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Antimicrobial compound deel II > Permeability and efflux pumps: > Flashcards

Permeability and efflux pumps: Flashcards

1
Q

Cell enveloppe gram negative

A
  • Gram negative higher intrinsic resistance
  • It’s less permeable for hydrophobic and hydrophyls through double membrane
  • Antibiotics can only enter via pores
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2
Q

LPS

A
  • Is endotoxin and very toxic for us
  • Anchored in outer membrane with fatty acids of lipid A
  • Core-polysacharide > Bacteria can’t cross because it’s charged
  • O- portion polysacharide > Very specific and variable
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3
Q

Hydrofylic drugs

A

Beta lactams/ Aminoglycosides/ Glycopeptides

Can not pass lipid bilayer of outer membrane

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4
Q

Hydrophobic drugs

A

Fluoroquinolones/ Macrolides/ Rifampicin

Pass outer membrane of gram negatives through difusion

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5
Q

First innate defence of gram negatives: Outer membrane

A
  • Amphipathic:
    Inside hydrofyl with phospholipiden
    Outside hydrofoob with lipopolisachariden and LPS
  • Because LPS is charched, it’s a barrier for hydrophobic drugs.
    &laquo_space; Only hydrophobic drugs that like lipid environment can diffuse
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6
Q

Amphipathic

A

Hydrofyl and hydrophobic

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7
Q

2 ways of innate defence of gram negatives

A
  • Outer membrane

- Efflux pumps by overexpression of these pumps (also gram positives)

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8
Q

Integral membrane proteins : Two types

A

Span the membrane and are in contact with two compartments

  1. Cytoplasmic membrane (sec protein): Transmembrane alpha helical structures highly hyddrophobic
  2. Outer membrane: Transmembrane beta sheets are amphipathic
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9
Q

Why differs structure between outer and inner membrane?

A
  • Hydrophobic alpha helices would get stuck in inner membrane.
  • The transport channel for OMP (the sec- translocon) considers OMP as soluble proteins (amphipathic strands)
  • Some OMP form channels (general diffusion protein)
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10
Q

Two classes of OMP

A

They allow passage of nutrients and metabolites important for cell growth
1. General diffusion porins (OMPF): A- selective, make holes in membrane
- Size limitation:
Hydrophilics <600Da can diffuse freely, diffusion not regulated
-Porin function regulated by physico chemical parameters like pH (can regulate rate and close)
-Ligand binding (can block porin

  1. Specific channels (like FhuA) : Recognition of special molecules such as iron transporter
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11
Q

Consideration for new drug development

A

New drugs

  • Need an hydrophobic compound to pass the lipid bilayer and is not to hydrophobic for LPS
  • Hydrophilic drugs shouldn’t be to big so they can pass porins.
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12
Q

Second innate defence of gram negative and positive bacteria
Efflux pumps
5 classes

A
  • Highly efficient in drug extrusion
  • Broad substrates specificities
    5 classes:
    1. RND family &laquo_space;Only in gram neg.
    Resistance Nodulation Division
    2. MFS familiy
    Major Facilitator Super-family
    3. ABC super family
    ATP-Binding Cassette
    4. DMT super family
    Metabolite transporter: with Small Multidrug Resistance (SMR)
    5. MATE family
    Multidrug And Toxid compound Extrusion
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13
Q

Poly amines

A

Produced by bacteria or host > Are ligands that can block porins

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14
Q

Resistance against porin drugs:

Mechanisms of resistance related porin funciton

A
  1. Regulate expression of porin (down regulate)
    Loss / Severe reduction of porins
    Replacement of one / two major porins by another (more selective) porin
  2. Altering porin function > Mutant porin function with reduced permeability
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15
Q

Efflux pumps energy sources

A
  1. Proton motive force:
    - Aearobic pathway
    - Proton gradient (pH) : Protons move to side with the lowest concentration (periplasm) by the E- transport chain&raquo_space; Resulting in a protin gradient and a Electochemical gradient
    - Electrochemical gradient can be used energy for innermembrane and to produced ATP by ATP synthase or of other cellular processes such as transport of molecules
  2. ATP hydrolysis
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16
Q

