Serum resistance-associated protein blocks lysosomal targeting of trypanosome lytic factor in Trypanosoma brucei

Abstract

Trypanosoma brucei brucei is the causative agent of nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I (apoA-I), apolipoprotein L-I (apoL-I), and haptoglobin-related protein (Hpr) provides this innate protection against T. b. brucei infection. This HDL subfraction, called trypanosome lytic factor (TLF), kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosoma brucei rhodesiense, which is morphologically and physiologically indistinguishable from T. b. brucei, is resistant to TLF-mediated killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a resistance-associated protein (SRA) that is able to ablate TLF killing. To examine the mechanism of TLF resistance, we transfected T. b. brucei with an epitope-tagged SRA gene. Transfected T. b. brucei expressed SRA mRNA at levels comparable to those in T. b. rhodesiense and was highly resistant to TLF. In the SRA-transfected cells, intracellular trafficking of TLF was altered, with TLF being mainly localized to a subset of SRA-containing cytoplasmic vesicles but not to the lysosome. These results indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine the organism's susceptibility to TLF.

FIG. 1.

Diagram of SRA gene construct and PCR analysis of transfectants. (A) Diagram of SRA gene indicating the locations of the Ty epitope tag, signal sequence, proposed α-helical domains, and a proposed glycosylphosphatidylinositol (GPI) anchor site. SRA-Ty was cloned into the transfection vector pURAN, consisting of a pUC9 backbone, the neomycin resistance gene (Neo^r), and sequences for targeting the rRNA promoter (ribosomal [marked with a flag]) following cleavage with the restriction enzyme SunI (25). Correct RNA processing and stability were provided by α-tubulin splice acceptor (α-tub sa) and αβ-tubulin intergenic (αβ-tub ir) sequences. The Ty epitope was cloned into the SRA gene at a unique HindIII site at 848 bp (accession no. AF097331) (2). (B) PCR amplification of SRA and tubulin genes from genomic DNAs isolated from trypanosomes transfected with pURAN vector alone (T. b. brucei 427, 667, and 927) or with pURAN containing SRA (T. b. brucei 427-SRA, 667-SRA, and 927-SRA). The PCR products for the SRA and tubulin genes run at 1.4 kb and 0.7 kb, respectively.

FIG. 2.

Transfection of three T. b. brucei lines with SRA confers resistance to TLF. Lysis assays were conducted with T. b. brucei transfected with empty vector (427 [▪], 667 [•], and 927 [▴]) and with SRA-Ty vector (427-SRA [□], 667-SRA [○], and 927-SRA [▵]). The percentages of trypanosomes lysed by increasing amounts of TLF under standard assay conditions were determined following a 4-h incubation at 37°C.

FIG. 3.

Expression of SRA mRNA and SRA protein in transfected T. b. brucei. (A) Northern blot analysis of total cell RNA from transfected and nontransfected T. b. brucei 427. RNA samples from T. b. brucei transfected with either empty vector (427) or SRA-containing vector (427-SRA) or from human-serum-resistant T. b. rhodesiense [T. b. r. (R)] were separated in 1% agarose gels and hybridized with probes specific for the VSG mRNA from T. b. brucei 427 (VSG-221) or wild-type T. b. rhodesiense (VSG-R) and for SRA mRNA. Ethidium bromide staining of the agarose gel is shown in the top panel. (B) Protein gel and Western blot of T. b. brucei 427. The Coomassie-stained gel and Western blot analysis show results for protein extracts from T. b. brucei transfected with vector alone (427) or with SRA (427-SRA-Ty). The Western blot was probed with antibodies to the Ty epitope, and proteins of approximately 59 and 65 kDa were detected in extracts from T. b. brucei 427-SRA-Ty (arrows).

FIG. 4.

Cellular localization of SRA in T. b. brucei 427-SRA-Ty. Immunofluorescence analysis was done with cells treated with antibodies to Ty epitope-tagged SRA and to the ER marker BiP (A to D), the endocytic marker TL (E to H), or the lysosomal marker p67 (I to L). The positions of the nucleus (N) and the kinetoplast (K) were visualized by staining with DAPI (blue). (A, E, and I) Cells visualized by bright-field (BF) microscopy overlaid with DAPI-stained DNA. (B) Cell stained with anti-BiP (red). (F) Cell following uptake of Alexa-conjugated TL (red). (J) Cell stained with anti-p67 (red). (C, G, and K) Cells stained for SRA with anti-Ty (green). (D, H, and L) Overlays of images of cells stained for DNA/BiP/SRA, DNA/TL/SRA, or DNA/p67/SRA, respectively.

FIG. 5.

Cellular localization of TL and TLF taken up by wild-type T. b. brucei 427 (A to D) and T. b. brucei 427-SRA-Ty (E to H). The positions of the nucleus and the kinetoplast were visualized by staining with DAPI (blue). (A and E) Cells visualized by bright-field (BF) microscopy overlaid with DAPI-stained DNA. Cells are shown following incubation with Alexa-conjugated TLF (red) (B and F) or Alexa-conjugated TL (green) (C and G). (D and H) Overlays of images of cells stained for DNA and visualized for the appearance of Alexa-conjugated TLF and TL. The arrows in panel D indicate the sites of TLF and TL colocalization in wild-type T. b. brucei.

FIG. 6.

Cellular localization of TLF relative to p67 in T. b. brucei 427 (A to D) and T. b. brucei 427-SRA-Ty (E to H) in the presence of chloroquine. The positions of the nucleus and the kinetoplast were visualized by staining with DAPI (blue). (A and E) Cells visualized by bright-field (BF) microscopy overlaid with DAPI staining. (B and F) Cells incubated with Alexa-conjugated TLF (red). (C and G) Cells stained with anti-p67 (green). (D and H) Overlays of images of cells incubated with Alexa-conjugated TLF and stained for DNA and p67.

FIG. 7.

Colocalization of TLF-1 and SRA in T. b. brucei 427-SRA-Ty. The positions of the nucleus and the kinetoplast were visualized by staining with DAPI (blue) (A to H). (A and E) Cells visualized by bright-field (BF) microscopy overlaid with DAPI staining of the nucleus and kinetoplast. (B and F) Cells incubated with Alexa-conjugated TLF (red). (C and G) Cells stained with anti-Ty (green) to visualize SRA. (D and H) Overlaid images of cells treated with Alexa-conjugated TLF and stained for DNA and SRA.