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.     

Inhibition and killing of fungi by the polyamine oxidase-polyamine system

Both components of the polyamine oxidase (PAO)-polyamine system are known to be present in phagocytes and have thus been postulated to contribute to the antimicrobial activity of these cells. Therefore, the effects of the PAO-polyamine system on three medically important opportunistic fungi were examined. Yeasts of Cryptococcus neoformans, but not Candida albicans blastoconidia or Aspergillus fumigatus conidia, were efficiently killed by the system. Two putative end products of the system, hydrogen peroxide and acrolein, both killed C. neoformans at concentrations attainable with the whole system. However, catalase failed to inhibit activity of the whole system, making hydrogen peroxide an unlikely mediator of killing. Although C. albicans blastoconidia and A. fumigatus conidia were not killed by the PAO-polyamine system, germ tube formation by the former, and hyphal growth by the latter, were markedly inhibited. These data establish that the PAO-polyamine system possesses antifungal activity.     

Molecular basis for the reverse reaction of African human trypanosomes glycerol kinase

The glycerol kinase (GK) of African human trypanosomes is compartmentalized in their glycosomes. Unlike the host GK, which under physiological conditions catalyzes only the forward reaction (ATP‐dependent glycerol phosphorylation), trypanosome GK can additionally catalyze the reverse reaction. In fact, owing to this unique reverse catalysis, GK is potentially essential for the parasites survival in the human host, hence a promising drug target. The mechanism of its reverse catalysis was unknown; therefore, it was not clear if this ability was purely due to its localization in the organelles or whether structure‐based catalytic differences also contribute. To investigate this lack of information, the X‐ray crystal structure of this protein was determined up to 1.90 Å resolution, in its unligated form and in complex with three natural ligands. These data, in conjunction with results from structure‐guided mutagenesis suggests that the trypanosome GK is possibly a transiently autophosphorylating threonine kinase, with the catalytic site formed by non‐conserved residues. Our results provide a series of structural peculiarities of this enzyme, and gives unexpected insight into the reverse catalysis mechanism. Together, they provide an encouraging molecular framework for the development of trypanosome GK‐specific inhibitors, which may lead to the design of new and safer trypanocidal drug(s).     

Inhibition of lymphocyte growth by spermidine in medium containing fetal bovine serum

The effect of spermidine and fetal bovine serum on DNA, RNA, and protein synthesis in phytohemagglutinin-stimulated human lymphocytes was investigated. At 10(-4) M spermidine, DNA, RNA, and protein synthesis ceased and 70% of the original cell population died within 62 hr. Lower spermidine concentrations had no significant effect on DNA and protein synthesis, but caused an early, unexplained increase in the rate of RNA synthesis. Heating at 56 degrees C for 30 min had no effect on the plasma amine oxidase activity in fetal bovine and horse sera but abolished the activity in human plasma. It is concluded that low amounts of aminoaldehydes and acrolein produced by plasma amine oxidase at spermidine concentrations below 10(-4) M do not noticeably alter lymphocyte metabolism. However, the aminoaldehydes and acrolein produced become abruptly cytotoxic at 10(-4) M spermidine.     

Comparison of the cytolytic effects in vitro on Trypanosoma brucei brucei of plasma, high density lipoproteins, and apolipoprotein A-I from hosts both susceptible (cattle and sheep) and resistant (human and baboon) to infection.

