hydroxyproline, which contributes to the stability of the collagen fibers.
Dehydroascorbate is formed when Vitamin C donates electrons during the process, highlighting its role as a cofactor in collagen synthesis.
Regulation of HIF-1α (Hypoxia-Inducible Factor 1-alpha) under normal oxygen conditions
HIF-1α Regulation Under Normoxia
PHD Enzymes:
Under normal oxygen conditions (normoxia), HIF-1α is targeted by prolyl hydroxylase domain (PHD) enzymes.
The PHD enzymes hydroxylate HIF-1α, a reaction that requires:
O₂ (oxygen)
αKG (α-ketoglutarate)
Fe²⁺ (ferrous iron)
Ascorbate (Vitamin C) as a cofactor
The reaction converts α-ketoglutarate into succinate and CO₂.
Hydroxylation and Ubiquitination:
After hydroxylation, HIF-1α is recognized by the von Hippel-Lindau protein (pVHL), which tags it for ubiquitination (Ub).
Ubiquitination leads to the proteasomal degradation of HIF-1α, preventing it from initiating a hypoxic response.
Under Hypoxia
When oxygen levels are low (hypoxia), the activity of PHD enzymes decreases.
HIF-1α is stabilized, translocates into the nucleus, and dimerizes with HIF-1β.
The HIF-1 complex binds to specific DNA sequences, activating the transcription of genes involved in:
Angiogenesis (formation of new blood vessels)
Glycolysis (to adapt to low oxygen availability)
Mitophagy (removal of damaged mitochondria)
Survival mechanisms for adaptation to hypoxic stress
Role of Vitamin C
Vitamin C is essential for the activity of PHD enzymes by maintaining Fe²⁺ in the reduced state.
Inadequate Vitamin C impairs the hydroxylation of HIF-1α, leading to increased stability and accumulation of HIF-1α, even under normal oxygen levels, which could falsely initiate a hypoxic response.
Macrocytic anemia in pregnant women
Macrocytic Anemia (Large Cell Anemia)
Macrocytic anemia, also referred to as megaloblastic anemia, is characterized by the presence of abnormally large red blood cells.
The condition occurs because:
Cells cannot produce DNA quickly enough for proper cell division, which leads to a delay in cell division.
As a result, cells grow too large before they are able to divide.
One cause of megaloblastic anemia is a deficiency in folate (Vitamin B9), which is crucial for DNA synthesis.
Wills Factor
Lucy Wills discovered that a nutritional factor in yeast could prevent and cure macrocytic anemia in pregnant women. This substance was later identified as folate, a B-group vitamin.
Folate is essential for proper DNA synthesis, and its deficiency can lead to impaired cell division, causing the development of large, immature red blood cells (megaloblasts), characteristic of macrocytic anemia.
Folic Acid (Folate, Vitamin B9)
Structure of Folic Acid
Folic acid, also known as pteroylglutamic acid, consists of three major components:
Pteridine Ring (in pink):
A bicyclic, heterocyclic ring structure known as a pteridine ring.
The term pteron means “wing” in Greek, and this ring structure is found in a large class of biological pigments, particularly those in butterfly wings.
p-Aminobenzoic Acid (PABA) (in black):
This is a part of the folic acid structure involved in linking the pteridine ring to the glutamate group.
Glutamic Acid (in blue):
Glutamic acid is an amino acid that forms the terminal portion of the folic acid structure.
Pteroylglutamic Acid (Folic Acid)
The full structure of folic acid is called pteroylglutamic acid, which is crucial for various metabolic processes in the body, including DNA synthesis and repair.
Conversion of folic acid to tetrahydrofolate (THF)
Conversion of Folic Acid
First Reduction:
Folic acid is reduced to dihydrofolate (DHF).
The reducing agent in this reaction is NADPH, which donates electrons, and NADP⁺ is produced.
Second Reduction:
Dihydrofolate is further reduced to tetrahydrofolate (THF), the active form of folate.
Again, NADPH serves as the electron donor in this reaction.
