Jayne Raper PhD, Assistant Professor

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A species of African trypanosomes, Trypanosoma brucei, are unicellular
parasites that cause sleeping sickness and are transmitted by the bite of a tsetse fly. Trypanosomes thrive in the bloodstream of their mammalian host, evading the adaptive immune response by switching one surface coat protein for another. T. brucei sub species constitute a significant health and economic problem in sub-Saharan Africa. T. b. brucei cannot infect humans because it is lysed by normal human serum, while T. b. rhodesiense and T. b. gambiense are human pathogens because they are resistant to lysis.

	Fig. 1 Geographical distribution of T. b. rhodesiense and T. b. gamibiense

Trypanosome lytic factors (TLFs) are circulating lipoprotein complexes in human blood that confer innate protection against infection by Trypanosoma brucei brucei. We have identified two structurally distinct TLF complexes, which due to the presence of apoA-I, are both subclasses of high-density lipoprotein (HDL) [Raper 1999; Lugli 2004].

Fig. 2 TLFs are multicomponent particles

We have directed our attention to the analysis of the putative receptors for the TLFs and the mechanism of lysis. The hypotheses to be tested are as follows:

1) TLFs bind to a lipoprotein scavenger receptor that selectively acquires lipids to supply the trypanosome's needs:
We have biochemical evidence that trypanosomes use a lipoprotein scavenger receptor to fulfill their obligate need for lipids. Heather Green, Pre-doctoral student (2000-2004) has shown that TLFs, HDL and LDL are taken up by trypanosomes through a receptor with a function similar to the eukaryotic class B scavenger receptor [Green 2003]. Like currently identified class B eukaryotic scavenger receptors, the trypanosome scavenger receptor exhibits selective lipid uptake from HDL, whereby the uptake of HDL lipid components exceeds that of HDL protein components.

Fig. 3 Trypanosome selective lipid uptake

Selective lipid uptake from HDL is exhibited by two Trypanosoma brucei subspecies, T. b. brucei (TLF sensitive) and T. b. rhodesiense (TLF resistant). Using fluorescent microscopy we find that polar HDL lipids are distributed immediately upon lipoprotein/cell contact, while HDL protein components and neutral lipids are endocytosed.

Trypanosomes are lipid auxotrophs, and the lipoprotein receptor may be the essential means to obtain lipid from lipoprotein particles in human serum. These findings provide the insight and reagents to purify and/or clone this scavenger receptor; there are only two endocytic receptors that have been molecularly cloned from trypanosomes. The cloning of this receptor is being undertaken by Dr. Vinh Philip Pham, MD, Ph.D. (2003-). This achievement would represent an important advance in the investigation of trypanosome biology that would likely open new avenues of research, including a new potential therapeutic target.

2) TLF mediated lysis is initiated by membrane lesions that cause an osmotic imbalance.
Trypanosome lytic factor 1 (TLF1) is a subclass of human high-density lipoprotein composed of lipids and at least six different proteins [Lugli, 2004].

TLFs must be endocytosed via the flagellar pocket (FP) and delivered to an acidic organelle (V) to kill trypanosomes, but the mechanism of lysis has not been elucidated. Dr. Maria del Pilar Molina Portela, Ph.D.(2000-) has shown that TLF 1 forms cation-selective pores in trypanosome membranes, which results in the loss of osmoregulation, leading to cytoplasmic vacuolization, cell swelling and ultimately parasite lysis.

	Fig. 4 Electron micrograph of trypanosomes incubated with TLF at (A) 0, (B and C) 45, and (D) 90 min. FP-flagellar pocket; V-vacuole/endosome/lysosome; N-nucleus; F-flagellum

Understanding whether TLF functions by a pore forming toxin-like mechanism will have implications for its role in human biology. This new direction of study has been undertaken by Dr. Gary Zeitlin, MD (2004-) and Wanda Almodovar, Pre-doctoral student (2004-).

3) Primate Lytic Factors that Kill Human Infective Trypanosomes have a unique unidentified lytic component.
Trypanosome lytic factors are found exclusively in Baboons, Gorillas, Mandrils, Sooty Mangabeys and all humans; the reason for this is unknown.