The African trypanosome, Trypanosoma brucei brucei causes a fatal wasting disease in livestock but does not ordinarily infect humans, apparently because this unicellular parasite is lysed by high density lipoproteins (HDL) in human serum. To assess whether there is a specific active constituent in trypanolytic HDL, we have systematically compared the cytotoxic action on T.b.brucei in vitro of native and delipidated HDL, and of individual apolipoproteins, from nonpermissive hosts (human and baboon) with their counterparts from susceptible hosts (cattle and sheep). When suspensions of trypanosomes were incubated for 2 h at 37 degrees C with human or baboon plasma most cells were lysed, but not with bovine or sheep plasma. Similarly, HDL isolated from human and baboon plasma were trypanolytic (typically about 95% and 60% lysis, respectively, at 1 mg protein/ml), whereas bovine and sheep HDL were benign (less than 8% lysis). Subfractionation of human HDL by serial isopycnic ultracentrifugation and by heparin-Sepharose affinity chromatography established that the denser and smaller particles had greater trypanolytic activity both in vitro and in vivo. When human HDL was delipidated, the trypanocidal activity was associated with the water-soluble protein (apolipoprotein) fraction and not with the lipid constituents. Bovine apolipoproteins were also weakly trypanolytic in free solution (20-40% lysis), but not when complexed with cholesterol-phospholipid liposomes (less than 10% lysis). The major apolipoprotein of human HDL, apolipoprotein (apo) A-I had full trypanolytic activity (89-95% lysis at 1 mg protein/ml) when purified, whether in solution or incorporated into liposomes, but other apolipoproteins isolated from human HDL, including apoA-II, apoC, and apoE, were nontrypanolytic. Purified baboon apoA-I was also trypanolytic, though less potent than human apoA-I, but apoA-I from permissive hosts (cattle and sheep) was inactive when presented in liposomes. Incubation of bovine or sheep HDL with purified human apoA-I, and subsequent separation of the HDL by ultracentrifugation, produced chimeric HDL containing significant amounts of the human apolipoprotein; these particles showed appreciable trypanolytic activity. By contrast, human HDL particles in which about 70% of the apoA-I had been displaced with apoA-II had markedly reduced lytic properties compared to the native HDL (30% versus 80% lysis at 0.6 mg total protein/ml). We tentatively conclude that the trypanolytic activity of native human or baboon plasma resides in the apoA-I content of the HDL particles and that, conversely, bovine and sheep plasma are inactive because the apoA-I polypeptide present in their HDL lacks trypanocidal activity.     

The Plasma Membrane of Bloodstream-form African Trypanosomes Confers Susceptibility and Specificity to Killing by Hydrophobic Peptides

Trypanosoma brucei is the causative agent of both a veterinary wasting disease and human African trypanosomiasis, or sleeping sickness. The cell membrane of the developmental stage found within the mammalian host, the bloodstream form (BSF), is highly dynamic, exhibiting rapid rates of endocytosis and lateral flow of glycosylphosphatidylinositol-anchored proteins. Here, we show that the cell membrane of these organisms is a target for killing by small hydrophobic peptides that increase the rigidity of lipid bilayers. Specifically, we have derived trypanocidal peptides that are based upon the hydrophobic N-terminal signal sequences of human apolipoproteins. These peptides selectively partitioned into the plasma membrane of BSF trypanosomes, resulting in an increase in the rigidity of the bilayer, dramatic changes in cell motility, and subsequent cell death. No killing of the developmental stage found within the insect midgut, the procyclic form, was observed. Additionally, the peptides exhibited no toxicity toward mammalian cell lines and did not induce hemolysis. Studies with model liposomes indicated that bilayer fluidity dictates the susceptibility of membranes to manipulation by hydrophobic peptides. We suggest that the composition of the BSF trypanosome cell membrane confers a high degree of fluidity and unique susceptibility to killing by hydrophobic peptides and is therefore a target for the development of trypanocidal drugs.     

Role of phospholipids in the cytotoxic action of high density lipoprotein on trypanosomes.

Host range among the African trypanosomes, protozoa that cause fatal diseases both in humans and livestock, may be, in part, regulated by toxic properties associated with host high density lipoproteins (HDL). High density lipoproteins from hosts resistant (baboon, human) or susceptible (rabbit, rat) to Trypanosoma brucei infection were isolated and their trypanocidal activity was determined in in vitro cell lysis assays. Rabbit and rat HDL were not cytotoxic while baboon and human HDL rapidly lysed trypanosomes within 2 h at 37 degrees C. Analysis of the phospholipid composition of HDL preparations from these species suggested a correlation between trypanocidal activity and low phosphatidylinositol content. Phospholipase digestion of HDL resulted in a loss of trypanocidal activity, indicating the importance of native phospholipids in maintaining this biological activity of HDL. Cell lysis and loss of trypanosome infectivity induced by baboon HDL could be inhibited either by addition of rabbit or rat HDL to the incubation medium or by addition of purified phospholipids, phosphatidylinositol being the most effective inhibitor. Although the mechanism by which HDL lyses trypanosomes remains to be elucidated, these results suggest an important role for phospholipids in determining the specificity of this cytotoxic property of HDL.     

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 α₂-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.     