Both reduction steps are catalyzed by the enzyme dihydrofolate reductase (DHFR), which specifically uses NADPH as the cofactor. This enzyme reduces the pyrazine part of the pteridine ring of folic acid.
Role of NADPH
NADPH acts as the electron donor (reducing agent) during these two reduction reactions, making it crucial for the activation of folic acid into its biologically functional form.
Forms of Tetrahydrofolate
Monoglutamate Form: The initial structure formed after the reduction steps.
Polyglutamate Form: In liver cells, additional glutamic acid residues are added to form a poly-γ-glutamate tail. This polyglutamate form is more efficient in retaining THF within cells and is better suited for its role in one-carbon metabolism.
Importance of THF
Tetrahydrofolate is essential for various cellular processes, especially DNA synthesis and repair, as it serves as a carrier of one-carbon units needed in nucleotide biosynthesis.
Folate Absorption and Conversion in Humans
Dietary Intake:
Humans source folates from their diet.
Intestinal Processing:
In the intestine, folate is usually found in the polyglutamate form.
The polyglutamate form is converted to a monoglutamate form for absorption.
Active Transport: This monoglutamate form is absorbed into cells by active transport.
Conversion in Cells:
Once inside the cells, folic acid is converted to tetrahydrofolate (THF) through a reduction reaction catalyzed by dihydrofolate reductase (DHFR). THF is the active form used in various metabolic processes.
Storage in the Liver:
Excess folate is stored in the liver.
In the liver, folate is converted back into the polyglutamate form, which allows it to be retained more effectively in cells and utilized when needed.
Role of Folic Acid in One-Carbon Metabolism
Role of Folic Acid in One-Carbon Metabolism
Tetrahydrofolate (THF), derived from folic acid, acts as a coenzyme that participates in the generation and utilization of single carbon functional groups, including:
Methyl (CH₃)
Methylene (CH₂)
Formyl (HCO)
These single carbon units are crucial in various metabolic reactions, including the synthesis of nucleotides and amino acids.
Key Reactions Involving Folate
Amino Acid Metabolism:
Serine and glycine are important sources of one-carbon units. The conversion between these amino acids involves THF as a cofactor.
The conversion of homocysteine to methionine also involves a one-carbon transfer, with THF acting in the methylation process.
Nucleotide Synthesis:
5,10-Methylene-THF donates a one-carbon unit to convert dUMP to dTMP, which is critical for DNA synthesis.
Purine synthesis also relies on folate derivatives as donors of formyl groups, which are essential for building purine rings.
Histidine Metabolism:
Formiminoglutamate (FIGLU), derived from histidine, requires THF for further processing.
Metabolic Intermediates and End Products
Formate is an important one-carbon donor in this pathway.
5,10-Methylene-THF and 5-formyl-THF are key intermediates in the folate cycle.
The slide highlights key substrates and products:
Orange circles indicate major sources of one-carbon units (e.g., serine, glycine).
Pink circles indicate major end products of one-carbon metabolism (e.g., purines, methionine).
Importance of Folates
Folate derivatives are essential for cell growth and tissue development, particularly in rapidly dividing cells.
Since mammals cannot synthesize folate derivatives de novo, they must obtain folate from exogenous sources (i.e., diet).
Folic acid is vital for one-carbon metabolism, which is crucial for synthesizing nucleotides (purines and pyrimidines) and certain amino acids. This makes folate an essential vitamin for DNA synthesis, cell growth, and repair. Deficiency can lead to issues such as megaloblastic anemia and neural tube defects during fetal development.
Role of Folic Acid in One-Carbon Metabolism
Tetrahydrofolate (THF), derived from folic acid, acts as a coenzyme that participates in the generation and utilization of single carbon functional groups, including:
Methyl (CH₃)
Methylene (CH₂)
Formyl (HCO)
These single carbon units are crucial in various metabolic reactions, including the synthesis of nucleotides and amino acids.
Key Reactions Involving Folate
Amino Acid Metabolism:
Serine and glycine are important sources of one-carbon units. The conversion between these amino acids involves THF as a cofactor.
