Mycobacteria 2 Flashcards
What experiment tells you that mycobacteria live in phagosomes with low pH?
Measure pH of the phagosomes by incubating m avium or control in macrophages. Add IgG bead and pH sensitive probe. Found that with pH sensitivity at pH 7 it prevented phagosome maturation. IgG beads pulled phagosomes at pH5.5
Where does m tuberculosis live in host?
Within macrophage phagosomes.
What do m avium phagosomes lack compared to IgG beaded phagosomes?
Vacuolar ATPase
How do mycobacteria prevent phagosome maturation? What experiment can you do?
- take 67 m tb transposon mutants with attenuated virulence in mice
- infect macrophages for 72hrs
- pulse with fluorescent dextran for 10 min and it will label early phagosomes
- measure colocalization of dextran in mtb.
- WT: high % colocalization
- mutants: low %
- identify ESX-1
What is colocalization?
In fluorescent microscopy, it’s the observation of the spatial overlap between 2 or more different fluorescent labels to see if the different targets are located in the same area of the cell or very near to one another.
What does the high percentage of colocalization in wt mycobacteria tb vs low % in mutants tell you about the virulence of tb? Keep in mind that the transposon mutants attenuated virulence in mice.
It tells you that in the strains that attenuate virulence in mice (the transposon mutants) didn’t allow much phagosome maturation. So phagosome maturation plays a key role in mtb habitation
How was ESX-1 found and what is it?
It was found through fluorescent microscopy in looking at phagosome maturation in comparison to virulence of mtb.
ESX-1 is a gene that encodes for a type VII SS (T7SS)
It’s encoded in the region of difference 1 (RD1) that differs between WT mtb and the vaccine strain BCG.
As part of mtb’s mechanism for invading the host immune system, what types of molecules does it mimic?
Host lipids:
- PI
- PIM
- ManLAM
What does ManLAM do?
It’s a glycolipid produced by mtb and a virulence factor
ManLAM inactivates macrophages and scavenged oxidative radicals. The inactivation of macs allow for mtb to disseminate to other parts of the body. Destruction of oxidative radicals are used to rid ourselves of infection so when they’re destroyed by mtb the bacteria can survive.
What does PIM do?
PIM is a lipid made by mtb in cell wall. Antibodies to PIM in mice help to defeat the infection.
PIM reduces acidification of phagosomes.
How do you know PIM’s role in infection by mtb?
Coat bead with PIM or have Uncoated bead. Put into macrophages. Measure lysosome association through presence of lysosome. Found that with PIM, only 16% lysosome presence compared with wt.
What is the role of mycobacterial lipids in preventing phagosome maturation?
ESX-1, PIM, and ManLAM block maturation from early phagosome to late phagosome.
After how long does mtb escape the phagosome?
2 days.
BCG can’t escape cuz it depends on ESX-1
How does the host defend against infection of mtb as far as phagosome maturation goes?
Promote phagosome maturation.
- IFNgamma promotes maturation
- look for genes up regulated by IFNgamma. Identified p47 GtPase!
- in the host cell it’s called LRG-47
- mice without LRG-47 don’t survive infection
What is p47 or LRG-47?
It’s a gene that encodes for a GtPase in host cells of mtb infection that is upregulated by IFNgamma. IFNgamma promotes maturation of phagosomes which is a host defense mech against mtb infection.
