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. 2023 Jan 27;10(2):18-35.
doi: 10.15698/mic2023.02.790. eCollection 2023 Feb 6.

The role of invariant surface glycoprotein 75 in xenobiotic acquisition by African trypanosomes

Alexandr Makarov  1 , Jakub Began  2 , Ileana Corvo Mautone  1   3 , Erika Pinto  1 , Liam Ferguson  1 , Martin Zoltner  1   4 , Sebastian Zoll  2 , Mark C Field  1   5
Affiliations

The role of invariant surface glycoprotein 75 in xenobiotic acquisition by African trypanosomes

Alexandr Makarov et al. Microb Cell. .

Abstract

The surface proteins of parasitic protozoa mediate functions essential to survival within a host, including nutrient accumulation, environmental sensing and immune evasion. Several receptors involved in nutrient uptake and defence from the innate immune response have been described in African trypanosomes and, together with antigenic variation, contribute towards persistence within vertebrate hosts. Significantly, a superfamily of invariant surface glycoproteins (ISGs) populates the trypanosome surface, one of which, ISG75, is implicated in uptake of the century-old drug suramin. By CRISPR/Cas9 knockout and biophysical analysis, we show here that ISG75 directly binds suramin and mediates uptake of additional naphthol-related compounds, making ISG75 a conduit for entry of at least one structural class of trypanocidal compounds. However, ISG75 null cells present only modest attenuation of suramin sensitivity, have unaltered viability in vivo and in vitro and no alteration to suramin-invoked proteome responses. While ISG75 is demonstrated as a valid suramin cell entry pathway, we suggest the presence of additional mechanisms for suramin accumulation, further demonstrating the complexity of trypanosomatid drug interactions and potential for evolution of resistance.

Keywords: CRISPR/Cas9; drug accumulation; drug metabolism; invariant surface glycoprotein; suramin; trypanosome; xenobiotics.

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Conflict of interest statement

