Mechanism of Trypanosoma brucei gambiense (group 1) resistance to human trypanosome lytic factor

Abstract

Human innate immunity against most African trypanosomes, including Trypanosoma brucei brucei, is mediated by a minor subclass of toxic serum HDL, called trypanosome lytic factor-1 (TLF-1). This HDL contains two primate specific proteins, apolipoprotein L-1 and haptoglobin (Hp)-related protein, as well as apolipoprotein A-1. These assembled proteins provide a powerful defense against trypanosome infection. Trypanosoma brucei rhodesiense causes human African sleeping sickness because it has evolved an inhibitor of TLF-1, serum resistance-associated (SRA) protein. Trypanosoma brucei gambiense lacks the SRA gene, yet it infects humans. As transfection of T. b. gambiense (group 1) is not possible, we initially used in vitro-selected TLF-1-resistant T. b. brucei to examine SRA-independent mechanisms of TLF-1 resistance. Here we show that TLF-1 resistance in T. b. brucei is caused by reduced expression of the Hp/Hb receptor gene (TbbHpHbR). Importantly, T. b. gambiense (group 1) also showed a marked reduction in uptake of TLF-1 and a corresponding decrease in expression of T. b. gambiense Hp/Hb receptor (TbgHpHbR). Ectopic expression of TbbHpHbR in TLF-1-resistant T. b. brucei rescued TLF-1 uptake, demonstrating that decreased TbbHpHbR expression conferred TLF-1 resistance. Ectopic expression of TbgHpHbR in TLF-1-resistant T. b. brucei failed to rescue TLF-1 killing, suggesting that coding sequence changes altered Hp/Hb receptor binding affinity for TLF-1. We propose that the combination of coding sequence mutations and decreased expression of TbgHpHbR directly contribute to parasite evasion of human innate immunity and infectivity of group 1 T. b. gambiense.

Fig. 1.

T. b. brucei resistance to TLF-1 correlates with decreased uptake and reduced levels of TbbHpHbR mRNA. (A) Northern blot hybridization analysis with specific probes for VSGs and β-tubulin. Lane 1, T. b. brucei 427–221^S; lane 2, T. b. brucei 427–800^R; lane 3, T. b. brucei 427–060^R. β-Tubulin was used as a control. (B) In vitro susceptibility of the T. b. brucei cell lines to TLF-1. Two-hour lysis assays were carried out with increasing concentration of TLF-1 [expressed as units (2)]. T. b. brucei 427–221^S is marked by a black triangle; T. b. brucei 427–800^R a red circle; T. b. brucei 427–060^R a green square. (C) Analysis of Alexa Fluor–488–conjugated TLF-1 uptake by FAC. T. b. brucei 427–221^S minus TLF-1 is blue; T. b. brucei 427–221^S plus TLF-1 is black; T. b. brucei 427–800^R plus TLF-1 is red; and T. b. brucei 427–060^R plus TLF is green. Each cell line was designated by the expressed VSG and its susceptibility to TLF-1. (D) Northern blot analysis of total RNA with probes specific for TbbHpHbR (Hp/Hb receptor) and β-tubulin mRNAs. Lanes are as in A.

Fig. 2.

Spontaneous reexpression of the TbbHpHbR gene restores susceptibility to TLF-1 in susceptible T. b. brucei 427–060^R. (A) Third round of FACS for T. b. brucei 427–060^R cells that bound Alexa Fluor–488–conjugated TLF-1. Successive rounds of sorting enriched for a subpopulation of cells that bound TLF-1, T. b. brucei 427–060^S. T. b. brucei 427–221^S minus TLF-1 is blue; T. b. brucei 427–221^S plus TLF-1 is black; T. b. brucei 427–060^R plus TLF-1 is green; and T. b. brucei 427–060^R (third FACS sort) plus TLF-1 is orange. SI Materials and Methods provides details of FACS. (B) Analysis of Alexa Fluor-488–conjugated TLF-1 uptake by FAC. T. b. brucei 427–221^S plus TLF-1 is black; T. b. brucei 427–060^R plus TLF-1 is green; clonal line from the TLF-1–positive subpopulation in the third round of FACS T. b. brucei 427–060^S plus TLF-1 is red. (C) In vitro TLF-1 lysis assay: percentage of cells lysed following incubation with TLF for 2 h at 37 °C. T. b. brucei 427–221^S is marked as a black triangle; T. b. brucei 427–060^R a green square; T. b. brucei 427–060^S a red circle. (D) Northern blot analysis of total RNA with probes specific for VSG 060, TbbHpHbR (Hp/Hb receptor), and β-tubulin mRNAs. Lane 1, T. b. brucei 427–060^R; lane 2, T. b. brucei 427–060^S.

