Molecular Studies on Plasmodium During Development in the Mosquito

Abstract

Among the factors that regulate transmission of malaria are host-derived immune factors which can inhibit the progression of Plasmodium through the mosquito. This thesis addresses whether members of a family of LCCL/lectin Adhesive–like Proteins (LAPs) could act as feasible targets to elicit an antibody-mediated blockade in parasite transmission. The LAP family comprises six putatively secreted multidomain proteins which appear conserved among Plasmodium species and other apicomplexan parasites. Studies utilise the Plasmodium berghei rodent malaria parasite as an experimental system to evaluate the potential transmission-blocking activity of antibodies raised against LAPs. Regions of the proteins were selected for heterologous expression in Escherichia coli. Eight recombinant proteins were successfully expressed and were used as antigens to generate antisera in mice. Following immunogenicity tests, antisera against regions of LAP1, LAP3, LAP4 and LAP6 were selected for further characterisation and tested for antimalarial transmission-blocking activity. A combination of in vitro and in vivo assays revealed that the presence of anti-LAP antibodies did not inhibit parasite development. Whilst not conclusively excluding their potential, this work provided no further evidence to support the inclusion of LAPs as candidates for transmission-blocking vaccines. Immunolocalisation studies using anti-LAP1 antibodies revealed that the protein is expressed in cytoplasmic regions of female gametocytes and to a lesser extent in female gametes. Although it has been proposed that LAP expression may cease following fertilisation, PbLAP1 was detected in the cytoplasm of developing zygotes and intriguingly was subsequently found to concentrate in compartments of the P. berghei ookinete corresponding to the crystalloids. Antisera against regions of LAP1, LAP3, LAP4 and LAP6 were all found to similarly label the distinct electron dense cellular compartments. Furthermore, observation of Pblap mutant ookinetes by light microscopy indicated deficiencies in the formation of crystalloids. A transient cellular compartment, formed in the ookinete and subsequently fragmenting during early stages of oocyst development, roles of the crystalloid remain unknown. The association between LAPs and the crystalloids however leads to potential insights into the biological roles of the LAP family, the crystalloids, and the cellular processes of Plasmodium sporogony. Through microarray analysis, a comparison of the transcriptional profiles of P. berghei Δlap1 and wild-type ookinetes was made, detecting 274 differentially expressed genes and thereby indicating that the absence of PbLAP1 and resulting deficiency in crystalloid formation may have several knock-on effects on cellular processes important to Plasmodium development in the mosquito.