Weak Bases as Inhibitors of the Trypanocidal Activity of Human Serum

Among the African trypanosomes, the Trypanosoma brucei group is subdivided into the three subspecies T. b. brucei, T. b. rhodesiense and T. b. gambiense. The latter two are the causative agents of human sleeping sickness. Trypanosoma b. brucei and T. b. rhodesiense are practically indistinguishable using either morphological or biochemical criteria. However, the two subspecies can be differentiated on the basis of their sensitivity to the lytic action of normal human serum (NHS).

The phenomenon of the trypanolytic action of NHS has been known for more than 80 years, when Laveran¹ was able to cure mice temporarily infected with T. b. brucei by injecting small quantities of NHS. Many attempts were made to identify and characterize the serum factor, before Rifkin² identified a high-density lipoprotein (HDL) fraction as containing the trypanocidal activity. She also proposed later a model whereby the factor leads, via its interaction with the trypanosome surface, to an alteration of the cell membrane permeability and a subsequent damage of the cell by osmotic shock.³

A few years ago we decided to study the mechanism of this cytotoxic action of NHS more closely. Encountering difficulties in the isolation of a trypanolytic HDL fraction, an alternative method of isolating the factor had to be designed. In parallel, we looked for ways to analyse the lytic mechanism in more detail. Assuming that the trypanolytic activity (TLA) might occur intracellularly, acidotropic agents were used and their modifying effects on the lysis process studied.

Effects of Weak Bases on the Trypanolytic Action

5 × 10⁶ freshly isolated bloodstream forms of either the human serum-sensitive T. b. brucei strain STIB 345-A or the human serum resistant stock T. b. rhodesiense STIB 704 BABC were resuspended in 1 ml of MEM with 40% horse serum or 10% horse serum and 30% NHS. The extent of lysis was determined by taking aliquots of the cell suspensions incubated at 37 °C and counting the motile cells in a haemocytometer every hour. The percentage of lysis was calculated by taking the initial cell number as 100%. Under these conditions 100% of T. b. brucei are lysed within 6 hours, whereas T. b. rhodesiense showed no signs of lysis. Supplementing the medium with different chloroquine concentrations in the range of 10-25 mM resulted in a complete inhibition of the lytic action. Higher concentrations were toxic for both T. b. brucei and T. b. rhodesiense. Similar effects were found with other weak bases (Table 1). The concentrations of the weak bases needed to inhibit the trypanolytic activity were in similar ranges to the concentrations reported to affect a variety of different intracellular processes associated with acidic compartments.^4,5

To test whether a preconditioning of trypanosomes with the weak bases would render them more resistant to the Lytic activity of NHS, T. b. brucei were preincubated for 3, 2 and 1 hours in media containing either 30% horse serum or 30% NHS, both supplemented with 10 mM chloroquine. As a control, trypanosomes were incubated only in media with 30% horse serum alone. After the preincubation period, all trypanosomes were pelleted and resuspended in 30% NHS without chloroquine. No significant inhibition of Lysis was observed in any of the samples. On the contrary, a slight enhancement of Lysis was observed with the cells preincubated in NHS and chloroquine for 2-3 hours. These data seem to indicate that the trypanolytic activity can be efficiently inhibited only when the weak bases are continuously present in the medium. To prove that the weak bases were taken up, T. b. brucei and T. b. rhodesiense were incubated in the presence of different concentrations of chloroquine for three hours, washed once in PBS and kept frozen until chloroquine was determined with a quantitative high-precision thin-layer chromatographic method.⁶ It could be shown that the trypanosomes accumulated chloroquine (approximately 50-100 fold accumulation), but no concentration dependent uptake was found in the range of 0.5 to 20 mM. This can probably be explained by the fact that after three hours an uptake plateau is reached. Chloroquine determinations at much earlier time points might well show a concentration dependence.

Attempts to cultivate T. b. brucei in NHS for longer time periods in the presence of chloroquine on a Microtus montanus feeder layer failed.⁷ All trypanosomes died. Chloroquine probably accumulated intracellularly to toxic levels, as has been shown by others.⁸ In contrast, it was possible to maintain T. b. brucei in 20 mM ammonium chloride for more than four days in the presence of normal human serum. The cells did not show any sign of lysis.

Characteristics of the Trypanolytic Factor

The trypanolytic activity in more detail it was necessary to isolate the trypanolytic factor. A new method to isolate the trypanolytic factor not involving ultracentrifugal flotation was established (Figure 1) using affinity chromatography of whole human serum on Blue Sepharose, followed by two anion-exchange steps and a final purification on Superose.⁹

The known characteristics of the factor are summarized in Table 2. The factor consists of a macromolecular complex with a molecular weight over 1,000,000. Under non-reducing conditions a single polypeptide is found, which is separated into 3 4 peptides under reducing conditions. The isolated active factor was shown to be different from typical high density lipoproteins.

Conclusions

The trypanolytic activity of human serum can be inhibited by coincubation of bloodstream forms of T. b. brucei with weak bases. It is proposed that the Lytic activity is mediated via a receptor-ligand interaction leading to endocytosis and a perturbation of the normal intracellular processing of receptor-ligand complexes. The perturbation of the processing of the receptor-ligand complexes might be prevented by the uptake of weak bases. Electron microscopic data indicate that significant structural changes occur in the presence of normal human serum in the region between the flagellar pocket and the nucleus, where endosomes and Lysosomes and the Golgi complex are known to be localized. For a detailed analysis of the cellular biochemistry of serum-resistant and susceptible trypanosomes, it is now necessary to find out precisely to which class of proteins the isolated trypanolytic factor belongs.

References

1. LAVERAN, A. 1902. Comptes rendus de l'Académie des Sciences 134: 735-739.

5. SCHWARTZ, A.L., A. BOLOGNESI and S. FRIDOVICH. 1984. J. Cell Biol. 98: 732-738.

7. BRUN, R. and M. SCHOENENBERGER. 1981. Zeitschrift für Parasitenkunde 66: 17-24.

8. KROGSTAD, D.J. and P.H. SCHLESINGER. 1987. Am. J. Trop. Med. Hyg. 36: 213-220.

Table 1. Optimal concentration ranges of different weak bases to inhibit the trypanolytic activity

Compound	Inhibitory concentrations (m M)
Chloroquine	10-30
Amantadine	50_500
Tributylamine	1-10
Ammonium sulphate	3-10
Ammonium chloride	5-50

· a complex of high molecular mass (>1000 kd) determined using gel filtration
· a single molecule under non-reducing conditions
· the factor is separated into three to four peptides under reducing conditions
· the major peptide has a molecular mass of » 80 kd
· the factor is present in human serum at a concentration of » 50 mg/l
· the isolated factor is not identical with high-density lipoprotein

Normal human serum

Blue Sepharose (albumin depletion)

Q-Sepharose anion-exchange

Mono Q anion exchange (FPLC)

Polishing on Superose 6 (FPLC)

ACTIVE TRYPANOCIDAL FACTOR