Trypanocidal Factor Research Articles

Trypanosoma brucei brucei and high-density lipoproteins: Old and new thoughts on the identity and mechanism of the trypanocidal factor in human serum

Nature has provided humans with a surprising means of protection against the African trypanosome Trypanosoma brucei brucei There is consensus, in that this singular trypanocidal factor is serum high-density lipoproteins (HDL). which the trypanosomes engulf through a physiological, receptor-mediated pathway for delivery to acidic intracellular vesicles. There is also controversy, however, in that the active particles and their essential cytotoxic elements are disputed, in part reflecting the ill-defined mechanism by which the parasites are finally killed. Here Patrick Lorenz, Bruno Betschart and Jim Owen discuss the possibilities for resolving these discrepancies and speculate on the prospects of exploiting this unexpected property of human HDL for protecting livestock.

Human serum-sensitive Trypanosoma brucei rhodesiences: a comparison with serologically identical human serum-resistant clones

Trypanosoma brucei rhodesience clones, which are susceptible to lysis by normal human serum, were isolated from 3 different human serum-resistant clones originally derived from strain ETat 1.10. Serologically, these pairs of serum-sensitive and serum-resistant clones displayed the same variant surface glycoprotein (VSG) on their surface. Acquisition of human serum sensitivity correlated with susceptibility to lysis by human high density lipoprotein, a trypanocidal factor in normal human serum. Analysis of these paired populations by two-dimensional gel electrophoresis of whole trypanosomes and various subcellular fractions failed to reveal any differences in mobility of VSG and other proteins. Northern blot analysis of mRNAs from serum-sensitive and serum-resistant clones showed no differences when probed with a previously described resistance-specific probe. In addition, the ethanolamine membrane transport system and the overall membrane lipid fluidity did not reveal any detectable biochemical or biophysical differences in membrane properties. If resistance to lysis in indeed mediated by membrane changes at the enzymatic or structural level, the data presented suggest that the gene product(s) responsible for this change in human serum sensitivity may be present in very small quantities.

African buffalo serum contains novel trypanocidal protein.

The high ability of African buffalo, as compared to domestic cattle, to control infections with Trypanosoma brucei brucei ILTat 1.4 organisms did not correlate with the timing or magnitude of parasite surface coat-specific antibody responses and may have resulted from the constitutive presence in buffalo blood of a novel trypanocidal factor. Buffalo plasma and serum contained material that killed bloodstream stage T. b. brucei, T. b. rhodesiense, T. b. gambiense, T. evansi, T. congolense, and T. vivax organisms during four h of incubation at 37 degrees C in vitro. Serum from eland was also trypanocidal whereas serum from oryx, waterbuck, yellow-back duiker, cattle, horse, sheep, goat, mouse, rat, and rabbit was not trypanocidal. The buffalo serum trypanocidal material was not lipoprotein, or IgG, and had the following properties: 1) a density of > 1.24 g/ml determined by flotation ultracentrifugation; 2) insolubility in 50% saturated ammonium sulphate; 3) non-reactivity with anti-bovine IgM, and anti-bovine IgG; 4) non-reactivity with protein G, and protein A; 5) a relative molecular mass of 152 kDa determined by chromatography on Sephacryl S 300, and of 133 kDa determined by chromatography of the 50% SAS cut of IgG-depleted buffalo serum on Superose 12; 6) no associated cholesterol; and 7) inactivation by digestion with proteinase K that was immobilized on agarose.

High-density lipoprotein-mediated lysis of trypanosomes

Nearly 90 years after the discovery that certain African trypanosornes were killed by normal human serum, we still do not understand how this innate trypanocidal factor works. Biochemical studies have provided us with an unlikely candidate: human high-density lipoprotein (HDL). This trypanosome lytic factor (TLF) from human serum is important since its activity restricts the host range of Trypanosoma brucei brucei, and the expression of this natural killing factor in cattle would represent a novel approach to the control of bovine tryponosomiasis. Here, Steve Hajduk, Kristin Hager and Jeffrey Esko discuss evidence for the TLF being a minor subclass of serum HDL and propose a mechanism for lysis based on the binding, endocytosis and lysosomal targeting of TLF.

