S.J. BLACK and V. VANDEWEERD
International Laboratory for Research on Animal Diseases
P O Box 30709, Nairobi, Kenya
References
Trypanosoma brucei brucei, a causative agent of trypanosomiasis in domestic livestock, and T. b. rhodesiense and T. b. gambiense, the causative agents of human sleeping sickness, are tsetse-transmitted flagellated protozoa that multiply extracellularly in infected mammalian hosts. Variation of the surface glycoprotein (VSG) of T. brucei organisms! prevents most hosts from clearing the parasites and leads to chronic and often debilitating infections. The pathologic consequences of trypanosomiasis caused by T. brucei and other pathogenic African trypanosomes are manifold, including: anaemia, raised Ig levels, immune complex disease, progressive destruction of lymphoid organs and other tissues, reduced fertility, cachexia and neurologic disorders. The severity of these disease symptoms varies among hosts, which, by this definition, are referred to as more trypano-resistant or more trypano-susceptible.
Immunity to T. brucei organisms is VSG-specific and is mediated by antibodies that react with exposed-epitopes on parasite VSG.² It has been observed in model studies that prolonged infection with African trypanosomes leads to a general inability to mount humoral immune responses to trypanosomes and third-party antigens.³ A widely held view arising from these observations is that the level of susceptibility of a given host to infections with African trypanosomes is likely to reflect the rapidity of development and severity of parasite-induced immunodepression. In this formulation of the host susceptibility problem, the interaction between trypanosomes and the humoral limb of the immune system assumes centre stage and the attention of the audience/investigator is focused on parasite products that drive or disengage the circuits underlying immune responses.
Recent studies provide reasonable grounds to doubt the above concept.4 An extensive analysis of plasma-cell responses has been performed in resistant mice, which mount serologically detectable VSG-specific antibody responses and cause clearance of the first T. brucei parasitaemic wave, and in susceptible mice, which do not. In both strains of mice plasma cells arose with the same kinetics, reached similar numbers in all lymphoid organs examined and synthesized and secreted similar amounts of antibodies of the same Ig classes, including antibodies specific for exposed VSG-epitopes on the infecting organisms.4
It was shown that the 2.5- to 10-fold higher peak levels of parasitaemia reached in the infected susceptible, as compared to the resistant, mice could result in the removal by the trypanosomes of large amounts of antibody. Hence it was inferred that there was less antibody bound per trypanosome, leading to failure of the susceptible mice to clear parasites from the bloodstream. VSG-specific antibody absorbed by trypanosomes is endocytosed and degraded (D. Russo, P. Webster and S. Black, unpublished). Failure to clear trypanosomes from the bloodstream leads to a prolonged parasitaemic wave, rapid destruction of lymphoid organ architecture and concomitant loss of ability to mount efficient humoral immune responses.
Accessory studies showed that the higher levels of parasitaemia reached in infected susceptible, as opposed to resistant, mice correlated with slower parasite differentiation to committed non-dividing trypanosomes in the bloodstream,5 an event that is probably controlled by antibody-independent host responses. The rate of parasite differentiation to committed non-dividing T. brucei in the bloodstream of infected mice can be accelerated by treatment of mice with Propionibacterium acnes.6 Biological mediators induced by P. acnes have no direct effect on the parasites in vitro.6 The mediators examined include IL-1, IL-2, TNF, INF, PGE1, PGE2, PGF2, mitogen-induced mixtures of mediators derived from T cells, B cells, macrophages and mixed populations of cells (early or late after stimulation), fibroblast growth factors, endothelial cell growth factors, nerve growth factors, platelet-derived growth factor, insulin and insulin-like growth factors, tumor-derived growth factors and tumor-promoting factors6 (S. Black and J. Newson, unpublished). The observations led to the idea that regulation of trypanosome multiplication and commitment to non-dividing forms might be mediated by secondary physiological effects of immune mediators that regulated the availability or nature of host-supplied growth nutrients/growth inhibitors.
