Permeability and efflux pumps: Flashcards
Cell enveloppe gram negative
- Gram negative higher intrinsic resistance
- It’s less permeable for hydrophobic and hydrophyls through double membrane
- Antibiotics can only enter via pores
LPS
- 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
Hydrofylic drugs
Beta lactams/ Aminoglycosides/ Glycopeptides
Can not pass lipid bilayer of outer membrane
Hydrophobic drugs
Fluoroquinolones/ Macrolides/ Rifampicin
Pass outer membrane of gram negatives through difusion
First innate defence of gram negatives: Outer membrane
- Amphipathic:
Inside hydrofyl with phospholipiden
Outside hydrofoob with lipopolisachariden and LPS - Because LPS is charched, it’s a barrier for hydrophobic drugs.
«_space; Only hydrophobic drugs that like lipid environment can diffuse
Amphipathic
Hydrofyl and hydrophobic
2 ways of innate defence of gram negatives
- Outer membrane
- Efflux pumps by overexpression of these pumps (also gram positives)
Integral membrane proteins : Two types
Span the membrane and are in contact with two compartments
- Cytoplasmic membrane (sec protein): Transmembrane alpha helical structures highly hyddrophobic
- Outer membrane: Transmembrane beta sheets are amphipathic
Why differs structure between outer and inner membrane?
- 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)
Two classes of OMP
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
- Specific channels (like FhuA) : Recognition of special molecules such as iron transporter
Consideration for new drug development
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.
Second innate defence of gram negative and positive bacteria
Efflux pumps
5 classes
- Highly efficient in drug extrusion
- Broad substrates specificities
5 classes:
1. RND family «_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
Poly amines
Produced by bacteria or host > Are ligands that can block porins
Resistance against porin drugs:
Mechanisms of resistance related porin funciton
- Regulate expression of porin (down regulate)
Loss / Severe reduction of porins
Replacement of one / two major porins by another (more selective) porin - Altering porin function > Mutant porin function with reduced permeability
Efflux pumps energy sources
- Proton motive force:
- Aearobic pathway
- Proton gradient (pH) : Protons move to side with the lowest concentration (periplasm) by the E- transport chain»_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 - ATP hydrolysis
2 classes of Efflux pumps most important clinically
- 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 - 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
Evolutional mechanism
2
- 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 - Quorum sensing and Biofilm formation:
Acting when enough bacteria.
- Biofilm: Provides a profitable environment for the pathogen
- Makes bacteria much less sensitive towards antimicrobials.
Regulatory network of efflux pumps
- 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
Mycobacteria Special cell envoloppe
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.
Functional rotation model
In innermembrane: To allow efficient drug efflux.
Three different binding sites»_space; One bind antibiotic, one drug extrusion, one proton transport.
Quorum sensing : Secretion of auto inducers (possibly also by efflux pumps)
- 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
Quorum sensing : Secretion of auto inducers (possibly also by efflux pumps)
- 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
Mechanism of upregulation to confer drug resistance
- Mutations in local repressor gene
- Mutations in global regulatory gene
- Mutations in promotor region of efflux gene
- Insertion elements upstream of efflux pump gene
Example P. aeruginosa regulatory the genes
- 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»_space; Expression gene
Efflux pump good target for antibiotics
2 reasons
- Used for many different bacteria
- It’s a major determinant for resistants
Tuberculosis mycobacteria
- 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.
Antibiotic cocktail tuberculosis
- Isoniazid
- Rifamspin
- Ethambutol
- Pyrazzinamide
Sec translocon:
- Transport channel for OMP and IMP
IMP directly to Inner membrane
Most studies to AcrAB-TOLC system in E.coli
RND pump
- Outermembrane: TolC is activated by AcrAB transporter and rotation of alpha helices
- Innermembrane: AcrB
Trimere, one monomere bound drug»_space; Functional rotation model
