Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei

Abstract

Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N'-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis.

Keywords: Trypanosoma brucei; cell death; innate immunity; osmotic swelling; oxidative stress; parasite; trypanosome.

FIGURE 1.

Oxidative stress is involved in TLF-1-induced lysis. A, TLF-1 lysis assay (8.8 nm), 2 h at 37 °C following preincubation with DPPD (blue diamonds) for 30 min at 37 °C. Gray squares indicated no TLF-1. B, kinetics of ROS production measured in T. brucei brucei pretreated for 30 min with 30 μm DPPD (blue) or nothing (black). In the indicated samples, 50 μm H₂O₂ (solid lines) or 5 μl of H₂O (dashed lines) was added at 0 min (solid lines). n = 4 for peroxide-treated samples with standard deviation bars, whereas untreated samples are in duplicate with no error bars shown. C, TLF-1 lysis assay at concentrations shown on the x axis, 2 h at 37 °C. TLF-1 treatment only (blue diamonds). TLF-1 treatment with concurrent addition of 40 μm tertbutyl hydroperoxide (+OOH) (red circles). D, TLF-1 lysis assay (8.8 nm), 2 h at 37 °C with concurrent addition of DEM (blue diamonds). Gray squares indicate no TLF-1. E, thiol determination assay. DEM at the indicated concentrations was preincubated with T. brucei brucei for 110 min at 37 °C before thiol determination on flow cytometry. F, TLF-1 lysis assay (3 nm) for 2 h at 37 °C with the indicated concentrations NEM added 1 h after TLF-1 addition (blue diamonds). Gray squares indicate no TLF-1. G, TLF-1 lysis assay (1.7 nm) for 2 h at 37 °C with the indicated concentrations BrBi added 1 h after TLF-1 addition (blue diamonds). Gray squares indicated no TLF-1. The error bars for lysis assays show standard deviations of three or four counts from one representative lysis assay.

FIGURE 2.

Hemoglobin causes peroxidation of TLF-1 lipids. A, in vitro TLF lipid peroxidation assay. TLF-1 (125 nm) was incubated for 30 min with native Hb, or Fe³⁺, Fe²⁺ Zn²⁺, or apo-globin in pH 4.8 buffer with 70 μm H₂O₂. All globins except Hb were prepared from acid-acetone precipitation of globin and reconstitution with free porphyrins. Malondialdehyde production was measured by the thiobarbituric acid reactive substances assay. Average and standard deviation error bars are from two samples each from two independent experiments. B, native PAGE gel mobility shift assay for globin binding to Hp1-1. A representative gel is shown. Human Hp1-1 was incubated with increasing mol eq of Hb, Fe³⁺, or Zn²⁺-globins. Brightness and contrast adjusted on each gel in Microsoft PowerPoint. Hb samples run on two separate gels. C, measurement of TLF-1 uptake by flow cytometry. 20-min uptake assay at 37 °C in serum-free medium with the indicated globin. Averages of three independent experiments with standard deviation error bars are shown. n = 2 for TLF-1 and apo-globin. One of the three replicas labeled as Hb is actually reconstituted Fe²⁺ globin.

FIGURE 3.

Hb-mediated peroxidation is not required for TLF-1 lysis of T. brucei brucei. A, TLF-1 lysis assay for 2 h at 37 °C in serum-free medium with 100 mol eq of either Hb (red circles), Zn²⁺globin (green triangles), Fe³⁺globin (blue diamonds), apo-globin (yellow circles), or no hemoglobin (gray squares). No differences between Hb and Zn-globin or Fe-globin had a p value less than 0.01. B, TLF-1 lysis assay (2.5 nm) for 2 h at 37 °C in serum-free medium with TLF-1 (10 nm) and 100 equivalents of the Hb (red circles), Zn²⁺globin (green triangles), and Fe³⁺globin (blue diamonds). T. brucei brucei were preincubated for 1 h with either DPPD (30 μm) prior to TLF-1 addition. DIDS (300 μm) was added concurrently with TLF-1. The error bars show standard deviation of three counts from one representative lysis assay.

FIGURE 4.

Lysosomal lipid peroxidation is not required for TLF-1 lysis of T. brucei brucei. A, TLF-1 lysis assay for 2 h at 37 °C in serum-free medium, after preincubation for 1.5 h with holotransferrin (100 μm FeTf, red triangles), apotransferrin (100 μm apo-Tf, green circles), or serum-free medium alone (blue diamonds). B, TLF-1 lysis assay in WT cells (3.75 nm) for 2 h at 37 °C, with Trolox added concurrently with TLF-1. C, TLF-1 lysis assay for 2 h at 37 °C with T. brucei brucei Px KO cells. The cells were washed from 100 μm Trolox and resuspended in medium with the indicated Trolox concentration. Lysis was determined with 1.5 nm TLF-1 (purple squares), 3 nm TLF-1 (red circles), no TLF-1 (black circles), or 1.5 μm Hp1-1 with 50 equivalents Hb (gray triangles). D, TLF-1 lysis assay for 2 h at 37 °C with WT and Px KO cells. Cells were washed from 100 μm Trolox and resuspended in medium with no Trolox and WT cells (black squares), 25 μm Trolox and WT cells (blue triangles), 100 μm Trolox and WT cells (green circles), 25 μm Trolox and Px KO cells (red triangles), or 100 μm Trolox and Px KO cells (purple circles). E, survival assay with WT and Px KO T. brucei brucei. A legend is shown in the inset. All error bars show standard deviation of three counts from one representative lysis assay.

FIGURE 5.