Conflict of Interest: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1. FIGURE 1: ISG75 binds suramin and trypan blue directly.
(A) Left panel – Isothermal titration calorimetry of the interaction of suramin with ISG75. 400 µM suramin was titrated into 16 µM ISG75 solution within the calorimetric cell. The upper left panel shows the raw data of sequential suramin injections, the lower panel the integral after subtraction of the heat of suramin dilution (dots) together with the fit (line). Right panel – Surface plasmon resonance of the ISG75:suramin interaction. Avi-tagged ISG75, site-specifically biotinylated at the C-terminus was used as ligand and immobilised onto the CAPture sensor chip surface. Suramin was used as analyte and flowed over the chip at 1, 2, 4, 8, 16 and 32 µM. Data were reference-subtracted and fitted using steady state (upper right) and one-to-one kinetic analysis (lower right). For fitting, data points from two independent measurements (circles, upper panel; solid/dashed lines, lower panel) were used. (B) Isothermal titration calorimetry of the interaction of trypan blue with ISG75. 500 µM trypan blue was titrated into 16 µM ISG75 solution within the calorimetric cell. The upper left panel shows the raw data of sequential trypan blue injections, the lower panel the integrated heats after subtraction of the heat of trypan blue dilution (dots) together with the fit (line). (C) Kinetic and thermodynamic parameters of the ISG75:suramin and ISG75:trypan blue interaction. KD, dissociation constant; Kon, association rate; Koff, dissociation rate; N, stoichiometry; ΔH, change in enthalpy; ΔS, change in entropy; T, temperature in Kelvin; t1/2=complex half-life time.
Figure 2
Figure 2. FIGURE 2: CRISPR-Cas9 strategy for obtaining ISG75 knockout.
(A) ISG75 locus map in T. brucei Lister 427. (B) Following delivery and selection for cells stably expressing a chosen gRNA, Cas9 was induced for 7 days and the population selected with suramin (70µm) for 4 days, then subcloned and individual clones subjected to Western blot analysis (WB) (in C). Clone 4 termed ISG75Crc4 and expressing ∼5% of ISG75 parental levels was subjected to an additional 14 days of Cas9 induction, subcloned again and individual clones again subjected to Western blot analysis. Clones ISG75Crc4 and ISG75Crc4.6 were analysed by whole genome sequencing and proteomics to assess respectively the genomic architecture of the edited ISG75 locus and remaining protein levels. (C) Western blot and proteomics analysis of first- and second-generation clones reveals ISG75Crc4 and ISG75Crc4.6 as 95% knockdown and effective knockout clones respectively. Numbers given are Western blot densitometry values for 1st and 2nd-generation clones and proteomics label free quantification for clones ISG75Crc4 and ISG75Crc4.6. (D) Cumulative growth curves over six days of cultivation for parental 2T1 (green), mutant ISG75Crc4 (blue) and ISG75Crc4.6 (purple) cell-lines. Three replicates for each line were grown in parallel. Error bars, standard deviations. (E) Average doubling times for 2T1, ISG75Crc4 and ISG75Crc4.6, calculated from (D).
Figure 3
Figure 3. FIGURE 3: Impact of ISG75 on drug sensitivity.
(A) EC50 curves showing shift in resistance to suramin and pentamidine in ISG75Crc4 and ISG75Crc4.6 cell-lines. (B) EC50 curves showing shift in resistance to trypan blue, ponceau S and cibacron blue in ISG75Crc4 compared to VSGsur. (C) Dynamite-plot showing respective shifts in EC50. Error bars, 95% confidence interval (CI). Asymmetric confidence intervals (95% CI) were calculated where possible in GraphPad PRISM and statistical significance of each EC50 shift assessed by confidence interval overlap – overlapping CI indicated absence of statistically significant shift. (D) Structures of drugs studied. Highlighted in light blue are naphthol moieties in suramin, trypan blue and ponceau S in red are single aromatic benzene rings and in dark blue are azo-groups. (E) Flow cytometry gating strategy (top) and representative flow cytometry histogram with bar-plots (bottom) showing LDL uptake upon knockout of ISG75 in ISG75Crc4.6. Bar plot error bars, standard errors from biological replicas, *** indicates p-value < 0.01.
Figure 4
Figure 4. FIGURE 4: Changes in protein expression levels in suramin-treated cells.
Volcano plot of protein level changes in parental cells with and without suramin (top). Coloured respectively are proteins significantly up- or down-regulated (p-value < 0.05) only in parental, only in ISG75Crc4 and in both as indicated after exposure to suramin. Additionally highlighted in coloured groups (top to bottom, lighter to darker blue shades) are proteins involved in proline catabolism, mitochondrial activation and Krebs' cycle, early BSF/PCF differentiation markers, and proteins involved in threonine metabolism as indicated. Single asterisk (*) marks PIP39 and PPDK that display similar upregulation as shown previously, but did not reach statistical significance (p-values of 0.052 and 0.103 respectively); and NRKB and threonine synthase that displayed similar trends in parental and ISG75Crc4, but have low significance levels in one or both samples (NRKB, 1.5-fold increase in both parental and ISG75Crc4 with p-values of 0.034 and 0.093 respectively; TS, 1.9-fold increase with p-value 0.059 in parental and 1.7-fold increase with p-value 0.094 in ISG75Crc4). Correlation plot of proteins increased or decreased in parental and ISG75Crc4 cell-lines (bottom). Coloured dots are significantly altered proteins. Overall Pearson correlation coefficient r = 0.87.
Figure 5
Figure 5. FIGURE 5: Changes in protein expression levels in ISG75 knockout cells.
(A) Volcano plot showing distribution of protein level changes in in ISG75Crc4 cells. Proteins and protein groups increased 1.2-fold, 1.5-fold to 2-fold and 2-fold or more with p-values < 0.05 are shown respectively in light blue, turquoise and dark blue filled circles, protein groups decreased 2-fold or more - in red, non-significantly shifting protein groups – in grey unfilled circles. (B) FGSEA output showing most enriched GO-terms among all proteins in A. Top five groups are shown in orange. Unfilled circles all proteins, filled circles proteins with increased expression (Table 1 and 2). Labelled circles with coloured outlines are proteins with most increased expression in these groups. (C) Parasitaemia levels in Balb/C mice blood on days three and four post infection with 104 ISG75Crc4 cells and days four to six post infection with 102 cells, top and bottom respectively. Error bars, standard error. (D) Western Blot analysis of lysates of 2T1 and ISG75Crc4 cells extracted on day four terminal bleeds of mice infected with 104 cells shows persistent depletion of ISG75 in trypanosomal cell when in host environment – similar to that observed in vitro.
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