Identification and Characterization of Glycosomal and Cytosolic Nudix Hydrolases with Polyphosphate Hydrolyzing Activity in Trypanosoma brucei

Abstract

Trypanosoma brucei is a protozoan parasite that causes the neglected tropical disease African trypanosomiasis, also known as sleeping sickness. T. brucei infection in humans leads to death if untreated and in cattle causes a disease called Nagana, which has considerable socioeconomic impact in developing nations of sub‐Saharan Africa. T. brucei is well adapted to fight the host immune response as well as various stress conditions it endures in its various hosts. The parasite relies only on glycolysis for ATP production when inside the mammalian host. It activates oxidative phosphorylation once in the fly vector. We are interested in understanding the metabolism and functions of the inorganic phosphate polymer polyphosphate (polyP) in trypanosomes. Previous work from our laboratory reported the importance of polyP for parasite homeostasis and survival. In this work, we identified and characterized the activity of two Nudix hydrolases, NH2 and NH4 that can degrade polyP. We found that NH2 is an exopolyphosphatase with higher activity on short chain polyP, while NH4 is an endopolyphosphatase that has similar activity on polyP of various chain sizes. Both enzymes have higher activity at around pH 8.0. NH2 has a glycosomal targeting signal and it was found previously in the proteome of glycosomes, peroxisome‐related organelles where glycolysis occurs. We confirmed NH2 localization using specific antibodies while endogenously tagged NH4 was localized to the cytosol. We also found that only NH2 can dephosphorylate ATP and ADP. However, NH2 has a higher affinity for polyP, which suggests that polyP could be relevant for glycosomal function. Additionally, NH2 could also participate in regulation of ATP/ADP levels. Furthermore, using the polyP‐binding domain (PPDB) of yeast exopolyphosphatase we localized long chain polyP to the glycosomes of trypanosomes. Our results suggest that Nudix hydrolases can participate in polyP homeostasis and therefore may help control polyP levels in glycosomes and cytosol.Support or Funding InformationNIH (5R01AI077538‐09)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.