African trypanosomes: intracellular trafficking of host defense molecules

Trypanosoma brucei brucei is the causative agent of Nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL), containing apolipoprotein A-I (APOA1), apolipoprotein L-I (APOL1) and haptoglobin-related protein (HPR) provides this innate protection against T. b. brucei infection. Both HPR and APOL1 are cytotoxic to T. b. brucei but their specific activities for killing increase several hundred-fold when assembled in the same HDL. This HDL is called trypanosome lytic factor (TLF) and kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosome lytic factor is activated in the acidic lysosome and facilitates lysosomal membrane disruption. Lysosomal localization is necessary for T. b. brucei killing by TLF. Trypanosoma brucei rhodesiense, which is indistinguishable from T. b. brucei, is resistant to TLF killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a human serum resistance associated protein (SRA) that is able to ablate TLF killing. When T. b. brucei is transfected with the SRA gene it becomes highly resistant to TLF and human serum. In the SRA transfected cells, intracellular trafficking of TLF is altered and TLF mainly localizes to a subset of SRA containing cytoplasmic vesicles but not to the lysosome. These findings indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine susceptibility.     

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.     

Inhibition of lymphocyte growth by spermidine in medium containing fetal bovine serum

Summary The effect of spermidine and fetal bovine serum on DNA, RNA, and protein synthesis in phytohemagglutinin-stimulated human lymphocytes was investigated. At 10⁻⁴ M spermidine, DNA, RNA, and protein synthesis ceased and 70% of the original cell population died within 62 hr. Lower spermidine concentrations had no significant effect on DNA and protein synthesis, but caused an early, unexplained increase in the rate of RNA synthesis. Heating at 56°C for 30 min had no effect on the plasma amine oxidase activity in fetal bovine and horse sera but abolished the activity in human plasma. It is concluded that low amounts of aminoaldehydes and acrolein produced by plasma amine oxidase at spermidine concentrations below 10⁻⁴ M do not noticeably alter lymphocyte metabolism. However, the aminoaldehydes and acrolein produced become abruptly cytotoxic at 10⁻⁴ M spermidine. This work was supported in part by the Cystic Fibrosis Foundation.     

Trypanosoma brucei: polyamine oxidase mediated trypanolytic activity in the serum of naturally resistant cattle

Trypanosoma brucei brucei are lysed when incubated in vitro in a mixture of bovine serum and polyamine. Normal bovine serum alone or polyamine alone does not show any trypanocidal activity. The bovine serum in the mixture can be replaced by purified polyamine oxidase, and addition of polyamine oxidase inhibitors blocks trypanolysis. Using this in vitro lysis test, it is shown that West African cattle which are resistant naturally to trypanosomiasis have a higher trypanolytic activity in their serum than do trypanosensitive cattle (P less than 10(-5]. Seric trypanolytic activity of individual animals remains stable when tested over a period of 18 months; moreover, it is not modified by trypanosome infection. Higher levels of seric polyamine oxidase in resistant cattle were demonstrated also by enzymatic analysis. The factors responsible for trypanolysis have been analyzed. Oxidation of spermidine by polyamine oxidase leads to the production of unstable aldehydes, acrolein, ammonia, O2-, HO, and H2O2. Acrolein and H2O2 show strong trypanolytic activity while the other products do not appear to be toxic for trypanosomes. The physiological importance of polyamine oxidase mediated trypanolysis is unclear; even at peak parasitemia in cattle (10(7) organisms/ml) it can be calculated that trypanosomes would not release enough spermidine for the generation of sufficient quantities of toxic degradation products. Additional polyamines could be released in serum from tissues damaged as a result of the infection.     

Gonococci in vivo and in vitro. Further studies on the host and bacterial determinants of gonococcal resistance to killing by human serum,and by phagocytes

A small M, heat and acid labile, host inducer(s) of gonococcal resistance to complement mediated killing by fresh human serum (-FHS), being purified from red blood cell (RBC) extracts, produced changed in lipopolysaccharide (LPS) structure, surface antigens and proteins; and acquirement of resistance related to loss of a target antigen for bactericidal IgM, possibly LPS components. A 20 kDalt, lipoprotein with a high content of glutamic acid isolated from outer membranes of a gonococcal strain selected in vivo is a determinant of gonococcal resistance to killing by human phagocytes. Somic extracts of gonococci may contain a cytotoxin for human phagocytes.At the 4th International Pathogenic Neisseriae Conference, we reported (Parsons et al. 1985) that conditions in vivo induced phenotypic change leading to gonococcal resistance to complement-mediated killing by human serum; and, also, selected gonococcal types which showed a greater resistance to intracellular killing by human phagocytes than laboratory strains. Furthermore, evidence was presented that not only was resistance to complement mediated killing important in gonococcal pathogenesis, but also resistance to phagocytic defences. This paper describes the continuance of our studies on the determinants of induced serum resistance and of resistance to killing by phagocytes including toxicity to these cells. Each section begins by summarising previous work that was referenced in Parsons et al. (1985).     

