Fig 1.
UDA binding to the surface coat of T. brucei.
A) 3D microscopy images of T. brucei BSF live cells labeled with UDA-FITC conjugates for different time periods. B) Accumulated growth of T. brucei BSF parasites after exposure to different concentrations of UDA for 24 h. C) Nuclei (N) and kinetoplasts (K) were stained with DAPI and categorized according to their number in 1N1K, 1N2K, 2N2K in addition to a population with an aberrant number of nuclei and kinetoplast denoted by XNXK. D) Microscopy images showing DAPI staining and cell morphology after UDA exposure for 10 h. Bars, 10 μm.
Table 1.
EC50 values for UDA in parental (BSF) and UDA-resistant T. brucei bloodstream lines.
Fig 2.
Analysis of the VSG expressed in UDAa and UDAb resistant lines.
A and B) VSG221 expression in parental and UDA-resistant parasites was monitored by immunofluorescence microscopy using a polyclonal antibody against VSG221. Nuclear and kinetoplast DNA was stained with DAPI. Bars, 10 μm. C and E) sVSG were purified from parental and resistant lines as described [29,30] and analyzed by SDS/PAGE and Coomassie blue staining. D and F) sVSG samples were digested with Endo H (that removes oligomannose N-linked glycans) or PNGase F (that removes all N-linked glycans), and the products of the reaction were subjected to SDS/PAGE and Coomassie blue staining.
Fig 3.
Assessment of the VSG glycosylation status by lectin blotting.
Purified sVSG of UDA15a (A) and UDA15b (B) were probed with tomato lectin (TL). Ponceau staining was used as loading control and chitin hydrolysate (inhibitor) was used as a specificity control for TL.
Fig 4.
Relative expression of TbSTT3 genes in UDAa and UDAb-resistant cell lines.
mRNA were purified from parental and resistant lines in the presence or the absence of UDA selective pressure (p-Rem 3M), quantified by RT-qPCR and normalized to the expression of PI3K-like gene. Myosin 1B gene expression was also introduced in the analysis as control. Values were calculated from triplicates of three independent experiments. The asterisks show significant differences by the Student’s t-test (n = 3). *, p < 0.05, **, p < 0.01 and ***, p < 0.001 vs the parental strain.
Fig 5.
Survival of mice inoculated with UDA-resistant parasites.
A) Kaplan-Meier survival analysis of mice infected with parental (Tb BSF), and UDA15a and UDA15b resistant parasites. B) SDS/PAGE analysis of sVSG expressed in UDA15b parasites isolated from mouse #4 (UDA15b/M4) at the final point of infection. Coomassie blue stained bands were identified by MALDI-TOF analysis. C) Relative expression of TbSTT3 genes in UDA15b/M4 was determined by RT-qPCR. The asterisks indicate significant differences according to the Student’s t-test (n = 3). *, p < 0.005 vs the UDA15b strain. D) PCR analysis of full-length and chimeric TbSTT3 genes in genomic DNA from (1) Tb BSF, (2) UDA15b/M4 and (c) HHA20 as control [19].
Fig 6.
Changes in the glycosylation profile of UDA-resistant cell lines.
Whole cell extracts obtained from UDA15a (A) and UDA15b (B) after hypotonic lysis were probed with TL or ConA. Ponceau staining was used as loading control. Chitin hydrolysate (inhibitor) and α-methylmannopyranoside were used as a specificity control for TL or ConA, respectively.
Fig 7.
Resistant parasites exhibit deficient binding and uptake of UDA-FITC.
A) Plot showing binding of UDA to resistant lines grown in the presence or after 3 months of lectin removal (p-Rem 3M). Data were represented as the percentage of binding relative to the labeling of the parental line. B) Microscopy images of fixed cells showing the decrease in UDA-labeling of UDA15a and UDA15b cells in comparison to parental T. brucei BSF. Nuclear and kinetoplast DNA were stained with DAPI. Bars, 10 μm. C and D) Plots showing the uptake of transferrin in UDA15a and UDA15b lines, respectively. E and F) Uptake of ConA in UDA15a and UDA15b lines, respectively. G) Uptake of dextran. H) Live cell microscopy illustrates the “big-eye” phenotype resulting from an endocytosis block after treatment of parental T. brucei BSF with 1.125 μM UDA for 10 hours. Uptake percentages were calculated relative to the maximum uptake observed in the parental line (Tb BSF). The asterisks show significant differences by the Student’s t-test (n = 3). *, p < 0.05, **, p < 0.01 and ***, p < 0.001 vs the parental strain.
Table 2.
EC50 values and cross-resistance (R) indices of UDA15a and UDA15b resistant lines.