2 classes of Efflux pumps most important clinically

A
  1. RND family in gram negative
    - In E.coli / Salmonella/ Pseudonomas aeruginose
    - More complex system (spans two membranes). Composed of three different proteins.
    - AcrAB-TolC system in E.coli
  2. MFS family:
    - Staphylococcus aureus/ Streptococcus pneumoniae
    - Crystal structures of different MFS members in different conformations , form homo dimers
    - Cytoplasmic membrane pumps
    Antibiotic in > Protons used the other way around > Flipping mechanism > Antibiotic out
17
Q

Evolutional mechanism

2

A
  1. Detoxification: Extrusion of host derived antimicrobials during infection.
    - Mutations in efflux pumps have effect on virulence
    - AcrAB-TolC mediates toxic bile salts > So E.coli can survive in intestinal tract
  2. Quorum sensing and Biofilm formation:
    Acting when enough bacteria.
    - Biofilm: Provides a profitable environment for the pathogen
    - Makes bacteria much less sensitive towards antimicrobials.
18
Q

Regulatory network of efflux pumps

A
  • By absence of sustrates expression of pumps is low > Otherwise important cells (metabolites) for the bacteria are pumped out
  • If repressor binds to promotor > Gene not expressed
19
Q

Mycobacteria Special cell envoloppe

A

It’s outer membrane :

  • No general diffusion proteins
  • Has special lipids (mycolic acids and no LPS)
  • Highly impermeable for hydrophilic molecules
  • Has good efflux pumps.
20
Q

Functional rotation model

A

In innermembrane: To allow efficient drug efflux.

Three different binding sites&raquo_space; One bind antibiotic, one drug extrusion, one proton transport.

21
Q

Quorum sensing : Secretion of auto inducers (possibly also by efflux pumps)

A
  • Preconditioning by protein moleculs
  • Cell deposition
  • Cell adsorption
  • Cell-to-cell signaling and onset of exopolymer production
  • Secretion of polysacharide matrix
  • Detachment erosion and sloughing
22
Q

Quorum sensing : Secretion of auto inducers (possibly also by efflux pumps)

A
  • Preconditioning by protein moleculs
  • Cell deposition
  • Cell adsorption
  • Cell-to-cell signaling and onset of exopolymer production
  • Secretion of polysacharide matrix
  • Detachment erosion and sloughing
23
Q

Mechanism of upregulation to confer drug resistance

A
  • Mutations in local repressor gene
  • Mutations in global regulatory gene
  • Mutations in promotor region of efflux gene
  • Insertion elements upstream of efflux pump gene
24
Q

Example P. aeruginosa regulatory the genes

A
  • MexZ is the repressor of RND efflux family MexXy.
    Co factor MexZ is unknown.
  • Mutations in DNA binding domain of MexZ > Lack promotor binding > Overexpression of MexXY pump and bacteria will survive.
  • TetR (tetracyclin) is repressor. Binds to promotor region and blocks expression of genes.
  • If substrate binds to TetR > TetR laat promotor los&raquo_space; Expression gene
25
Q

Efflux pump good target for antibiotics

2 reasons

A
  • Used for many different bacteria

- It’s a major determinant for resistants

26
Q

Tuberculosis mycobacteria

A
  • Extremely drug resistants and deadliest bacterial pathogen
  • Only treated with specific antibiotics for > 6 months
  • Concerning risk of MDR/ XDR TB
  • Problems in antibacterial discovery: Many antibiotics do not enter the cell wall and they have good efflux pumps.
27
Q

Antibiotic cocktail tuberculosis

A
  • Isoniazid
  • Rifamspin
  • Ethambutol
  • Pyrazzinamide
28
Q

Sec translocon:

A
  • Transport channel for OMP and IMP

IMP directly to Inner membrane

29
Q

Most studies to AcrAB-TOLC system in E.coli

RND pump

A
  • Outermembrane: TolC is activated by AcrAB transporter and rotation of alpha helices
  • Innermembrane: AcrB
    Trimere, one monomere bound drug&raquo_space; Functional rotation model

Antimicrobial compound deel II (12 decks)

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