Title Identification of the Genetic Factors Which Control Tropism in
Leishmania
Research Focus: Leishmaniasis
Research Type: Basic Research
Agency DoD Study Location Ft.
Detrick
Study Start Date 7/1/94
Estimated Completion Date 7/1/98
Project Status Ongoing DoD-9
Summary
OVERALL PROJECT OBJECTIVE: To
identify the gene(s) that control tropism in Leishmania and
determine its (their) sequence and
function.
SPECIFIC AIMS: The goal of this
DOD/VA research is to identify the gene(s) that control tropism
in
Leishmania. The identification of
a "tropism" gene will:
(a) Enable the development of
specific gene
probes (primers) to be used in a
patient screening program to identify those at risk of
reactivation of
latent infections;
The identification of the genetic factor(s)
involved in the visceralizaiton of
Leishmania will require us to develop in vitro and in vivo models
of
tropism to facilitate the study of
viscerotropic leishmaniasis (diagnosis, therapy, prevention) and
Leishmania tropism.
METHODOLOGY:
A. Develop in vitro
and in vivo models of Leishmania tropism to use for the
determination of the genetic
factors controlling tropism:
B. Create Leishmania
mutants with altered tropism in
the in vitro and in vivo models of tropism:
C. Use recently developed molecular genetic techniques to
restore the original (wild-type)
phenotype to the Leishmania temperature and tropism mutants by
transfection with the appropriate
Leishmania cosmid DNA libraries.
D. Identify the gene(s)
controlling
temperature sensitivity and
tropism present in the cosmid(s) which restore(s) a wild-type
phenotype
to the Leishmania cells. E. Retest
the function of these genes in both models using transfection
methodology.
EXPECTED PRODUCTS (MILESTONES):
1.
Develop an in vitro temperature sensitivity model that
will correlate with tropism in
vivo - 1995.
STATUS/RESULTS TO DATE: Ongoing.
1. Developed an in vitro promastigote temperature model
and determined that temperature
sensitivity in vitro correlates with Leishmania tropism in vivo.
PUBLICATIONS Callahan HL, Portal
IF, Grogl M. Development of an in vivo Leishmania
temperature sensitivity model:
preliminary studies with strains with aberrant
tropism in vivo. 44th Annual
Meeting of Am Soc Tropical Med & Hygiene
53(2):215, 1995.
Callahan HL, Portal IF, Bensinger
SJ, Grogl M. The temperature sensitivity of
Leishmania promastigotes in vitro:
a model for tropism in vivo. 1996. Accepted,
Exp. Parasit.
Callahan HL, Grogl M. Development
of animal models to study Leishmania tropism
at the molecular level. 1995, in
preparation for submission to the Am J Tropical
Med.
A-19
(b) Address the
fundamental question of infectious disease pathophysiology,
namely why an organism infects a
particular cell;
(c) Optimize treatment regimens according to
Leishmania species and the immune
status of the host.
i) a promastigote temperature
sensitivity
model; and
ii) a cutaneous and a
visceral animal model of Leishmania tropism.
i) temperature
sensitive
parasites changed to resistant and
visa versa; and
ii) cutaneous parasites (in the animal model)
changed to visceral and visa
versa.
2. Develop animal models which can be used to select
for
altered tropism - 1995.
3. Obtain
Leishmania mutants in vitro - 1995 - 1996.
4. Obtain Leishmania
mutants in vivo - 1996 - 1997.
5.
Create cosmid DNA libraries - 1996 - 1997.
6. Reconstitute the
wild-type phenotype (tropism)
utilizing a wild-type Leishmania DNA cosmid library - 1997-1998.
7.
Molecular characterization:
a)
Identify the gene(s) present in the cosmid(s) which restore(s) a
wild-type phenotype to the
Leishmania mutants.
b) Sequence the gene(s) - 1998.
c) Determine
homologies with genes in the
database - 1998.
2.
Determined that there is no
absolute temperature requirement for dermotropism, but that there
is a
minimum temperature resistance
requirement for visceralization.
3. Determined that parasite
strains
that have an unusual tropism in
the human host (to include the viscerotropic L. tropica from
Desert
Storm) also show an unexpected
temperature sensitivity in the model.
4. Developed two animal
models where the tropism of the
Leishmania strain is known, uniform, and reproducible (all
organisms are found in one
location, either visceral or cutaneous) when inoculated in the
skin (sc) to
mimic a sand fly bite: Cutaneous
model - no visceralization; and Visceral model - no skin lesions.
5.
Obtained Leishmania temperature
sensitive and resistant mutants.
6. Developed and standardized in
the laboratory transfection and
Leishmania cosmid DNA library techniques.
7. Selected a L.
donovani and two L. tropica
temperature-resistant strains in vitro.
8. Chemically mutagenized
a L.
mexicana strain and selected a
tropism mutant in our hamster model.
9. Made wild-type DNA cosmid
librarios of all the Leishmania
strains (4) that will be mutagenized and selected for altered
tropism in
our hamster model.