The molecular interplay of proteins expressed in the sexual stages and the induction of gamete formation in the malaria parasite Plasmodium falciparum

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Transmission of the malaria parasite from man to the mosquito requires the formation of sexual parasite stages, the gametocytes. The gametocytes are the only parasite stage that is able to survive in the mosquito midgut and to undergo further development – gamete
formation and fertilization. Numerous sexual stage-specific proteins have been discovered, some of which play crucial roles for parasite transmission. However, the functions of many sexual stage proteins remain elusive. Amongst the sexual stage-specific proteins
are the proteins of the PfCCp proteins family, which exhibit numerous adhesion domains in their protein structures. For four members of the protein family, PfCCp1 to PfCCp4 gene-disruptant parasite lines had been already studied. Amongst these, PfCCp2 and PfCCp3 showed an important role for development of the parasites in the
mosquito. In the present work the study of gene-disrupted parasites of the PfCCp Protein family was completed. PfCCp5-KO and PfFNPA-KO parasite lines were characterized
to a great extent and many properties were similar to those of other PfCCp proteins. The co-dependent expression previously reported to be a phenomenon of PfCCp
proteins was also observed in these two mutants, although to lesser extent. When either PfCCp5 or PfFNPA were absent, all other proteins were detected in reduced abundance only. Co-dependent expression manifests exclusively on the protein level. Transcript
levels were not altered as RT-PCR showed. Amongst PfCCp proteins numerous proteinproteins interactions are taking place. The previously described multimeric protein
complexes also include further sexual stage-specific proteins like Pfs230, Pfs48/45 and Pfs25. Recently, a new component of PfCCp-based multimeric protein complexes had
been identified. The protein was named PfWLP1 (WD repeat protein-like protein 1) due to its possession of several WD40 repeats. In the present study expression of this uncharacterized protein was investigated via indirect IFA. It was expressed in asexual blood stages and gametocytes. Upon gamete formation and fertilization its expression ceased. Another sexual stage protein studied in this work was PfactinII. It was shown to be exclusively
expressed in sexual stages. In gametocytes it co-localizes with Pfs230 and correct localization of PfactinII depends on presence of Pfs230. Transcript analysis by means of RT-PCR revealed the expression of several components of the IMC in gametocytes. Furthermore, five or six myosin genes encoded in the
P. falciparum genome were detected in gametocytes. Gametocyte egress was studied on the ultrastructural level via transmission electron microscopy and an inside-out type of egress was observed. Firstly, the membrane of the parasitophorous vacuole (PVM) was lysed and only thereafter the membrane of the red blood cell (RBCM) ruptured. Furthermore, a new inductor of gametogenesis was identified: The K+/H+ ionophore nigericin induced gametocytes activation in the absence of xanthurenic acid (XA), which is responsible for gamtetocyte activation in the mosquito midgut. Selective permeabilization of RBCM and PVM by the mild detergent saponin, showed that in the absence of these membranes male gametocytes were still able to perceive both XA and the drop in temperature. Thus, the receptors for both factors signaling the parasite transmission to the mosquito, seem to be of parasitic origin. LC/MS/MS analysis confirmed the ability
of RBCs to take up XA. With malaria eradication on the agenda of malaria research targeting the sexual stages
becomes a crucial part of intervention strategies. The sexual stages are especially attractive target as they represent a population bottleneck. The here reported findings on P. falciparum gametocytes provide several potential candidate proteins for developing tools to interrupt transmission from man to mosquito. Such tools might include Transmission blocking vaccines and drugs.