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Molecular Microbiology > Lecture 4 Mycobacterial cell envelope transport > Flashcards

Lecture 4 Mycobacterial cell envelope transport Flashcards

1
Q

Why is tuberculosis still so deadly?

A
  1. No effective vaccine available

2. M. tuberculosis is intrinsically resistant to most antibiotics

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2
Q

What is the biggest issue for why tuberculosis is resistant?

A

Antibiotic treatment: regimen of >6 months using 4 specific antibiotics. Biggest issue = people don’t finish their cure (lack of compliance or non-availabiliy): resistance

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3
Q

Most of the mycobacteria species are/are not pathogenic. Most pathogenic bacteria are/are not slow growing.

A

are pathogenic & most pathogenic are slow growing

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4
Q

Typical characteristics of mycobacteria?

A
  • Big difference in growth speed among Mycobacterium species. From 1 to 300 hours doubling time
  • Characteristic colony morphology. Looks sort of fungal, ‘myco’ = fungal.
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4
Q

Typical characteristics of mycobacteria?

A
  • Big difference in growth speed among Mycobacterium species. From 1 to 300 hours doubling time
  • Characteristic colony morphology. Looks sort of fungal, ‘myco’ = fungal.
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4
Q

Typical characteristics of mycobacteria?

A
  • Big difference in growth speed among Mycobacterium species. From 1 to 300 hours doubling time
  • Characteristic colony morphology. Looks sort of fungal, ‘myco’ = fungal.
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5
Q

Why is the Mycobacterial cell envelope an Efficient barrier for antimicrobial compounds ?

A

> 60% of cell envelope consists of lipids
Permeability for hydrophilic molecules is 10x lower than that of Gram-negative bacteria.
Impermeability is an important virulence factor.

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6
Q

Gramstaining mycobacterium will result in..

A

unclear: Gram-variable.

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7
Q

Waar berust gramkleuring op?

A

Difference in stain dependent on peptidoglycan layer.

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8
Q

Mycobacteria belong to the..

A

Actinomycetes (high D-C, G+ bacteria)

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9
Q

Why do mycobacteria belong to gram+ bacteria when gram staining is unclear?

A

On DNA-level: Mycobact. Looks more like Gram-positive bacteria, but when looking at the build-up of the cell envelope they look more like the Gram-negative bacteria.

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10
Q

Mycobacterial outer membrane: two characteristics?

A
  • No LPS

- Responsible for impermeable barrier

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11
Q

Composition of the mycobacterial cell envelope?

A
  • Normal inner membrane
  • Slightly different peptidoglycan: different side group because of sugar molecules: NAC is the same, NAM is slightly different (not major reason why cell envelope is so different)
  • Has a unique structure: arabinogalactan. Also a sugar structure, connected to the peptidoglycan.
  • On top of this, you have the OM composed of mycolic acids. Major components of second lipid layer, connected to arabinogalactan. Some are also ‘free’ lipids, like trehalose dimycolate
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12
Q

What are myolic acids?

A
  • Very long fatty acids (C70-C90)
  • Tend to fold in certain shapes
  • Many of them are glycosylated
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13
Q

What bacteria have myolic acids?

A

Corynebacteriales (subgroup of actinobacteria)

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14
Q

OM channels in gram-negatives are crucial for uptake of nutrients and other metabolites. They are to be subdivided into two groups. What groups are they and what are they used for?

A
  • General diffusion porins: mediate influx/efflux, forms holes in OM. Free diffusion.
  • Specific channels: molecules cannot readily diffuse in/out, recognition required.
15
Q

MspA mycobacterial porins were discovered. What type of porins are these and what was opmerkelijk about this discovery?

A
  • Forms an octamer, very stable complex
  • Forms a beta-barrel (atypical)
  • General diffusion porin: passive diffusion of small hydrophilic molecules (nutrients)

BUT:

1) Mycobacteria do not have a Bam complex
2) Only fast-growing mycobacteria have MspA-like porins

16
Q

Extracellular proteins play a crucial role in the virulence of pathogenic mycobacteria. What type of secretion systems do they have?

