Supplementary Material and Methods.

Plasmids constructs for recombinant metacaspase expression. Full length and truncated versions of metacaspase genes were tagged at the N-terminus with a 6 x His-HA tag and at the C-terminus with a 3 x Flag epitope. For all metacaspase-5 constructs, the first 18 amino acids were not included since they encode a putative signal peptide. N-terminal tags were introduced by PCR in two consecutive steps, whereas the C-terminal tag was added by cloning the final PCR product in frame with a 3 x Flag present in the arabinose-inducible plasmid vector pBAD24.1 In a first PCR, TcMCA3 and TcMCA5 were amplified from genomic DNA (GenBank DQ015868.1) or a previously reported clone respectively.2 Primer sequences are provided in Supplementary Table 1. Forward primers introduced the HA tag (italic) upstream the first triplet (bold) of the amplified region within the open reading frame and reverse primers removed the translation stop codon and introduced enzyme restriction sites (underlined; NcoI for TcMCA3, EcoRI site for TcMCA5). The second PCR was performed using the product of the first reaction as a template and one forward primer for all the reactions (SDKOZAKHISHA Fwd) encoding six histidine residues (italic) before the artificially added ATG start codon (bold) and the NheI site (underlined). The reverse primer was the same as the one used for each construct in the first PCR andallowed the subcloning of the final product in frame with the 3 x Flag in the pBAD24 vector.

For TcMCA3, forward primer contained a NotI site (underlined) upstream the translation start codon (bold) and reverse primer contained a MluI site (underlined) downstream the translation stop codon (bold) and a partial sequence priming the flag epitope. The resulting PCR products were digested with NotI and MluI, and cloned into pTcINDEX digested with the same enzymes.

For the generation of the new pBAD24 constructs (#3 and #7), TcMCA5 and the truncated form were amplified from the original clone DQ015868 using a forward primer containing a NheI restriction site (underlined) for cloning into pBAD24 followed by a NotI site (dashed) for next subcloning into pTcINDEX, just before the translation start codon (bold) and nucleotides encoding the putative signal peptide. The reverse primers were exactly the same as the ones used for bacterial expression (allow in frame addition of the Flag epitope present in the pBAD24). Using pBAD24 constructs as templates, the different versions of metacaspase-5 were amplified and subcloned into pTcINDEX as described for TcMCA3.

Supplementary Table 1.Primers for generation of recombinant enzymes.

Construct name / PCR / DNA / Primer forward / Primer reverse
pBAD24-His6-HA-TcMCA3(1-358)-3xFlag / 1st / gDNA / TcMCA3HAFULL Fwd
5'-TAT CCG TAT GAT GTG CCG GAT TAT GCGATG GGC TTT GAT TTT GGC TGT C-3' / TcMCA3FULL Rev
5'-C CAT GGC TGT GGC GAC GGG TGG-3'
2nd / 1st PCR / SDKOZAKHISHA Fwd
5'-GC TAG CAG GAG GTT ACT ACC ATG CAC CAC CAC CAC CAC CAC TAT CCG TAT GAT GTG-3' / TcMCA3FULL Rev
5'-C CAT GGC TGT GGC GAC GGG TGG-3'
pBAD24-His6-HA-TcMCA3(85-358)-3xFlag / 1st / gDNA / TcMCA3HADC Fwd
5'-TAT CCG TAT GAT GTG CCG GAT TAT GCGGCC CTT TTC ATC GGC ATC AAC-3' / TcMCA3FULL Rev
5'-C CAT GGC TGT GGC GAC GGG TGG-3'
2nd / 1st PCR / SDKOZAKHISHA Fwd
5'-GC TAG CAG GAG GTT ACT ACC ATG CAC CAC CAC CAC CAC CAC TAT CCG TAT GAT GTG-3' / TcMCA3FULL Rev
5'-C CAT GGC TGT GGC GAC GGG TGG-3'
pBAD24-His6-HA-TcMCA5(19-442)-3xFlag / 1st / DQ015868 / TcMCA5HAFULL Fwd
5'-TAT CCG TAT GAT GTG CCG GAT TAT GCGTTT GTT GCT GGT GTT GGA AGG-3' / TcMCA5FULL Rev
5'-GAA TTC CAG GGG CTT TCC TAC-3'
2nd / 1st PCR / SDKOZAKHISHA Fwd
5'-GC TAG CAG GAG GTT ACT ACC ATG CAC CAC CAC CAC CAC CAC TAT CCG TAT GAT GTG-3' / TcMCA5FULL Rev
5'-GAA TTC CAG GGG CTT TCC TAC-3'
pBAD24-His6-HA-TcMCA5(19-336)-3xFlag / 1st / DQ015868 / TcMCA5HAFULL Fwd
5'-TAT CCG TAT GAT GTG CCG GAT TAT GCGTTT GTT GCT GGT GTT GGA AGG-3' / TcMCA5DC Rev
5'-GAA TTC ATG AAT GAG ACT GGT G-3'
2nd / 1st PCR / SDKOZAKHISHA Fwd
5'-GC TAG CAG GAG GTT ACT ACC ATG CAC CAC CAC CAC CAC CAC TAT CCG TAT GAT GTG-3' / TcMCA5DC Rev
5'-GAA TTC ATG AAT GAG ACT GGT G-3'
pBAD24-His6-HA-TcMCA5(63-336)-3xFlag / 1st / DQ015868 / TcMCA5HADC Fwd
5'-TAT CCG TAT GAT GTG CCG GAT TAT GCGGCG CTG TTT GTA GGC ATC AAC-3' / TcMCA5DC Rev
5'-GAA TTC ATG AAT GAG ACT GGT G-3'
2nd / 1st PCR / SDKOZAKHISHA Fwd
5'-GC TAG CAG GAG GTT ACT ACC ATG CAC CAC CAC CAC CAC CAC TAT CCG TAT GAT GTG-3' / TcMCA5DC Rev
5'-GAA TTC ATG AAT GAG ACT GGT G-3'