The conversion of homocysteine to methionine also involves a one-carbon transfer, with THF acting in the methylation process.
Nucleotide Synthesis:
5,10-Methylene-THF donates a one-carbon unit to convert dUMP to dTMP, which is critical for DNA synthesis.
Purine synthesis also relies on folate derivatives as donors of formyl groups, which are essential for building purine rings.
Histidine Metabolism:
Formiminoglutamate (FIGLU), derived from histidine, requires THF for further processing.
Metabolic Intermediates and End Products
Formate is an important one-carbon donor in this pathway.
5,10-Methylene-THF and 5-formyl-THF are key intermediates in the folate cycle.
The slide highlights key substrates and products:
Orange circles indicate major sources of one-carbon units (e.g., serine, glycine).
Pink circles indicate major end products of one-carbon metabolism (e.g., purines, methionine).
Importance of Folates
Folate derivatives are essential for cell growth and tissue development, particularly in rapidly dividing cells.
Since mammals cannot synthesize folate derivatives de novo, they must obtain folate from exogenous sources (i.e., diet).
Folic acid is vital for one-carbon metabolism, which is crucial for synthesizing nucleotides (purines and pyrimidines) and certain amino acids. This makes folate an essential vitamin for DNA synthesis, cell growth, and repair. Deficiency can lead to issues such as megaloblastic anemia and neural tube defects during fetal development.
Role of Dihydrofolate Reductase (DHFR)
Role of Dihydrofolate Reductase (DHFR)
DHFR is an enzyme crucial for converting folic acid to its active form, tetrahydrofolate (THF).
THF is necessary for the synthesis of nucleotides, which are essential for DNA replication and cell division.
Antimetabolites as Drugs
An antimetabolite is a synthetic compound structurally related to a natural metabolite, designed to interfere with its function.
Drugs Targeting DHFR
Anticancer Agents:
Aminopterin and Methotrexate are structurally similar to folic acid and act as competitive inhibitors of human DHFR.
These drugs inhibit the conversion of dihydrofolate to tetrahydrofolate, thus reducing nucleotide synthesis and inhibiting cell proliferation.
Methotrexate, for instance, is widely used in cancer treatment to slow down rapidly dividing cancer cells.
Antibacterial Agents:
Trimethoprim is used to inhibit bacterial DHFR, specifically targeting bacterial cells without affecting human cells.
This inhibition interferes with bacterial folate metabolism, thereby preventing bacterial growth and replication.
Antiparasitic Agents:
Pyrimethamine is used to inhibit protozoan DHFR, which is essential for the survival of the parasite.
This makes pyrimethamine useful in treating parasitic infections such as malaria.
Mechanism of Action
The drugs listed (e.g., aminopterin, methotrexate, trimethoprim, pyrimethamine) all mimic the structure of folic acid, competitively binding to the active site of DHFR, which prevents the enzyme from catalyzing its normal reaction.
This competitive inhibition blocks the production of tetrahydrofolate, ultimately hindering the synthesis of nucleotides needed for DNA replication.
Summary
Antimetabolites are powerful drugs that leverage the structure of natural metabolites to inhibit enzymes like DHFR.
Methotrexate and aminopterin are used as anticancer agents to slow down cell division by inhibiting human DHFR.
Trimethoprim is an antibacterial that targets bacterial DHFR, while pyrimethamine targets protozoan DHFR, making it useful against parasitic infections.
Protective role of folate against the development of neural tube defects (NTDs):
Neural Tube Defects (NTDs)
NTDs are birth defects that occur due to improper formation of the neural tube during embryonic development.
The neural tube is the precursor to the central nervous system and starts as a plate of cells that folds on itself to form a tube.
Failure of closure of the neural tube leads to NTDs, which can involve either the cranial or caudal end of the tube:
If the cranial end fails to close, it may lead to anencephaly (absence of major parts of the brain, skull, and scalp).
If the caudal end fails to close, it can cause spina bifida (incomplete closure of the spine and spinal cord).
