Mutant/mutation
The mutant expression a C-terminal 3xFLAG-tagged version of B9 and expresses GFP under the control of the HSP70 promoter. The b9 gene is tagged in mutant RMgm-1026 that expresses GFP under the control of the HSP70 promoter. The GFP gene is introduced into the silent 230p locus using the GOMO method of transfection.

Because B9 is predicted to be glycosylphosphatidylinositol (GPI) anchored, the 3xFlag tag was inserted towards the C-terminus of the protein, downstream of the putative 6-cys domains but upstream of the predicted omega site (aspartate residue at position 826). 

Protein (function)
The B9 protein contains a 4-cysteine domain with a structure that is highly similar to the structure of 4-cysteine domains in the known proteins of the 6-Cys family and is identified as a 6-Cys-related protein. B9 is predicted to be glycosylphophatidylinositol (GPI) anchored. 

Phenotype
Normal/wild-type sporozoite development and infectivity, indicating that the 3xFlag epitope had no detrimental effect on B9 function. 
B9-Flag detected in salivary gland sporozoites, with a distribution pattern typical of a micronemal protein. It distributes in numerous vesicles localized on each side of the nucleus. B9 was detected in Western analysis as a single band around 80 kDa. Upon stimulation of microneme secretion, B9 was also recovered in the supernatant fraction as a slightly smaller band, indicating that B9 is secreted from sporozoites upon activation, possibly after proteolytic processing. B9 was not detected on the surface of sporozoites by immunofluorescence, irrespective of parasite activation, suggesting that following microneme secretion, B9 is mainly released as a shed protein.

Additional information
B9 did not co-localize with the apical membrane antigen 1 (AMA1), suggesting that the two proteins are present in distinct microneme subsets in salivary gland sporozoites

Evidence in the paper is presented that: 
- B9 is required for sporozoite invasion
- B9 is secreted from the sporozoite micronemes
- B9 contains a CyRPA-like beta propeller domain, required for B9 function
- The propeller domain of B9 interacts with P36 and P52

Other mutants