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Multidrug Transporters (Drug Efflux Pumps & Other Efflux Proteins (ABC…

- - - - This is how all substrates in the periplasm are removed directly out of the cell. Other pumps move substrates into the periplasm
      - The substrates are shown to sit between phosphate head groups and the upper regions of the lipid tail (Siarheyeva, 2006, Biochemistry)
      - The model of how it initiates transport is either as an aqueous pore, a flippase (moving substrates from the inner to outer membrane) or as a hydrophobic vacuum cleaner (excising substrates from the inner leaflet into the periplasm and then extruding them)
        
        The vacuum cleaner model seems to be most favoured as a result of fluorescent quenching experiments using TMA-DPH which show it only removes substrates embedded in the inner membrane
        
        Crystal structures show a movement similar to a clothes peg, drawing in substrates from the inner membrane and then releasing them in the inner membrane
        
        The RDS of drug entry is passing through the outer membrane, making this mechanism effective
        
        This movement is caused by the rotation of helices though (so not quite like a peg) which causes the binding pocket to collapse and extrude the substrate(s) (Gutmann, 2010, Trend Biochem Sci)
      - Polyspecificty shows that ABCB1 may be able to bind multiple drugs at the same time (Aller, 2009, Science)
      - ABCC1 has been shown to have separate large pockets containing hydrophilic or hydrophobic residues that can either bind a few large or many small molecules (Johnson & Chen, 2017, Cell)
      - AcrB is a trimer through which substrates pass. It has three pockets, which alternate between open, binding and release conformations - this is a peristaltic pump mechanism (Seeger, 2006, Science)
      - Have an electrochemical and ATP-driven action
    - - These are important in drug elimination pathways. ABCC1, 2 & 3 are best at PhIII of metabolism - exporting conjugate molecules out of cells
        
        The integration of each determines their effect in the drug elimination pathway (Szakacs, 2013, Drug Discovery Today)
      - ABCC1 has a high level of polyspecificity (Johnson, 2017, Cell)
  - - - Lecturer does some research on AcrD
      - All work on a subset of all drugs (Nishino, 2009, Biochim Biophys Acta)
      - They confer in vivo bile acid resistance (Ma, 1995, PNAS) which results in them having very high IC50 value for sodium cholate and sodium taurodeoxycholate
- - - - Can be tracked through genomic sequencing to determine where mutations have arisen (C. difficile - He, 2012, Nature Genetics)
      - This has led to a severe lack of investment in the space
    - - CTX-M enzymes are now the most common, may be related to sewage treatment but this is contentious (Amos, 2014, J Antimicrob Chemother)
    - - Trimethoprim used to inhibit dihydrofolate reductase and exert a selective pressure
        
        Fitness of the population is driven by mutants that are sufficiently fit and arise sufficiently close to the advancing front, showing that mutants need to occur in the right environment for them to proliferate
        
        It also showed that adaptation to low drug concentrations facilitated adaptation to higher ones (Doing increases in three steps led to better survival than one)
        
        Found that some of these mutations resulted in lower fitness but that they picked up compensatory mutations to improve this later on. Highly resistant mutants were also sometimes spatially trapped
    - - IC50 increases dramatically over time no matter what antibiotic is used (showing resistance will always develop in right conditions
      - Populations under the same stresses travel similar evolutionary pathways
      - Different drugs affect different genes to different amounts - TMP causes mutations predominantly folic acid synthesis, DOX mutations are mostly in transcription & translation, CHL are tyipcally in membrane proteins (acrB and promoter of Cmr), thought to be because it has double the partition coefficient of DOX
        
        There appears to be some order in which this arises, as is shown in TMP resistance
        
        CHL and DOX resistance is somewhat complementary
    - - Shows that populations can survive higher doses than isolates can and that populations are dominated by less resistant isolates
      - DNA gyrase-inhibiting norfloxacin was used
      - Antibiotic challenge results in an increase in indole production - this is only seen in the highly resistant isolates, seen in gel shift assays as a band corresponding to tryptophanase production
        
        Adding indole to the less resistant isolates increased resistance in them. This is because indole stimulates antibiotic-tolerance mechanisms in the population to allow non-resistant individuals to survive
        
        This charity work allows for greater biodiversity in the surviving population
      - Look into what they have done since
  - - - Sensor kinase (VanS) binds vancomycin and phosphorylates the response regulator (VanR) which then stimulates transcription of the vancomycin resistance operon
        
