Identifying and exploiting bacterial differences

State the significant difference between bacteria and viruses when it comes to replicating within host cells

• Bacteria maintain structural integrity during replication

• Do not require release of genome so the host can replicate pathogen proteins using host machinery

• Makes own ATP and proteins, uses host for resources, environment and host machinery is just for more efficient / faster replication, or synthesis of nutrients it needs

Differences in components of bacterial cells to mammalian

• No membrane-bound organelles, except mitochondria

• Cell wall, but basic cytoskeleton

• Pili as hooks

• Rotary flagella for movement

What is bacteria still containing mitochondria (despite no other membrane bound organelles) indictive of?

• Evolutionary relationship between bacteria and eukaryotes

What are the differences in bacterial reproduction compared to mammalian cells?

• Binary fission instead of mitosis

• Sexual reproduction uses horizontal gene transfer, rather than meiosis

What are the differences in bacterial replication compared to mammalian cells?

• mRNA is polycistronic and unstable (has no introns, cap or tail)

• Regulate translation by regulating transcription initiation (they are coupled)

Why do bacteria have a cell wall?

• Membrane alone is not strong enough to prevent very hypertonic cytoplasm from rupturing by osmotic shock

Which type of bacteria has thicker cell walls?

• Gram positive

How is the cell wall so strong?

• Enzymatic cross-linking of glycan strands by transglycosylase

• Enzymatic cross-linking of peptide strands by transpeptidase

• Covalent connectivity in meshwork provides mechanical strength

• Resist outward osmotic forces

Simply, what are the four steps of peptidoglycan synthesis?

• UDP-NAM-tripeptide

• UDP-NAM-pentapeptide

• Linkage of UDP-NAM to UDP-NAG and association with outer leaflet of bilayer

• Cross linking of chains between membranes by transpeptidases

Describe fully the first step of peptidoglycan synthesis, and the antibiotic that can inhibit this

• UTP bound covalently to NAG, forms UDP-NAG

• Bound covalently to tripeptide

• Occurs inside cell

• Fosfomycin irreversibly inhibits enzyme that transfers tripeptide

Describe the second step of peptidoglycan synthesis (so far, UDP-NAG-tripeptide is made), and the antibiotic that can inhibit this

• Addition of D-alanyl-D-alanine to form UDP-NAM-pentapeptide

• D-cycloserine, structural analogue of D-alanine, inhibits synthases enzyme and also possibly the ligase involved in this step

What are the different ways peptidoglycan synthesis can be targeted with antibiotics? Give examples

• Synthesis of UDP-NAG and UDP-NAM monomers

  • Fosfomycin, D-cycloserine

• Transpeptidase action

  • Penicillin G (beta-lactams, carbapenems, cephalosporins), vancomycin (glycopeptide)

Describe fully the gram-staining procedure

• Loop of water on slide

• Smear colony into water to fix

• Dry

• Stain crystal violet, 1 min, wash

• Stain Gram’s iodine, 1 min, wash

• Gram’s decolouriser, 5-10s, wash

• Safranin counterstain 45s, wash

• Dry

• 100x objective lens with immersion oil

State the results of gram staining

• Gram negative: pink

• Gram positive: violet

Explain how Gram staining works

• Iodine staining will be washed away with gram negative cells as it is unable to penetrate the outer membrane to reach peptidoglycan, and this peptidoglycan is thin anyway

• Gram positive has no outer layer so immediate access to peptidoglycan, and thick peptidoglycan so stain accumulates

Explain why we counterstain in Gram staining and what it does

• Safranin binds negative charge, which bacteria are surrounded with

• Stains negative pink, but no effect on positive as iodine too dark

• Allows visualization of gram negative cells present in the mixture

• Confirms that transfer of bacterial colony to the slide was successful (when gram positive are absent)

State the two medically important bacteria that do not Gram stain

• Mycobacteria

• Chlamydia

Why do mycobacteria not gram stain?

• Waxy lipid coat hard to stain, even harder to decolourise

• Difficult to adapt procedure as doubling time is very long, little investigation

Why does chlamydia not gram stain?

