Trypanosoma brucei brucei causes Nagana in cattle but is non-infectious to humans because of its susceptibility to the cytotoxic activity of normal human serum. This innate immune activity is due to a minor subclass of high-density lipoprotein (HDL) termed the Trypanosome Lytic Factor (TLF). TLF contains apolipoprotein A-1 (apoA-1), a protein found in all HDLs, and two primate specific proteins, haptoglobin related protein (Hpr) and apolipoprotein L-1 (apoL-1). Both Hpr and apoL-1 have been shown to be necessary for maximal trypanosome killing in vitro and in a transgenic mouse model. Hpr is a hemoglobin (Hb) binding protein and Hb is an essential co-factor for trypanosome killing by TLF. The cellular pathway of TLF mediated lysis of T. b. brucei initiates with binding of TLF to a high affinity receptor (Tb927.6.440) located in the flagellar pocket. Following binding TLF is rapidly taken up and localized to the lysosome. Within the acidified lysosome TLF is activated and kills T. b. brucei by destabilization of the lysosomal membrane. The human sleeping sickness parasite Trypanosoma brucei rhodesiense has evolved a defense against TLF. T. b. rhodesiense produces a potent inhibitor of TLF, Serum Resistance Associated protein (SRA), which binds apoL-1 and neutralizes the activity of TLF. Trypanosoma brucei gambiense also infects humans but lacks SRA and, as shown in this proposal, resists TLF killing by down regulation of the TLF receptor. In this proposal, we outline a number of experiments that will provide important information on the mechanism of assembly of TLF and how its composition might be altered to produce """"""""variant TLFs"""""""" with activity against the human sleeping sickness parasites (Aim 1). To better understand the biophysical basis for membrane destabilization by native TLF and its constituent proteins we will use model liposomes of defined composition in fluorescence based assays to measure TLF induced changes in lipid dynamics (Aim 2). Finally, we will investigate the cellular, molecular and biochemical basis of SRA-dependent and SRA-independent resistance to TLF. In addition to resolving a longstanding question concerning how SRA inhibits TLF activity we will investigate the mechanism of T. b. gambiense resistance to TLF (Aim 3). Our long-term goals are to develop a better understanding of the mechanism of TLF killing and how the human sleeping sickness parasites evade this activity. This may allow us to develop novel approaches for the treatment of this important human disease.

Public Health Relevance

African sleeping sickness is a re-emerging human disease in sub- Saharan Africa. It is currently estimated that over 35 million people are at risk and the number of infected people may exceed 300,000. There is no vaccine for African sleeping sickness and most of the drugs have serious toxicity problems. The proposed studies on the mechanism of action of the innate immune complex, TLF, may lead to the identification of novel approaches for therapeutic development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI039033-14
Application #
8474678
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Rogers, Martin J
Project Start
1996-02-15
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
14
Fiscal Year
2013
Total Cost
$345,485
Indirect Cost
$112,835
Name
University of Georgia
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Greene, Amy Styer; Hajduk, Stephen L (2016) Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei. J Biol Chem 291:3063-75
Szempruch, Anthony J; Sykes, Steven E; Kieft, Rudo et al. (2016) Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia. Cell 164:246-57
Sykes, Steven; Szempruch, Anthony; Hajduk, Stephen (2015) The krebs cycle enzyme α-ketoglutarate decarboxylase is an essential glycosomal protein in bloodstream African trypanosomes. Eukaryot Cell 14:206-15
Harrington, John M; Nishanova, Tuiumkan; Pena, Savannah Rose et al. (2014) A retained secretory signal peptide mediates high density lipoprotein (HDL) assembly and function of haptoglobin-related protein. J Biol Chem 289:24811-20
Capewell, Paul; Clucas, Caroline; DeJesus, Eric et al. (2013) The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense. PLoS Pathog 9:e1003686
DeJesus, E; Kieft, R; Albright, B et al. (2013) A single amino acid substitution in the group 1 Trypanosoma brucei gambiense haptoglobin-hemoglobin receptor abolishes TLF-1 binding. PLoS Pathog 9:e1003317
Stephens, Natalie A; Kieft, Rudo; Macleod, Annette et al. (2012) Trypanosome resistance to human innate immunity: targeting Achilles' heel. Trends Parasitol 28:539-45
Kieft, Rudo; Stephens, Natalie A; Capewell, Paul et al. (2012) Role of expression site switching in the development of resistance to human Trypanosome Lytic Factor-1 in Trypanosoma brucei brucei. Mol Biochem Parasitol 183:8-14
Harrington, John M; Scelsi, Chris; Hartel, Andreas et al. (2012) Novel African trypanocidal agents: membrane rigidifying peptides. PLoS One 7:e44384
Bullard, Whitney; Kieft, Rudo; Capewell, Paul et al. (2012) Haptoglobin-hemoglobin receptor independent killing of African trypanosomes by human serum and trypanosome lytic factors. Virulence 3:72-6

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