Targeting Trypanosome Cation Pumps and Channels in Trypanosomiasis Control

Morehouse School Of Medicine, Atlanta GA

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Abstract

Human African Trypanosomiasis (HAT, sleeping sickness) is a neglected but fatal vector borne disease[unreadable] caused by Trypanosoma brucei ssp. (T. brucei). Sixty million people are at risk of infection with HAT, and[unreadable] 50,000 new case's and an equal number of deaths are reported annually in subsaharan Africa. The only[unreadable] available anti-trypanosomal drugs on the market are extremely toxic, and result in post-treatment[unreadable] encephalopathy in treated patients. The ideal anti-trypanosomal agents will target vital physiological[unreadable] processes and/or non-variant parasite-derived molecules without adversely affecting the human host. We[unreadable] and others have shown that that cytosolic calcium ion concentration [Ca2+ ]i in blood stages of T. brucei is 4-[unreadable] 10 orders of magnitude below that encountered in host extracellular milieu and that parasites require effective[unreadable] mechanisms to maintain [Ca2+ ]\ homeostasis, survival, and proliferation in their host. In our previous grant,[unreadable] we identified and characterized two key plasma-membrane-like cation pumps (ATPases; TBCA1 and TBCA2)[unreadable] utilized by T. brucei for survival and generated antibodies to immunolocalize them in bloodstage and insect[unreadable] stage parasites. We determined their functional role in insect and bloodstage parasites using transient RNAi[unreadable] inhibition technology and synthetic inhibitor assays. We subsequently constructed recombinant anti-pump[unreadable] vaccines based on a novel bacterial ghost vaccine delivery technology, developed at Morehouse School of[unreadable] Medicine, which partially protected against parasite challenge in mice. Our results revealed that TBCA1[unreadable] resembled a fungal K+/Na+-ATPase while TBCA2 was a plasma-membrane-like Ca2+ ATPase. RNAi[unreadable] inhibition of these targets resulted in increased parasite mortality. Furthermore, we determined by inhibition[unreadable] studies that Ca2+ homeostasis in T. brucei is not only regulated by the Ca2+ ATPases but also by L-type[unreadable] calcium ion channels. Since targeting the Ca2+ pumps by RNAi inhibition increased parasite mortality and[unreadable] vaccination with TBCA2 significantly reduced parasitemia and survival of infected mice, we propose that[unreadable] targeting the key cation pumps (TBCA1 and TBCA2) as well as L-type Ca2+ channels together with either[unreadable] synthetic inhibitor drugs or vaccines, will be sufficient to inhibit proliferation of T. brucei and provide complete[unreadable] protection against T. brucei infection. In this competitive renewal proposal, we have gone a step further to[unreadable] hypothesize that trypanosomes utilize cation pumps and channels to mediate establishment in[unreadable] mammalian host blood and that simultaneous inhibition by target specific drugs and blocking by[unreadable] vaccination will prevent T. brucei proliferation and development. Two specific aims are proposed: In[unreadable] Specific aim 1. we will functionally characterize and localize the L-type Ca2+ channel in T. brucei and[unreadable] determine its role in Ca2+ homeostasis. In Specific aim 2. we will construct and test various Ca2+[unreadable] pump/channel gene constructs as antigens in a novel bacterial ghost based vaccine system against[unreadable] infections by T. brucei and determine their levels of protection against T. brucei infection. Our longterm[unreadable] goal is to build on our established proof of principle to develop and deliver a novel class of small[unreadable] molecule drugs and/or immunotherapeutics capable of inhibiting the essential Ca2+ pumps and channels of T.[unreadable] brucei during development. We also plan to generate enough data, through this application, to apply for an[unreadable] RQ-1 type grant to fund future studies on anti-trypanosome drugs.