• Lack substantial cell wall so do not take up enough stain to visualise

• Hard to culture and insensitive to many AB so nearly impossible to make non-pathogenic for secure lab studies in how to adapt procedure

Describe the chemical reactions involved in the transpeptidase reeaction

• Attack of active site serine onto amide bond of D-Ala4-D-Ala5

• D-Ala5 released

• Acyl transfer to neighbouring pentapeptide

• Cross links the two strands

• Regenerates non-acetylates Ser in active site

How do beta-lactams work? Name two

• Long-term inactivation of transpeptidases

• Mistaken for yet-to-be-crosslinked peptidoglycan chain

• Beta-lactam ring of drug opens inside enzyme AS, very slow to hydrolyse

• If cell is replicating, autolysin activity that would normally be re-shaping peptidoglycan for division now induces lytic death as demand for the new wall is not being met

• Penicillin G, imipenem (carbapenem)

How do glycopeptide antibiotics work? Give an example

• Binds to terminating D-Ala4-D-Ala5 of uncrosslinked peptidoglycan

• Transpeptidase cannot bind

• No cross links lose integrity of wall

• Osmotic rupture

• Vancomycin

What sort of selectivity are antibiotics that target peptidoglycan using, and give examples

• Differential presence (no peptidoglycan in mammalian)

• Penicillins e.g. penicillin G

• Cabapenems e.g. imipenem

• Glycopeptides e.g. vancomycin

The full process of prokaryotic translation initiation

• 30S associates with IF1 and IF3 (initiation factors)

• 30S:IF1:IF3 associates with mRNA at shine-delgano by 16sRNA bp’ing with SD

• IF2-GTP-citRNA associates, holding citRNA at AUG

• 50S joins

• IF2 intrinsic GTPase activity activated

• Releases all initiation factors

• 70S initiation complex formed

• IF2 exchanges GDP with GTP, recycling

Proteins important for interactions with the host are typically…

• Exported through the periplasm for assembly on bacterial surface

• Use sec dependent or independent transport

Describe Sec-dependent transport

• N-terminus secretion signal

• ATPase activity of sec transporter pushes molecule through channel into periplasm

• Sec has protease activity, cleaves off secretion signal

• Creates a reservoir in periplasm

What bacteria has a good example of sec-dependent transport?

• UPEC

Describe sec-dependent transport in UPEC

• Glycoprotein subunits for pili and pili-terminus adhesion proteins are pumped from cytosol to periplasm by sec

• Periplasmic chaperones capture subunits, take to outer membrane USHER pores

• Terminus ahesion protein secreted first, followed by joining of many pili subunits for long pili projections

Simply, what molecules are sec-dependently transported in UPEC?

• Glycoprotein subunits for pili and pili-terminus adhesion proteins

Describe sec-independent transport

• Single step

• Pore transverses inner membrane, periplasm and outer membrane all at once

• Export of toxins

Does C. Botullinium secrete BoNT sec dependent or independently?

• Dependent, accumulates in periplasm first

Describe different ways of combating bacterial diseases

• Reduce exposure (disinfect, treat water, pasteurization, quarantine)

• Reduce susceptibility (vaccine)

• Chemotherapy (antibiotics)

State 3 antibiotics that act on the ribosome

• Tetracycline

• Erythromycin

• Chloramphenicol

How do we know so specifically where antibiotics bind to the ribosome?

• X-ray crystallography

Describe how tetracyclines bind the prokaryotic ribosome

• Bind reversibly to 30S subunit (prok. excl)

• Inhibits entry of aminoacyl-tRNA into A site of overall 70S

• Current peptide being formed is prematurely released, terminating synthesis

What S subunits are for prok and euk. ribosomes?

• Prok: 30 50 (70S)

• Eu: 40 60 (80S)

What can reduce activity of tetracycline?

• Ca2+ and Mg2+ binding

How does erythromycin and other macrolides bind the prokaryotic ribosome?

• Bind at entrance to E site of 50S (prok. specific)

• 6 to 8 aa maximum can be joined before elongation is blocked and prematurely terminated

What is the default alternative antibiotic when someone is allergic to peniciillins?

• Erythromycin / macrolides

How does chloramphenicol interact with the ribosome?

• Blocks aminoacyl-tRNA interaction with A site of 50S

• Premature translation termination

Describe the pros and cons of chloramphenicol

• Broad spectrum (ribosome targetting), bacteriostatic

• Low therapeutic index, causes bone marrow suppression

• Used in eye drops in conjunctivitis (localised, cannot access bone marrow)

What are three ways that antibiotics can act on DNA replication and gene expression, and give an example for each. Which has the lowest therapeutic index and why?

• DNA topoisomerases e.g, fluoroquinolones

• RNA synthesis e.g. rifampin

• dsDNA e.g. ethidium - lowest TE as very hard to make specific, differential accumulation to an extent

Describe how fluoroquinolones inhibit bacterial DNA replication and gene expression, give examples

• Topoisomerases cause ds nicks to enable relaxation and uncoiling during replication

• Lock topoisomerase onto DNA, prevents nicks, DNA remains supercoiled so DNA pol cannot replicate further

• Selective for bacterial topoisomerase isoforms

• Ciprofloxacin (gram pos and neg), gatifloxacin (more potent against gram pos)

Describe how rifampin inhibits bacterial DNA replication and gene expression

• Bind non-covalently but very tightly to allosteric subunit on beta subunit of bacterial DdRp

• Inhibits synthesis of new RNA, but no effect on already-progressing RNA synthesis

• Not effective immediately

• Bactericidal

Describe how ethidium inhibits bacterial DNA replication and gene expression

• Planar, inserts between stacked base pairs in dsDNA helix

• Intercalates

• Causes partial unwinding that changes the length of major and minor binding grooves so polymerases and TFs cannot access some recognition sequences

• Very low therapeutic index due to low selectivity

How have some antibiotics been repurposed as anti-cancers?

