Mechanisms by which Factor H protects Trypanosoma cruzi from the alternative pathway of complement

Abstract

Chagas disease, a chronic disabling disease caused by the protozoan Trypanosoma cruzi, has no standardized treatment or preventative vaccine. The infective trypomastigote form of T. cruzi is highly resistant to killing by the complement immune system. Factor H (FH), a negative regulator of the alternative pathway (AP) of complement on cell surfaces and in blood, contains 20 short consensus repeat domains. The four N-terminal domains of FH inactivate the AP, while the other domains interact with C3b/d and glycan markers on cell surfaces. Various pathogens bind FH to inactivate the AP. T. cruzi uses its trans-sialidase enzyme to transfer host sialic acids to its own surface, which could be one of the approaches it uses to bind FH. Previous studies have shown that FH binds to complement-opsonized T. cruzi and parasite desialylation increases complement-mediated lysis of trypomastigotes. However, the molecular basis of FH binding to T. cruzi remain unknown. Only trypomastigotes, but not epimastigotes (non-infective, complement susceptible) bound FH directly, independent of C3 deposition, in a dose-dependent manner. Domain mapping experiments using 3-5 FH domain fragments showed that domains 5-8 competitively inhibited FH binding to the trypomastigotes by ~35% but did not decrease survival in complement. FH-Fc or mutant FH-Fc fusion proteins (3-11 contiguous FH domains fused to the IgG Fc) also did not kill trypomastigotes. FH-related protein-5, whose domains bear significant sequence identity to all known polyanion-binding FH domains (6-7, 10-14, 19-20), fully inhibited FH binding to trypomastigotes and reduced trypomastigote survival to < 24% in the presence of serum. In conclusion, we have elucidated the role of FH in complement resistance of trypomastigotes.

Keywords: Factor H; Factor H-related protein-5; Trypanosoma cruzi; alternative pathway; evasion strategy.

Figure 1

Factor H binds to epimastigotes in a C3-dependent manner. 3x10⁶ epimastigotes were incubated with 0.2% BSA/HBSS⁺⁺ with (A) varying concentration of Factor H (FH), (B) 5% NHS or 5% FH-dpl serum, or (C) 5% NHS with or without polyclonal anti-C3 Ab for 30 minutes at 37°C. The complement reaction is stopped by adding 200 μl of 0.2% BSA/HBSS⁺⁺ containing 10 mM EDTA for (B) and (C). FH binding was determined by FACs as described in section 2.5 and was plotted relative to background for (A) and (B). For (C), FH binding was plotted as % bound FH relative to 0 μg/ml anti-C3 (100%). Results shown for (A) and (B) were representative of 2 independent experiments and was graphed as mean and standard deviation of duplicates. For (C), results are shown as mean and standard deviation from two independent experiments with duplicates. Significant differences in FH binding were assessed by unpaired t test; p<0.0001****, p<0.05*. For (C) there were no significant differences between 100 μg/ml of anti-C3 Ab and background (represented by the dotted line).

Figure 2

Factor H binds to trypomastigotes directly, in the absence of complement activation. 3x10⁶ Tulahuen and Peru strain trypomastigotes were incubated in 0.2% BSA/HBSS⁺⁺ with (A) varying concentrations of Factor H (FH) for 30 minutes at 37°C and with (C) varying concentrations of polyclonal anti-C3 Ab for 30 minutes at 37°C followed by incubation with 25 μg/ml FH, at 37°C for 30 minutes. FH binding was determined by FACs as described in section 2.5 and was plotted as relative to background for (A) and plotted as % bound FH relative to 0 μg/ml anti-C3 (100%) for (C). The data shown for (A) was representative of 2 independent experiments and was graphed as the mean and standard deviation of duplicates. (B) Histogram of FH binding at 300 μg/ml FH, as compared to the negative controls from (A). The data shown for (C) for both strains were representative of 2 independent experiments and were graphed as mean and standard deviation of duplicates.

Figure 3

Assessment of Factor H individual 3 domain fragments in their ability to inhibit FH binding and increase susceptibility of Tulahuen and Peru strain trypomastigotes to alternative pathway-mediated killing. (A) 3x10⁶ Tulahuen and Peru strain trypomastigotes were incubated in 0.2% BSA/HBSS⁺⁺ with 5 μM of overlapping 3 domain recombinant fragments of Factor H (rH), spanning regions 2-20 for 30 minutes at 37°C followed by incubation with 32 nM of Factor H (FH) at 37°C for another 30 minutes. Negative control with FH fragments and parasites alone (no FH) was added. FACS was carried out and FH binding was determined as described in section 2.5 and graphed. FH binding was determined by FACs and plotted as % bound FH relative to FH alone (100%). (B) 5x10⁵ Tulahuen strain and Peru strain trypomastigotes were incubated at 37°C for 60 minutes with varying concentrations of rH 5-7, rH 6-8, or rH 19-20 and 0 or 60% NHS under AP conditions (NHS + 5mM Mg EGTA) or under inactive complement conditions (NHS + 10mM EDTA). 400 μl of cold media was added and % survival of the parasites was determined as described in 2.6. The results for (A) were graphed as mean and standard deviation of duplicates of 2 independent experiments. Significant differences in % bound FH for samples with FH fragments were assessed by one-way ANOVA with Tukey’s multiple comparison test. Only significant reduction in the presence of FH fragments as compared to FH alone samples are shown; p<0.0001 ****, p<0.01 **, p≥ 0.05 non-significant (not shown). All comparisons to the negative control were significant; p<0.0001 **** (not shown). For (B), the highest and lowest doses tested on the Peru strain represent the mean and standard deviation from duplicates in two independent experiments. For the remaining doses, the data are from a single experiment. The results for the Tulahuen strain are plotted similarly, except for rH 19-20, where all doses reflect the mean and standard deviation from duplicates in two independent experiments. Survival was plotted relative to 0 μM rH fragments (100%).