A
  • Sec & Tat system for IM
  • Specialized protein secretion systems 1-6 not present
  • Type VII secretion in Mycobacteria: they have 5 types of VII secretion systems, called ESX-1 to ESX-5
  • > Mediate protein secretion while maintaining cell envelope impermeability
17
Q

Model of mycobacterial type VII secretion?

A
  • Five conserved membrane components
  • Outer membrane component remains unknown
  • Substrates are secreted as folded heterodimers
18
Q

In what bacteria is ESC-5 system present + what is it used for?

A

• ESX-5 is only present in slow-growers.

• ESX-5 is essential for growth, as mutants cannot be made of the gene (knock-out)
(conditional mutant: regulational promoter). This compound being ATc. Bacteria could only grow in presence of ATc.

19
Q

Hypothesis:
No secretion systems = accumulation of proteins in the cytosol = toxic?

How to find the toxic substrate?

A

By random transposon mutagenesis

  1. Make a transposon mutant library (>10.000 clones)
  2. Screen library for mutants in which ESX-5 could be knocked out

Two mutants were isolated

Both with transposon in PDIM (free lipid of OM) biosynthesis gene.

20
Q

Why is it the case that If you delete this biosynthesis of this lipid, ESX-5 is not essential anymore?

A

PDIM mutants are known to be more susceptible to antibiotics -> more permeable outer membrane?

21
Q

How was the PDIM mutants that gained a more permeable outer membrane hypothesis tested?

A

Test: increase OM permeability and see if deletion of ESX-5 could still result in viable mutants. Approach used to permeabilize the OM: by introducing MspA of M. smegmatis.
Presence of MspA circumvents ESX-5 essentiality!

Link between ESX-5 essentiality and outer membrane permeability.

22
Q

Next hypothesis: Is ESX-5 involved in the uptake of specific nutrients? What kind of nutrient should you look for?

A

Identify a carbon source that is not able to pass through MspA, but is dependent on ESX-5 for uptake.
-> Tween-80: hydrophobic molecule not able to pass through MspA

23
Q

ESX-5 mutant + tween80 could not grow. ESX-5 is thus..

A

important for metabolism of at least this C-source.

Another experiment: Uptake of fluorescent fatty acid (Bodipy-C12).
ESX-5 is involved in the uptake of fatty acids, and possibly also of other nutrients.

24
Q

What is the difference in the Model for nutrient uptake in nonpathogenic vs. pathogenic mycobacteria?

A

Fast-growing/non-pathogenic = general/efficient uptake of nutrients

Slow-growing/pathogenic = Selective uptake of nutrients.

25
Q

How was it analyzed whether ESC5-membrane complex had a one or two-step mechanism of secretion?

A

Electron Microscopy to analyze: single particle analysis/Cryo-electron tomography.
-> Averaging of a large number of complexes to obtain high(er) resolution structures.

26
Q

How to isolate T7SS components for elucidation of the ESX-5 membrane complex?

A
  • Isolate membranes from M. marinum
  • Solubilization of membrane complexes by mild detergent
  • BN-PAGE (blue native): Separation of protein complexes on size by replacing SDS by coomassie
  • Westernblotting (antibodies) to detect T7SS components
27
Q

How to indicate if the proteins are in the same complex together or all are in different complexes of the same size?

A

Other way to state this question:

How to verify an interaction between two proteins?

Isolate ESX-5 membrane complex using a recombinant affinity tag (strep-tag). Then you see which other proteins are interact with your protein. (EM)

28
Q

Are beta-barrel proteins of mycobacteria in the OM the same as in gram- bacteria?

A

no

29
Q

Overview

A

ESX-5 might mediate insertion of selective outer membrane porins in the outer membrane, important for nutrient uptake.
• The elucidated ESX-5 membrane channel shows its insertion solely in the inner membrane and unusual flexibility of the ATPase subunits. (which might be a mechanism of substrate recognition and transport)

Molecular Microbiology (12 decks)

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