antibiotic resistance Flashcards
Basic mechanisms for antibiotic resistance
Alter target
Alter drug
Get rid of drug
Ways to alter the target
Reduce binding.
Titration.
Metabolic bypass.
Ways to alter the drug
Degrade antibiotic.
Sequester antibiotic.
Inactivate antibiotic.
Reducing target binding
1) Mutate target binding site
2) Shield target binding site.
Shielding target binding site.
Reducing target binding, altering target.
Example. In TB MfpA binds gyrase and prevents it from forming the gyrase-DNA complex that fluoroquinolones bind.
Mutating target binding site
Reducing target binding, altering target.
Examples: vancomycin and quinolone resistance.
Quinolone resistance by mutating binding site.
A subunit of DNA gyrase, coded by gyrA has lowered affinity.
Vancomycin resistance
D-ala-D-ala to D-ala-D-lac leads to 1000 fold reduction in affinity. Conversion to D-ala-D-Ser leads to 7 fold reduction.
Ways to decrease drug concentration.
Reduce uptake
Pump out antibiotics.
Example of titration.
Altering drug. Trimethoprim - mutate DHFR gene promoter region to increase transcription. Overwhelm drug.
Metabolic bypass
Altering drug. Trimethoprim and sulfonamides.
Metabolic bypass - trimethoprim.
Trimethoprim targets DHFR. Use plasmid encoded DHFR with lower affinity for trimethoprim. Used in Neisseria species. Or mutate binding site of chromosomal copy. Several species discovered which do both.
Resistance - sulfonamides.
Either mutation of active site of dihydropterate synthetase to decrease affinity, or overproduction of p-aminobenzoic acid.
Sulfonamides mode of action.
Compete with p-aminobenzoic acid for the dihydropterate synthetase active site.
Altering drug - general notes.
Tends to occur more for naturally derived antibiotics, which are likely to be similar to molecules used in housekeeping reactions. T
Altering drug - general notes.
Tends to occur more for naturally derived antibiotics, which are likely to be similar to molecules used in housekeeping reactions. Thus B-lactams and aminoglycosides are usually targeted this way, but trimethoprims are very rarely targeted thus.
Degradation of antibiotics
Within cell or outside cell.
Degradation of antibiotics within cellular structures
B-lactamases open the lactam ring to prevent this binding glycopeptides transpeptidase, a PBP. B-lactamases have a similar mode of action and structure to PBPs; they share an ancestral core.
Degradation of antibiotics outside the cell.
Proteases used against antimicrobial peptides. E.g. Strep pyogenes and Pseudomonas.
Enzymatic modification - types
Addition of acyl groups, phosphoryl groups, thiol groups, nucleotidyl groups, ADP-ribosyl groups and glycosyl groups.
Enzymatic modification example.
Bleomycin is modified by N-acetyltransferase dimer. Tunnel in Ntd accommodated both acetyl CoA at one end and Bm at the other. The proximity leads to efficient acetylation of Bm.
Sequestration of antibiotic
Inside or outside cell.
Sequestration of antibiotics - inside cell.
Bleomycin by actinomycetes, which make it. Dimeric bleomycin binding protein binds 2 molecules co-operatively.
Sequestration of antibiotics - inside cell. 2 examples.
Bleomycin by actinomycetes, which make it. Dimeric bleomycin binding protein binds 2 molecules co-operatively.
In some bacterial strains, evidence of monomeric muropeptides ending D-ala-D-ala which sequester vancomycin.
Sequestration of antibiotics - outside cell. 3 examples.
1) Staphylokinase binding a-defensins.
2) Free AMPs
3) Polymixins.
Sequestration of free AMPs outside cell.
Free AMPs scavenged by polysaccharides, plasmid DNA, some polyanionic species and glucose aminoglycans (from bacteria, or due to degradation of host cells by bacteria).
Sequestration of polymixins outside cell.
Klebsiella pneumonia reported to shed capsular polysaccharides to bind polymixins.
Ways to reduce uptake
Alter lipid membrane.
Modify porins.
Altering uptake by altering lipid membrane
LPS is negatively charged, so takes up cationic peptides and antimicrobial peptides.
Decrease negative charge.
Decreasing negative charge on LPS - E. coli and Salmonella.
E. coli and salmonella.
1) alter lipid A moiety with phosphoethanolamine.
2) Substitute phosphate groups for L-ara4N, decreasing net charge to 0.
Uptake across membrane; destabilisation.
Polymixin and possibly aminoglycosides destabilise cross-linking by cations to promote their own uptake.
Decreasing negative charge on outer leaflet - Staph aureus.
Gain of function mutation in MprF leads to increased lysinylation and flipping so makes charge repulsive milieu for Ca++ complexed daptomycin.
Similar effect by D-alanylation of teichoic acid on cell wall.
Decreasing uptake - extracellular matrices
May provide phyisical barrier to larger antimicrobial peptides.
Why antimicrobial peptides do not target host cells.
Different membrane composition - e.g. more cholesterol and zwitterionic lipids in erythrocytes.
Porins
Present in E. coli and many others. Have B-barrel motif, large pore size, some selectivity for ionic charge and high open probability.
E. coli porins
OmpF, OmpC, PhoE.
