1
Q
Spectrum of activity of antibiotics
A
- Antimicrobials are classified according to activity
- Some agents have narrow spectrum, some have broad
- Some antibiotics kill a bacterium
○ bactericidal - Some prevent growth of bacterium but do not kill it
○ Bacteriostatic
○ Effective: suspends growth, buys the immune system time
2
Q
measuring drug susceptibility
A
- Antibiotic effectiveness depends on
○ Organism being treated
○ Attainable tissue levels of the drug, not all penetrate as well as others
○ Route of administration- In Vitro we measure the min inhibitory concentration (MIC of an antibiotic against its target)
○ Serial dilution in a 96-well plate
○ E-strips (MIC strip tests)
○ Kirby-Bauer disk diffusion assay
Downside to these tests: takes time, patients may die
- In Vitro we measure the min inhibitory concentration (MIC of an antibiotic against its target)
3
Q
Antibiotic mechanisms of action
A
- Classic targets include
○ Cell wall synthesis
○ Cell membrane integrity
○ DNA synthesis
○ RNA synthesis
○ Protein synthesis
Metabolism
4
Q
Cell wall antibiotics
A
-Sugar molecules NAM and NAG made in cytosol
-Linked by a transglucosylase enzyme at cell wall
-Side chains of NAM molecules cross-linked by transpeptidase to provide rigidity
5
Q
Beta-lactam antibiotics
A
- Derived from fungi, has beta lactam ring structure to which R groups can be added
- Transpeptidase and transglucosylase enzymes involved in cell wall building are aka penicillin binding proteins
- Resistance to these (2 mechanisms)
- Inheritance of a gene that. Encodes for beta-lactamase gene
i. Can be overcome by inhibitors - Inheritance of a gene that codes for an altered PBP (penicillin binding protein) that does not bind the antibiotic
- Inheritance of a gene that. Encodes for beta-lactamase gene
- Microbes are quick to adapt and become resistant
Some antibiotics are only used in worst case scenarios to slow down development of resistance
6
Q
Other Target cell-wall synthesis antibiotics
A
- Bacitracin: binds to bactoprenol lipid carrier, inhibits transport of the peptidoglycan monomers to the growing chain (toxic)
- Cycloserine: inhibits the two enzymes that make a precursor peptides of the NAM side-chain
- vancomycin binds to D -Ala- D -Ala terminal end of the disaccharide unit & prevents bindings of transglucosylases and transpeptidases
Some bacteria have incorporated D-lactate into the D-Ala terminus to develop resistance
7
Q
Drugs that affect bacterial membrane integrity
A
- Gramicidin: cyclic peptide that inserts into bacterial membrane
- Polymyxin: binds to outer and inner membranes of G-bacteria, disrupts inner membrane like a detergent
- Daptomycin: aggregated gram positive bacterial membranes to form channels
Effective against MRSA
7
Q
Drugs that affect synthesis and integrity
A
- Sulfa drugs: interfere with nucleic acid synthesis by preventing synthesis of THF that is a cofactor in the synthesis of nucleic acid precursors
○ All organisms use THF to synthesize nucleic acids
○ Sulfa drugs are selectively toxic to bacteria because bacteria is the only ones that can make it, incorporated into pathway that shuts the pathway down- Quinolones: target microbial topoisomerases, enzymes essential for changes in DNA to allow replication and transcription
○ These drugs are toxic to mitochondria because mitochondria uses the same mechanisms and enzymes to replicate genomes, independently of cells genome
- Quinolones: target microbial topoisomerases, enzymes essential for changes in DNA to allow replication and transcription
8
Q
RNA synthesis inhibitors
A
- Binds to exist tunnel of bacterial RNA polymerase, blocks RNA from leaving polymerase structure
- Stops transcription
- Turns bodily secretions orange while in use
Examples: rifampicin and actinomycin
8
Q
Drugs that affect DNA synthesis and integrity
A
- Ex: Metronidazole, example of a pro-drug
○ Activated on reduction by microbial flavodoxin or ferredoxin, found in microaerophilic and anaerobic bacteria
○ Nicks DNA at random when activated
Not effective against aerobic bacteria
9
Q
Protein synthesis inhibitors
A
- Binds and interfere with bacterial rRNA functions
- Bacterial ribosomes and eukaryotic ribosomes have fundamentally different properties
