Lecture 4 - Adhesion Flashcards
Why may a bacteria want to adhere to a surface?
environment with rapid flow, want to stay where you are
if surface is a nice nutrient
How does Vibrio cholera attach to zoaplankton?
uses the chitin as carbon and nitrogen source with a chitinase
What are problematic places for bacteria to adhere to?
Epithialial cells
Hospital equipment such as a catheter
What are the differences between Gr+ and Gr-? examples
Gr+ has one membrane with a thick peptidoglycan layer on top - s aureas
Gr- have a very thin peptidoglycan layer between and inner and outer membrane - e coli
Background of S aureus?
20% carriers
Causes harm when moves from nasal to other cavities
What is SasG and what is its structure?
A cell wall attached adhesin of Staph aureus
long filaments 92nm long
What are the two functions of SasG?
Extends out 10% further than the microbe to adhere to cells
Masks it from other surfaces so no adherance
What assay did RM Corrigan perform tp find out if SasG is important?
Took parent strain, introduced SasG in two different ways: on a plasmid and on a suicide plasmid so they could recombine it onto the chromosome. On the chromosome it is single copy whereas the plasmid is multicopy, better to test both.
Showed introducing more SasG increases attachment.
What is an LPS?
lipopolysaccharide on the outside of a Gr- bacterium
What is GbpA?
A binding protein of V.cholera which mediates attachment to chitin and host epithilial cells (mucin) using multidomains.
Describe the steps to Cholera causing disease
Attaches to zoaplankton
gets into drinking water
adherance to epithilium
quorum sensing leads to detachment by release of proteases to break down GbpA
Background of Type 1 fimbriae
encoded by fim operon
seen in all E.coli
What is seen in 30% of E.coli?
pap fimbriae (pyelonephritis associated pili)
What does a Type 1 fimbriae look like (from the top)
Adhesins, (FimH,G,F)
Adaptor proteins (FimA)
outer membrace proteins (FimD)
Chaperone and Usher
Why are a chaperone and usher needed?
If the protein went straight into the periplasm they would not fold properly at all
Chaperone forces correct folding
Usher takes them to the outside
What are type 1 Fimbriae also known as and why?
Mannose sensitive fimbriae.
Tested long time ago with yeast, usually clumped up but when mannose added they did not as the binding sites bind to mannose instead of each other
How did they test the importance of FimH in bladder cell adhesion?
Took a bladder cell line, attached urinary tract E coli cell line.
FimH mutant did not attach
As increase mannose, adhesion goes down.
with heptylmannose get much higher blocking of adhesion
could heptylmannose be a treatment for disease?
What are some challenges for giving a mouse a UTI?
Which bacteria you have and load cannot damage UT or will cause inflammation mouse ut very small mouse strain age of mouse
What are the experimental problems with fimbriae?
many fimbrial operons with slightly different FimH and different affinity
pap have slightly different PapG and different affinity for Gal4 (could be reason for different susceptibility to uti)
How can you get different amounts of protein from one RNA?
Different ribosome binding sites
differential stability of different parts of mRNA in long transcript (happens in pap cluster with PapG)
What environmental factors have an effect on the fim operon?
amino acids temperature (only expressed at 37 degrees) sialic acid n-acertylglucosamine slyA (virulence regulator)
What environmental factors have an addect on pap operon?
glucose
temperature
What did they find when they sequenced the whole gene of the pap operon?
263 more bp beyond G, swim vs attach system.
PapX - a regulatory protein to inhibit flagellar expression so you dont swim anymore when you attach
Who’s is the paper on fiimbrial regulation in Salmonella?
Sterzenbach
How many fimbrial operons does Salmonella have and how many types grow in the lab?
11
Only the Type 1 fimbrium
In Salmonella which Fimbriae is turned on when the Type 1 is turned off and what is it important for?
PefA
Virulence
What are the key players in fimbrial regulation in Salmonella?
CsrA - RNA binding protein
CsrB and C - sRNAs that bind to A
Two 5’ untranslated regions fim and pef
SirA - part of the 2 component system controlling A and B
Why may you have fimbrial regulation?
Have a lot of operons, dont want them to compete with each other
fimbriae are expensive to make
How did Sterzenbach show CsrA regulates expression of PefA?
Did an EMSA (electron mobility shift assay) run a gel with labelled primer, if it binds its a higher molecular mass so moves higher in the gel.
Seen that CsrA is binding to the regulatory region of PefA.
What does fim bind to?
CsrA
What did Sterzenbach use qRT-PCR to show?
The 5’ UTR of the fimA transcript regulates gene expression
What was the strange thing happening with pefA?
Experiments showed that it was a virulence factor however it was not expressed in the host
What does the fim 5’ UTR do?
Binds to a lot of CsrA, allowing pefA transcript to be expressed
What other fimbriae does the CsrA system affect?
Type 3 secretion
What percentage of bacterial infections does the CDC estimate involve biofilms?
65%
What does the CDC define a biofilm as?
Structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface
What are 3 examples of human infections associated with biofilms?
Otitis media - H. influenzae
Cystic fibrosis - Pseudomonas aeruginosa
Chronic bacterial prostatitis - E . coli
What did Donlan & Costerton add to the definition of biofilms in 2002?
a microbially derived sessile community, typified by cells that are attached to a substratum, interface, or to each other, are embedded in a matrix of extracellular polymeric substance, and exhibit an altered phenotype with regard to growth, gene expression and protein production
What is multi photon laser scanning microscopy
Have a glass flow cell, allow bacteria to grow through it, image it and can see layers (stacks of bacteria only a micron thick)
What is George O’Toole discover?
If you put Pseudomonas in a 96 well plate and put culture in, then take culture out and stain what is left with crystal violet.
Psuedomonas leaves a ring around the edge. Can they resuspend this and get an OD measurement for how much biofilm.
Also mutagenesis for biofilm genes
What is the matrix of a biofilm made of in Pseudomonas?
Nutrients,
alginate,
Pel and PsI polysaccharides (carbohydrates)
DNA in CF patients
What can affect the structure of a biofilm?
Abundance of carbon and nitrogen sources
What tests did Hentzer perform with alginate?
Overexpressed algS causing alginate overproduction - lead to flow culture taking a different structure
Why doesn’t a change in a biofilm gene prevent biofilm formation completely?
Many different things that will adhere to surfaces
what is CsgD?
A major biofilm regulator that has effects on curli fibres
What is RpoS?
A stationary phase sigma factor - incorporates info about nutrient status in E. coli through sRNAs
