Lecture 4 Flashcards
What types of surfaces do bacteria need to be able to adhere to?
Abiotic surfaces
Host Tissue
Describe the membrane of a gram positive bacterium
A cytoplasmic membrane is topped with a thick layer of peptidoglycan
Describe the membrane of a gram negative bacteria
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
Name and describe the main adhesion molecule for S. aureus
SasG - a cell wall attached adhesin
~92nm long and very rigid fibrils
How was SasG shown to be important?
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
Name and describe the main adhesion molecule in V. cholera
GlcNac binding protein A
Multidomain protein
Can bind chitin (for section of life cycle in the sea) and mucin (in host)
How was the GbpA gene found?
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.
What is a his-tag?
Why is it useful?
What are the assumptions when using it?
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.
How was the binding of cholera on chitin shown to be dependent on GbpA?
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
What is one possible drawback of flourescence based assays?
Many things have natural flourescent
How was it shown that GpbA also binds to GlcNac?
Which binding is stronger?
Same experiment using beeda coated in GlcNac
Binding to GlcNac is stonger - increasing soluble GlcNac in solution causes less binding to chitin beads
Is GbpA important for pathogenicity?
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.
What other role has GbpA been shown to play?
Stimulation of mucin production in host
What type of adhesion molecule are mostly commonly seen on gram -ve bacteria?
Fimbriae (aka pili)
Which three fimbriae are the focus?
Type 1 fimbriae - encoded by the fim operon
PAP - encoded by the pap operon
Curli
How are fimbriae stained for visualisation?
Negatively stained
What was used in initial investigation into the roles of the proteins in the fimbriae?
Gold staining
How are subunits for the fimbriae transported to the outside for assembly?
Chaperone proteins, FimC or PapD, transport the subunits through the periplasm and then through usher proteins, FimD or PapC, out of the cell membrane.
Which proteins are the adhesins for each type?
FimH and PapG
Which proteins make up the majority of the fimbriae?
FimA or PapA
How many FimA subunits are needed per 1.5 helical repeat?
Why is this important?
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)
What does FimH attach to?
Mannose type groups on specialized bladder epithelium
What must be considered when using a murine model?
- Mouse strain
- Mouse age
- Procedure artifacts (damage during procedure)
- Bacterial innoculum
- Type of Assay
- Still not a human
Has FimH been shown to be important for virulence?
In vitro: Loss of FimH=less adhesion. Mannose can out compete adhesion
In vivo: Soluble mannose: fewer colony forming units from mouse
What environmental factors control each fimbriae type?
Type 1: Branched amino acids and alanine, temperature, sialic acid and N-acetylglucosamine, SlyA
PAP: Glucose and temperature
What is stick or swim?
When Pap is being expressed, one of it’s genes, X, will repress flagellar expression
And vice versa
What is different about curli structure?
What controls this?
They will fold into an amyloid fibre.
Due to CsgB nucleation protein.
What feature curli makes them nice to use for visualisation?
They will bind congo red and be stained
What role has curli recently been shown to have?
Anti-inflammatory factor
What has been shown at curli by clinical isolet screenings?
They are also formed at 37°C, ie in host, rather than just in the environment as thought
What is a biofilm?
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
Why could transmission EM not be used to prove a biofilm?
What is used instead?
Requires the sample to be dired out and hence the matrix would no longer be visible.
Multu-photon microscopy
How are 96-well plates used for biofilms screens?
Stain bacteria and can see whether or not form biofilm in the well. Can see which mutants stop biofilm formation,
What other method is used to study biofilms?
Flow cell - tube with flowing media rather and stationary in 96-well plate screen
Where do biofilms tend to form?
On the interferace between liquid and air
What % of bacterial infections are thought to have biofilms?
65-80%
What disease is hijacked by P. aeruginosa?
Cystic Fibrosis