• Topoisomerase inhibitors can be made specific for human enzymes instead

• dsDNA intercalators used in late-stage, last-resort chemotherapy, uses differential accumulation for tumour cells but lack of selectivity makes very toxic

Describe how sulfamethoxazole and trimethoprim are antibiotics

• Bactericidal

• Block folic acid synthesis

• Sulfamethoxazole blocks DHPS, trimethoprim blocks DHFR, both key enzymes in folic acid synthesis pathway

• Folic acid required for DNA synthesis

• Differential importance as euk can scavenge folic acid from diet, bacteria entirely reliant on synthesis

Give examples of antimetabolites that have to be used together

• Sulfamethoxazole, trimethoprim

What are four targets of bacteria for antibiotics? Give one example for each

• Peptidoglycan, penicillin G

• 70S ribosome, rifampin

• dsDNA, ethidium

• Folic acid synthesis, sulfamethoxazole with trimethoprim

What is a potential reason that there are few bacterial vaccines in wide use?

• Efficacy and low cost of antibiotic treatments

• Prevention methods e.g. water cleaning, antibac spray easier to implement on a larger scale (compared to safe sex or harm reduction for lots of viral transmissions), therefore disincentivizes funding

Simply, what are the two antibacterial vaccines in widespread use?

• DPT (diptheria, pertussis, tetanus)

• Ty21a (salmonella)

Describe the DPT vaccine

• Inactivated diptheria and tetanus toxin

• Pertussis with pure antigens not attached

• Safe for immunocompromised

• Long term IgG generated

Describe the Ty21a vaccine

• Live attenuated (replicated, no virulence) salmonella typhi

• Genetic recombinant to limit polysaccharide production, cell wall is unstable so can’t accurately produce pili or injector needles for invasion

• Not safe for immunocompromised

• 3 capsules taken on alternate days

• 3 years protection, given prophylactically before entering high risk area, requires boosters

How could Salmonella vaccines be used for more than just salmonella?

• Research into attenuated Salmonella to present non-salmonella antigens

• Even if antigen still binds receptor, the internalisation / toxin secretion that would happen in either non-modified bacteria cannot occur as they do not have the correct machinery

Describe intrinsic resistance to a drug. How can it sometimes be overcome?

• Never were and never would be sensitive to a drug in the first place

• Lacks receptor for drug, or appropriate drug target e.g. a specific enzyme

• Combine with polyene antibiotics that disrupt cell envelope to facilitate drug entry

Describe how mycobacterium TB shows intrinsic resistance to beta lactams and glycopeptide antibiotics. How can this be overcome?

• High mycolic acid content in lipid bilayer

• Prevents access to peptidoglycan

• Combine with isoniazid, which inhibits mycolic acid synthesis

Describe acquired resistance to a drug

• Populations that are initially sensitive to a drug have modified so they become either less or completely insensitive

Describe acquired resistance of staph. aureus

• Now extremely resistant to vancomycin and erythromycin

• Cause of hospital-acquired catheter and surgical wound infections, so under high selection pressure against AB

Multiple drug vs mutlidrug resistance

• Multiple: Expression of various different resistance mechanisms using multiple different genes, each specific for a drug or class of drugs

• Multi: one resistance mechanism makes organism resistant to multiple drugs (e.g. efflux pump)

Comment on the type of drugs that AMR via enzymatic inactivation seems to target more

• Present against several natural-product drug classes (beta lactams), not yet major resistance route against completely synthetic antibiotics (sulfamethoxazole)

• Could reflect time of exposure for bacteria to natural products (hundreds of millions of years) compared to 70 or less for man-made synthetic ABs

How can drugs be resistant to beta-lactam ABs?

• Beta-lactamases

• E.coli can excrete 10^5 beta-lactamases with the ability to hydrolyse 1000 beta-lactam molecules a minute, [AB] required to overcome this would exceed toxic conc, and would inevitably just push resistance higher with time

• As secreted, can hydrolyse beta-lactams before they reach and bind transpeptidases

What are the beta lactam antibiotics

• Penicillins

• Cephalosporins

• Carbapenems

Why aren’t beta-lactamases a target for drug development with combination therapy?

• Oer 190 different known now, with 4 different classes

How have ESBLs arisen?