Axenic culture systems7 were exploited to identify host-derived macromolecules required for the multiplication of T. brucei. Two different serodemes of T. brucei were adapted to grow under axenic culture conditions and the adapted parasites grew equally well in both in vivo and in vitro environments. No major biochemical adaptations were thus required to transit between the two environments and hence the in vivo and in vitro T. brucei growth nutrient requirements were similar.8 The trypanosomes multiplied under axenic culture conditions in medium supplemented with 10% foetal bovine serum (FBS). In contrast, lipoprotein-depleted-FBS (LPD-FBS; density 1.25 gm/ml) did not support parasite multiplication unless supplemented with FBS lipoproteins (density 1.21 gm/ml). High-density lipoproteins (HDL; density 1.06-1.21 gm/ml) and low-density lipoproteins (LDL; density 1.006-1.06 gm/ml), prepared by sequential flotation ultracentrifugation, were equally able to supplement LPD-FBS to support T. brucei multiplication. Chylomicrons (density 0.96 gm/ml) and very low-density lipoproteins (VLDL; density 0.96-1.006 gm/ml) were unable to support T. brucei multiplication. Removal of HDL or LDL-lipids by alcohol/ether extraction abrogated the ability of the lipoproteins to support T. brucei multiplication. Both HDL and LDL from a number of different species, including cattle, African buffalo, eland, rabbits and rats, were as able as FBS-HDL or FBS-LDL to support T. brucei multiplication.8 The observations fit well with the published requirement of bloodstream T. brucei for exogenous lipids9 and suggest the presence of a lipid scavenging mechanism suited to a parasite with a wide host range.
Foetal bovine serum (FBS), rabbit and rat HDL and LDL were labelled with 125I on the apolipoprotein content, or with ³H cholesterol, ³H cholesteryl linoleate or ³H dipalmitoyl phosphatidyl choline, or with combinations of 125I and ³H labels. It was shown that both culture-adapted T. brucei 8 and T. brucei isolated from the blood of infected mice (including organisms that had not been culture-adapted) (V. Vandeweerd and S. Black, unpublished) took up lipoprotein-lipids without talking up or degrading apolipoproteins. Uptake of the lipoprotein-lipids occurred at 37 °C but not at 0 °C to 4 °C, was saturable and was several thousand times more efficient than uptake expected to occur by fluid endocytosis. The uptake process did not discriminate between HDL and LDL, was independent of exogenous divalent ions and was not influenced by exogenous weak bases (20 mM ammonium chloride, 20 m M chloroquine8). The uptake mechanism was thus utterly different from receptor-mediated endocytosis of LDL as practiced by mammalian cells. 10
Uptake by T. brucei, X63 mouse myeloma cells and normal mouse spleen cells of lipoprotein-associated ³H cholesterol occurred to a similar extent. It resulted from desorption of the ³H cholesterol from the lipoproteins and its diffusion into the plasma membranes of the target cells. In contrast, uptake of lipoprotein-associated ³H dipalmitoyl phosphatidyl choline and ³H cholesteryl linoleate differed markedly among the three cell types. Trypansoma brucei obtained these lipids from both HDL and LDL. X63 obtained the lipids from LDL only and normal mouse spleen cells did not take up the lipids.8
Uptake by T. brucei of lipoprotein-associated ³H dipalmitoyl phosphatidyl choline was inhibited by including LPD-FBS in the incubation mixture. The active ingredient in the LPD-FBS was likely to be albumin. LPD-FBS stimulated rather than inhibited the uptake by T. brucei of lipoprotein-associated ³H cholesterol linoleate. This observation suggests that lipoprotein-derived phospholipids and cholesterol esters might enter T. brucei by different processes and that the phospholipid ³H dipalmitoyl phosphatidyl choline) is free of the carrier lipoprotein prior to entry into T. brucei.
In contrast, bile acids and conjugated and unconjugated bile salts inhibited the uptake by culture-adapted and normal bloodstream T. brucei of lipoprotein-associated ³H cholesteryl linoleate but not lipoprotein-associated ³H dipalmitoyl phosphatidyl choline. Different cholesterol conversion products had differing efficiencies to inhibit T. brucei lipoprotein-cholesterol ester uptake. The observed order, derived from inhibition of HDL-associated ³H cholesterol linoleate uptake using culture-adapted T. brucei as: lithocholic acid > chenodeoxycholic acid > deoxycholic acid > cholic acid = glycochenodeoxycholic acid > taurochenodeoxycholic acid (V. Vandeweerd and S. Black, unpublished).