Genetic and Environmental Influences
NTDs are believed to result from a combination of genetic predisposition and environmental influences.
Protective Effect of Folate
Folate (Vitamin B9) plays a significant role in preventing NTDs. It is crucial during early pregnancy for proper cell division and tissue formation, particularly for the neural tube.
Adequate folate intake before and during early pregnancy reduces the risk of NTDs significantly.
Illustration
The diagram shows a developing neural tube and highlights areas where failure of closure can occur, resulting in NTDs such as spina bifida.
In summary, folate is essential for the correct formation of the neural tube during embryogenesis. Ensuring adequate intake of folate during pregnancy is crucial in minimizing the risk of neural tube defects, which are serious birth defects involving the brain and spinal cord.
two major types of neural tube defects (NTDs): anencephaly and spina bifida
Neural Tube Defects (NTDs) Overview
Neural tube defects occur when the neural tube, the precursor to the brain and spinal cord, fails to close properly during early embryonic development.
There are two main types of NTDs depending on whether the cranial or caudal end of the neural tube is involved.
Cranial Defect: Anencephaly
Anencephaly is the most significant cranial NTD:
The cerebral cortex fails to develop properly.
One-third of NTD cases are anencephaly.
It is invariably lethal, with affected infants either dying before or shortly after birth.
The image illustrates that in anencephaly, there is no proper brain formation, especially in the upper parts of the brain.
Caudal Defect: Spina Bifida
Spina bifida is the primary caudal NTD:
It results from abnormal development of the spinal cord.
Accounts for about two-thirds of NTDs.
Causes paralysis of the lower extremities, as well as impaired bladder and bowel function.
Unlike anencephaly, spina bifida is not usually fatal unless it is accompanied by other severe conditions.
The image shows how the spinal cord is exposed, which can lead to nerve damage and physical disabilities.
Summary
Anencephaly and spina bifida are the two main forms of NTDs, involving cranial and caudal ends of the neural tube, respectively.
Anencephaly is lethal and affects brain formation, while spina bifida affects the spinal cord, often leading to physical impairments.
Folic acid supplementation in early pregnancy significantly reduces the risk of NTDs, emphasizing the importance of folate in neural tube development.
Lipid (fat)- soluble vitamins
Structurally more similar than water-soluble
Lipid (Fat)-Soluble Vitamins Overview
Fat-soluble vitamins include Vitamin A (Retinol), Vitamin D₂ (Calciferol), Vitamin E (α-Tocopherol), and Vitamin K₁.
These vitamins have properties distinct from water-soluble vitamins due to their lipid-soluble nature.
Key Characteristics:
Structural Similarity:
These vitamins are structurally more similar to one another than to water-soluble vitamins.
They are isoprenoid compounds, meaning they share structural elements derived from isoprene units, which are lipid-like in nature.
Function:
Unlike many water-soluble vitamins, which function primarily as activated carriers or coenzymes in metabolic reactions, fat-soluble vitamins tend to act more like hormones or serve structural or antioxidant functions.
For example:
Vitamin D acts as a hormone that regulates calcium metabolism.
Vitamin A is involved in vision and gene expression.
Vitamin E functions as an antioxidant.
Vitamin K is crucial for blood clotting.
Storage and Accumulation:
Fat-soluble vitamins can accumulate in the body, particularly in adipose tissue and the liver.
This capacity for accumulation contrasts with water-soluble vitamins, which are generally not stored to the same extent and are excreted more readily.
Accumulation of fat-soluble vitamins can lead to toxicity if consumed in excessive amounts, unlike most water-soluble vitamins.
Structural Examples:
The slide provides structures for several fat-soluble vitamins, illustrating their lipid-like isoprenoid backbones:
Vitamin A (Retinol): Involved in vision, immune function, and cellular differentiation.
Vitamin D₂ (Calciferol): Essential for calcium absorption and bone health.
Vitamin E (α-Tocopherol): An antioxidant that protects cell membranes from oxidative damage.
