No abstract

Malaria is a life-threatening disease caused by one of the five species of Plasmodium, among which Plasmodium falciparum cause the deadliest form of the disease. Plasmodium species are dependent on a vertebrate host and a bloodsucking insect vector to complete their life cycle. Plasmodium chitinases belonging to the GH18 family are secreted inside the mosquito midgut, during the ookinete stage of the parasite. Chitinases mediate the penetration of parasite through the peritrophic membrane, facilitating access to the gut epithelial layer. In this review, we describe Plasmodium chitinases with special emphasis on chitinases from P. falciparum and P. vivax, the representative examples of the short and long forms of this protein. In addition to the chitinase domain, chitinases belonging to the long form contain a pro-domain and chitin-binding domain. Amino acid sequence alignment of long and short form chitinase domains reveals multiple positions containing variant residues. A subset of these positions was found to be conserved or invariant within long or short forms, indicating the role of these positions in attributing form-specific activity. The reported differences in affinities to allosamidin for P. vivax and P. falciparum were predicted to be due to different residues at two amino acid positions, resulting in altered interactions with the inhibitor. Understanding the role of these amino acids in Plasmodium chitinases will help us elucidate the mechanism of catalysis and the mode of inhibition, which will be the key for identification of potent inhibitors or antibodies demonstrating transmission-blocking activity.

Transmission blocking monoclonal antibodies, 4B7 and 1245, targeting Pfs25, have been demonstrated to block the developmental stages of the malarial parasite inside the mosquitoes. In this work, anti-plasmodium antibody fragments of 4B7 and 1245, designated as diabodies, were expressed in bacteria and refolded using a standardized method, with a yield of 0.1 and 0.4 mg per litre of culture, respectively. Dimeric species was detected for 4B7-Db, but not for 1245-Db, using glutaraldehyde cross-linking experiment. The quality of the purified refolded fraction was assessed with differential scanning fluorimetry (DSF). Refolded 4B7-Db, with a melting temperature of 59 degrees C, recognized Pfs25 expressed on the surface of ookinete and zygote stages of Plasmodium in an immunofluorescence-based assay, whereas, 1245-Db, exhibiting a skewed melt profile, showed weak recognition. Further, refolded 4B7-Db recognized the linear epitope, on purified Pfs25 protein, both in the denatured and native state. Differential scanning fluorimetry can be potentially employed as a qualitative measure of functionality, to evaluate refolded proteins, with applicability for antibody engineering in passive immunization.