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Bacterial Pathogenesis > Lecture 4 > Flashcards

Lecture 4 Flashcards

1
Q

What types of surfaces do bacteria need to be able to adhere to?

A

Abiotic surfaces

Host Tissue

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2
Q

Describe the membrane of a gram positive bacterium

A

A cytoplasmic membrane is topped with a thick layer of peptidoglycan

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3
Q

Describe the membrane of a gram negative bacteria

A

A cytoplasmic membrane topped with a thin layer of peptidoglycan (these two are the periplasm) and then the outer membrane, an 8nm thick membrane with polysaccharides and lipopolysaccharides sticking out into extracellular space

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4
Q

Name and describe the main adhesion molecule for S. aureus

A

SasG - a cell wall attached adhesin

~92nm long and very rigid fibrils

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5
Q

How was SasG shown to be important?

A

Bacterial count per cell 100 nasal swab cells

Use of tetracycline, an inducer for SasG, caused more cells to be attached.

Shown that other adhesins used; had attachment when had no SasG expression

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6
Q

Name and describe the main adhesion molecule in V. cholera

A

GlcNac binding protein A

Multidomain protein

Can bind chitin (for section of life cycle in the sea) and mucin (in host)

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7
Q

How was the GbpA gene found?

A

Random mutagenesis screen found loss of a gene which decreased attachment HT29 cells.

Complement with vector carrying his-tagged version of this gene restored attachment.

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8
Q

What is a his-tag?

Why is it useful?

What are the assumptions when using it?

A

Histidine tag - 6 repeats of histidine tag on C-terminus allows for protein purification on nickel column for affintity chromatography. Assumes the histidine tag doesn’t affect the function of the protein.

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9
Q

How was the binding of cholera on chitin shown to be dependent on GbpA?

A

Binding of GFP expressing cholera to chitin beads almost completely knocked out in ΔgpbA strain. Ability to bind not fully restored by complementing with gbpA-his vector

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10
Q

What is one possible drawback of flourescence based assays?

A

Many things have natural flourescent

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11
Q

How was it shown that GpbA also binds to GlcNac?

Which binding is stronger?

A

Same experiment using beeda coated in GlcNac

Binding to GlcNac is stonger - increasing soluble GlcNac in solution causes less binding to chitin beads

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12
Q

Is GbpA important for pathogenicity?

A

Loss of GbpA= lower competetiveness index than wild type- which is resotred by plasmid complementation.

Immune serum raised against GbpA and given to mice. Mice given the serum has higher survival percentage than non-treated mice.

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13
Q

What other role has GbpA been shown to play?

A

Stimulation of mucin production in host

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14
Q

What type of adhesion molecule are mostly commonly seen on gram -ve bacteria?

A

Fimbriae (aka pili)

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15
Q

Which three fimbriae are the focus?

A

Type 1 fimbriae - encoded by the fim operon

PAP - encoded by the pap operon

Curli

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16
Q

How are fimbriae stained for visualisation?

A

Negatively stained

17
Q

What was used in initial investigation into the roles of the proteins in the fimbriae?

A

Gold staining

18
Q

How are subunits for the fimbriae transported to the outside for assembly?

A

Chaperone proteins, FimC or PapD, transport the subunits through the periplasm and then through usher proteins, FimD or PapC, out of the cell membrane.

19
Q

Which proteins are the adhesins for each type?

A

FimH and PapG

20
Q

Which proteins make up the majority of the fimbriae?

A

FimA or PapA

21
Q

How many FimA subunits are needed per 1.5 helical repeat?

Why is this important?

A

40

Lots of FimA but little of the other proteins in the operon; highlights becteria has way of controlling amount of the othe proteins (e.g. differential stability)

22
Q

What does FimH attach to?

A

Mannose type groups on specialized bladder epithelium

23
Q

What must be considered when using a murine model?

A
  • Mouse strain
  • Mouse age
  • Procedure artifacts (damage during procedure)
  • Bacterial innoculum
  • Type of Assay
  • Still not a human
24
Q

Has FimH been shown to be important for virulence?

A

In vitro: Loss of FimH=less adhesion. Mannose can out compete adhesion

In vivo: Soluble mannose: fewer colony forming units from mouse

25
Q

What environmental factors control each fimbriae type?

A

Type 1: Branched amino acids and alanine, temperature, sialic acid and N-acetylglucosamine, SlyA

PAP: Glucose and temperature

26
Q

What is stick or swim?

A

When Pap is being expressed, one of it’s genes, X, will repress flagellar expression

And vice versa

27
Q

What is different about curli structure?

What controls this?

A

They will fold into an amyloid fibre.

Due to CsgB nucleation protein.

28
Q

What feature curli makes them nice to use for visualisation?

A

They will bind congo red and be stained

29
Q

What role has curli recently been shown to have?

A

Anti-inflammatory factor

30
Q

What has been shown at curli by clinical isolet screenings?

A

They are also formed at 37°C, ie in host, rather than just in the environment as thought

31
Q

What is a biofilm?

A

Structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to a substratum, interface or each other, and exhibit an altered phenotype in regard to grwoth, gene expression and protein production

32
Q

Why could transmission EM not be used to prove a biofilm?

What is used instead?

A

Requires the sample to be dired out and hence the matrix would no longer be visible.

Multu-photon microscopy

33
Q

How are 96-well plates used for biofilms screens?

A

Stain bacteria and can see whether or not form biofilm in the well. Can see which mutants stop biofilm formation,

34
Q

What other method is used to study biofilms?

A

Flow cell - tube with flowing media rather and stationary in 96-well plate screen

35
Q

Where do biofilms tend to form?

A

On the interferace between liquid and air

36
Q

What % of bacterial infections are thought to have biofilms?

A

65-80%

37
Q

What disease is hijacked by P. aeruginosa?

A

Cystic Fibrosis

Bacterial Pathogenesis (9 decks)

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