Vitamin K₁: Involved in blood clotting and bone metabolism
Vitamin A Overview
Vitamin A Overview
Vitamin A (retinol) is a fat-soluble vitamin that plays a vital role in vision, immune function, and cellular growth and differentiation.
Humans cannot synthesize vitamin A and must obtain it through their diet.
Dietary Sources of Vitamin A:
Carotenoids from Plant Sources:
Carotenoids, especially β-carotene, are precursors to Vitamin A found in plants.
Common sources include:
Carrots
Sweet potato
Pumpkin
Green leafy vegetables
β-carotene is a provitamin that is converted into retinol in the body.
Esterified Retinol from Animal Sources:
Esterified retinol (retinol bound to fatty acids) is obtained from animal sources such as liver, fish oils, and dairy products.
These forms of Vitamin A are more readily absorbed by the body compared to plant-derived carotenoids.
Role of β-Carotene in Plants
Photosynthesis is the process by which plants convert light energy into chemical energy, ultimately producing carbohydrates that serve as food for the plant and, indirectly, for other organisms.
Primary Pigments in Photosynthesis:
The primary pigments involved in light absorption are chlorophylls, particularly chlorophyll a.
Chlorophyll a is crucial for capturing light energy and converting it into chemical energy during photosynthesis.
Chlorophyll pigments absorb light and transfer the energy to the reaction center within the chloroplast.
β-Carotene as an Accessory Pigment:
β-Carotene functions as an accessory pigment in photosynthesis.
Accessory pigments like β-carotene help expand the range of light wavelengths that plants can use.
While chlorophylls primarily absorb blue and red light, β-carotene absorbs light in the blue-green part of the spectrum.
This broadens the spectrum of light available for photosynthesis, allowing plants to capture more energy.
Diagram Explanation:
The diagram shows how chloroplasts contain various pigments, including antenna chlorophylls and carotenoids (e.g., β-carotene).
Light energy is absorbed by these pigments and transferred through a series of molecules until it reaches the reaction center, where the photochemical reaction converts the energy into a separation of charge, initiating electron flow.
β-carotene and other accessory pigments work alongside chlorophylls to optimize the capture of light energy and maximize the efficiency of photosynthesis.
All-trans-Retinal:
All-trans-retinal is produced by cleaving β-carotene.
It can then be reduced to form all-trans-retinol, also known as Vitamin A.
All-trans-Retinol (Vitamin A):
This is the primary circulating form of Vitamin A in the human body.
It serves as a precursor to other active forms of Vitamin A, including retinal and retinoic acid.
All-trans-Retinoic Acid:
Retinoic acid is another active form derived from retinol.
It acts as a hormone-like molecule and binds to retinoic acid receptors (RAR).
These receptors regulate gene expression and are involved in development, immune function, and cell differentiation.
11-cis-Retinal:
All-trans-retinal can be converted to 11-cis-retinal, which forms a Schiff base with the opsin protein to create rhodopsin.
Rhodopsin is crucial for vision, particularly in low-light conditions.
The diagram shows 11-cis-retinal linked to lysine in opsin, which is necessary for the visual cycle.
Functions of Vitamin A and Its Derivatives:
All-trans-retinol (Vitamin A) is important for:
Vision: Retinal is essential for forming rhodopsin in the photoreceptor cells of the retina.
Growth and Development: Retinoic acid regulates gene expression involved in cellular growth, differentiation, and embryonic development.
Immune Function: Vitamin A supports the immune system by maintaining epithelial integrity and supporting immune cell function.
Retinal and Rhodopsin
Rhodopsin is a light-sensitive receptor protein found in the rod cells of the retina, which are responsible for vision in low-light conditions.
Retinal, a form of Vitamin A, is a crucial component of rhodopsin, acting as the light-absorbing molecule.
Formation of the Schiff Base
11-cis-Retinal binds to lysine within the opsin protein to form a Schiff base.
The Schiff base involves a double bond between the nitrogen atom of lysine and the aldehyde group of retinal, forming a linkage that allows for the proper functioning of rhodopsin.
