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. 2009 Dec;5(12):e1000685.
doi: 10.1371/journal.ppat.1000685. Epub 2009 Dec 4.

C-terminal mutants of apolipoprotein L-I efficiently kill both Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

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C-terminal mutants of apolipoprotein L-I efficiently kill both Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

Laurence Lecordier et al. PLoS Pathog. 2009 Dec.

Abstract

Apolipoprotein L-I (apoL1) is a human-specific serum protein that kills Trypanosoma brucei through ionic pore formation in endosomal membranes of the parasite. The T. brucei subspecies rhodesiense and gambiense resist this lytic activity and can infect humans, causing sleeping sickness. In the case of T. b. rhodesiense, resistance to lysis involves interaction of the Serum Resistance-Associated (SRA) protein with the C-terminal helix of apoL1. We undertook a mutational and deletional analysis of the C-terminal helix of apoL1 to investigate the linkage between interaction with SRA and lytic potential for different T. brucei subspecies. We confirm that the C-terminal helix is the SRA-interacting domain. Although in E. coli this domain was dispensable for ionic pore-forming activity, its interaction with SRA resulted in inhibition of this activity. Different mutations affecting the C-terminal helix reduced the interaction of apoL1 with SRA. However, mutants in the L370-L392 leucine zipper also lost in vitro trypanolytic activity. Truncating and/or mutating the C-terminal sequence of human apoL1 like that of apoL1-like sequences of Papio anubis resulted in both loss of interaction with SRA and acquired ability to efficiently kill human serum-resistant T. b. rhodesiense parasites, in vitro as well as in transgenic mice. These findings demonstrate that SRA interaction with the C-terminal helix of apoL1 inhibits its pore-forming activity and determines resistance of T. b. rhodesiense to human serum. In addition, they provide a possible explanation for the ability of Papio serum to kill T. b. rhodesiense, and offer a perspective to generate transgenic cattle resistant to both T. b. brucei and T. b. rhodesiense.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Lytic activities of apoL1.
A. Schematic representation of apoL1 and apoL1 variants analyzed in this work, with indication of the amino acid residues delineating the three protein domains (see ref. 4 for details). B. Colicin-like activity of WT and mutant apoL1. pCDF-DUET plasmids encoding apoL1 variants provided with a C-terminal V5-His6 tag were transfected into E. coli BL21(DE3). In the case of the deletion (del) variants, an N-terminal bacterial signal peptide (pelB) was added. After overnight incubation at 37°C the bacterial plating efficiency was scored comparing expression induction by IPTG addition or non-induction by glucose (Glc) addition, or comparing the effect of pH. The ΔH9–H10 apoL1 mutant lacks the region encoding helices 9 and 10 of the pore-forming domain. C. Trypanolytic potential of various apoL1 variants, as determined on T. b. brucei after 24 h–incubation in vitro (ctrl = control without apoL1).
Figure 2
Figure 2. SRA interaction with apoL1.
A. Double expression of apoL1 and SRA in E. coli. The scheme of the plasmid construct is shown above Western blot data illustrating the detection of the two tagged proteins from the lysis supernatant (sup = supernatant) and their recovery in the fractions bound to nickel beads. ApoL1 and SRA were revealed by anti-apoL1 and anti-S tag antibodies, respectively. The pH of the extraction buffer was between 6 and 7, as indicated. B. Influence of apoL1 and/or SRA expression on bacterial plating efficiency determined by the ratio of colonies counted following induction versus non-induced controls, as illustrated in Fig. 1B. C. Evaluation of the level of protein association with the nickel beads. SRA binding to apoL1 was expressed as SRA binding percentage divided by the percentage of apoL1 binding to the nickel beads, which varied depending on the type of mutation/deletion performed on the protein. This ratio was considered as 100% for SRA binding to WT apoL1. In E. coli co-expressing WT apoL1 and SRA, the typical yield of each protein was respectively 3 μg and 100 ng/1010 cells. In this and the following figures, the values resulted from three independent experiments each performed in triplicate.
Figure 3
Figure 3. Effects of mutations in the C-terminal leucine zipper of apoL1.
A. The upper panel shows the sequence alignment of the leucine zipper within the human apoL family. The lower panels show hydrophobic cluster analysis of this region, in WT and various mutant apoL1s. B. SRA binding to WT or mutant apoL1s, provided with a C-terminal V5-His6 tag. C. Quantification of SRA binding to WT or mutant apoL1s. D. Bacterial plating efficiency of various mutant apoL1s. E. Trypanolytic potential of various apoL1 variants (ctrl = control).
Figure 4
Figure 4. Trypanolytic activity of Papio cynocephalus.
A. Trypanolytic activity of Papio serum on NHS-resistant (R) or sensitive (S) clones of T. b. rhodesiense, and effect of DIDS on this activity. B. Trypanolytic potential of Papio and human serum, and effect of haptoglobin (Hp) on this potential. C. Phenotype of T. b. rhodesiense NHS-resistant (R) or sensitive (S) clones incubated with human or Papio serum. D. Western blot analysis with anti-apoL1 (goat anti-apoL1 N20 antibody, from Santa Cruz, diluted 1∶100), of human or Papio serum and of serum fractions bound to either anti-apoA1 or SRA.
Figure 5
Figure 5. ApoL1-like sequences in Papio.
Upper panel: Alignment of Papio apoL1-like sequences reconstituted from information present in current Papio genome databases. The apparent deletion in exon 3 occurs in a region dispensable for the pore-forming activity of human apoL1 . The arrowhead identifies a frameshift predicted in the two apoL1-like genes. Arrows indicate the position and the orientation of the 10 primers used for RT-PCR analysis. Lower panel: Detail of the frameshift: nucleotides of human gene deleted in the Papio sequence are boxed.
Figure 6
Figure 6. Effects of mutations in the C-terminus of apoL1.
A. The upper panel shows the sequence of the various mutants. The lower panels show hydrophobic cluster analysis of this region, in WT and two mutants of apoL1. B. SRA binding to WT or mutant apoL1s, provided with a C-terminal V5-His6 tag. C. Quantification of SRA binding to WT or mutant apoL1s. D. Bacterial plating efficiency of various mutant apoL1s. E. Trypanolytic activity of various apoL1 variants (30 μg/ml), as determined on NHS-resistant (R) clones of T. b. rhodesiense, T. b. brucei and T. b. gambiense (ctrl = control).
Figure 7
Figure 7. Effect of transient transgenic expression of WT or mutant apoL1 on trypanosome infection in mice.
A. Detection of WT apoL1 at days 1, 4 and 8 after hydrodynamic injection of the plasmid construct, monitored by incubation of Western blots of mouse serum proteins with rat anti-apoL1 antibodies. Mice 1 and 2 were injected with control (empty) plasmid. The lane labelled NHS shows the result obtained with normal human serum. B. Detection of the different apoL1 variants at day 1 post-injection of DNA (CTRL = control empty plamid). The mice whose sera were analyzed here were used for the trypanosome infection experiments reported in panel C (S = T. b. rhodesiense ETat 1.2S; R = T. b. rhodesiense ETat 1.2R; g = T. b. gambiense LiTat 1.2). Normal human serum (NHS) was used for comparison. Loading control by Ponceau red staining of albumin is shown below each panel. C. 24 h post-injection of DNA, 106 parasites of the indicated strains were inoculated intraperitoneally into mice. Parasitemia was measured 3 days after parasite inoculation (control: empty plamid).

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