• Beta-lactam resistance meant penicillins couldnt’ be used

• Led to development of extended-spectrum cephalosporins and carbapenems

• ESBLs arose by plasmid transfer due to frantic over uses of extended spectrum antibiotics in response to penicillin resistance, and lack of knowledge we have now on how acquired resistance arises and how intrinsic resistance can be exploited

How do aminoglycoside-modifying enzymes lead to acquired resistance?

• Covalently modify -OH and -NH2 on aminoglycoside antibiotics (gentamicin) using O-phosphoryl transfer or N-acetylation

• Interferes with antibiotic’s ability to hydrogen bond with 30S of ribosome

What are two methods of AMR via enzymatic inactivation

• Beta lactamase secretion against penicillins (and then cephalosporins and carbapenems)

• Aminoglycoside-modifying enzymes against e.g. gentamicin

Describe how MRSA arose

• Methicillin introduced in 1950 for gram positive beta-lactam resistant staphylococcus aureus infections

• Can reach incidence of 20-40% in US hospitals, especially burn centres, due to selection pressure of the medical environment

Describe the resistance mechanisms of MRSA

• Acquired MecA gene that encodes new transpeptidase that is completely insensitive to beta lactams

• Acquired fem gene that modifies peptidoglycan strands so they are a better substrate for cross linking by mecA transpeptidase, now even less susceptible to other peptidoglycan-targeting ABs e.g. vancomycin

How have pneumococci developed macrolide resistance?

• Methylation of specific adenine in 50S alters macrolide binding site, reducing affinity

• Also reduces affinity for some other classes

• Does not effect 50S function

Give two example mechanisms of resistance that have used modification of the drug target

• MRSA (mecA and fem gene, new transpeptidase and peptidoglycan)

• Macrolide resistant pneumococci (methylate specific site in 50S)

What type of drug is AMR via modification of influx useful against?

• Large, hydrophilic drugs that don’t passively diffuse across bilayer and must rely on influx pumps

Describe aminoglycoside resistance based on influx / efflux

• Rely on facilitated diffusion through porins

• Decreased porin expression decreases influx

• If do enter periplasm, actively transported into cytoplasm by oligopeptide transporters

• Modifications in oligopeptide transporters to reduce affinity to aminoglycosides, decreases flux

What type of drug is AMR via modification of efflux useful against?

• Small, hydrophobic, non-polar drugs that can passively diffuse across membranes

Describe drug efflux pumps

• Some dedicated to single drug or class, others can be broader

• Usually co-transporters with Na+/H+

• Difficult to deal with clinically

• In gram negative, be associated with secondary protein that spans periplasm to link inner and outer membrane drug pumps, so doesn’t even enter the periplasm

State three mechanisms of AMR

• Enzymatic inactivation

• Modification of drug target in a way that maintains original function

• Reduced influx / enhanced efflux

State 5 examples of AMR

• Beta lactamase (beta lactams)

• Aminoglycoside-modifying enzymes (aminoglycosides)

• MRSA (beta lactams)

• Methylation in pneumococci (macrolides)

• Decreased porin expression and oligopeptide transporter modification (Aminoglycosides)

Aminoglycoside antibiotic example

• Gentamicin

What are two main difficulties of treating bacterial infections with antibiottics?

• AMR

• Opportunistic infection post-treatment

Describe a common example of post-antibiotic treatment opportunistic infection

• Clostridium difficile colonises colon following eradication of normal gut microflora during antibiotic treatment (especially common with tetracycline)

• Uses dead microflora for nutrients

• Especially a problem in hospitals where someone is already immunocompromised

Why is tetracycline a common cause of post-antibiotic treatment opportunistic infections?

• Binds 30S subunit so specific to prok ribosomes in general, but not specific bacteria

• Also kills gut microflora

What is the treatment for post-antibiotic opportunistic infections?

• Other ABs e.g. vancomycin

• Rehydration therapy

• Dietary adjustments to try and restore gut microflora to prevent reinfection after vancomycin treatment

What are three strategies for overcoming AMR?

• New drug targets and new drugs

• Inhibiting or bypassing resistance mechanisms

• Combination therapy

Give an example of how inhibiting or bypassing resistance mechanisms has been a good strategy for overcoming AMR

• Clavulanate discovered from studying streptomyces

• Not itself an effective antibiotic, but is a beta lactamase inhibitor, so used in combination with beta lactams

• Also allowed some antibiotics that would be too toxic alone to be used clinically, as clavulanate increases susceptibility so a much lower concentration is needed - amoxicillin

• Synergistic

Why is combination therapy a good strategy for overcoming AMR?

• Works on different targets at the same time

• Hard to gain mutations against all targets simultaneously whilst maintaining treatment with those drugs

• Usually also synergistic (e.g. clavulanate and amoxicillin)