Trypanosoma cruzi (T. cruzi) causes Chagas Disease (e.g. American Trypanosomiasis) inhumans. This infection is endemic to Latin America; however, due to immigration from endemicareas Chagas Disease is found in both Europe and the United States. What is striking aboutInfection with T cruzi is the development of chronic infection with disease symptoms manifestingdecades after the acute infection. Research on T. cruzi has been limited by the difficulties ingenetic manipulation. We used a modified pTREX vector with a ligand-controlled destabilizationdomain (ddFKBP) to regulate a gene/protein of interest. This vector system allows rapid andreversible protein expression and efficient functional analysis of proteins in different T. cruzi lifecycle stages. Using this technique, we found that two mitogen activated protein kinases(MAPK), TcMAPK1 and TcMAPK3, are essential for T. cruzi. We plan to develop a conditionalgene deletion system based on our ddFKBPpTREX vector system. In addition, quickly over-expressing a lethal gene in a regulated fashion should be feasible in the ddFKBP system andthis can be done in multiple T. cruzi isolates using the same vector construct without any needto genetically modify the isolates. Such an inducible lethal phenotype T. cruzi would be veryuseful for pathogenesis studies allowing elimination of the organism at various time points afterinfection to dissect the mechanisms of disease causation. Such parasites could also representa new vaccine strain and would provide controlled immune stimulation. We, therefore, propose to: (1) develop a robust conditional knockout vector system usingTcMAPK1 and TcMAPK3, essential genes for T. cruzi growth, based on our ddFKBPpTREXvectors. This system should be useful for the manipulation of other essential genes thisparasite; and (2) we will also create vector systems for T. cruzi that allow the regulatedexpression of toxin genes that will kill this parasite when these genes are expressed. Thesetransgenic parasites will be potentially valuable to define pathogeneses or as vaccine strains.
The overall project is to develop a robust conditional knockout vector system usingTcMAPK1 and TcMAPK3 which are essential genes for T. cruzi growth and to create T.cruzi strains carrying toxin genes that will kill this parasite when these genes are induced;based on our ddFKBPpTREX vectors. The conditional knockout vector system shouldbe useful for the manipulation of other essential genes in this parasite. The transgenicparasites will be potentially valuable to define pathogeneses or as vaccine strains.
| Mukherjee, Shankar; Mukhopadhyay, Aparna; Andriani, Grasiella et al. (2015) Trypanosoma cruzi invasion is associated with trogocytosis. Microbes Infect 17:62-70 |
| Ma, Yanfen; Weiss, Louis M; Huang, Huan (2015) Inducible suicide vector systems for Trypanosoma cruzi. Microbes Infect 17:440-50 |