Fig. 3.

Group 1 T. b. gambiense shows reduced TLF-1 uptake and expression of the TbgHpHbR gene. (A) Uptake of TLF-1 examined by fluorescence microscopy. Images are typical examples showing phase contrast, DAPI staining of the nucleus and kinetoplast, uptake of Alexa Fluor–488–conjugated TLF-1, and identification of the lysosome by lysotracker. (1) T. b. brucei, strain STIB247; (2) T. b. rhodesiense, strain Baganzi; (3) group 1 T. b. gambiense, strain Eliane. (B) Relative expression of Hp/Hb receptor genes was determined using real-time RT-PCR. T. b. brucei STIB247 black; T. b. rhodesiense, strain Baganzi, is green; and the following are all red: T. b. gambiense A1 (group 1 T. b. gambiense strain Eliane, in vitro; Côte d'Ivoire), T. b. gambiense A2 (group 1 T. b. gambiense strain Eliane from mice; Côte d'Ivoire), T. b. gambiense B (group 1 T. b. gambiense strain from mice, “Tobo”; Côte d'Ivoire), T. b. gambiense C (group 1 T. b. gambiense strain “Isti”; from mice, Côte d'Ivoire), T. b. gambiense D (group 1 T. b. gambiense strain “Bim”; from mice, Cameroon), T. b. gambiense E (group 1 T. b. gambiense strain “Mos”; from mice, Cameroon), T. b. gambiense F (group 1 T. b. gambiense strain “Pa”; from mice, Democratic Republic of Congo; ± 1 SD). (C) Analysis of Alexa Fluor-488–conjugated TLF-1 uptake by FAC. T. b. brucei, strain STIB247 minus TLF-1 is orange; T. b. brucei, strain STIB247 plus TLF-1 is black; T. b. brucei 427–800^R plus TLF-1 is blue; T. b. rhodesiense KETRI2482 plus TLF-1 is green; and T. b. gambiense group 1, strain Eliane, plus TLF-1 is red.

Fig. 4.

Ectopic expression of the TbgHpHbR gene fails to rescue TLF-1 susceptibility to T. b. brucei 427–060^R. (A) Schematic showing the predicted mRNAs from the endogenous TbbHpHbR gene and the complete ORF for either the TbbHpHbR or TbgHpHbR genes ectopically expressed from the tubulin locus with actin A 5′UTR and α-tubulin 3′UTR. (B) RT-PCR analysis of mRNAs from endogenous TbbHpHbR (Top) or ectopically expressed TbbHpHbR or TbgHpHbR (Middle). Lane 1, T. b. brucei 427–221^S; lane 2, T. b. brucei 427–060^R; lane 3, T. b. brucei 427–060_T. b. brucei rescue^S; lane 4, T. b. brucei 427–060_T. b. gambiense rescue^R. Enolase expression was analyzed as a loading control (Bottom). (C) In vitro TLF lysis assay: percentage of cells lysed following incubation with TLF-1 for 2 h at 37 °C. T. b. brucei 427–221^S is shown as a black triangle; T. b. brucei 427–060^R a blue triangle; T. b. brucei 427–060_T. b. brucei rescue^S a red triangle; and T. b. brucei 427–060_T. b. gambiense rescue^R a green triangle. (D) Analysis of Alexa Fluor-488–conjugated TLF uptake by FACS. Samples are as in C.