Role of the Trypanosoma brucei natural cysteine peptidase inhibitor ICP in differentiation and virulence

Abstract

ICP is a chagasin-family natural tight binding inhibitor of Clan CA, family C1 cysteine peptidases (CPs). We investigated the role of ICP in Trypanosoma brucei by generating bloodstream form ICP-deficient mutants (Deltaicp). A threefold increase in CP activity was detected in lysates of Deltaicp, which was restored to the levels in wild type parasites by re-expression of the gene in the null mutant. Deltaicp displayed slower growth in culture and increased resistance to a trypanocidal synthetic CP inhibitor. More efficient exchange of the variant surface glycoprotein (VSG) to procyclin during differentiation from bloodstream to procyclic form was observed in Deltaicp, a phenotype that was reversed in the presence of synthetic CP inhibitors. Furthermore, we showed that degradation of anti-VSG IgG is abolished when parasites are pretreated with synthetic CP inhibitors, and that parasites lacking ICP degrade IgG more efficiently than wild type. In addition, Deltaicp reached higher parasitemia than wild type parasites in infected mice, suggesting that ICP modulates parasite infectivity. Taken together, these data suggest that CPs of T. brucei bloodstream form play a role in surface coat exchange during differentiation, in the degradation of internalized IgG and in parasite infectivity, and that their function is regulated by ICP.

Fig. 1. Targeted replacement of TbICP

A. Schematic representation of the TbICP locus and the plasmid constructs used for gene replacement. Upper panel: ORFs are shown as arrows; intergenic and flanking DNA sequences are shown as boxes. The predicted sizes of StuI/SphI-digested DNA fragments from both native and modified TbICP locus are shown. Lower panel: Schematic representation of the re-integration of ICP into the tubulin locus of the Δicp line. DHFR, dihydrofolate reductase gene; BSD, blasticidin-resistance gene; HYG, hygromycin-resistance gene. B and C. Southern blot analysis. Genomic DNA was digested with StuI and SphI, separated on a 0.8% agarose gel, blotted onto a nylon membrane and hybridized with ³²P-labelled DNA probes; 5′ FR of TbICP(B) and TbICP ORF (C). Lane 1, WT T. brucei; lane 2, BSD-resistant heterozygote; lane 3, HYG-resistant heterozygote; lane 4, Δicp; lane 5, Δicp:ICP.

Fig. 3

CP expression and activity. A. Five micrograms of BSF lysates was tested for peptidase activity using 5 μM of Z-Phe-Arg-MCA as a substrate. The activity sensitive to inhibition by 10 μM of E-64, which corresponds to CP activity, is shown. The experiments were performed in quadruplicate and are represented as mean values with standard deviations (SD). The analysis of significance was performed using anova, and the asterisk indicates the scores that are statistically significantly at P < 0.05. B and C. Western blot analysis of BSF lysates (equivalent to 5 × 10⁵ parasites per lane) using antiserum to (B) brucipain or to (C) T. brucei cathepsin B. Antibodies to anti-EF1α were used to visualize loading controls (bottom panels).

Fig. 2. Δicp lacks CP inhibitory activity

A. T. cruzi epimastigote lysates and T. brucei WT BSF lysates were boiled and tested (8.5 μg protein ml⁻¹) for inhibitory CP activity by pre-incubation with 3 nM papain for 20 min. The residual activity of the enzyme was measured using 15 μM Z-Phe-Arg-MCA. B. BSF lysates (50 μg protein) were boiled and tested for the inhibition of papain by pre-incubating with 2 nM papain for 20 min, followed by determination of residual peptidase activity using 5 μM of Z-Phe-Arg-MCA. Asterisk shows scores statistically significant from buffer at P < 0.05.

Fig. 4. Growth of T. brucei

A. BSF parasites were seeded at 5 × 10³ ml⁻¹ and grown for 5 days in vitro, with culture density being analysed daily. At the third day, the cultures were diluted to 5 × 10³ ml⁻¹. The mean densities at days 4 (WT: 7 × 10⁵ ml⁻¹; Δicp: 2.3 × 10⁵ ml⁻¹; Δicp:ICP: 7 × 10⁵ ml⁻¹) and 5 (WT: 9.6 × 10⁵ ml⁻¹; Δicp: 3.5 × 10⁵ ml⁻¹; Δicp:ICP: 8.6 × 10⁵ ml⁻¹) were multiplied by the respective dilution factors for presentation. The assay was performed in triplicate and four independent times (***P< 0.001, in comparison with WT and Δicp:ICP). Keys: closed square, wild type; open square, Δicp:ICP; triangle, Δicp. B. BSF parasites were purified from the blood of infected mice and injected in Balb/c mice (10³ parasites per animal). The parasitemia was determined by counting the number of parasites in 5 μl of blood from days 3 to 6 of infection. Five mice were used per group, and the graph is representative of two independent experiments (***P < 0.001, in comparison with WT and Δicp:ICP). White bars, WT parasites; black bars, Δicp; grey bars, Δicp:ICP.

Fig. 5. Δicp has increased resistance to a CP inhibitor

BSF parasites were inoculated at 5 × 10⁴ ml⁻¹ in culture medium in the presence of varying concentrations of K11777, and cultivated for 2 days at 37°C. The controls were cultivated in the presence of 0.5% DMSO. The culture densities were monitored at 12, 24 and 30 h, and the numbers of cell divisions by 30 h are given. The experiments were performed in triplicate, two independent times, and are reported as mean and standard deviations of the six replicates. The analysis of significance was performed using two-way anova and the Bonferroni post-test at a significance of 5%. Single asterisks represent scores that are statistically significant at P < 0.05, and triple asterisks show scores statistically significant at P < 0.01. White bars, WT parasites; black bars, Δicp; grey bars, Δicp:ICP.

Fig. 6

Increased degradation of anti-VSG IgG by Δicp. BSF parasites were incubated for 30 min on ice with rabbit anti-VSG 221 IgG, prior to incubation at 37° for 5–30 min to detect IgG degradation (left panels). Cells were treated with 20 μM of K11777 (VSPh) or E64d prior to incubation with IgG and were chased for 30 min at 37°C (right panels). Whole-cell lysates were prepared, and equivalent of 5 × 10⁶ cells were loaded in a SDS-PAGE gel, transferred to nitrocellulose membranes, followed by incubation with anti-rabbit IgG HRP conjugate. The densitometry indicates the ratio of the intensity of IgG to EF1-α. A. WT parasites. B. Δicp. C. Δicp:ICP.

Fig. 7

Differentiation from bloodstream to PCF. BSF were incubated in SDM-79 medium at 27°C, in the presence of 6 mM cis-aconitate and samples analysed at the indicated times. The exchange of the cell surface coat from VSG (A) to procyclin (B) was quantified by flow cytometry and the percentage of fluorescence cells plotted. The experiments were performed in triplicate on three separate occasions. The graph shows the means plus standard deviations of the nine replicates. The analysis of significance was performed using two-way anova and the Bonferroni post-test at a significance of 5%. Single asterisks represent scores that are statistically significant at P < 0.05, and double asterisks represent scores statistically significant at P < 0.01. White bars, WT parasites; black bars, Δicp; grey bars, Δicp:ICP.

Fig. 8

Involvement of CPs in the differentiation from BSF to PCF. Differentiation of Δicp BSF to PCF in the presence of the irreversible CP inhibitor K11777 was analysed as described in Fig. 7. A and B. WT parasites. White bars, WT parasites; hatched grey bars, WT parasites + 0.25 μM K11777. C and D. Δicp. Black bars, Δicp; hatched grey bars, Δicp+ 2 μM K11777.