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

Abstract

Human innate immunity against the veterinary pathogenTrypanosoma 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.

The Cell Biology of APOL1

“…That is, does APOL1 directly stimulate autophagic pathways or does APOL1-mediated cell death trigger secondary activation of autophagic pathways? APOL1-dependent cytotoxicity results from ion fluxes after its insertion into the plasma membrane 38, 39 , similar to mechanisms of trypanolysis reported by some but not all investigators 13, 14 . Pore forming toxins of pathogens can activate autophagy in cells 47, 48 suggesting that the observed induction of autophagy by APOL1 expression systems may be a cellular response to APOL1 mediated channel activity.…”

“…The initial report showed lysosomal membrane targeting resulting in chloride influx 11 . However others demonstrated APOL1 targets the plasma membrane through endosomal recycling and functions as a non-selective, cation channel 13,14,18 . In contrast to data that have demonstrated low pH allows ion fluxes, other investigators have found that APOL1, inserted into membranes in acid environments, remains inactive, only permitting ion flux when exposed to physiological pH 18 .…”

“…Lysosomal swelling may be the proximate cause of trypanosomal death 11 . Other data suggest sodium influx through APOL1 pores in the plasma membrane generates osmotic stress with cytoplasmic swelling and trypanolysis 13,14,18 APOL1-depedent osmotic lysis can be blocked by the anti-oxidant DPPD, suggesting APOL1-dependent oxidative stress also results from its pore-forming activity 14 . Trypanolysis has been dissociated from lysosomal swelling 15 , suggesting the lysosomal localization of APOL1 may reflect the lysosome’s role as a detoxifying pathway for the parasite rather than key effector of parasite lysis.…”

ApoL1 Overexpression Drives Variant-Independent Cytotoxicity

“…10 Its ability to form channels in lipid bilayers is central to its trypanolytic activity. [11][12][13][14] The APOL1-G2 variant confers enhanced protection from infection with the trypanosomal subspecies, Trypanosoma brucei rhodesiense, and the APOL1-G1 variant is associated with less severe clinical disease after infection with another trypanosomal subspecies, Trypanosoma brucei gambiense. 15 These trypanosomal subspecies cause African sleeping sickness in humans and a single APOL1-G1 or -G2 allele confers these selective advantages, which have likely driven their high frequencies in black populations.…”

“…26,28,30 Lysosomal damage, a proposed mechanism of trypanolysis, 12 has also been implicated in APOL1-mediated mammalian cytotoxicity. 26 Finally, similar to other trypanolytic mechanisms, [11][12][13][14] APOL1-mediated cell death can result from its channel activity when it inserts into the plasma membrane, which allows loss of cellular ion gradients, osmotic swelling, and ultimately cell death. 28,30 A key question that remains outstanding is whether the APOL1-G1 and -G2 cause differential effects on cytotoxicity compared with APOL1-G0, and whether cytotoxicity is the mechanism driving the pathogenesis of APOL1-associated kidney diseases.…”