Paper of the Month: Gene cloning leads to molecular discovery

Gene cloning leads to molecular discovery of an essential pyruvate transporter for Trypanosoma brucei parasite survival

This month's featured paper is from the lab of Marco Sanchez, Ph.D. It is titled, "Molecular Identification and Characterization of an Essential Pyruvate Transporter from Trypanosoma brucei." The paper was published in The Journal of Biological Chemistry.

"This month's paper highlights Marco Sanchez's discovery of an important pyruvate transport system that allows the parasite, T. brucei, to survive in mammalian hosts. Sanchez's characterization of this gene employed a full range of molecular and genetic tests which led to the identification of a family of related transporter genes that are promising targets for anti-parasitic drugs that could inhibit survival of this parasite in mammalian hosts."

- - Mary Stenzel-Poore, Ph.D.Senior Associate Dean for Research

May 28, 2013

Dr. SanchezTrypanosoma brucei (T. brucei) is a flagellated protozoan parasite that causes African trypanosomiasis, also known as "sleeping sickness,"in humans and nagana in animals in Saharan and sub-Saharan Africa.The parasite is transmitted by the bite of the tsetse fly, and is fatal if left untreated causing an economical and medical burden for African countries.

"Anti-trypanosomal therapy is far from ideal," said Marco Sanchez, Ph.D., an assistant professor in the department of molecular microbiology & immunology. "It is expensive and toxic, and there is increasing incidence of drug resistance."

Vaccination against these parasites is unlikely to be effective in the near future. According to Dr. Sanchez, this is due to "antigenic variation" of the variant surface glycoproteins. "There is an urgent need for new drug targets to develop more suitable anti-trypanosomal drugs," he said.

For the last 20 years, Dr. Sanchez's research has focused on the plasma membrane transporters of Leishmania sp and T. brucei parasites—especially on purine transporters. In 2005, he initiated a research project on the study of a type of T. brucei transporter called pyruvate transporter. This includeda thorough biochemical, genetic, cellular, functional and pharmacological analysis of the pathway.

"Pyruvate transport is an essential cellular process in the bloodstream life cycle stage of the parasite T. brucei," said Dr. Sanchez. "It mediates the secretion of pyruvate, the final product of glucose metabolism, which would otherwise accumulate to toxic levels inside the parasite and ultimately cause cellular death. Consequently, plasma membrane pyruvate transporters are important for detoxification and survival of the parasite in the mammalian host."

Previous biochemical experiments employing whole parasites have shown the presence of pyruvate transporters in the plasma membrane of T. brucei. However, the molecular identification of these important transporters has eluded discovery for over twenty years.

21_coverIn a paper titled, Molecular Identification and Characterization of an Essential Pyruvate Transporter from Trypanosoma brucei, published recently in The Journal of Biological Chemistry, Dr. Sanchez furthered the science behind this mystery by cloning a gene vital to understanding the molecular nature and importance of a pyruvate transporter.

In his study, Dr. Sanchez cloned a bona fide T. brucei pyruvate transporter, employing genetic complementation in Saccharomyces cerevisiae deficient in pyruvate transport. Pyruvate transport complementation allowed for the identification and cloning of a TbPT0 gene, important for encoding the transporter's molecular makeup.

The results were significant. "Functional analysis of TbPT0 in yeast revealed that it is a monocarboxylate transporter with specificity for pyruvate, acetate, and lactate but with much higher affinity for pyruvate," said Dr. Sanchez.

Furthermore, in silico analysis in the Sanchez lab revealed that TbPT0 belongs to a multigene family consisting of five members clustered together in the genome. All members of the family encode similar but not identical transporters, which probably have similar transport function.

To demonstrate that the TbPT gene family is essential for the bloodstream form parasite survival, the expression of the TbPT gene family was inhibited by RNA interference (RNAi), resulting in a striking lethal phenotype.

"Functional and genetic studies of this transporter underscore its critical function for survival and potential as a drug target against the mammalian life cycle stage of T. brucei," said Dr. Sanchez.

In future experiments, possibly in collaboration with OTRADI or NIH, Dr. Sanchez will be engaged in developing a yeast-based high-throughput screen to identify inhibitors of TbPTs, which would be potential anti-parasitic lead compounds.


Pictured above: Marco Sanchez, Ph.D.

About the Paper of the Month

The School of Medicine newsletter spotlights a recently published faculty research paper in each issue. The goals are to highlight the great research happening at OHSU and to share this information across departments, institutes and disciplines. The monthly paper summary is selected by Associate Dean for Basic Science Mary Stenzel-Poore, Ph.D., and Associate Dean for Clinical Science Eric Orwoll, M.D.

More Published Papers

The entire list of OHSU papers published this month is here.