○ (ribosomes catalyze linkage between amino acids during translations)
- Bacterial ribosomes and eukaryotic ribosomes have fundamentally different properties
10
Q
Targeting the 30S subunit
A
- Aminoglycosides: bind 16S ribosomal RNA and causes translation misreading of mRNA
○ Results: jumbled peptides- tetracyclines binds and distorts ribosomal A site
Interferes with bone development
- tetracyclines binds and distorts ribosomal A site
11
Q
Targeting te 50S subunit
A
- Macrolides and lacosamide’s: inhibit translocation of the growing peptide chain
- Chloramphenicol: inhibit peptidyl transferase activity
○ Can depress production of blood cells in the bone marrows - Oxazolidinones: binds to the 23S rRNA component of the 50S ribosome, prevent formation of 70S complex
Streptogramins: 2 types, both bind to peptidyl transferase site
- Chloramphenicol: inhibit peptidyl transferase activity
12
Q
Targeting aminoacyl tRNA synthetases
A
- mupirocin binds to bacterial enzymes that attach amino acids to the end of tRNA molecules, stops protein synthesis
○ Used in creams to treated infections caused by gram positive bacteria
Cannot be used internally, degraded in bloodstream
13
Q
Problem of antibiotic resistance
A
- Many antibiotics derived from nature
○ Not an issue in soil microbes that make them use them very sparingly
Antibiotic resistance has become an issue in medicine since we have consistently used high concentrations of this for long periods of time
14
Q
Antibiotic resistance strategy 1: keep antibiotics out of the cell
A
- Destroy antibiotic before it can enter the cell
○ Ex: beta-lactamases- Decrease membrane permeability across the outer membrane (express narrower pores), but not great for microbe, reduces amount of nutrients for it
○ Ex: fluoroquinolone resistance - Pump the antibiotic out of the cell via specific transporters
○ Ex: tetracycline resistance
○ Efflux pump the resistant microbe makes can be used by cell to pump the antibiotic out, has no effect since it isn’t there long enough to inhibit
Can be dangerous if the efflux pump can export many types of antibiotics despite differing antibiotic structures
- Decrease membrane permeability across the outer membrane (express narrower pores), but not great for microbe, reduces amount of nutrients for it
15
Q
Antibiotic resistance strategy 2: prevent antibiotics from binding to their target
A
- Modify target so it no longer binds the antibiotic
○ Ex: modify shape of PBPs or ribosomal proteins
Add modifying groups to the antibiotic so that the antibiotic is inactivated
16
Q
Antibiotic resistance strategy 3: dislodge an antibiotic bound to its target
A
- Ribosome protection or rescue
Some gram positive organisms can make proteins that bind to ribosomes and dislodge or prevent binding of antibiotics that bind near the peptidyl transferase site
17
Q
How antibiotic resistance spreads
A
- Mutation
- Vertical transmission: when cell divides (binary fission) daughter clels will carry same resistance
- Horizontal transmission: cell with element can conjugate with another cell, and transfer the plasmids with the resistance element
18
Q
Antibiotics and your microbiome
A
- Antibiotics have the potential to destroy the ecological balance the microbiome exists in
○ Collateral damage
○ A huge number of diseases are now recognized to be associated with a lack of diversity in the gut microbiome
We should still take them as they are lifesaving drugs, used appropriately
18
Q
Influenza virus: example of a pathogen
A
Negative strand RNA virus
- Influenza A: most common in western world
- Influenza B:
○ narrower host range than A
○ Can cause serious disease, but mutates slower - Influenza C
○ Narrower host range than A
○ Mild disease, not easily spread - Influenza D
○ Never detected in humans
Associated with swine and cattle
19
Q
Differences between flus and colds
A
Cold:
- Usually a cough
- Sore throat, congestion
- Sometimes a headache, muscle aches and pains
- Not often a fever, no exhaustion or GI symptoms
- Approx 1 week
Influenza
- Usually a cough
- Sore throat, fever, severe headache, muscle aches and pains, extreme exhaustion
- Sometimes congestion and GI symptoms
- 2-3 weeks or longer
19
Q
Virion structure
A
- No geometrical capsid