Figure 4

Assessment of various Factor H recombinant proteins in trypomastigote binding and alternative pathway-mediated killing. 5x10⁵ Tulahuen strain trypomastigotes were incubated at 37°C for 45 minutes with: (A) 20 μM of Factor H (FH)-Ig fusion proteins (i.e., human 5-8 + mouse IgG2b (black bar), human FH 6,7/18-20 + human IgGFc1 (white bars), human FH 6,7/18-20 + human IgGFc3 (light grey bars) or recombinant FH domains (rH) 5-7, rH 6-8 or rH 19-20 (dark grey bars) followed by incubation with 60% NHS under AP conditions (NHS + 5mM Mg EGTA) or when all 3 complement pathways active (NHS only) at 37°C for 60 minutes; (C) 5 μM of FH 6,7/18-20, rH 6-8 and rH 18-20 followed by incubation with 60% NHS under AP conditions (NHS + 5mM Mg EGTA) at 37°C for 60 minutes. 400 μl of cold media was added and % survival of the parasites was determined as described in section 2.6, and survival was plotted relative to 0 μM rH fragments or fusion proteins (100%, dotted line). (B) 3x10⁶ Tulahuen or Peru strain trypomastigotes were incubated in 0.2% BSA/HBSS⁺⁺ with 5 μM of recombinant Factor H (rH) 6,7/18-20, rH 6-8 or rH 18-20 for 30 minutes at 37°C followed by incubation with 32 nM of FH at 37°C for 30 minutes. Negative control with FH fragments and parasites alone (no FH) was added. FH binding was assessed by FACs as described in section 2.5 and plotted as % bound FH relative to FH alone (100%). For (A) and (C), the results are representative of 2 independent experiments and were graphed as mean and standard deviation of duplicates. For (B), results were graphed as mean and standard deviation of duplicates of 2 independent experiments. Significant differences in % bound FH for samples with FH fragments were assessed by one-way ANOVA with Tukey’s multiple comparison test; p<0.0001 ****, p<0.001 ***, p<0.01 **, p≥ 0.05 non-significant (not shown). All comparisons to the negative control were significant; p<0.0001 **** (not shown). *, p<0.05.

Figure 5

Factor H-Related Protein-5 completely inhibits the binding of Factor H to trypomastigotes. 3x10⁶ Tulahuen or Peru strain trypomastigotes were incubated in 0.2% BSA/HBSS⁺⁺ with: (A) Factor H-related proteins (FHRs): FHR-1, FHR-3, FHR-4 and FHR-5 and (B) varying concentrations of FHR-5 for 30 minutes at 37°C followed by incubation with 32 nM of FH at 37°C for 30 minutes. Factor H (FH) binding was determined by FACs and plotted as % bound FH relative to FH alone (100%). The data for (A) and (B) were representative of 2 independent experiments and were graphed as mean and standard deviation of duplicates. For (A) significant differences in % bound FH for samples with FHRs as compared to FH alone samples were assessed by one-way ANOVA with Tukey’s multiple comparison test. Only significant reduction in the presence of FHRs as compared to FH alone samples are shown; p<0.0001 ****, p≥ 0.05 non-significant (not shown). For (B): only p≥ 0.05 non-significances were shown.

Figure 6

Assessment of FHR-5 and Factor H 6-8/10-14/18-20 with a D1119G mutation on trypomastigote susceptibility to complement-mediated killing. 5x10⁵ Tulahuen strain or Peru strain trypomastigotes were incubated at 37°C for 45 minutes with (A) and (B) varying concentrations of Factor H-related protein-5 (FHR-5) and (C) 500 nM of Factor H 6-8/10-14/18-20 with a D1119G mutation (rH 6-8/10-14/mut18-20) or FHR-5. For (A) and (C), samples are incubated then with 60% NHS under AP conditions (NHS + 5mM Mg EGTA) and for (B) when all 3 complement pathways are active. Samples treated with 0% NHS were added as controls. 400 μl of cold media was added and % survival of the parasites was determined as described in section 2.6. Survival was plotted relative to 0 μM FHR-5 (100%) for (A) and (B) and relative to 0 μM rH 6-8/10-14/mut18-20 (100%) for (C). For (A), the data for Tulahuen strain were representative of 3 independent experiments and the data for Peru strain was representative of 2 independent experiments, and both were graphed as mean and standard deviation of duplicates. For (B), the data for both strains were graphed as mean and standard deviation of duplicates of 2 independent experiments. For (C), the data for both strains were graphed as mean and standard deviation of duplicates of an independent experiment.