When protonated, this Schiff base helps stabilize the binding of retinal to the protein, making it sensitive to light changes.
Light Activation and Signal Transduction
Absorption of Light:
When a photon of light hits rhodopsin, 11-cis-retinal isomerizes to all-trans-retinal. This change triggers a conformational shift in rhodopsin, activating the protein.
Signal Transduction Pathway:
The conformational change in rhodopsin activates a G-protein called transducin.
Transducin exchanges GDP for GTP, which then activates phosphodiesterase.
Phosphodiesterase converts cGMP to GMP, reducing the levels of cGMP.
cGMP is necessary to keep cGMP-gated ion channels open. When cGMP levels drop, these ion channels close, leading to a reduction in the inward flow of Na⁺ and Ca²⁺ ions, which ultimately causes hyperpolarization of the photoreceptor cell.
This change in membrane potential is transmitted to the brain, allowing for the perception of light.
Structure of Rhodopsin
The image on the left illustrates the three-dimensional structure of rhodopsin, showing how retinal is embedded within the opsin protein, which is a seven-transmembrane helix receptor.
vit A transport and storage
Absorption in the Intestine:
Dietary Sources:
β-Carotene and retinol are absorbed in the small intestine through the brush border cells.
Conversion and Esterification:
β-Carotene is cleaved to form retinal, which is then converted into retinol.
Retinol can be esterified to form retinyl ester, which is the storage form of Vitamin A.
Transport via Chylomicrons:
Formation of Chylomicrons:
Retinyl esters are incorporated into chylomicrons, which are lipid particles that transport dietary fats and fat-soluble vitamins through the lymphatic system.
Transport to the Liver:
Chylomicrons enter the bloodstream and transport retinyl esters to various tissues.
In the liver, chylomicron remnants undergo receptor-mediated endocytosis (step 2) for processing.
Storage in the Liver:
Storage as Retinyl Esters:
Retinyl esters are stored in the liver, which acts as the major storage site for Vitamin A. This storage helps maintain a steady supply for the body’s needs.
Retinol Binding Protein (RBP):
When Vitamin A is required by peripheral tissues, retinol is bound to retinol-binding protein (RBP) in the liver for distribution into the bloodstream.
Transport to Peripheral Cells:
Retinol Transport to Target Cells:
Retinol bound to RBP is transported to target peripheral cells through the bloodstream.
STRA6-Mediated Uptake:
STRA6 is a membrane receptor that facilitates the uptake of retinol from RBP into the target cells (step 3).
Once inside the cell, retinol can be stored, converted into retinoic acid for signaling, or utilized directly.
Functions of Vitamin A:
Retinoic acid acts as a hormone-like signaling molecule that regulates gene expression related to cell growth, immune function, and development.
Retinal, an active form of Vitamin A, is essential for vision.
Summary:
Vitamin A is absorbed as β-carotene or retinol in the intestine, converted to retinyl esters, and transported via chylomicrons to the liver for storage.
The liver releases retinol bound to RBP for transport to peripheral cells, where it can be taken up through STRA6 receptors.
This transport and storage mechanism ensures that Vitamin A is available for critical functions, including vision and gene regulation, highlighting its importance in maintaining various physiological processes.
transport of retinol across cellular membranes, focusing on the role of STRA6 and its interactions with retinol-binding protein (RBP4) and other intracellular elements
STRA6-Mediated Retinol Transport:
STRA6 Receptor:
STRA6 is a transmembrane receptor responsible for the uptake of retinol from the extracellular space into the cell.
It binds to retinol-binding protein 4 (RBP4), which is the main carrier of retinol in the bloodstream.
Retinol Transport Mechanism:
RBP4 delivers retinol to the STRA6 receptor on the cell surface.
Upon binding to STRA6, retinol is released from RBP4 and transported across the membrane into the cell.
Inside the cell, retinol binds to cellular retinol-binding protein I (CRBPI), which protects retinol and facilitates its further metabolism.
Intracellular Retinol Processing:
Retinyl ester formation: The enzyme LRAT (Lecithin Retinol Acyltransferase) converts retinol into retinyl esters for storage within the cell, mainly in the endoplasmic reticulum (ER).