Supplementary Table 2. Primers for generation of recombinant active site mutants.

Construct name / Template / Primer forward / Primer reverse
pBAD24-His6-HA-TcMCA3H168A(1-358)-3xFlag / pBAD24-His6-HA- TcMCA3(1-358)-3xFlag / TcMCA3 His168Ala Fwd
5'-G CAG TAT TCC GGT GCC TGC ACG CAG ACC-3' / TcMCA3 His168Ala Rev
5'-GGT CTG CGT GCA GGC ACC GGA ATA CTG C-5'
pBAD24-His6-HA-TcMCA3C223A(1-358)-3xFlag / pBAD24-His6-HA-TcMCA3 (1-358)-3xFlag / TcMCA3 Cys223Ala Fwd
5'-C GTA TTT GAC TGC GCC CAC TCT GGC TCC-3' / TcMCA3 Cys223Ala Rev
5'-GGA GCC AGA GTG GGC GCA GTC AAA TAC G-3'
pBAD24-His6-HA-TcMCA5H146A(19-442)-3xFlag / pBAD24-His6-HA-TcMCA5(19-442)-3xFlag / TcMCA5 His146Ala Fwd
5'-CAC TAC TCT GGG GCT GGG ACA GAG ACC-3' / TcMCA5 His146Ala Rev
5'-GGT CTC TGT CCC AGC CCC AGA GTA GTG-3'
pBAD24-His6-HA-TcMCA5C201A(19-442)-3xFlag / pBAD24-His6-HA-TcMCA5(19-442)-3xFlag / TcMCA5 Cys201Ala Fwd
5'-GCT GTC TTT GAC TGT GCT CAC TCC GCC TCG CTG-3' / TcMCA5 Cys201Ala Rev
5'-CAG CGA GGC GGA GTG AGC ACA GTC AAA GAC AGC-3'

Mutation in bold

Supplementary Table 3. Plasmids constructs for parasite transfections.

