Download Characterization of the mycobacterial lipids presented by

Training offer_M2 2018
Team : Dr J.Nigou
Characterization of the mycobacterial lipids presented by CD1b molecules
Biological context:
Mycobacteria, including Mycobacterium tuberculosis (Mtb) - a causative agent of
tuberculosis, are characterized by exceptionally high content of lipids in their cell walls.
Several mycobacterial lipid antigens have been found to be associated with group I CD1
molecules - non-polymorphic, MHC class I-like glycoproteins. Mtb glycolipids presented
in the context of CD1 molecules can get recognized by the T cells, which in turn triggers
their activation and contributes to mounting the immune system response against the
pathogen. Yet, the full spectrum of mycobacterial lipids associated with CD1b, one of the
CD1 molecules known to present several Mtb glycolipids, is to be characterized.
Goal of the training:
In order to analyze the mycobacterial lipids presented by CD1b molecules on the
antigen presenting cells (APCs) we will pulse the cells with mycobacterial lipids, purify
the CD1b-bound lipids to consequently analyze them by mass spectrometry. This
involves generation of the APCs expressing recombinant CD1b molecules with the
protease cleavage site introduced between their luminal and transmembrane parts. Mtb
glycolipid-pulsed APC will be lysed, and the CD1b proteins will be digested with the
protease in order to purify the antigen-binding domains. Next, the lipids will be isolated
and analyzed by mass spectrometry (MS).
The specific goal of this internship project will be to set up the conditions for
extraction of the lipids bound by CD1b and their characterization by mass spectrometry
(MALDI, ESI).
CD1b-espressing cells pulsed with mycobacterial glycolipids will be subjected to the
protease digestion followed by purification of the CD1b extracellular domains. Next,
extraction of the lipid antigens from the CD1b molecules will be achieved using a
modified Blight and Dyer method. Finally, identification of the epitopes associated with
CD1b will be performed by MS.
Methodology/techniques:
- Eukaryotic Cell culture (CD1b-expressing cell lines)
- Analysis of the protein extracts (SDS-PAGE + Western blot)
- Affinity column purification
- Lipid extraction
- HPLC – separation of the molecules
- Mass spectrometry (MALDI, ESI, MS/MS)
References:
1. Yuan et al. , J Immunol. 2009 Apr 15;182(8):4784-91.
2. De Libero et al, Front Immunol. 2014; 5: 219. (REVIEW)
3. de la Salle et al, Science. 2005 Nov 25;310(5752):1321-4., August 2015(DOI:
10.1002/9780470015902.a0020182.pub2)
Supervision
Dr Martine Gilleron : [email protected]
IPBS, 205 route de Narbonne, Toulouse