Two possibilities present themselves. The cholesterol conversion products might prevent the uptake by T. brucei of lipoprotein-associated cholesterol ester by inhibiting cleavage to cholesterol that can readily diffuse across the cell membrane. Alternatively, the cholesterol conversion products might compete with an interaction between lipoprotein-associated cholesterol ester and a T. brucei cholesterol ester binding molecule. To examine these possibilities, purified lipoproteins were labelled with ³H cholesterol ether ³H cholesterol oleoyl ether) and incubated with culture-adapted T. brucei in the presence or absence of an inhibitory concentration of chenodeoxycholic acid. In the absence of the bile acid, the T. brucei took up ³H cholesterol ether by a process that was similar in all respects to uptake of ³H cholesterol ester, e.g., uptake occurred only at 37 °C, was saturable, was inhibited by unlabelled lipoproteins and was enhanced by LPD-FBS. Uptake of the cholesterol ether was inhibited by the bile acid (Vandeweerd and Black, unpublished). Because cholesterol esterases are unable to cleave the ether bond11 and because cholesterol ether cannot diffuse through cell membranes, we conclude that T. brucei have a cholesterol ester/ether binding molecule that is blocked by cholesterol conversion products.
Both chylomicrons and VLDL contain phospholipids and cholesterol ester yet do not support T. brucei multiplication and do not inhibit the capacity of LDL or HDL to support T. brucei multiplication in vitro.8 It therefore seems possible that size constraints prevent interactions between the largest lipoprotein molecules and the T. brucei. This conclusion leads to the idea that lipoprotein/T. brucei interactions occur in the T. brucei flagellar pocket, from which the larger lipoproteins might be excluded. Alternatively, cholesterol esters and phospholipids might be sequestered in chylomicrons and VLDL in such a way that they cannot interact with T. brucei organisms. As a preliminary step to distinguishing between these possibilities, it is of some importance to define the maximum size of molecules that can enter the T. brucei flagellar pocket.
Uptake by T. brucei of HDL-associated ³H cholesteryl linoleate is inhibited by including puromycin or cycloheximide (20 m g/ml medium) in the incubation mixture. In contrast, uptake by T. brucei of HDL-associated ³H dipalmitoyl phosphatidyl choline is not inhibited by including puromycin or cycloheximide in the incubation mixture. We therefore consider it likely that uptake of the H cholesteryl linoleate is mediated by binding to a protein, whereas uptake of H dipalmitoyl phosphatidyl choline is not. Based on these observations and the ability of LPD-FBS (albumin) to prevent uptake by T. brucei of lipoprotein-associated ³H dipalmitoyl phosphatidyl choline, we speculate that the ³H phospholipid is released from the lipoprotein particle by a process that does not require protein synthesis by the parasite and thereafter diffuses into the parasite membrane. It is tempting to suggest that release of the lipoprotein-associated phospholipid occurs in the T. brucei flagellar pocket as a result of mechanical disruption of the lipoprotein particle. The uptake by T. brucei of lipoprotein-associated cholesterol occurs as a result of simple desorption; uptake of phospholipid possibly occurs by mechanically induced release and diffusion. Uptake by T. brucei of lipoprotein-associated cholesterol ester may therefore be the only component of the parasite lipid scavenging mechanism amenable to specific chemotherapeutic or immunological attack.
Concentrations of bile acids (5 to 15 m M), and conjugated and unconjugated bile salts (50 to 100 m M), which inhibit uptake by T. brucei of lipoprotein-associated cholesterol ester/ether in the absence of LPD-FBS, are close to toxic concentrations. Although the inclusion of LPD-FBS in the incubation mixture reduces the short-term toxicity of cholesterol conversion products, it does not completely abrogate their effects. Concentrations of bile acids (100 m M) can be chosen that prevent multiplication of T. brucei in long-term cultures supplemented with 10% FBS. Because conjugated bile salts and bile acids are found in normal plasma, it is an attractive idea that these molecules might have a role to play in protection against African trypanosomes. Clearly, quantitative data are required on the bile acid, and conjugated and unconjugated bile salt concentrations in the plasma and interstitial fluids of normal and infected trypano-susceptible and trypano-resistant hosts. Equally clearly, quantitative data are required on the sensitivity of different trypanosome clones, serodemes and species to the toxic effects of cholesterol conversion products in the presence or absence of blood.
We hope that the above overview will stimulate further studies on trypanosome nutrient uptake, on the host/trypanosome interface as manifested in the T. brucei flagellar pocket and on the mechanisms used by infected hosts to control parasite growth.
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4. NEWSON, J., S.M. MAHAN and S.J. BLACK. Submitted (ILRAD publication No. 682).
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6. BLACK, S.J., M. MURRAY, S.Z. SHAPIRO, R. KAMINSKY, N.K. BOROWY, R. MUSANGA and F. OTIENO. Submitted (ILRAD publication No. 532).
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8. VANDEWEERD, V. and S.J. BLACK. Submitted (ILRAD publication No. 631).
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