A Survey for a Trypanocidal Factor in Primate Sera

The sera of 21 different species of primates were surveyed for the presence of a trypanocidal factor to a monomorphic human serum-sensitive clone of Trypanosoma brucei gambiense (T.b.g.); human, gorilla, baboon (2 species), and the mandrill were found to contain this factor. The factor in all the sera is in the high density lipoprotein (HDL) fraction, and has similar modes of biological action. It has been shown that the human and gorilla trypanocidal factor share cross-reactive antigenic epitopes, but do not share similar cross-reactive epitopes with the baboon and mandrill factor. There was no relationship between the presence or absence of this factor and the primate's position on the phylogenetic tree. In addition, there was also no obvious correlation between the animals' preferred diet, and the presence or absence of trypanocidal activity. The evidence to date suggests that only African ground-dwelling primates that live in tsetse endemic areas contain the trypanocidal factor. It is assumed that this factor is involved in resistance of these primates to T.b.b. We believe that the host has developed trypanocidal substances as a result of selective evolutionary pressure by the African trypanosomes.

Nature of the Trypanocidal Factor in Human Serum

The chemical nature of the trypanocidal factor in human serum was investigated. The results show that although the trypanocidal factor is contained within the high density lipoprotein (HDL) fraction of human serum, it is apparently not one of the major apolipoproteins of the HDL complex such as apolipoprotein A-I, A-II, or apolipoprotein B. The factor would appear to be a minor component of the HDL fraction whose chemical nature is still uncertain.

A new method for the isolation of the trypanocidal factor from normal human serum

A novel procedure to isolate the trypanocidal factor from normal human serum (NHS) was developed, using four chromatographic steps: one affinity chromatography, then two anion exchange columns, and a final gel filtration. The isolated fraction showed the same lytic activity against Trypanosoma brucei brucei strains as NHS, whereas NHS-resistant trypanosomes of the Trypanosoma brucei rhodesiense subspecies remained unaffected. The factor is a protein complex of high molecular mass (> 1000 kDa) comprised of four peptides which were visualised under reducing conditions using SDS-polyacrylamide gel electrophoresis. No high density lipoproteins were detectable in the active fraction. Capillary stabilates of the human serum sensitive Trypanosoma brucei brucei clone STIB 345-A (kmdly provided by R. Brun, Swiss Tropical Institute, Basel), or of the Trypanosoma brucei rhodestense stock STIB 704-BABA (prepared following four mouse passages after cloning), were used to infect female ICR mice. 72 h after infecuon, blood was taken by cardiac puncture. Trypanosomes were isolated (Brun and Schoenenberger, 1981), resuspended in phosphate-buffered saline-glucose, pH 8.0, and used for the trypanocidal assay. Assays were performed in 14 ml stoppered plastic tubes, using a modified Minimum Essential Medium with Earle's salts (Jenni and Brun, 1982; Brun and Jenni, 1985). Trypanosomes were incubated at a concentration of 5 x 10 6 cells/ml at 37°C in 2 ml medium containing 10% horse serum and 30% NHS or the volume-equivalent serum fraction. Controls were carried out in the presence of 10% horse serum alone. Normal human serum (NHS) used in these experiments was a pool of sera from five healthy volunteers. Albumin was removed from NHS by affinity chromatography on Blue Sepharose CL-6B (Pharmacia) (Travis et al., 1976). The albumin-depleted serum was concentrated by ultrafiltration through a PM-10 membrane (Amicon) and then chromatographed on a Q Sepharose Fast Flow anion exchange column. Fractions were

