Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Sep 14;107(37):16137-41.
doi: 10.1073/pnas.1007074107. Epub 2010 Aug 30.

Mechanism of Trypanosoma Brucei Gambiense (Group 1) Resistance to Human Trypanosome Lytic Factor

Affiliations
Free PMC article

Mechanism of Trypanosoma Brucei Gambiense (Group 1) Resistance to Human Trypanosome Lytic Factor

Rudo Kieft et al. Proc Natl Acad Sci U S A. .
Free PMC article

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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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 427–221S; lane 2, T. b. brucei 427–800R; lane 3, T. b. brucei 427–060R. 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 427–221S is marked by a black triangle; T. b. brucei 427–800R a red circle; T. b. brucei 427–060R a green square. (C) Analysis of Alexa Fluor–488–conjugated TLF-1 uptake by FAC. T. b. brucei 427–221S minus TLF-1 is blue; T. b. brucei 427–221S plus TLF-1 is black; T. b. brucei 427–800R plus TLF-1 is red; and T. b. brucei 427–060R 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.
Fig. 2.
Spontaneous reexpression of the TbbHpHbR gene restores susceptibility to TLF-1 in susceptible T. b. brucei 427–060R. (A) Third round of FACS for T. b. brucei 427–060R 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 427–060S. T. b. brucei 427–221S minus TLF-1 is blue; T. b. brucei 427–221S plus TLF-1 is black; T. b. brucei 427–060R plus TLF-1 is green; and T. b. brucei 427–060R (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 427–221S plus TLF-1 is black; T. b. brucei 427–060R plus TLF-1 is green; clonal line from the TLF-1–positive subpopulation in the third round of FACS T. b. brucei 427–060S 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 427–221S is marked as a black triangle; T. b. brucei 427–060R a green square; T. b. brucei 427–060S 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 427–060R; lane 2, T. b. brucei 427–060S.
Fig. 3.
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 427–800R 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.
Fig. 4.
Ectopic expression of the TbgHpHbR gene fails to rescue TLF-1 susceptibility to T. b. brucei 427–060R. (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 427–221S; lane 2, T. b. brucei 427–060R; lane 3, T. b. brucei 427–060_T. b. brucei rescueS; lane 4, T. b. brucei 427–060_T. b. gambiense rescueR. 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 427–221S is shown as a black triangle; T. b. brucei 427–060R a blue triangle; T. b. brucei 427–060_T. b. brucei rescueS a red triangle; and T. b. brucei 427–060_T. b. gambiense rescueR a green triangle. (D) Analysis of Alexa Fluor-488–conjugated TLF uptake by FACS. Samples are as in C.

Similar articles

See all similar articles

Cited by 47 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback