Vsg-switching experiments & analysis of switch variants:

Goal: to determine vsg switching frequencies & mechanisms

Created T brucei 221pGFphyhTK cellline (puromycin, eGFP gene downstream the
promoter, 70 bp repeats, hygtk fusion protein, 221 gene) + H1 RNAi construct
Parental & H1+ cells were maintained under puromycin & hygromycin solution to select for
a homogeneous population expressing VSG221 ES;
Slowed to switch for 48 hours
+tetracycline (1 ug/mL) = to induce RNAi of histone H1 (48 hrs)
Cells plated in 3-fold serial dilutions in the presence of ganciclovir (

t brucei =sophisticated VSG coat

Glycoproteins= integral membrane proteins, where they play a role in cellcell interactions.
VSG = thick immunogenic surface coats of a single polypeptide species; used to evade the host's
immune system by mean of antigenic variation.
Antigenic variation =mechanism; an infectious agent such as a protozoan, bacterium or virus
alters its surface proteins in order to evade a host immune response.

the trypanosomes never enter the host cell, and instead move freely in tissue fluids. (blood,
lymph, CSF)
host immune response vs trypanosome
strong selection pressure;
complex interaction between host immune response & parasite enabled the African
trypanosomes to develop sophisticated mechanisms to evade immune killing = antigenic
variation

*only 1 VSG gene is expressed at a time (1 antigen coat at a time),

VSG switching produces new variant antigenic types --> different proteins --> different coats -->
evade B and T cell recognition
--> also exhausts the host immune system, for the benefit of the tyrpanosome
w/ more than 1 000 VSG coats, they can display new coat antigens and adapt to the hosts
immune responses
*types of VSG switching events:

~
method;
-fig 8. used thymidine kinase (TK) gene fused to hygromycin resistance gene integrated in the
VSG221 ES

-selected for VSG switching events that cause silencing or loss of the TK gene using Gangiclovir
(GCV) - GCV resistance can arise from VSG switching or due to mutations in the TK gene