Lecture 6 - Intracellular bacteria Flashcards
Whats the difference between endocytosis and pinocytosis?
Endocytosis is receptor mediated
What is the difference between endocytosis and phagocytosis?
Phago when the molecule is bigger than 0.2 microns
Average bacteria is 1.2 microns
What are the first vesicles after endocytosis called and what does it mature into next?
Early endosome matures into Late endosome
What is the final step in endocytosis called?
Endolysosome
What is the final stage of phagocytosis?
Phagolysosome
What is the fundamental different between the early phagosome, late phagosome and phagolysosome?
The lipids and the proteins on the peripheral membrane
How many proteins does a lysosome have?
1600
What protein defines the early compartments in cytosis?
Rab5
EA1
What proteins define the late compartments in cytosis?
Rab7
LAMP1 and 2 (Lysosomal associated membrane proteins)
What are the rough pH’s of each step?
Early -7.0
Late - 5.5
Lyso - 4.5
What changes the endosome to acidic and how does it do it?
V-ATPase pumps protons in
What produces reactive oxygen species?
NADPH oxidase
What does lactoferrin do and why?
Chelates iron in the cell which bacteria need to replicate
How can a bacteria break down hydrolases in the phagolysosome?
Produce proteases
What does Listeria do?
Produces listeria lysonome which degrades the phagosomal membrane so it escapes into the cytosol
What do Legionella do when it has escaped the phagolysosome
Fuses with the ER and mimics ER as it is never sent to the lysosome
What does Mycobacterium tuberculosis do to evade degradation?
Avoids delivery to the lysosome
What experiment did Helaine et al (2014) do?
Injected mice with fluorescent Salmonella and looked for nonreplicating bacteria (ones with no dilution of fluorescence)
They then added antibiotics to the cells.
What did Helaine’s experiment show?
Cells did not divide inside mouse but did persist, outside of mouse they did divide.
Some bacteria persisting in the cells were resistant to antibiotics.
What else did Helaine do? (part 2)
Put Salmonella in bone marrow macrophages for certain amounts of time then took them out and tested them with antibiotic Cefotaxime
What did part two of Helaine’s experiment show?
Even after 15 minutes of being in bone marrow macrophage the bacteria became persisters resistant to Cef.
What does Vecinomycin do?
Stops V-ATPase and so stops acidification
What happened when Helaine added vecinomycin and what did this show?
Decrease in persisting bacteria.
Showed that the acidity is important for switching on the mechanism for persisting
What is selective fusion?
Inhibit some fusion events but allow others
How does Salmonella control it’s uptake into lower intestinal cells?
Upregulates T3SS to cause active uptake.
Transcytoses across epithialial barrier, then into the basement membrane, then into a macrophage.
What is the structure of the T3SS? (from top)
Translocon
Tip
Needle
How is the T3SS assembled?
Basement proteins put into the membrane
Tip is formed by same mechanism by which the proteins that are to be secreted into the cell are secreted.
How did Lower + Schneider (2009) show if a protein is going to be secreted by the T3SS?
Put a cylase on every protein - if it was secreted the cyclase would bind calmodulin and increase cAMP.
Then did a series of truncations on proteins.
What did Lower and Schneider conclude?
As the protein was truncated, cAMP decreased.
8-50 residues on end of proteins involved in targeting them to the base of T3SS
What was often encoded right next to a secreted protein?
A chaperone
What was interesting about the chaperones?
Amino acid sequence different but all have a similar structure
How is a protein secreted in Salmonella?
Inve strips protein from chaperone and it is completed unfolded and passed through needle.
Translocase formed at membrane of euk cell to refold protein on entry
How do we identify virulence genes?
Signature tagged mutagenesis
Where on the Salmonella serovar Typhimurium’s (typhoid fever) genome is the T3SS?
Pathogenicity island 1
What are the steps in signature tagged mutagenesis?
Have DNA that binds to 2 primers with a restriction site.
In the middle have NK20 (2 bases repeated 20 times).
Incorporate tagged DNA into transposons which insert randomly once into DNA (suidide plasmid).
Put into 96 well plate, pool bacteria together then PCR.
Radioactively tag middle then add restriction enzyme to cut off both variable arms.
Now have radioactively tagged DNA labels, make single stranded and will bind to complementary DNA.
Inject into mouse, recover them and see which ones survive.
If transposon inserted into virulence gene, bacteria will die and does not smear.
Then just look which piece of DNA it was by sequencing it
What kind of pathogen is Chlamydia and what does this mean?
An obligate pathogen.
Cannot survive outside the cell
What is the life cycle of chlamydia?
Elementary bodies phagocytosed by cell, into endosome, replicates, forms reticulate bodies which then differentiate into elementary bodies which exocytose and reenter other cells
What enzyme does chlamydia produce and what is its function?
Chlamydia deubiquitinase which inhibits ubiquitination
What did Wang et al do?
Fused an E coli plasmid and a chlamydia plasmid. With beta lactamase.
What does beta-lactamase do?
Breaks down pennicillin
What is Salmonella plasmid virulence gene C (spvC)?
A phosphothreonine lyase in the map Kinase pathway
What does spvC do?
Takes off the oxygen from phosphothreonine so the molecule cannot be phosphorylated anymore to switch off MAPK
What happens when you overexpresss SpvC and what does this show?
Cells have enlarged lysosomes.
MAPK pathway is involved in membrane trafficking
What kind of secretory system does Mycobacterium tuberculosis use?
Type 7
What are the known secreted systems of tuberculosis?
PtpB phosphorylates ERK.
PtpA inhibits HOPS complex (involved in tethering)