Trypanosoma brucei gambiense: growth in vitro of bloodstream forms inhibited by dichlorodiammineplatinum (II) compounds and hypolipidemic drugs

Infectious bloodstream forms of Trypanosoma brucei gambiense were grown in microcultures of murine bone marrow cells in 96-well tissue culture plates. Limiting dilution studies showed that fewer than 10 cultured trypanosomes developed into populations of about 5 X 10(4) parasites per well in a week. Bloodstream parasites were reisolated with high efficiency from mice infected with cultured parasites; fewer than 10 bloodstream parasites successfully established a trypanosome population in a microculture. Both the cis and trans isomers of dichlorodiammineplatinum (II) (cisplatin and transplatin) and a hypolipidemic agent, Wy 14643, were found to have activity against T. b. gambiense growing in microcultures. The minimum concentration of drug necessary to completely eliminate parasites from microcultures was 4 microM for cisplatin, 40 microM for transplatin, and 500 microM for Wy 14643. A preformed complex of cisplatin and bovine serum albumin and another hypolipidemic agent, chlofibric acid, were inactive. This culture system should be useful for rapid screening of large numbers of compounds for trypanocidal activity.     

Cysteine proteinase inhibitors kill cultured bloodstream forms of Trypanosoma brucei brucei

Trypanosoma brucei brucei is a causative agent of bovine trypanosomiasis (nagana), a disease of considerable economic significance in much of Africa. Here we report investigations on the effects of various irreversible cysteine proteinase inhibitors, including vinyl sulfones (VS), peptidyl chloromethylketones (CMK), diazomethylketones, and fluoromethyl ketones, on the major lysosomal cysteine proteinase (trypanopain-Tb) of T. b. brucei and on in vitro-cultured bloodstream forms of the parasite. Many of the tested inhibitors were trypanocidal at low micromolar concentrations. Methylpiperazine urea-Phe-homoPhe-VS was the most effective trypanocidal agent, killing 50% of test populations at a work ing concentration of 0.11 microM, while carbobenzoxy-Phe-Phe-CMK was the most trypanocidal of the methylketones with an IC50 of 3.6 microM. Labelling of live and lysed T. b. brucei with biotinylated inhibitor derivatives suggests that trypanopain-Tb is the likely intracellular target for these inhibitors. Kinetic analysis of the inhibition of purified trypanopain-Tb by the inhibitors showed that most had kass values in the 10(6) M-1 s-1 range. We conclude that cysteine proteinase inhibitors have potential as trypanocidal agents and that a major target of these compounds is the lysosomal enzyme trypanopain-Tb.     

Trypanocidal activity of peptidyl vinyl ester derivatives selective for inhibition of mammalian proteasome trypsin-like activity

Nine vinyl ester tripeptides selective for inhibition of mammalian proteasome trypsin-like activity were tested for in vitro activity against Trypanosoma brucei. Interestingly, two compounds showed trypanocidal activity in the low micromolar range without displaying cytotoxicity against human cells. However, the compounds did not inhibit the trypsin-like activity of the trypanosome proteasome although their effect correlates with inactivation of the chymotrypsin-like activity. This finding shows that the inhibitor sensitivities between mammalian and trypanosome proteasome are distinct. This difference may be exploited for rational anti-trypanosomal drug development.     

IncP-1 R plasmids decrease the serum resistance and the virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO1 was compared to PAO1 strains containing an IncP-1 R plasmid (RP1, R68, or R68.45) in an experimental mouse burn infection model. All R plasmids tested caused a 10- to 400-fold increase in mean lethal dose (LD50). The decrease in virulence produced by plasmids R68 and R68.45 was significantly greater than the decrease caused by the closely related plasmid RP1. All plasmids also led to an increased sensitivity of strain PAO1 to human serum bactericidal activity. Virulence and serum resistance of strain PAO1 were restored by curing of the entire plasmid R68.45 but not by deletions in the plasmid's transfer gene regions.     