	Fig. 5 Primate trypanosome lytic factors. Group A contain Hpr and apoL-I. Group B contain Hpr.

Trypanosome lytic factors (TLFs) are protein/lipid complexes that contain apolipoprotein A-I, and are therefore a class of high density lipoproteins (HDLs). Dr. Elena Lugli, Ph.D. (2000-2004) showed that haptoglobin-related protein (Hpr) is a unique protein component of all trypanolytic primate HDLs. Furthermore, a polyclonal antiserum to Hpr can neutralize the lytic activity. However, transgenic mice, which express human Hpr do not have any trypanolytic activity in their serum or purified HDL [Hatada, 2002].

ApoL-I, a candidate lytic component of human serum (TLFs), was not immunologically or genetically detectable in two primate species with lytic activity. Polyclonal antiserum to apoL-I also did not neutralize TLF activity in a total human HDL preparation [Lugli 2004]. These findings suggest that apoL-I is not essential in all primate TLFs, and apoL-I alone is not sufficient for optimal trypanosome lytic activity in human TLF. Wanda Almodavor (2004-) is currently analyzing the levels of Hpr and apoL-I and their relative contribution/correlation to trypanolytic activity in 300 different sera from Africa in collaboration with Dr. Jeremy Sternberg, University of Aberdeen, UK.

Most importantly, primate TLFs without apoL-I are able to kill human infective trypanosomes. Dr. Maria del Pilar Molina Portela, Ph.D. is currently analyzing these TLFs in collaboration with Dr. Michael Ferguson, (University of Dundee, Scotland) to identify novel components, which confer lytic activity.

4) The IgM-HDL complex (TLF2) has atherogenic potential.
Dr. Maria del Pilar Molina Portela, Ph.D. evaluated the interaction of the polyclonal IgM (see Figure 2) found in TLF2. When we separate the components of TLF2 on non-reducing 4 M urea polyacrylamide gels, we find that 99% of the complex dissociates into the individual components, IgM (950 kDa), Hpr (45 kDa) and ApoA-I (28 kDa), indicating that the complex is maintained by non-covalent interactions. We are currently analyzing if the epitopes that IgM recognizes are due to the oxidation of TLF components, as would occur in atherosclerosis.

Selected Publications

1. Molina-Portela, MP, Lugli, E., Recio-Pinto, E. and Raper. J. Trypanosome lyitc factor, a subclass of high-density lipoprotein, forms cation-selective pores in membranes. Mol. Biochem. Parasitol. 2005 Dec;144(2):218-26
2. Lugli EB, Pouliot M, Portela Mdel P, Loomis MR, Raper J.
   Characterization of primate trypanosome lytic factors.
   Mol Biochem Parasitol. 2004;138(1):9-20.
3. Green HP, Molina Portela MP, St. Jean EN, Lugli EB, Raper J.
   Evidence for a Trypanosoma brucei Lipoprotein Scavenger Receptor.
   J. Biol. Chem. 2003;278: 422-427. (#J53458)
4. Raper J, Portela Molina MP, Redpath M, Tomlinson S, Lugli E, Green H. Natural immunity to human African trypanosomiasis: trypanosome lytic factors and the blood incubation infectivity test.
   Trans R Soc Trop Med Hyg. 2002;96 S145-50. (#J53937)
5. Raper J, Portela MP, Lugli E, Frevert U, Tomlinson S.
   Trypanosome lytic factors: novel mediators of human innate immunity.
   Curr Opin Microbiol. 2001;4:402-8. (#J28981)
6. Molina Portela MP, Raper J, Tomlinson S. An investigation into the mechanism of trypanosome lysis by human serum factors.
   Mol Biochem Parasitol. 2000;110:273-82. (#J23231)
7. Raper J, Fung R, Ghiso G, Nussenzweig V and Tomlinson S. The characterisation of a novel trypanosome lytic factor in human serum. Infection and Immunity. 1999;67:1910-1916. (#J00157)