Inhibition protein secretion systems Flashcards
Virulence factors
- Components of pathogens not essential for in vitro growth, but for infection of the host.
- Examples: Toxins, adhesins, nutrient acquirisiton «_space;Block these and inhibit infection
Antivirulence drugs
- Block virulence factors
- Are antibodies and enzymes that aren’t small molecules.
Why target virulence instead of growth?]
5 reasons
- Rapid action f.i. by blocking toxins
- Will reveal new chemical classes not exploited before.
- No effect on existing microbial flora
- Not toxic for host
- Les sujective pressure for resistance than drugs that inhibit growth.
But didn’t reache the farma yet.
Microbiome
Microbial flora of host
Ways to block virulence
5 ways
- Block attachment to host cells
- Prevent biofilm formation (block attachment to each other or surface)
- Disrupt (regulation of) expression of virulence factors
- Block function of secreted virulence factors (toxins)
- Focus mostly on block secretion of virulence factors.
Normal antibiotics working
Kill pathogen > Killed pathogene is removed by immune cells
Pathogen secrete factors in cell via : Secretion system:
Different proteinaceous machines for secretion of effector proteins into the extra cellular milieu.
Classification is based on order of discovery
Type VII
Tuberculosis
One step or two step
Anti-virulence drugs:
Disarm pathogen and pathogen can’t secrete virulence factors in cell.
Inhibit system 8 ways:
- Inhibit chaperone activity
- Inhibit substrate maturation (folding)
- Inhibit release of substrate into periplasm
- Inhibit energize transport (ATPase)
- Inhibit BSS components assembly
- Inhibit transport of BSS substrate
- Inhibit expression of BSS substrate
- Inhibit expression of BSS components
One step mechanism
- No chaperones / no signal peptides
- Type I/ III/ IV
Two step mechanism
- Chaperones and signal peptides in periplasm needed
- Type II/V/IV can work in one or two steps
Major systems present on bacterial surface:
They are adapted/ evolved from macromolecular structures present on the bacterial surface:
- Pili, Flagella, Conjugation (Transfer DNA via plasmids)
Type I
Substrate is unfolded during transit
- ATP dependent
Inner membrane:
- TM helices
- ATP binding & Hydrolysis
Periplasm:
Fuses IM with HlyD complex to OMP
Outer membrane:
- TolC OMP channel: Tremeric betha channel in OMP
Transport of:
- Proteins : toxins/ enzymes
- Polysacharides / sugars
- Antibiotics / Harmfull chemicals (SDS)
Secretion occurs after: Recognistion of secretion signal at C terminus. By ABC transporter
- No structural features.
Target antibiotics:
- TolC OMP channel: Part of efflux pump
Type II
Outer membrane:
- Pore complex: Secretin
Transport of:
- Toxins (cholera toxin)
- Lipolytic enzymes
- Proteases
Characteristics:
- ATP dependent type IV pilus (can move)
- Scafold of pilus: Works as piston
- Two step
Target antibiotics: - Virstatin: Impairs type IV pilus biofilms & Impairs dimerisation of ToxT (=transcriptional activator) > Cholera toxin isn't made
Type III
One step related to flagella
Needle spans 3 membranes > Proteins directly in host.
Inhibition ATPase
Inhibition Cytoplasmid chaperone
Inhibition adherence of tip complex to host cell receptor
Target antibiotics:
Found: Salicylidene acylhydrazide: Mechanism inhibition unclear.
And found 3 mechanism:
- Inhibition ATPase
- Inhibition chaperone
- Inhibition adherence to host cel receptor
Type IV
One and two step used for horizontal gene transfer, related to conjugation systems
Spans three membranes > proteins directly in host
Transport of : Effector proteins
Target antibiotics:
After structure based design found compounds:
- CHIR1/2/3 To prevent spread of resistance genes. They inhibit ATPase activity.
»
- Anti virulence
- Prevents resistance through DNA transfer
Type V
Two steps mechanism. SPATE family.
Target of antibiotics:
- Block HbP (= heamoglobine protease)
- Block automembrane channel
Type VI
One step: Cholera
In cytosol, piston/ pilus like structure
Push throug inner and outer membrane
Look like bacteriophages: Deliver toxin in host cell.
Two types of enzymes inject in periplasm:
- VrG derivates : Actin polymerization
- Secreted toxin: Muaminidase and amidase»_space; Degrade peptidoglycan of neighbouring cells
Measurements with light
In vitro
- Test inhibition transcription activity
Found new inhibitors: Whole cell based luciferase activity assay. Potential compound has effect on transcription > Miss promotors»_space; No light > = potential compound.
In vivo
- Test preventing spread
Light in other cell goes on when genes spread to ontvangende cell.
Bacterial peritonitis model
E. coli
Heamoglobin protease (Hbp): Steals iron from heamoglobin. Has as outer transporter V5 secretie systeem.
SPATE family
Serine protease auto transporters of Enterobacteria
Essay to find type V inhibitors: Auto-transporter pathway
- Have to find a compound that can block outer membrane
- When heamoglobine gets stuck in periplasm»_space; Stress for bacteria and they sense ProE protease.
- Bind ProE with GFP and find the cause of stress
- This cause is your new compound to block outer membrane
Resistance of anti-virulence drugs:
- Not evolution proof drugs
- Selection depends on function of inhibited virulence factor.
- Spread of resistance depends on function of virulence factors at site of infection.
Future studies
- Focus on mode of action
- Testing complex infection models
- More compounds optimized to drug like compounds
Virulence factor: Resistance pressure
High pressure:
- Essential cell viability
Pressure unknown but selection for resistance:
- Not essential for vialbility, but is beneficial
Selection against resistance:
- Not beneficial
- Collectively benificial
