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 [1] wild-type T. b. brucei [2], TgsGP−/+ T. b. brucei [3], wild-type T. b. gambiense, [4] TgsGP−/0 T. b. gambiense [5] and negative control. (B) RT-PCR amplification of HpHbR followed by HpyCH4V restriction digestion of [1] wild-type T. b. brucei, [2] wild-type T. b. gambiense and [3] TbbHbHpR−/+TgsGP−/0 T. b. gambiense.
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 5×105 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.
Uptake of TLF-1 across strains.
Uptake of TLF-1 after one hour in [1] wild-type T. b. brucei [2] wild-type T. b. gambiense and [3] 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.
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 [1] TY-TgsGP−/+ T. b. brucei and [2] TbbHbHpR−/+ TY-TgsGP−/+ T. b. gambiense. The flagellar pocket (revealed by Concanavalin A and kinetoplast position) is indicated with a white arrow.