Construct name / Template / Primer forward / Primer reverse
1 / pTcINDEX-
His6-HA-TcMCA3(1-358)-
3xFlag / pBAD24-
His6-HA-TcMCA3(1-358)-3xFlag / TcMCA3pTcINDEX Fwd
5'-GC GGC CGCATGCAC CAC CAC CAC CAC CAC TAT CCG TAT GAT GTG-3' / TcMCA3pTcINDEX Rev
5'-ACG CGTCTA GAG ATC GTC ATC CTT G-3'
2 / pTcINDEX-
His6-HA-TcMCA3C223A(1-358)-
3xFlag / pBAD24-
His6-HA-TcMCA3C223A(1-358)-3xFlag / TcMCA3pTcINDEX Fwd
5'-GC GGC CGCATGCAC CAC CAC CAC CAC CAC TAT CCG TAT GAT GTG-3' / TcMCApTcINDEX Rev
5'-ACG CGTCTA GAG ATC GTC ATC CTT G-3'
3 / pBAD24-
TcMCA5(1-442)-3xFlag / DQ015868 / TcMCA5pTcINDEX Fwd
5’-GC TAG C GC GGC CGCATG GAC CTG CTT CTT GGC GTC-3’ / TcMCA5FULL Rev
5'-GAA TTC CAG GGG CTT TCC TAC-3'
4 / pTcINDEX-
TcMCA5(1-442)-3xFlag / pBAD24-
TcMCA5(1-442)-3xFlag / TcMCA5pTcINDEX Fwd
5’-GC TAG C GC GGC CGCATG GAC CTG CTT CTT GGC GTC-3’ / TcMCApTcINDEX Rev
5'-ACG CGTCTA GAG ATC GTC ATC CTT G-3'
5 / pBAD24-
TcMCA5 C201A (1-442)-3xFlag / pBAD24-
TcMCA5(1-442)-3xFlag / TcMCA5 Cys201Ala Fwd
5'-GCT GTC TTT GAC TGT GCT CAC TCC GCC TCG CTG-3' / TcMCA5 Cys201Ala Rev
5'-CAG CGA GGC GGA GTG AGC ACA GTC AAA GAC AGC-3'
6 / pTcINDEX-
TcMCA5 C201A (1-442)-3xFlag / pBAD24-
TcMCA5 C201A (1-442)-3xFlag / TcMCA5pTcINDEX Fwd
5’-GC TAG C GC GGC CGCATG GAC CTG CTT CTT GGC GTC-3’ / TcMCApTcINDEX Rev
5'-ACG CGTCTA GAG ATC GTC ATC CTT G-3'
7 / pBAD24-
TcMCA5(1-336)-3xFlag / DQ015868 / TcMCA5pTcINDEX Fwd
5’-GC TAG C GC GGC CGCATG GAC CTG CTT CTT GGC GTC-3’ / TcMCA5DC Rev
5'-GAA TTC ATG AAT GAG ACT GGT G-3'
8 / pTcINDEX-
TcMCA5(1-336)-3xFlag / pBAD24-
TcMCA5(1-336)-3xFlag / TcMCA5pTcINDEX Fwd
5’-GC TAG C GC GGC CGCATG GAC CTG CTT CTT GGC GTC-3’ / TcMCApTcINDEX Rev
5'-ACG CGTCTA GAG ATC GTC ATC CTT G-3'
9 / pTcINDEX-eGFP / pTEX-eGFP / eGFP ClaI Fwd
5’-ATC GATATG GTG AGC AAG GGC GAG GAG-3’ / eGFPBamHI Rev
5'-GGA TCCTCA CTT GTA CAG CTC GTC ATG CC-3'
10 / pTcINDEX-
TcMCA3(1-358)-eGFP / gDNA / TcMCA3NotI Fwd
5’-GC GGCCGCATG GGC TTT GAT TTT GGC TGT C-3’ / TcMCA3ClaI Rev
5'-ATCGATTGT GGC GAC GGG TGG-3'
11 / pBAD24-
TcMCA3(1-358)-3xFlag / gDNA / TcMCA3NheINotIFwd
5’-GC TAG C GC GGC CGC ATG GGC TTT GAT TTT GGC TGT C-3’ / TcMCA3FULL Rev
5'-C CAT GGC TGT GGC GAC GGG TGG-3'
11 / pTcINDEX-
TcMCA3(1-358)-3xFlag / pBAD24-
TcMCA3(1-358)-3xFlag / TcMCA3NheINotIFwd
5’-GC TAG C GC GGC CGC ATG GGC TTT GAT TTT GGC TGT C-3’ / TcMCApTcINDEX Rev
5'-ACG CGTCTA GAG ATC GTC ATC CTT G-3'

References:

1.Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995Jul.; 177(14): 4121–4130.

2.Kosec G, Alvarez VE, Agüero F, Sánchez D, Dolinar M, Turk B, et al. Metacaspases of Trypanosoma cruzi: possible candidates for programmed cell death mediators. Molecular & Biochemical Parasitology. 2006 Jan.;145(1):18–28.