Lysis of African trypanosomes by human plasma lipoproteins

are the causative agents of African sleeping sickness, whilst the closely related T. brucei is one of several that cause nagana in cattle. In man, T. brucei brucei is not normally infective and the haemoflagellate is lysed upon exposure to human plasma. Rifkin (1978~) has identified the trypanocidal factor present in human plasma as high-density lipoprotein (HDL), but neither the particular sub-class involved nor the mode of action has been defined (Rifkin, 1983). The present findings suggest that the smaller HDL particles are the lytic agents to T. brucei brucei, apparently because they are rich in apoplipoprotein (apo) A. A capillary stabilate of T. brucei brucei, derived from a clone of strain MIAG 427 MI Tat 1.6 (kindly provided by Dr. R. Klein, M.R.C. Parasitology Unit, Molten0 Institute, University of Cambridge, Cambridge, U.K.) and containing approx. 2 x lo6 trypanosomes, was used to infect individual mke. Blood was collected 72 h later during the rising phase GI' parasitaemia and, after centrifugation, the buffy coat was resuspended in phosphate-buffered saline/glucose, pH 8.0, I = 0.109, and applied to a column of DEAE-cellulose (DE-52, Whatman) (Lanham & Godfrey, 1970). Trypanosomes were eluted free of other cells with the buffer, washed three times and their proteins labelled by incubation at 2 x lo7 trypanosomes/ml in leucine-free minimum essential medium containing 10% (v/v) heat-inactivated foetal calf serum and 5 pCi of ~-[4,5-'H]leucine/rnl for 30 min at 37°C (Rifkin, 19786). Cells were collected by centrifugation at 4°C washed three times and suspended in ice-cold minimum essential medium containing 1 % (w/v) defatted bovine serum albumin at 2 x lo7 trypanosomes/ml. Human and rabbit HDL (e 1.063-1.21) were obtained by sequential, preparative ultracentrifugation of fresh plasma (Owen ef a f., 1984). In some experiments HDL was separated into apo A-rich (HDL-A) and apo E-rich (HDL-E) fractions by affinity chromatography on heparin-Sepharose (Weisgraber & Mahley, 1980), whilst in other studies tyrosine residues in apolipoproteins of HDL-A were converted to 3-nitrotyrosine with tetranitromethane (Brinton et al., 1986). Lipoproteins were dialysed against phosphate-buffered saline, pH 7.4 containing 10 mM-glucose and 0.3 mM-EDTA and their concentrations expressed in terms of their protein content (Lowry ef al., 1951). Incubations of labelled trypanosomes and HDL were carried out for up to 2 h at 37°C and cell lysis assessed by measuring the release of acid-precipitable tritium into the culture medium as described in the legend to Fig. 1. This technique is known to correlate closely with direct estimates of lysis by microscopicexamination (Rifiin, 19786).

Effects of oxygen-centred free radicals on low-density lipoprotein structure and metabolism

The present study shows that trypanosomes are able to obtain cholesterol by taking up and degrading human LDL, a process which increases intracellular cholesterol esterification. Presumably other lipoprotein particles which show similar binding to that of LDL, such as E-HDL, may also be taken up by trypanosomes and their cholesterol utilized. In some host species LDL may be present only in low concentrations or even absent (Chapman, 1980) and such alternative lipoproteins may be the major source of cholesterol for the parasite. However, it is clear from the present results that human A-HDL is unlikely to serve as a significant source of cholesterol for the parasite. In man, T . hrucei hrucei is not normally infective and the parasite is lysed on exposure to human plasma. Rifkin (1978) has identified the trypanocidal factor present in human plasma as highdensity lipoprotein, presumably the major fraction, A-HDL. The mode of action of A-HDL is unknown, but the suggestion that it is taken up by trypanosomes, causing massive accumulation of cholesteryl esters and release of autolytic enzymes (Ormerod, 1985), may be discarded. Instead, since A-HDL is saturably bound by T . hrucei hrucei (Gillett & Owen, 1987), is poorly degraded and inhibits, rather than stimulates, intracellular cholesterol esterification it is more probable that the lethal action of this lipoprotein occurs directly at the trypanosome surface.