        VanH, VanX and VanA then contribute to add D-Ala-D-Lactate to the end of the peptidoglycan chain rather than D-Ala-D-Ala
      - Arises in Enterococcus faecalis, the drug is also ineffective against gram -ve bacteria
    - - Penicilinase (blaZ) is inducible and on a transposon
        Broad spectrum lactamases are typically plasmid encoded (TEM, SHV, PXA - in Pseudomonas)
        Extended spectrum (ESBLs) are typically related to the above ones
        ampC BLs are chromosomal and resistant to BLase inhibitor
        Carbapenemases are either metall-BLases or serine carabpenemases
        
        TEM-1 is the most common in gram negative bateria
    - - Mutations in dnaQ/DNAPolIII, most often at Q97(T/N/S), typically non-synonymous mutations
      - Several mutatinos also seen in folA/DHFR, this is the most commonly mutated gene
    - - Central regulatory role to this is carried out by three homologous TFs, MarA, SoxS and Rob (Chubiz, 2012, J Bacteriol)
    - - First described by Joseph Bigger (1944) - they appear to exist in that state already rather than being induced by the penicilin
      - This "persisten" population cease growth in favour of survival
      - A balance of duration of treatment and [antibiotic] can be used to model tolerant and resistant bacteria - tolerant survive for longer, resistant survive at higher [antibiotic] (Fridman, 2014, Nature) - this shows that a minimum dosage and duration of time is required for 99% of bacteria to be killed
      - These bacteria are described as 'tolerant' and can be induced by infrequent dosing with an antibiotic. Mutations are termed tbl for 'tolerance by lag'
        
        Underlying this is an extension in single-cell lag-times (time before the cells divide). These matched the duration of the antibiotic-exposure interval
        
        These tbl mutations are along two major modules. Toxin-antitoxin modules and aminoacyl-transfer RNA synthetase
        
        Toxin-antitoxin modules (Harms, 2018, Molecular Cell)
        
        (p)ppGpp
        
        This is relied upon heavily. It is increased as part of the "stringent response" and the persistence phenotype depends on it, as do other types of multidrug resistance in biofilms
        
        persistence also depends on Lon protease, Pi and toxin-antitoxins
        
        High [(p)ppGpp] inhibit exopolyphosphatase (PPX) activity. PPX stimulates Lon protease to degrade antitoxins. This allows antitoxins to function and inhibit mRNA synthesis/translation. Toxins also act as TFs for a gene that encodes both toxins and antitoxins, resulting in a dynamic equilibrium forming
        
        Toxins slow the cell cycle by acting on many parts of the cell - DNA gyrase, aa-tRNA synthetase, ribosomes.
        They also dissipate electrochemical proton gradients, causing depletion of the ATP pool
      - High persister (hip) mutants of myobacterium tuberculosis have been observed when exposing them to two antibiotics (Torrey, 2016, PLoS One)
        
        Chemical mutagenesis is carried out and then hip mutants are selected by treatment with rifampin and streptomycin
        
        Mutants had:
        Reduced PDIM cell wall lipid and virulence factor expression
        Mutations in regulators of feast and famine regulation
        Decreasde phospholipid biosynthesis and lipid catabolism - preventing cell division
        Upregulation of toxin-antitoxin module expression
      - Studying these in bacteria showed that some bacteria can survive inside macrophages (Helaine, 2010, PNAS) using conditional dyes to show whether cells are dead or dormant
        
        This is the result of a dormancy response - unclear how much the effect is the result of reduced [nutrients] or mutations
        
        Studies have looked at this in more detail, showing how efflux pump Rv1258c (MFS transporter) might confer rifampicin tolerance within macrophages (Adams, 2014/2011, J Infect Diseases/Cell)
      - May be the result of transporters being upregulated. Evidence suggests a link between the two. Increase in efflux pumps --> increase in signalling molecules released --> shortened growth phase (Yang, 2006, PNAS)
  - - - Resistance genes spread typically by first jumping to a high copy plasmid to create a large number of positive cells in a colony and then horizontally transferring to another species
- - - - Used against ABC efflux pumps
      - May be useful for treating cancers
      - Flavonoids were thought to work but were unable to enter cells in high enough concentrations to be effective
    - - ABCB1 modulators
        
        1st gen = existing therapies (verapamil, benzocaine etc)
        2nd gen = More potent analogues of these (verapamil --> dexverapamil, emopamil) however these showed unacceptable toxicity
        3rd gen = Based on pharmacophore models of 2nd generation modulators (Elacridar = anti ABCB1/G2, Zosuquidar = most potent) which has recently had its MoA shown (Alam, 2019, Science) although it is unclear why it is not transported