Reciprocal interactions of NO and TGFbeta in malaria

Description of projects available to graduate students:
Since the mid-1970s, malaria has re-emerged as a global health problem. It is becoming clear that anti-parasite defenses mounted by both mammalian hosts and the mosquito host Anopheles can suppress the growth of the parasite Plasmodium. Nonetheless, the parasite is able to escape complete elimination in vivo, perhaps by thwarting or co-opting these mechanisms for its own survival as do numerous other pathogens. My collaborator Shirley Luckhart (Virginia Tech) and I have demonstrated recently that nitric oxide (NO), produced by the action of an inducible A. stephensi NO synthase (AsNOS) may be central to the anti-parasitic arsenal of this mosquito. However, much still remains to be elucidated regarding the mechanisms by which Plasmodium may elude the potent anti-microbial NO, and how this defense response is regulated. In vertebrates, the inducible NOS (iNOS;NOS II) activity that suppresses the growth of pathogens can be modulated by members of the transforming growth factor-beta (TGF-beta) cytokine superfamily. TGF-beta is produced as an inactive precursor that is activated following dissociation of certain inhibitory proteins, a process that our studies indicate can be promoted indirectly by NO and directly by the NO reaction product nitroxyl as well as the blood degradation product hemin. Our studies indicate that active vertebrate TGF-beta can be detected up to 48h post-ingestion, that latent TGF-beta can be activated by midgut components in vitro, that mammalian TGF-beta is perceived as a cytokine by A. stephensi cells in vitro, and that mammalian TGF-beta can alter Plasmodium development in vivo. Bloodfeeding by Anopheles, therefore, results in the juxtaposition of evolutionarily conserved mosquito and mammalian TGF-beta superfamily homologs. We propose 1) to determine if mammalian TGF-beta is activated in the mosquito midgut by heme released during vertebrate blood digestion and by NO produced by the mosquito; 2) to determine whether signaling by TGF-beta is transduced through a serine/threonine kinase receptor and TGF-beta/activin Smads in A. stephensi cells and whether signaling alters midgut AsNOS expression in a manner that is consistent with effects on Plasmodium development; and 3) to determine whether the Anopheles homolog of TGF-beta,As60A, regulates Plasmodium development. If so, to determine whether As60A and mammalian TGF-beta activate intersecting signaling pathways to regulate expression of effector genes such as AsNOS. My laboratory is focused on Aim 1, though we are interacting with Dr. Luckhart's laboratory for all of the projects described above. For further details, see Luckhart, S.; Crampton, A. L.; Zamora, R.; Lieber, M. J.; Dos Santos, P. C.; Peterson, T. M. L.; Emmith, N.; Lim, J.; Wink, D. A.; Vodovotz, Y. Mammalian transforming growth factor-beta1 activated after ingestion by Anopheles stephensi modulates mosquito immunity. Infect. Immun. 2003. 71:3000-3009.

Techniques graduate student will learn:
Heme spectroscopy, ELISA assays, NOS enzyme activity assays