Interaction of high‐density lipoprotein with Trypanosoma brucei: Effect of membrane stabilizers

The specific lysis of bloodstream trypanosomes by serum from a nonpermissive mammalian host is the result of interaction between the serum trypanocidal factor (high-density lipoprotein) and the trypanosome surface. The studies described in this paper attempt to define further the mode of action of this cytotoxic lipoprotein. The binding of high-density lipoprotein to Trypanosoma brucei was instantaneous at 4 degrees C and readily reversible. Binding was not mediated by the surface glycoprotein as removal of the surface coat enhanced binding at 4 degrees C, and no stable glycoprotein-lipoprotein complex could be detected. Pretreatment of trypanosomes with the cross linker dimethylsuberimidate rendered cells resistant to lysis. Addition of membrane-stabilizing drugs, such as cytochalasins C, D, and E, and local anesthetics (dibucaine, tetracaine, and procaine), also inhibited high-density lipoprotein-induced cell lysis. The data presented support the idea that at 37 degrees C lateral diffusion of the variant surface glycoprotein, an integral membrane protein, allows maximal high-density lipoprotein-cell interaction in serum-sensitive cells, and that altered properties of the plasma membrane induced by low temperature or the addition of cytochalasins, local anesthetics, or zinc inhibit this interaction, possibly by increasing the shielding of the plasma membrane by more rigidly anchored surface glycoprotein molecules.

Carbohydrate alters the trypanocidal activity of normal human serum with Trypanosoma brucei.

Three categories of carbohydrates are distinguished based on their effects on the trypanocidal activity of normal human serum on Trypanosoma brucei. The first category includes carbohydrates such as glucose, mannose and fructose which are utilized by parasites as an energy source. These carbohydrates inhibit the trypanocidal activity in a concentration-dependent manner. The third category, represented by xylose and mannitol, are not energy sources to the parasites but inhibit the trypanocidal activity in a manner similar to the first category carbohydrate. However, at very high concentrations (approximately 140-150 mM), the inhibition is reversed. Glycerol is the only known carbohydrate belonging to the second category. Glycerol enhances the trypanocidal activity of normal human serum and of those sera which normally display no cytotoxic activity, although glycerol serves as an excellent energy source. On the basis of these results, the mechanisms by which these three categories of carbohydrate could alter the trypanocidal activity are discussed in terms of the interaction between a trypanocidal factor and the surface receptor.

Ca2+ is essential cofactor for trypanocidal activity of normal human serum.

Normal human serum has been known to exert a cytotoxic effect on Trypanosoma brucei subspecies for nearly 80 yr. But in spite of many attempts, no trypanocidal factor was found in human or baboon serum, until Rifkin demostrated a high density lipoprotein (HDL) in normal human serum with trypanocidal activity. The conclusion that this was the trypanocidal factor was supported by the report that serum from patients with Tangier disease, characterised by a severe deficiency of HDL, lacked trypanocidal activity. We report here that Ca2+ is an essential cofactor for the trypanocidal activity of normal human serum, in which alpha2 macroglobulin (alpha 2) might function as a Ca2+-carrier. We further show that D-glucose, D-fructose and D-mannose can suppress the trypanocidal action of normal human serum, whereas glycerol has the opposite effect.

Trypanosoma brucei: Some properties of the cytotoxic reaction induced by normal human serum

The trypanocidal activity of normal human serum has been studied in vitro using Trypanosoma brucei as the test organism. The variables affecting the rate and extent of lysis, such as time, temperature, serum concentration, and pleomorphism of trypanosomes, are described. Trypanocidal titers of serum and serum fractions were quantitatively determined under standardized incubation conditions. Inactivation and/or removal of components of both the classical and alternate pathways of complement activation had no effect on the trypanocidal properties of human serum. The active factor was nondialyzable, present in plasma at equivalent levels to that in serum, and not removed by absorption with IgG fractions of antisera against human IgM or α 2-macroglobulin. The trypanocidal factor could be inactivated by heat (65 C), dithiothreitol, urea, and trypsin. Gel filtration studies indicated that the trypanocidal activity eluted as a single protein with a molecular weight of about 500,000.