Retinyl esters are the storage form of Vitamin A, ensuring that retinol is readily available when needed.
Transport Across the Membrane (Panel B):
The figure on the right illustrates a possible mechanism for STRA6-mediated retinol uptake, involving interactions between various components to allow the passage of retinol into the cell.
The image shows the structural complexity of STRA6, which spans the membrane and facilitates retinol uptake while ensuring specificity and regulation of this crucial process.
Expression of STRA6:
The graph at the bottom shows the tissue distribution of STRA6 expression.
High levels of STRA6 are observed in tissues like the eye, where Vitamin A is essential for vision, particularly in the retina where rhodopsin is involved in phototransduction.
Expression in other tissues, like the lungs and brain, reflects the diverse roles of Vitamin A in different physiological processes, including immune regulation and neural development.
Summary:
Retinol is delivered to cells by RBP4 and is transported across the membrane by the STRA6 receptor.
Inside the cell, CRBPI binds retinol, and LRAT converts it into retinyl esters for storage.
STRA6 expression is highest in tissues like the eye, highlighting its key role in Vitamin A uptake for vision and other functions.
The transport and processing of Vitamin A are tightly regulated to maintain appropriate levels for visual function, growth, development, and immune response.
why there are two pathways by which Vitamin A is transported around the body?
Two Transport Mechanisms for Vitamin A:
Chylomicrons:
Chylomicrons are responsible for the transport of Vitamin A from the intestine to the liver.
After dietary Vitamin A (in the form of retinyl esters or β-carotene) is absorbed in the intestines, it is incorporated into chylomicrons.
These lipid particles carry retinyl esters through the lymphatic system and subsequently into the bloodstream.
Chylomicrons primarily transport Vitamin A to the liver for storage, but they also deliver some Vitamin A to target tissues for immediate use.
Retinol-Binding Protein (RBP):
Retinol-binding protein (RBP) is the main carrier for retinol (Vitamin A) in the bloodstream.
RBP is synthesized in the liver and binds to retinol to form a retinol-RBP complex.
This complex allows the transport of retinol from liver stores to target tissues, making it the major circulating source of Vitamin A.
RBP plays a critical role in:
Binding to receptors on target cells (such as STRA6), which allows retinol to be taken up into those cells.
Regulating the release of retinol from the liver, thus controlling which tissues have access to Vitamin A based on their physiological needs.
Why Two Transport Mechanisms?:
Chylomicrons are mainly involved in post-dietary absorption to deliver Vitamin A to the liver for storage and some target tissues directly after ingestion.
RBP, on the other hand, manages long-term distribution of stored retinol from the liver to target tissues, ensuring a steady supply of Vitamin A as needed.
These two mechanisms work together to maintain Vitamin A homeostasis:
Chylomicrons deal with dietary Vitamin A absorption and initial transport.
RBP ensures that stored Vitamin A can be delivered to tissues throughout the body as required, contributing to processes such as vision, immune function, and cell differentiation.
Summary:
The body uses two pathways for Vitamin A transport:
Chylomicrons for initial transport of dietary Vitamin A to the liver.
RBP for circulating retinol from liver stores to peripheral tissues.
These two mechanisms ensure efficient use and distribution of Vitamin A to maintain adequate levels for various physiological functions, including vision and immune system health.
Control of Peripheral Availability by Storage
Control of Peripheral Availability by Storage:
The body controls the amount of a substance available in the peripheral tissues by storing it in a major storage organ, such as the liver.
This mechanism helps maintain a reservoir of important nutrients or compounds that can be accessed when needed.
2. Signaling for Release from Storage:
The liver releases stored substances in response to signals that indicate changes in metabolic needs.
For instance, if the body’s demand for a particular substance increases (e.g., during growth, immune response, or stress), signals prompt the liver to release the stored substance into circulation.
3. Regulation of Transport Proteins:
The availability of a substance is also controlled by regulating the proteins required for its transport.