- Instead, a shell of matrix proteins (M1) that surround the 8 RNA chromosome fragments
- Matrix surrounded by a membrane envelope
○ Derived from the host cell during budding
Viral envelope proteins hemagglutinin (HA) and neuraminidase (NA) stud the surface of the virus
20
Q
Virion genome
A
8 negative sense RNA segments
○ Each coated with NPs (nucleocapsid proteins) that encodes 1 protein
○ 2 segments undergo splicing to encode further proteins
Each segment packaged with an RNA-dependent RNA polymerase complex
○ If you make your own polymerase and package with genome, as t=genome is released, you can make your RNA genome, churning out genomes, don’t have to wait
○ Uses their own machinery to make their own enzyme to speedily replicate
* During viral assembly I infected cell, segments are precisely packaged
○ Link to each other in order as they arrange themselves
○ Segments line like bundle of sticks
Tiny extensions connect them
21
Main advantage of a segmented genome
* Most dangerous aspect of flu virus= ability to continually change its antigenic determinants
○ Evades host acquired immunity
* Segmented genomes allow for re-assortment of genetic information, generating drastically new strains more quickly than viruses with non-segmented genomes
○ Other virus that have non-segmented genomes don’t change over time therefore immunity through exposure or vaccination is long-lasting
22
The 'H' bit
* H=hemagglutinin
* Forms a trimer complex each with an N-terminal fusion peptide
* Allows fusion of the viral membrane with the host cell membrane
1. HA C-terminal domain recognizes and binds to host cell sialic acid receptor
2. Triggers uptake of virion aby endocytosis
3. Endocytic vesicle acidifies and produces a conformational change that exposes the N-terminal fusion peptide
4. Fusion of host and viral membranes can take place
5. Trigger release of the genome cargo into the host cytosol
23
Avian, swine and human 'flu
Birds are the natural reservoir for influenza A virus
* Whether other animals are susceptible depends on
○ presence of a host cellular protease t cleave the HA and initiate infection
○ Nature of the cel-surface glycoproteins on host cells that bind the HA and allow endocytosis
* Influenza A virus binds of a sialic acid glycoprotein that is found on epithelial cells (lining lungs and intestines)
* Subtle differences of sialic acids between birds and humans separate susceptibility to given strain
1. Viral segments travel to nucleus and enter nuclear pores
2. Attached viral RNA polymerase synthesizes (+) strand RNA
3. mRNA travels to cytoplasm for translation to viral proteins-processed by the ER/Golgi and sent to the host cell membrane
23
The 'N' bit
Envelope proteins and viral genome packages travel to cell membrane for packaging into new virions
○ Within cell membrane, envelope proteins assemble around the genome and matrix proteins
○ Virion buds out of the host cell
○ Neuraminidase cut the virion loose from host glycoproteins to release it to the extra cellular space
1. Virion taken up by host cell
2. RNA (-) and RNA-dependent RNA poly are released, enters nucleus through nuclear pores
3. Transcription to mRNA
4. mRNA translated to viral proteins and envelope proteins enter Golgi
5. Transfer to host cell membrane
6. Other viral proteins redirected back to nucleus
7. Some viral genome is transcribed to RNA(+) and used as template to make more viral genomes
8. New viral genomes packaged and exported out of the nucleus using Nuclear Export protein
9. At host cell surface, new virions are packaged, and host cell envelope containing viral surface proteins are used to make the capsid
10. New virion buds out, neuraminidase cleaves the sialic acid receptor to release the virion
11. Host protease cleaves the haemagglutinin to activate the virion and allow it to go on to infect more host cells
24
Drifting and shifting
Drifting
○ Ability of influenza virus (A and B) to mutate and change slightly
○ Usually because RNA replication errors in HA and NA genes
Shifting
○ Change in structure of the fly virus
○ Can be Caused by jumping of the virus into a new species
○ Can be Caused by re-assortment of the genes from 2 different viruses mixing in a single host to make a new strain
