Bio:
I am a post-doc working primarily on the A. gambiae innate immunity
and its response to Plasmodium infection. I have previously studied
the blood-feeding behavior of
A.gambiae
female mosquitoes with response to light stimulation and have
characterized several molecular components involved in this machinery.
Current research:
1. Generation of Rel2-transgenic mosquito lines (work in
collaboration with Dr. Dong):
The innate immunity of mosquitoes is the primary line of defense
against the malaria parasite Plasmodium and other microbes. It mainly
comprises of the TOLL and IMD pathways where the two NF-kappa B-like
transcription factors, Rel1 and Rel2 translocates to the nucleus and
activates the transcription of several antimicrobial peptides and many other
effector genes. IMD pathway is the major player in regulating resistance of
several Anopheles species to several malaria parasites (Dong et al,
Plos Pathogen, 2006, 2(6):e52; Garver and Dimopoulos, unpublished data) and
is more likely appealing for the generation of genetically modified
mosquitoes that are resistant to Plasmodium species. The Rel2 gene
(orthologous to
Drosophila
Relish) of the malaria vector A. gambiae, has been shown
to control the expression of several immune genes (LRIM1, CLIPB14, KIN1, FBN
etc) or antimicrobial peptides like Cecropin, Gambicin and also regulate the
bacterial and Plasmodium infections. The A. gambiae Rel2-S (Rel2
short form lacking the inhibitory ankyrin repeats and death domain)
transcript has been cloned under the A. gambiae carboxypeptidase and
vitellogenin promoter(s) to generate blood-fed inducible Rel2 transgenic
mosquitoes in both A. gambiae and Anopheles stephensi.
Generation of Rel2 transgenic mosquito line under the
tetracycline/SRPN10 promoter to generate Rel2 transgenic A. stephensi
is under progress; the transcription of Rel2 can be induced by the addition
of tetracycline analog doxycycline (dox) and blood-feeding. All the
constructs have GFP cloned under some constitutive promoter, which will be
helpful to screen for selection of transgenic mosquito
line by looking at green fluorescent larvae/adults at G1
generation and further.
Prior to the generation of all these transgenic mosquito lines the
capacity of the recombinant A. gambiae Rel2 proteins to induce immune
response in the immune competent A. gambiae
Sua1B and A. stephensi MSQ43 cell lines will be
investigated; which will provide information on heterologous function of
these transcription factors. If the A. gambiae
Rel2 fail to function in the heterologous system, the A.
stephensi Rel2 will be cloned through a combination of PCR and RACE
method and investigate in the A. stephensi cell line system before
generating A. stephensi
Rel2 transgenic mosquitoes. Once we generate the Rel2
transgenic mosquito line(s), the regulatory role of Rel2 in mosquito innate
immunity through TOLL and IMD pathways will be explored and further look at
their susceptibility to bacterial and Plasmodium infections. The fitness,
longevity and fecundity of the Rel2 transgenic mosquito lines will also be
assayed.
2. Role of negative regulators of TOLL/IMD/JAK-STAT pathways
in regulating the Plasmodium falciparum infection level in
A. gambiae at different stages of infection.
Caspar, Cactus and Pias are negative regulators of Rel-1, Rel-2
and STAT, the transcription factors of three insect immune pathways; IMD,
Toll and JAK/STAT, respectively. All these three negative regulators have
been shown to play a role in
Plasmodium falciparum
and Plasmodium berghei killing (Dimopoulos lab,
unpublished data) in different mosquito species.
Figure 2: The three different immune pathways of Anopheline
mosquitoes and the different regulators and suppressors involved
in the pathways.
We are interested to investigate the regulatory role of
Cactus, Caspar and Pias at different stages of Plasmodium falciparum
development in the mosquito midgut, hemolymph and salivary
glands. Injection of dsRNA of these three negative regulators after P.
falciparum infection; 2 days (ookinete to early oocyst stage) and 6 days
(late oocyst stage) after feeding on infected blood have been completed. The
data shows that injection of all three dsRNAs on day 2 can significantly
reduce the Plasmodium oocyst level in the midgut however they fail to do so
if injected at the later stage of infection on day 6. Our next plan is to
inject the respective dsRNA very late after infected blood feeding, between
days 10-12 and look at the sporozoite load in the salivary gland.
Co-silencing of the inhibitors along with other effector molecules will also
be performed to investigate the specific role of the inhibitors at different
levels of the three pathways.
Suchismita Das PhD.
Current C.V.
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