IMMUNE EVASION BY PARASITES THAT CAUSE TROPICAL DISEASES 
John E. Donelson, Ph.D., Investigator 
More than a billion people in tropical areas of the 
world endure parasitic infections throughout most 
of their lives. The protozoan and helminthic para- 
sites that cause these infectious diseases possess so- 
phisticated molecular mechanisms for evading the 
immune response. Most of these immune evasion 
strategies are based on the appearance of unique 
proteins on the surface of the parasite at specific 
times during infection. Dr. Donelson's laboratory 
seeks to understand at the genetic level how para- 
sites regulate the production of these surface pro- 
teins during their different developmental stages. 
The information gained from the studies should con- 
tribute to eradication or better control of these 
diseases. 
Antigenic Variation by African Trypanosomas 
African trypanosomes are protozoan parasites that 
cause sleeping sickness throughout equatorial 
Africa. They are transmitted from tsetse flies to the 
bloodstream, where they continually confront the 
humoral and cellular immune systems as they grow 
and multiply. Each trypanosome in the bloodstream 
contains on its surface about 1 0^ copies of a single 
protein called the variant surface glycoprotein, or 
VSG. The trypanosome population survives the con- 
tinuous immune assault against it because individ- 
ual parasites occasionally switch spontaneously 
from the expression of one VSG to another — a pro- 
cess called antigenic variation. After a switch the 
changed parasite and its descendants remain tempo- 
rarily "one step ahead" of the host defenses while a 
new immune response is mounted. The trypano- 
some genome contains about 1 ,000 different genes 
encoding antigenically distinct VSGs. 
Usually only one VSG gene at a time undergoes 
transcription. Rearrangements and duplications of 
these genes are partly responsible for the selection 
of which VSG gene is to be transcribed. The rear- 
rangements maneuver specific VSG genes into and 
out of telomere-linked "expression sites" where 
transcription occurs. The process is complicated by 
the fact that several such expression sites exist in the 
genome, yet only one is normally activated at a time. 
Dr. Donelson's laboratory is currently examining a 
subset of 12 VSG genes that are expressed specifi- 
cally at the metacyclic stage, which is the final de- 
velopmental stage in the tsetse fly. These VSG genes 
are always adjacent to telomeres and have several 
unique characteristics that contribute to their ex- 
pression during this specific developmental stage. 
Nuclear run-on experiments have shown that 
transcription initiation of these metacyclic VSG 
genes occurs at developmental stages that do not 
possess a VSG, suggesting that the high steady-state 
levels of these transcripts at the metacyclic stage are 
regulated by post-transcriptional events. Placement 
of sequences flanking these metacyclic VSG genes 
into a plasmid containing a luciferase reporter gene, 
followed by DNA transfections of the recombinant 
plasmid, have shown which of the flanking DNA 
segments are necessary for the proper appearance of 
the VSG. Proteins that bind to these regulatory re- 
gions are now under investigation. 
Another distinctive feature of telomere-linked 
VSG genes is their high rate of mutation. Careful 
examination of four different bloodstream trypano- 
some clones that reexpress a specific metacyclic 
VSG via a gene duplication mechanism revealed that 
each of these trypanosome clones possesses a VSG 
containing a slightly different amino acid sequence. 
Recombinant cloning of the original VSG gene and 
its duplicated, expressed gene copy demonstrated 
that the duplicated VSG gene undergoes point muta- 
tions during the gene conversion (i.e., duplicative 
transposition) event. 
Additional examination of several VSG genes ex- 
pressed only in the bloodstream demonstrated that, 
unlike the metacyclic VSG genes, they do not accu- 
mulate point changes. Thus the metacyclic VSG 
genes appear to be in a distinctive chromosomal en- 
vironment that tolerates and/or facilitates point mu- 
tations. Such changes within the VSG genes would 
contribute to a greater diversity of the 1 2 VSGs, en- 
hancing the phenomenon of antigenic variation at 
the metacyclic developmental stage. 
Expression of Genes for a Surface 
Protease of Leishmania 
Leishmania parasites are protozoan organisms that 
are transmitted as promastigotes by sand flies. De- 
pending on the Leishmania species, they cause 
either visceral, cutaneous, or mucocutaneous 
leishmaniasis. These parasites have a different mech- 
anism of evading the immune response than the 
African trypanosomes. They are intracellular patho- 
gens that reside inside macrophages — cells of the 
immune system whose normal function is to engulf 
and destroy foreign pathogens and substances. In 
collaboration with Dr. Mary Wilson of the Depart- 
ment of Internal Medicine at the University of Iowa, 
Dr. Donelson's laboratory is examining how leish- 
GENETICS 183 
