Mutant/mutation
The mutant expresses a mutated form of PIMMS2 (the conserved Asp155, His222 and Ser414 residues of PIMMS2, which make up the catalytic triad of subtilases, were replaced with alanine residues by site-directed mutagenesis). In addition, it expresses GFP under the constitutive eef1a promoter.
The mutated pimms2 gene has been introduced in a mutant lacking the pimms2 gene.

Protein (function)
PBANKA_1106900, is mapped between genes encoding two known subtilisin-like proteases, SUB1 and SUB3, and encodes an 836 amino acid long protein. Bioinformatics analysis predicted with high probability two hydrophobic regions of alpha helix structure encompassing the amino acid residues 6-25 and 416-427, respectively. Additionally,  a cleavage site at amino acid residue 30 was predicted suggesting an N-terminal signal peptide and a transmembrane domain located at the center of the protein. Protein sequence analysis predicted a peptidase S8 domain (IPR000209) that ends shortly after the predicted transmembrane domain and is followed by a long C-terminal extension (CTE)  with no known domain homology. A highly significant (100% confidence) structural similarity was predicted of the predicted peptidase S8 domain region to the P. vivax SUB1, and P. falciparum SUB1. PIMMS2 sequence alignments with PvSUB1 and the bacterial subtilisin BPN’ identified several conserved amino acid residues within the predicted subtilisin-like domain including D155, H122 and S414, which correspond to the catalytic triad of subtilisin-like serine proteases. A conserved asparagine residue N328 that serves in stabilizing the putative oxyanion hole was also identified. However, the conserved motifs HGT and GTS, which encompass the catalytic His and Ser residues of subtilases, respectively, are not present in PIMMS2, suggesting that the putative catalytic triad is likely to be non-functional and thus PIMMS2 is a potential pseudoenzyme.
The subtilisin-like domain architecture of PbPIMMS2 is conserved only in PvPIMMS2; the  rest of the PIMMS2 orthologs appear to lack one or more of the conserved amino acid residues or motifs.

Phenotype
Analyses of a mutant lacking the wild type pimms2 gene (RMgm-4173) showed significantly reduced oocyst numbers/production. Evidence is presented that ookinetes fail to traverse the midgut epithelium in the absence of PIMMS2 expression (see below)

Analysis of the mutant expressing the mutated PIMMS2 showed the following:
Normal ookinete production. The Δpbpimms2::pimms2mut parasites exhibited oocysts numbers that were significantly less than those of control parasite lines parasites but higher than those of the Δpbpimms2 parasites (RMgm-4173). These data suggest that the amino acid residues that form the putative catalytic triad of subtilases are important but not essential for the function of PIMMS2.

Additional information


Other mutants
RMgm-4173 - a mutant lacking expression of PIMMS2.