A Single Amino Acid Substitution in the Group 1 Trypanosoma brucei gambiense Haptoglobin-Hemoglobin Receptor Abolishes TLF-1 Binding

Abstract

Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1). This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR) on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT), escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR) mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens. African trypanosomes are parasites that are able to infect a wide variety of mammals; however, only two sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense, are able to infect humans. A human innate immune molecule, trypanosome lytic factor-1 (TLF-1), is responsible for this selective protection. TLF-1 killing requires high affinity binding to the trypanosome haptoglobin-hemoglobin receptor (HpHbR), which initiates endocytosis and lysosomal localization of the toxin. T. b. gambiense infects humans because it does not bind TLF-1, and several amino acid changes in the HpHbR are conserved in group 1 T. b. gambiense. To better understand the mechanism of resistance in these parasites, we analyzed TLF-1 binding to trypanosomes expressing the T. b. gambiense HpHbR (TbgHpHbR) and variants in which single amino acids were changed. Our studies showed that a single, highly conserved, amino acid substitution in the TbgHpHbR was sufficient to ablate high affinity TLF-1 binding and contributed to TLF-1 resistance. This likely plays a key role in human infectivity by group 1 T. b. gambiense.