Distinct roles of haptoglobin-related protein and apolipoprotein L-I in trypanolysis by human serum

Abstract

Apolipoprotein L-I (apoL-I) is a human high-density lipoprotein (HDL) component able to kill Trypanosoma brucei brucei by forming anion-selective pores in the lysosomal membrane of the parasite. Another HDL component, haptoglobin-related protein (Hpr), has been suggested as an additional toxin required for full trypanolytic activity of normal human serum. We recently reported the case of a human lacking apoL-I (apoL-I(-/-)HS) as the result of frameshift mutations in both apoL-I alleles. Here, we show that this serum, devoid of any trypanolytic activity, exhibits normal concentrations of HDL-bound Hpr. Conversely, the serum of individuals with normal HDL-bound apoL-I but who lack Hpr and haptoglobin [Hp(r)(-/-)HS] as the result of gene deletion (anhaptoglobinemia) exhibited phenotypically normal but delayed trypanolytic activity. The trypanolytic properties of Hp(r)(-/-)HS were mimicked by free recombinant apoL-I, whereas recombinant Hpr did not affect trypanosomes. The lysis delay observed with either Hp(r)(-/-)HS or recombinant apoL-I could entirely be attributed to a defect in the uptake of the lytic components. Thus, apoL-I is responsible for the trypanolytic activity of normal human serum, whereas Hpr allows fast uptake of the carrier HDL particles, presumably through their binding to an Hp/Hpr surface receptor of the parasite.

Fig. 1.

Characteristics of apoL-I ^−/− and Hp(r) ^−/− human sera. NHS, apoL-I^−/−HS, and Hp(r)^−/−HS were fractionated by immunoaffinity chromatography on an anti-apoA-I column in the absence of detergents. (A) Coomassie blue staining of equivalent volumes of unfractioned serum (W, whole), apoA-I depleted serum (FT, flow-through), and bound fraction (BF). The identity of apoA-I was checked by immunodetection (data not shown). (B) Immunodetection of apoL-I, Hp α2 chain, and Hpr α chain.

Fig. 2.

Trypanolytic potential of various sera. The trypanosomes, isolated from mice, were incubated at 1.10⁵/ml in HMI-9 for 24 h with 10% of the indicated serum. After 24 h, living trypanosomes were counted in triplicate under the microscope. In this and Figs. 3–6, errors bars represent SD from three independent experiments, and the trypanosome concentrations are normalized to growth in 10% FCS. Growth of ETat 1.2R in 100% NHS was normalized to that in 100% FCS. (A) ETaT 1.2S (NHS-sensitive) trypanosomes were incubated in various sera containing or not containing 8.5 μg/ml free recombinant apoL-I or 150 μg/ml free recombinant Hpr. (B) ETaT 1.2S and ETaT 1.2R (NHS-resistant) trypanosomes were incubated in various serum concentrations. At serum concentrations <10%, FCS was added to maintain a final 10% serum concentration.

Fig. 3.

Characteristics of trypanolysis by various sera. (A) In situ immunofluorescence of the lysosomal membrane protein p67 detected by Alexa 488 (green)-coupled antibodies in ETaT 1.2S trypanosomes incubated in 30% FCS, NHS, or Hp(r)^−/−HS. Large and small blue dots are DAPI-stained nucleus and kinetoplast, respectively. The arrows point to the swollen lysosome. (B) Inhibition of ETaT 1.2S lysis by 50% NHS or Hp(r)^−/−HS after incubation [3 h for NHS, 6 h for Hp(r)^−/−HS] at 4°C or at 37°C in the presence or absence of 25 μM chloroquine (ClQ) or 1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS). (C) ETaT 1.2S lysis kinetics in various sera. (D) Survival percentage of ETaT 1.2S (solid curves) or ETaT 1.2R (dashed curve) trypanosomes incubated with log dilutions of the indicated sera for 24 h. In the undiluted FCS recombinant, apoL-I was supplemented at physiological concentration (8.5 μg/ml).

Fig. 4.

Involvement of Hpr in TLF binding and uptake. (A) ETat 1.2S trypanosomes were preincubated at 4°C or 37°C for various periods of time in 10% different sera, sometimes supplemented with 8.5 μg/ml recombinant apoL-I as indicated, and then, after repeated washing steps, resuspended in 10% FCS and incubated for 24 h at 37°C. (B) Survival percentage of ETat 1.2S trypanosomes incubated for 24 h with log dilutions of different sera supplemented or not with 200 μg/ml of Hp or Hpr.

Fig. 5.

Specific uptake of Hp in T. brucei. ETat 1.2S parasites were incubated at 37°C with 20 μg/ml Alexa-labeled Hp or BSA. Internalized Hp localized in the endocytic pathway between the kinetoplast and nucleus (small and large DAPI-stained spot, respectively). The fluorescence of the two proteins was equivalent, as measured on serial 2-fold dilutions of ≈20 mg/ml samples.

Fig. 6.

Evidence that Hpr does not synergize apoL-I activity. The time periods required for full lysis of ETat 1.2S parasites were plotted against those required for commitment to lysis by various dilutions of different sera [from left to right: 3.3, 10, 20, 50, 66, and 100 × dilution of NHS; 2.2, 3.3, and 6.7 × dilution of Hp(r)^−/−HS; 1 and 1.3 × dilution of FCS + apoL-I].