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Mycobacterium marinum promotes mitochondrial degradation in an ESX-1 dependent manner

Lovins, Victoria (2017) MOBN01 20171
Degree Projects in Molecular Biology
Popular Abstract
Investigating the role of mitochondrial damage in a Tuberculosis infection

Tuberculosis (TB) is a significant health risk that was responsible for 10.4 million infections and 1.8 million deaths in the year 2015 alone. The causative agent for TB is Mycobacterium tuberculosis (Mtb), which is an intra-cellular bacterium that primarily replicates within the white blood cell called the macrophage. To avoid detection and destruction by the macrophage, the bacterium has evolved a wide variety of evasion mechanisms. One type of evasion used by bacteria is damage to the mitochondria because the mitochondria is pivotal for cell survival. Two symptoms of mitochondrial damage that have been associated with Mtb include release of mitochondrial DNA... (More)
Investigating the role of mitochondrial damage in a Tuberculosis infection

Tuberculosis (TB) is a significant health risk that was responsible for 10.4 million infections and 1.8 million deaths in the year 2015 alone. The causative agent for TB is Mycobacterium tuberculosis (Mtb), which is an intra-cellular bacterium that primarily replicates within the white blood cell called the macrophage. To avoid detection and destruction by the macrophage, the bacterium has evolved a wide variety of evasion mechanisms. One type of evasion used by bacteria is damage to the mitochondria because the mitochondria is pivotal for cell survival. Two symptoms of mitochondrial damage that have been associated with Mtb include release of mitochondrial DNA and cytochrome c release. Release of mitochondrial DNA into the cytosol of the cell can be negative for the macrophage as it promotes production of IFNβ, which suppresses the immune response against the bacteria. IFNβ is classified as a cytokine meaning that it is secreted from immune cells and functions as a cellular messenger. Cytochrome c, on the other hand, is a protein that sits on the inner mitochondrial membrane and helps drive the production of energy for the cell. During infection, a pore called the mitochondrial transition permeability pore (mPTP) can open for a prolonged period leading to mitochondrial swelling, mitochondrial membrane rupture, and the release of cytochrome c. The mPTP is regulated by a protein called cyclophilin D (CyPD) in response to ion levels like increased Ca2+ in the mitochondria.
To test mitochondrial damage, macrophages were infected with Mycobacterium marinum (Mm) rather than Mtb because Mm is cheaper, safer, replicates faster, and causes disease similarly to Mtb. Macrophages were then checked for levels of mitochondrial DNA in the cytosol and it was found that early after infection, mitochondrial DNA was released into the cytosol at time-points that correspond to the production of IFNβ. This suggests that Mm infection can induce mitochondrial damage and that the release of mitochondrial DNA into the cytosol might be driving production of IFNβ. Next, we measured the release of cytochrome c in infected macrophages by adding an antibody which is a protein that has been designed to specifically bind to cytochrome c. This anti-cytochrome c antibody is then connected to a light-emitting chemical such that when more cytochrome c is present, more antibodies can bind releasing a greater light emittance that is measurable. Cytosolic cytochrome c was then washed away leaving only the mitochondrial associated cytochrome c available for binding by the antibody. In this way, we could detect that infection with Mm drives cytochrome c release because infected cells generated a weaker emittance. Next, we wanted to discern whether the mPTP could be responsible for the release of cytochrome c observed. To test this, macrophages were infected that lacked the mPTP regulator, CyPD, hoping to create a system in which if the pore cannot be induced to open, cytochrome c cannot be released. We detected less cytochrome c release in cells lacking CyPD than in those with in-tact CyPD, indicating that CyPD and the mPTP are important components of cytochrome c release. Finally, we wanted to examine the effect that CyPD dependent mitochondrial damage might have on cytokine production, namely IFNβ and the pro-inflammatory IL-1β. IFNβ was not affected by the presence or absence of CyPD suggesting that mitochondrial DNA release is not dependent on the mPTP. Pro-inflammatory IL-1β however, increased in CyPD deficient cells suggesting a possible suppressive role for CyPD dependent mitochondrial damage on IL-1β.

Master’s degree project in Biology/Molecular Biology/Medical Biology 45 credits 2016/7
Department of Biology, Lund University
Advisor: Fredric Carlsson Co-Advisor: Julia Lienard
Infectious Immunology BMC D14 (Less)
Please use this url to cite or link to this publication:
author
Lovins, Victoria
supervisor
organization
course
MOBN01 20171
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8925284
date added to LUP
2017-09-11 12:24:04
date last changed
2017-09-11 12:24:04
@misc{8925284,
  author       = {Lovins, Victoria},
  language     = {eng},
  note         = {Student Paper},
  title        = {Mycobacterium marinum promotes mitochondrial degradation in an ESX-1 dependent manner},
  year         = {2017},
}