Identification of the trypanocidal factor in normal human serum: high density lipoprotein.

The differentiation of Trypanosoma brucei from T. rhodesiense, the causative agent of human sleeping sickness, depends on their relative sensitivities to the cytotoxic effects of normal human serum. The molecule responsible for the specific lysis of T. brucei has now been isolated. Serum lipoproteins were fractionated and purified by ultracentrifugal flotation and chromatography on Bio-Gel A-5m. Trypanocidal activity was recovered in the high density lipoprotein fraction (density, 1.063-1.216 g/ml). Contamination by other serum proteins was checked by crossed immunoelectrophoresis and sodium dodecyl sulfate/acrylamide gel electrophoresis. Only a trace of beta-lipoprotein was found. The trypanocidal activity of pure human high density lipoprotein was identical to that of unfractionated serum when the following were tested: (i) time course of in vitro lysis of T. bruceli; (ii) in vivo destruction of T. brucei; (iii) relative resistance of T. rhodesiense to lysis. Rat or rabbit high density lipoprotein had no trypanocidal activity. Identification of the trypanocidal factor as high density lipoprotein was confirmed by the finding that serum from patients with Tangier disease, an autosomal recessive disorder characterized by a severe deficiency of high density lipoprotein, had no trypanocidal activity.

FURTHER STUDIES ON T. LEWISI INFECTION IN ALBINO RATS

T. lewisi infection in normal adult 3 month old albino rats raised from a single stock and maintained under identical conditions was studied. Daily quantitative estimates of the trypanosomes in the circulating blood were made and the course of the infection was studied. Bilateral suprarenalectomy in rats lowers the resistance to a subsequent infection with T. lewisi. About 70 per cent of these rats die in an average period of 5.8 days after injection. The multiplication of the parasites, in the circulating stream, however, is not more considerable in the suprarenalectomized than in the previously normal rats, nor is the duration of the disease in the surviving rats any longer than in the normal group. The removal of the suprarenal glands does not alter the immune reaction to the parasite, but lowers the natural resistance of the animal to the toxic effects of the protozoan infection. Bilateral suprarenalectomy does not lessen the immunity of rats recovered from T. lewisi infection to subsequent infection. Unilateral nephrectomy does not influence the course of a subsequent infection with T. lewisi infection. The mortality of splenectomized rats from Bartonella muris anemia increases from 30 to 100 per cent following the injection of T. lewisi at the height of the anemia 7 days after splenectomy. T. lewisi infection 48 days after splenectomy that is to say at a time when the Bartonella anemia is no longer present produces a more severe infection than in normal rats. The number of trypanosomes at the height of infection averages 3 times the ordinary and the infection endures twice as long. Both the immune substance that inhibits the reproduction of the parasite and the lytic factor are markedly depressed. Splenic autotransplantation performed 4 weeks prior to splenectomy raises the resistance of rats to a subsequent T. lewisi infection. Thymectomy in 6 week old rats diminishes the severity of a subsequent trypanosome infection and shortens its course. Both the formation of the immune substance which inhibits reproduction of the trypanosomes and formation of trypanolytic antibodies are stimulated by this procedure. In the adult rat thymectomy shortens the course of the infection but the severity is only slightly diminished. Bilateral gonadectomy in the adult increases the severity of the infection. The number of trypanosomes at the height of the infection is almost three times the normal. However, the duration of the infection is the same as in the normal rats. The reproduction-inhibiting factor is depressed by bilateral gonadectomy but not the trypanocidal factor. Unilateral gonadectomy does not influence the infection.