Hemoglobin Is a Co-Factor of Human Trypanosome Lytic Factor

Abstract

Trypanosome lytic factor (TLF) is a high-density lipoprotein (HDL) subclass providing innate protection to humans against infection by the protozoan parasite Trypanosoma brucei brucei. Two primate-specific plasma proteins, haptoglobin-related protein (Hpr) and apolipoprotein L-1 (ApoL-1), have been proposed to kill T. b. brucei both singularly or when co-assembled into the same HDL. To better understand the mechanism of T. b. brucei killing by TLF, the protein composition of TLF was investigated using a gentle immunoaffinity purification technique that avoids the loss of weakly associated proteins. HDL particles recovered by immunoaffinity absorption, with either anti-Hpr or anti-ApoL-1, were identical in protein composition and specific activity for T. b. brucei killing. Here, we show that TLF-bound Hpr strongly binds Hb and that addition of Hb stimulates TLF killing of T. b. brucei by increasing the affinity of TLF for its receptor, and by inducing Fenton chemistry within the trypanosome lysosome. These findings suggest that TLF in uninfected humans may be inactive against T. b. brucei prior to initiation of infection. We propose that infection of humans by T. b. brucei causes hemolysis that triggers the activation of TLF by the formation of Hpr–Hb complexes, leading to enhanced binding, trypanolytic activity, and clearance of parasites.

Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: Relevance to trypanosome lysis

“…The proposed model would also explain the morphological changes that are consistently demonstrated to precede trypanolysis, whether it is initiated by recombinant APOL1, TLF1/2, or normal human serum: these changes are characterized by a rounding of the cell body and swelling of the cytoplasm (12,23,25,26,40). In cases where swelling of the cell body proceeded slowly, and completion of lysis required several hours, swelling of the lysosome was also demonstrated (25,26), but lysosomal swelling was not obviously apparent when lysis was driven rapidly to completion in 2 h or less (12,23,40).…”

“…In cases where swelling of the cell body proceeded slowly, and completion of lysis required several hours, swelling of the lysosome was also demonstrated (25,26), but lysosomal swelling was not obviously apparent when lysis was driven rapidly to completion in 2 h or less (12,23,40). Therefore, it is our opinion that cytoplasmic swelling, as a result of osmolyte influx across the plasma membrane, is the defining feature of trypanosome lysis by APOL1 and that lysosomal swelling can occur as a consequence, but only if sufficient time is allowed before lysis occurs.…”

“…In addition, APOL1 transgenesis in mice (which normally lack both APOL1 and Hpr-encoding genes) is sufficient to prevent infection with human serum-sensitive, but not human serum-resistant, trypanosomes (11). Hpr-hemoglobin, in the context of TLF1 particles, may also contribute directly to killing via the generation of oxygen free-radicals in the parasite lysosome, particularly under conditions of intravascular hemolysis (12).…”

Structural basis for trypanosomal haem acquisition and susceptibility to the host innate immune system

“…Despite their structural similarity and common ability to bind Hb, Hp and Hpr serve quite different biological functions. Hp is responsible for binding and neutralizing the toxic Hb released from red blood cells and subsequent scavenging by macrophages via its interaction with the receptor CD163 (refs 9,39), whereas Hpr in complex with Hb mediates delivery of toxic TLF particles to trypanosome parasites 16,17 .…”

“…TLF1 particles enter the parasite via HpHbRmediated uptake 15 . The uptake of TLF1 requires Hb, which interacts with Hpr to form an Hpr-Hb complex 16 . As HpHbR, in contrast to CD163, also recognizes Hpr-Hb 5 , the entire TLF1 particle is endocytosed by the parasite 17 .…”