Inhibition protein secretion systems Flashcards

1
Q

Virulence factors

A
  • Components of pathogens not essential for in vitro growth, but for infection of the host.
  • Examples: Toxins, adhesins, nutrient acquirisiton &laquo_space;Block these and inhibit infection
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2
Q

Antivirulence drugs

A
  • Block virulence factors

- Are antibodies and enzymes that aren’t small molecules.

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3
Q

Why target virulence instead of growth?]

5 reasons

A
  • 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.

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4
Q

Microbiome

A

Microbial flora of host

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5
Q

Ways to block virulence

5 ways

A
  • 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.
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6
Q

Normal antibiotics working

A

Kill pathogen > Killed pathogene is removed by immune cells

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7
Q

Pathogen secrete factors in cell via : Secretion system:

A

Different proteinaceous machines for secretion of effector proteins into the extra cellular milieu.
Classification is based on order of discovery

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8
Q

Type VII

A

Tuberculosis

One step or two step

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9
Q

Anti-virulence drugs:
Disarm pathogen and pathogen can’t secrete virulence factors in cell.
Inhibit system 8 ways:

A
  • 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
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10
Q

One step mechanism

A
  • No chaperones / no signal peptides

- Type I/ III/ IV

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11
Q

Two step mechanism

A
  • Chaperones and signal peptides in periplasm needed

- Type II/V/IV can work in one or two steps

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12
Q

Major systems present on bacterial surface:

A

They are adapted/ evolved from macromolecular structures present on the bacterial surface:
- Pili, Flagella, Conjugation (Transfer DNA via plasmids)

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13
Q

Type I

A

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

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14
Q

Type II

A

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
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15
Q

Type III

A

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

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16
Q

Type IV

A

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

17
Q

Type V

A

Two steps mechanism. SPATE family.

Target of antibiotics:

  • Block HbP (= heamoglobine protease)
  • Block automembrane channel
18
Q

Type VI

A

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&raquo_space; Degrade peptidoglycan of neighbouring cells
19
Q

Measurements with light

A

In vitro
- Test inhibition transcription activity
Found new inhibitors: Whole cell based luciferase activity assay. Potential compound has effect on transcription > Miss promotors&raquo_space; No light > = potential compound.

In vivo
- Test preventing spread
Light in other cell goes on when genes spread to ontvangende cell.

20
Q

Bacterial peritonitis model

E. coli

A
Heamoglobin protease (Hbp): Steals iron from heamoglobin. 
Has as outer transporter V5 secretie systeem.
21
Q

SPATE family

A

Serine protease auto transporters of Enterobacteria

22
Q

Essay to find type V inhibitors: Auto-transporter pathway

A
  • Have to find a compound that can block outer membrane
  • When heamoglobine gets stuck in periplasm&raquo_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
23
Q

Resistance of anti-virulence drugs:

A
  • 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.
24
Q

Future studies

A
  • Focus on mode of action
  • Testing complex infection models
  • More compounds optimized to drug like compounds
25
Q

Virulence factor: Resistance pressure

A

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