For example, retinol-binding protein (RBP) transports Vitamin A in the bloodstream. The production of RBP can be adjusted to control the amount of retinol being transported to target tissues.
4. Tissue Specificity via Receptor Expression:
Tissue specificity is regulated by controlling the expression of receptors on target cells.
For instance, the STRA6 receptor facilitates the uptake of Vitamin A into specific cells. By regulating receptor expression, the body can determine which tissues receive the substance and in what quantities.
5. Removal and Degradation:
The body ensures proper removal or degradation of substances to prevent excessive accumulation.
This step is crucial for maintaining homeostasis and preventing toxicity, particularly for fat-soluble vitamins like Vitamin A, which can accumulate if not properly regulated.
excessive Vitamin A consumption
Excessive Vitamin A Consumption:
Hypervitaminosis A typically occurs when Vitamin A is ingested in its preformed state, such as from consuming:
Liver (especially from animals like polar bears, which have very high concentrations of Vitamin A).
Excessive Vitamin A supplements.
Important Note:
Excessive Vitamin A consumption does not occur when ingesting carotenoids like β-carotene, which is a precursor to Vitamin A and is more safely processed by the body. The conversion of carotenoids to Vitamin A is regulated, preventing toxicity.
Symptoms of Hypervitaminosis A:
Acute Symptoms:
Abdominal pain.
Nausea.
Vomiting.
Dizziness.
These symptoms can occur with sudden large intakes of preformed Vitamin A.
Chronic Symptoms:
Bone abnormalities and joint pain.
Visual disturbances.
Appetite loss.
Dizziness.
Peeling, oily, or itchy skin.
Increased risk of respiratory infections.
Chronic hypervitaminosis A is usually associated with long-term excessive intake of preformed Vitamin A.
Summary:
Hypervitaminosis A is typically the result of ingesting high levels of preformed Vitamin A (e.g., from animal liver or supplements), not from β-carotene or other carotenoid sources.
Acute and chronic symptoms range from gastrointestinal discomfort to serious issues like bone deformities and skin conditions, illustrating the importance of regulating Vitamin A intake to avoid toxicity.
metabolism and effects of Vitamin D in the body
A. Metabolism of Vitamin D:
Synthesis of Vitamin D3 (Cholecalciferol):
Vitamin D3 is synthesized in the skin from 7-dehydrocholesterol when exposed to UV-B radiation from sunlight.
Alternatively, Vitamin D can be obtained from dietary sources through the intestine.
Transport and Conversion:
After synthesis in the skin or absorption from the diet, Vitamin D is transported to the liver, where it is converted by 25-hydroxylase (25-OHase) into 25-hydroxyvitamin D (25(OH)D).
The 25(OH)D is then transported to the kidney, where 1α-hydroxylase (1α-OHase) converts it into the active form, 1,25-dihydroxyvitamin D (1,25(OH)₂D).
Transport in Blood:
Vitamin D is transported in the blood bound to Vitamin D-binding protein (VDBP), ensuring that it reaches target tissues where it is required.
B. Target Tissue Actions of Vitamin D:
Bone:
1,25(OH)₂D stimulates bone mineralization, promoting the deposition of calcium and phosphate into the bone matrix, which strengthens bone structure.
Intestine:
In the intestine, Vitamin D increases the absorption of calcium (Ca²⁺) and phosphate (Pᵢ), which are essential minerals for bone health and many other physiological functions.
Immune Cells:
Vitamin D also acts on the immune system, inducing the differentiation of immune cells, particularly enhancing the function of macrophages, dendritic cells, and T cells.
This regulation of the immune response plays a role in inflammation and autoimmunity.
Summary:
Vitamin D is synthesized in the skin or absorbed from the diet, and it undergoes activation in the liver and kidneys to its active form, 1,25(OH)₂D.
This active form acts on bone, intestine, and immune cells, promoting bone mineralization, enhancing calcium and phosphate absorption, and supporting immune cell differentiation.
Adequate Vitamin D is essential for bone health and plays a critical role in immune regulation.
