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. 2013;9(10):e1003686.
doi: 10.1371/journal.ppat.1003686. Epub 2013 Oct 3.

The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense

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The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense

Paul Capewell et al. PLoS Pathog. 2013.

Abstract

Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PCR amplification of TgsGP and RT-PCR of HpHbR in wild-type and transfected lines.
(A) Amplification of TgsGP and a control gene (cathepsin L) by PCR in wild-type T. b. brucei , TgsGP −/+ T. b. brucei , wild-type T. b. gambiense, TgsGP −/0 T. b. gambiense and negative control. (B) RT-PCR amplification of HpHbR followed by HpyCH4V restriction digestion of wild-type T. b. brucei, wild-type T. b. gambiense and TbbHbHpR −/+ TgsGP −/0 T. b. gambiense.
Figure 2
Figure 2. TgsGP is essential for resistance to human serum in T. b. gambiense.
The number of surviving cells after 24% human serum (open box), 10 μg/ml TLF-1 (dark grey box), 50 μg/ml recombinant APOL1 (light grey box) or a non-lytic 20% FBS control (black box). The dotted line indicates the starting concentration of ×ばつ105 cells. The cell lines assayed were wild-type T. b. brucei; TgsGP −/+ T. b. brucei; wild-type T. b. gambiense; TgsGP −/0 T. b. gambiense; TbbHbHpR −/+ T. b. gambiense; TbbHbHpR −/+ TgsGP −/0 T. b. gambiense and TbbHbHpR −/+ TgsGP −/+ T. b. gambiense. Standard error is shown, n = 4 for each data point.
Figure 3
Figure 3. Uptake of TLF-1 across strains.
Uptake of TLF-1 after one hour in wild-type T. b. brucei wild-type T. b. gambiense and TbbHbHpR −/+ TgsGP −/0 T. b. gambiense by co-localization of fluorescently tagged TLF-1 (green) with the lysosomal marker Lysotracker (red). The kinetoplast and nucleus were also stained using DAPI (blue).
Figure 4
Figure 4. Localisation of TY-TgsGP.
Localisation of TY-tagged TgsGP (red) relative to un-endocytosed FITC-labeled Concanavalin A bound to glycoproteins in the flagellar pocket (green) and DAPI stained nucleus and kinetoplast (blue) in TY-TgsGP −/+ T. b. brucei and TbbHbHpR −/+ TY-TgsGP −/+ T. b. gambiense. The flagellar pocket (revealed by Concanavalin A and kinetoplast position) is indicated with a white arrow.

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