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cmi12123-sup-0001-si.doc131K

Fig. S1. Related to Fig. 1. Phylogenetic analysis of the sialidase-encoding genes of T. vivax, T. congolense and T. brucei.

A. Analysis of the TvTS family.

B. Analysis of T. vivax, T. congolense and T. brucei sialidases. T. congolense sialidases (TcoTS) are indicated by a blue circle, T. brucei sialidases (TbSA and TbTS) are indicated by a green triangle, and T. vivax sialidases are indicated by a red square.

Phylogenetic trees were constructed using the neighbour-joining method on MEGA 5 software. Branch length is indicated.

cmi12123-sup-0002-si.doc357K

Fig. S2. Related to Fig. 2. Biochemical characterization of TvTS2.

A. Study of pH effect on SA activity. Enzyme activity was assayed at 37°C in AMT buffer at various pH.

B. Lineweaver-Burk plots of SA activity. The activity of the enzymes was measured at 37°C, pH 7.0, at varying concentrations of MUNeu5Ac.

C. Lineweaver-Burk plots of TS activity. Enzyme activity was measured at 37°C, pH 7.0, using fixed MU-Gal concentration and varying concentrations of sialyl lactose.

D. Kinetic parameters of T. vivax TvTS2 The apparent Vmax and Km were determined using the Lineweaver-Burk method.

Data are expressed as the mean of three independent experiments.

cmi12123-sup-0003-si.doc163K

Fig. S3. Related to Fig. 2. Western blot analysis of TvTS expression in T. vivax Y486 BSF. Blots were probed with anti-TvTS5 sera. Molecular weights (kDa) are indicated on the left.

cmi12123-sup-0004-si.tif768K

Fig. S4. Related to Fig. 6. Modification profiles of the mouse erythrocyte surface glycoproteins throughout T. vivax infection and in a bleeding-induced anaemic mouse model.

A. Lectin blot analysis of erythrocyte membrane glycoproteins throughout T. vivax infection with biotin-conjugated Peanut Agglutinin (PNA) specific for terminal galactose attached to galactosamide, visible after removal of terminal sialic acids.

B. Monitoring of haematocrit and reticulocytes in the bleeding-induced anaemic CD1 mouse used to analyse erythrocyte membrane profiles (G–H).

C. SNA lectin blot analysis of erythrocyte membranes throughout the bleeding-induced anaemia.

D. Western blot analysis of glycophorin A (GlyA) and integrin β1 (β1) expression throughout the bleeding-induced anaemia.

cmi12123-sup-0005-si.tif457K

Fig. S5. Effects of myricetin on SA activity in infected mice and on recombinant TvTS2 action on haematocrit.

A. Parasitaemia, haematocrit and SA activity were monitored during T. vivax Y486 infection in myricetin treated mice CD1 mice. Myricetin (1 mg kg−1) was injected intraperitoneally 1 day post infection (infection with 3–4·106 BSF of T. vivax Y486) for 4 consecutive days in five CD1 mice (treated mice). Haematocrit was expressed in PCV.

B. In vivo effect of injection of recombinant TvTS2 previously incubated with myricetin on mouse haematocrit. 650 μU of T. vivax TvTS2 were incubated with myricetin (30 μg) for 30 min then injected in three CD1 mice for 3 consecutive days (0, 1, 2). Haematocrit was monitored during 11 days. Each bar represents the mean of haematocrit values ± SD.

cmi12123-sup-0006-si.doc219K

Table S1. Related to Fig. 1. Identity percentage between TvTS family members and between TvTS1–5 and other trypanosomal sialidases.

A. Comparison of the five complete TvTS enzymes on the full-length protein (1), on the catalytic domain (2) and on the lectin-like domain (3).

B. Comparison of TvTS1–5 with one representative T. cruzi TcTS (Q26964) (1), with T. congolense TS family (TcoTS) (2) and with T. brucei SA and TS (TbTS and TbSA) (3).

cmi12123-sup-0007-si.doc62K

Table S2. Related to Fig. 2. Sialidase peptides identified by mass spectrometry in membrane preparations in T. vivax Y486 BSF. Y486 BSF membranes were prepared as described below and analysed by SDS-PAGE and silver staining. Protein bands from 60 to 200 kDa were analysed by mass spectrometry and the peptides identified were listed in the tables. The presence of the peptides in the TvTS family members is indicated by +.

Supplemental experimental procedures.

cmi12123-sup-0008-si.zip23KSupporting Information

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