Brugia Malayi - Clark Science Center - Smith College

Decoding the Promoter Region of the Thioredoxin Peroxidase-2 Gene in
Brugia malayi
Iju Shakya, Louise Hart Bodt and Krithika Venkataraman
With over a billion people at risk of infection, 1 Lymphatic filariasis (LF) is considered a leading cause of disability. LF is a
Neglected Tropical Disease caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori 1 . During the L3 stage, when these
parasites transition host from mosquito to human, the parasite is considered most vulnerable. To manipulate this stage, the
thioredoxin peroxidase-2 (tpx-2) gene was studied in our project. The thioredoxin peroxidases transcribed by this gene are crucial,
as they defend B. malayi from toxic oxygen radicals released by the host immune cells. Our study was based on mutating the tpx-
2 promoter, which allows the gene to be turned on and off. Using a luciferase assay, the efficiency of the mutated promoters
was measured, based on differences in fluorescence levels. This enables future identification of important regions of the tpx-2
promoter for determining future vaccine targets. For the purpose of this project, Brugia pahangi was used as a safe, reliable model
for investigation, as it does not infect humans, unlike Brugia malayi.
In order to transfect DNA into Brugia pahangi, the required DNA from transformed cells was streaked and inoculated. The
DNA (wildtype E8 and mutants 58, A8, and B8) was then isolated using a Qiagen maxi-prep kit and run on a gel (Figure 1). The
concentrations of the DNA were then measured with the Qubit.
Following this, approximately 1,000 B. pahangi were cultured using a calcium chloride precipitation technique. DNA was then
added to eight wells of 100 worms each, leaving two wells of 100 worms without added DNA to serve as the control.
After ten days, the worms were lysed and measured with a luciferase assay in the luminometer (Table 1). The data were
inconsistent with prior results, and inconclusive due to contamination and inconsistency with transfection.
The next step will be to repeat the experiment for more luminometer measurements. Additionally, an alternate assay technique
will be implemented, exploiting the properties of green fluorescent protein, instead of luciferase. (Supported by the Blakeslee
Fund in the Biological Sciences)
Advisor: Steven A. Williams
References:
1
CDC. (2010)/ Parasites - lymphatic filariasis. (2010). Retrieved from http://www.cdc.gov/parasites/lymphaticfilariasis/index.html.
2012
49