Ways to modify porins
Decrease expression.
Replace with channels with smaller lumens.
Mutate channels
Block porins
Ways to modify porins (enterobacter)
Decrease expression.
Replace with channels with smaller lumens.
Mutate channels
Block porins
Decreasing porin expression example.
OmpF in 4-quinolone resistance.
Mutate channels (modifying porins)
In constriction region, L3 loop.
Rapidly closing porins (modifying porins).
Binding molecules to porin e.g. cadaverin, spermine. Host or bacterial molecules.
Organisms with low OM permeability.
P. aeruginosa; key porin OprF is normally closed.
Pumps - powering
Chemical or electrical gradients, or ATPases. Many are antiporters.
Types of pumps
MFS, MATE, ABC, SMR
MFS pump example
QacA.
MFS general
Uses proton gradient, although some members of the family are passive. Very key in Gram positive.
MFS mechanism.
Alternating access.
MATE pump example
NorM
MATE mechanism
Alternating access. Antiporters, gnereally Na+ (common) or H+ (rare).
ABC pumps examples.
HlyB, LmrA, MacB. MsbA, a lipid flippase.
ABC pumps examples.
HlyB, LmrA, MacB.
ABC mechansism. 4 steps.
- Open accepts substrate
- Substrate bound form favours closed
- Closed form cannot release substance to periplasm; binding ATP causes twisting conformational change; substrate released.
- Hydrolysis of ATP powers reversion to open state.
Closed form cannot release substance to periplasm; binding ATP causes twisting conformational change; substrate released. EVIDENCE
As seen from crystallisation of MsbA with ADP and vanadate; protein bound by ADP-Vi (mimics ATP)
Hydrolysis of ATP powers reversion to open state. EVIDENCE
Seen from fact that open structures solved are either non-nucleotide bound, or ADP-bound.
SMR example
QacC
SMR substrates
Quaternary ammonium compounds.
SMR mechanism.
Not understood. Pumps have dual topology.
RND pumps
E.g. AcrABTolC.
Trimeric AcrABTolC mechanism.
Each monomer at a different stage of the cycle which goes open - closed - bound.
Powered by H+ gradient.
Inhibition of pumping mechanisms.
Alter expression Inhibit assembly Block TolC Collapse efflux energy Competitive inhibition of substrate efflux Change antibiotic design.
Inhibition of pumping mechanisms. Collapsing efflux energy.
This is toxic.
Inhibition of pumping mechanisms. Competitive inhibition of substrate efflux. Example and difficulties.
e.g. pyridopyrimidine molecule and MexAB. Derivatives inhibit AcrABTolC.
Difficulty; often these inhibitors are slowly exported, so with selective pressure applied, mutations increasing rate of transport of these drugs and hence resulting in resistance are likely to be selected for.
First identified antibiotic resistance.
1940, Abraham and Chain.
Reasons antibiotic resistance has spread
Widespread use in farming, medicine etc. Release into envirionment.
Horizontal transmission and modern transportation systems.
People who have recently highlighted the issues of antibiotic resistance.
Keiji Fukuda Assistant general for World security, WHO.
Sally Davies, David Cameron, Barack Obama.
Reasons antibiotic resistance has spread
Widespread use in farming, medicine etc. Release into envirionment.
Horizontal transmission and modern transportation systems.
TolC protein structure.
40Å β-barrel spanning the membrane
100Å α helical barrel forming a channel spanning the periplasmic space
Crystallography studies by the Koronakis laboratory.
ABC transporter structures.
2 nucleotide binding domains. Two transmembrane domains. Walker motifs are conserved throughout P-loop ATPases. ATP binding leads to dimerisation of NBDs and hence torque.
Models for TolC-associated tripartite efflux systems.
Model proposed by Symmons.
Model proposed by Du in 2014.
Symmons model of TolC associated tripartite efflux systems.
TolC makes direct contact both with pump and with inside face of helices of adaptor protein.
Du model of TolC associated tripartite efflux systems.
No contact between the pump and TolC, but proteins are bridged in periplasm by adaptor molecules which form a funnel like structure, interacting independently with the pump and the exit channel.
Conventional vaccinology
Cultivate pathogen, isolate candidate, develop vaccine.
Reverse vaccinology
Take pathogen genome, use computational biology to find in silico vaccine candidates, express and purify the antigen, test immunogenicity. If there is a protective response, develop vaccine.
Reverse vaccinology - computational biology. ORFs.
Use ORF prediction to on genetic sequence to identify potential ORFs. Use homology searches on these to identify potential proteins.
Reverse vaccinology - computational biology. ORFs with no hits for homology
Hypothetical proteins. Search for localisation prediction to see if secreted (OM, periplasmic, inner membrane etc) or if cytoplasmic.
Reverse vaccinology - computational biology. ORFs with hits for homology
Assign function. Homology with bacterial surface associated proteins leads to potential vaccine candidates.
Reverse vaccinology - expressing and purifying potential antigens.
PCR it up, then insert into plasmids, then express, purify and inject into mice.
Reverse vaccinology - expressing and purifying potential antigens.
PCR it up, then insert into plasmids, then express, purify and inject into mice. Check serum bactericidal activity.
