Bacterial Persistence

Main area:Microbiology
Target group:Biochemistry, Biology, Molecular Biomedicine
Educational level:Bachelor, Masters
Project description:
All bacteria, including major human pathogens such as Mycobacterium tuberculosis produce persister cells that are tolerant to multiple antibiotics. Persister cells are dormant but have a low rate of metabolism to stay alive. The proteins synthesized during this dormant phase are unknown but may be important as a target for elimination of the persisters.

Using E. coli as the model organism, we discovered a signaling pathway underlying the persistence phenomenon (Maisonneuve et al., Cell 2013, Maisonneuve & Gerdes, Cell, 2014). In short the regulatory molecule ppGpp varies stochastically such that a low fraction of a growing cell population has a high level of ppGpp. This high level of ppGpp in turn activates inhibitors of translation called toxin-antitoxin (TA) genes. Activation of such toxins inhibits cell growth and induces persistence. E. coli has 11 TA genes while M. tuberculosis has approximate 90 as shown in this Figure:

The Figure shows toxin – antitoxin ”persister” genes encoded by the chromosomes of E. coli and M. tuberculosis

The project:
The discovery of toxin – antitoxin genes as effectors of bacterial persistence has opened the door to investigate the physiology of persister cells because mutant strains lacking all TAs are now available for analysis. We will identify, for the first time, proteins that are highly expressed in persister cells using radioactive labelling and two-dimensional gel electrophoresis of proteins from wild type and mutant cells.

New follow up project

The first project succeded in identifying one or possibly a few proteins that are synthesized as the only detectable proteins in the persister state. In the new project the indetity of this or these protein (s) should be established. Therefore the project will
1) grow a sufficient amount of persister cells
2) purify persister cells from dead cells/cell debris
3) Separate the persister proteins on 2D gels
4) identify the relevant protein spot by mass spec.
If the project should be a master project, a broader survey of persister specific proteins may be attempted by SILAC.

If you are interested, please contact the supervisors by E-mail or personally.

Date of Announcement: July 1st 2014
Methods used:2D gel electrophoresis
Keywords:generation of persistant bacteria, mechanism for persistence, radioactive labeling of cells, 2D gel electrophoresis, mass spec/SILAC
Supervisor(s): Steen Pedersen, Kenn Gerdes
Email:steenp@bio.ku.dk