ApoL1 initiates lysis stimulated by oxidation. A, ApoL1 lysis assay for 6 h at 37 °C in HpHbR KO T. brucei brucei following preincubation for 1 h with 30 μm DPPD. B, lysis assay for 5 h 37 °C in HpHbR KO T. brucei brucei with TLF-1 and ApoL1 both ∼50 nm; DEM added 1 h after start of lysis for 0.2 or 0.4 mm DEM. C, lysis assay for 4 h at 37 °C in HpHbR KO T. brucei brucei with or without 40 μm +OOH added 1 h after the start of the assay. All error bars show standard deviation of three counts from one representative lysis assay.

FIGURE 6.

TLF-1 trafficking from the endosomes precedes peroxide sensitivity. A, TLF-1 lysis assay (0.4 nm) for 2 h at 37 °C. H₂O₂ (40 μm) added to aliquots every 10 min from the start of the assay (0 min) until 120 min. See diagram below figure for experimental set-up. B, TLF-1 (4 nm) was preloaded into the endosomes at 15 °C for 1 h, washed at 4 °C, and shifted to 37 °C at 0 min as shown in the diagram below the figure. H₂O₂ (40 μm) was added once to each aliquot of TLF-1-treated T. brucei brucei at the indicated times, and lysis was recorded after 100 min at 37 °C. All error bars show standard deviation of three counts from one representative lysis assay.

FIGURE 7.

TLF-1 treatment does not cause global oxidative damage to T. brucei brucei. A, kinetics of ROS production by cells treated with 10 nm TLF-1 (no Hb) added at 0 min (red lines) or without TLF-1 (black lines). Each graph shows a different sample from the same experiment, with an arrow indicating the time(s) when 50 μm H₂O₂ were added to the wells. The dotted lines show TLF-1 and control samples without H₂O₂ added as a reference in each graph. Less than 20% lysis caused by TLF-1 or H₂O₂ was measured at the end point of this assay, although TLF-1-treated cells had begun to swell. B, thiol determination assay. At the start of the assay, TLF-1 (2 or 20 nm) or DEM (0.6 mm) was added to cells at 37 °C. Flow cytometry analysis of free thiols was performed at the times shown on the x axis. A representative assay is shown (n = 1). C, percentage of PI-positive cells of total cells counted by flow cytometry in B.

FIGURE 8.

TLF-1 causes osmotic stress to T. brucei brucei. A, differential interference contrast images of T. brucei brucei treated at 37 °C in culture medium with either 10 nm TLF-1 (with Hb) for 75 min, 100 nm ApoL1 (in HpHbR KO cells) for 5 h, or 60% diluted hypotonic media for less than 5 min. B, 2-h lysis assay. The addition of sucrose to the isotonic growth medium saves T. brucei brucei from TLF-1 lysis if added concurrently with TLF-1 or 1 h into the assay. C, hypotonic assay (5 min). Aliquots of cells incubated with 8 nm TLF for the time indicated on the x axis were placed under hypotonic shock for 5 min and then counted. n = 1 for TLF-1-treated T. brucei brucei in isotonic buffer, and 60 and 70% hypotonic shock shows the averages of three independent assays. The error bars show standard deviation.

FIGURE 9.

Oxidation induces osmotic lysis of T. brucei brucei. A, hypotonic assay (5 min) with indicated concentrations H₂O₂ added concurrently with hypotonic shock into 60% hypotonic medium (red circles), 65% hypotonic medium (blue diamonds), 70% hypotonic medium (green triangles), or isotonic medium (gray squares). The error bars show standard deviation from six counts from three assays. B, kinetics of ROS production measured by DCF-DA fluorescence in T. brucei brucei diluted into either 50% DMEM (black lines) or water (red lines), with (solid lines) or without (dashed lines) concurrent addition of 50 μm H₂O₂. C, hypotonic assay (5 min) on T. brucei brucei preincubated for 1 h at 37 °C with DMSO (0.5%), or 60 μm DPPD. Control cells had no added DPPD. The error bars show the standard deviation of six counts from three assays. D, hypotonic assay (5 min) with 0.8 mm DEM (blue bars) preincubated with T. brucei brucei for 30 min. No DEM (black bars). The error bars show standard deviation from six counts from three assays. E, hypotonic assay (5 min) on cells preincubated for 30 min at 37 °C with NEM. Legend indicates the percentage of hypotonic medium. The error bars show standard deviation from six counts from three assays. F, hypotonic assay (5 min) on cells preincubated for 30 min at 37 °C with BrBi. The legend indicates the percentage of hypotonic medium. The error bars show standard deviation from six counts from three assays. Isotonic controls are 70% PBS rather than 100% media. G, hypotonic assay for 30 min in 50% hypotonic buffer. H₂O₂ (10 μm) was added at the beginning of the assay (0 min) and in 5-min intervals subsequently (black bars). Peroxide addition at 0 min in isotonic buffer is shown as a gray striped bar. The error bars show standard deviation for four counts from two assays.

FIGURE 10.

Model for oxidation-stimulated osmotic lysis of T. brucei brucei via ApoL1 trafficking to the plasma membrane. TLF-1 is endocytosed via the HpHbR in the flagellar pocket of T. brucei brucei. Inside an acidic endosomal vesicle, ApoL1 inserts into the membrane. The sodium channel activity of ApoL1 is inhibited in acidic conditions; thus, an × is drawn over the channel. The series of steps leading to trypanosome lysis are indicated in the figure. First, after recycling to the plasma membrane (PM) ApoL1 causes early ionic disruption and osmotic stress to T. brucei brucei at the plasma membrane (shown in purple). Next, peroxide-induced oxidation of cellular thiols stimulates osmotic deregulation (shown in blue). Finally, lytic cell death is caused by excessive influx of water through the plasma membrane of T. brucei brucei (shown in green).