Lysis of Trypanosoma brucei by a toxic subspecies of human high density lipoprotein

Trypanosoma brucei brucei is an important pathogen of domestic cattle in sub-Saharan Africa and is closely related to the human sleeping sickness parasites, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, T. b. brucei is non-infectious to humans. The restriction of the host range of T. b. brucei results from the sensitivity of the parasite to lysis by toxic human high density lipoproteins (HDL) (Rifkin, M. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3450-3454). We show in this report that trypanosome lytic activity is not a universal feature of all human HDL particles but rather that it is associated with a minor subclass of HDL. We have purified the lytic activity about 8,000-fold and have identified and characterized the subspecies of HDL responsible for trypanosome lysis. This class of HDL has a relative molecular weight of 490,000, a buoyant density of 1.21-1.24 g/ml, and a particle diameter of 150-210 A. It contains apolipoproteins AI, AII, CI, CII, and CIII, and monoclonal antibodies against apo-AI and apo-AII inhibit trypanocidal activity. In addition to these common apolipoproteins, the particles also contain at least three unique proteins, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Treatment of the particles with dithiothreitol resulted in the disappearance of two of the proteins and abolished trypanocidal activity. Two-dimensional gel electrophoresis showed that these proteins were a disulfide-linked trimer of 45,000, 36,000, and 13,500-Da polypeptides and dimers of the 36,000- and 13,500-Da polypeptides or of 65,000- and 8,500-Da polypeptides. Studies on the lysis of T. b. brucei by the purified particle suggest that the lytic pathway may involve the uptake of the trypanocidal subspecies of HDL by endocytosis.     

Midgut lectin activity and sugar specificity in teneral and fed tsetse

Abstract. . Midgut infection rates of Trypanosoma congolense in Glossina palpalis palpalis and of Trypanosoma brucei rhodesiense in Glossina pallidipes are potentiated by the addition of D+ glucosamine to the infective feed, but not to the levels of super-infection reported for G. m. morsitans. G. p. palpalis and G.pallidipes are shown to possess two trypanocidal molecules: a glucosyl lectin which can be inhibited by D+ glucosamine and a galactosyl molecule inhibited by D+ galactose. Addition of both D+ glucosamine and D+ galactose to the teneral infective feed promotes super-infection of the midguts of G.p.palpalis. The glucosyl lectin is specific for rabbit erythrocytes and is present in guts of fed G.m.morsitans and G.p.palpalis , titres of lectin activity do not increase substantially after the second bloodmeal. The galactosyl specific molecule does not show any erythrocyte specificity, although haemolytic activity is observed only in G.p.palpalis and not in G.m.morsitans. The presence of two trypanocidal molecules in some species of tsetse may account for the innate refractoriness of these flies to trypanosome infection.
As D+ glucosamine also inhibits the killing of procyclic trypanosomes taken as an infective feed, it is suggested that the midgut lectin is normally responsible for the agglutination of trypanosomes in the fly midgut by binding to the pro-cyclic surface coat, prior to establishment in the ecto-peritrophic space.     

Cyclical differentiation of Trypanosoma brucei involves changes in the cellular complement of calmodulin-binding proteins

The present study was undertaken to evaluate changes in the complement of calmodulin-binding proteins which accompany cyclical differentiation in Trypanosoma brucei. An [125I]trypanosome calmodulin overlay procedure was used to detect calmodulin-binding proteins with Mr of 126,000 and 106,000 that were present in homogenates of slender bloodstream froms but were absent in procyclic culture forms. Competition assays with unlabeled bovine brain or trypanosome calmodulins indicated that the developmentally regulated proteins associated with calmodulins from either source. Moreover, [125I]bovine brain calmodulin associated with the same proteins as trypanosome calmodulin. Homogenates of T. evansi exhibited the same pattern of calmodulin-binding activity as T. brucei slender bloodstream forms; however, T. cruzi and Leishmania tarentolae contained distinct patterns of calmodulin-binding activity. Mouse serum contained no detectable binding proteins while mouse brain contained predominantly proteins of Mr 210,000, 60,000, and 49,000 which were associated with the trypanosome calmodulin probe. The developmentally regulated calmodulin-binding proteins from T. brucei were in the 10,000g pellet. We conclude that the cellular complement of calmodulin-binding proteins varies during the trypanosome life cycle.