Malaria is one of the most lethal infectious diseases worldwide. Understanding the
biology of the causative agent Plasmodium will lead to better control of the disease.
The biogenesis and maintenance of the parasitophorous vacuole (PV) within the
infected erythrocyte is an essential factor for parasite survival. The PV has been
postulated to be involved in various pivotal functions, however little is known about
the PV contents and their respective functions. Our group had previously provided the
first PV’s proteome research and have continuously exposed more members of this
important compartment. The protein PfPV1 was a newly discovered PV localisation
protein, encoded by the PF11_0302 gene. In order to address the function of this
protein a gene knock-out strategy was applied. A search for the interacting proteins of
PfPV1 was also carried out using the GST pull-down assay.
The first attempt to knock out the encoding gene was the double-crossover strategy in
the presence of a negative selection. The knocked-out parasite was unable to obtain.
However, the integration into the PfPV1 locus did occur, evidenced by the presence of
both endogenous and knock-out band in the specific southern blot hybridisation. The
PfPV1 gene was therefore assumed to be essential for in vitro growth, thus the
targeting into the endogenous locus was accompanied by a duplication event for
maintaining expression of the gene.
The assumption was further validated by the second knock-out strategy, using the
complementing experiment. The result had been expected to be able to disrupt the
endogenous gene locus through the knock-out vector while concomitantly expressing
a copy of PfPV1 under the control of a foreign promoter from an episomal plasmid,
which should not recombine with the endogenous gene locus. However, the gene was
still resistant to be disrupted. Various clones were isolated from the double transfected
parasites. One of the clones has lost the episomal copy of the PfPV1 gene and showed
the same southern blot result as the single transfected parasite, indicating that the
parasite needs to maintain the expression of the endogenous gene. Other clones, if
keeping the episomal copy of the PfPV1, did not show the specific integration. The
result strongly suggests that the PfPV1 expression needs to be controlled by its
endogenous promoter to be fully active.
The data has also proved for the first time that in some cases of the negative selection
strategy, upon the integration, the Plasmodium parasite might rearrange the thymidine
kinase encoding sequence in order to inactivate its activity, therefore prevent the
lethal effect from converting ganciclovir to toxic metabolite.
In the GST pull-down assay, no interacting protein was obtained. However, the
experiment was carried out with the cell extract from trophozoite-stage parasites, thus
might not detect interactions at other stages.
In conclusion, the data suggest that PfPV1 is a conserved, unique protein with
unknown but essential function during the intraerythrocytic cycle.