| Summary: | <p>Abstract</p> <p>Background</p> <p>The <it>Plasmodium </it>Cysteine Repeat Modular Proteins (PCRMP) are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite <it>Plasmodium berghei </it>lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of <it>P. berghei </it>sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse.</p> <p>Methods</p> <p>In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the <it>Plasmodium </it>life cycle by generation and analysis of <it>P. berghei </it>gene deletion mutants, Δ<it>pcrmp</it>3 and Δ<it>pcrmp</it>4. The role of PCRMP members during the transmission and hepatic stages of the <it>Plasmodium </it>lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells <it>in vitro </it>and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Δ<it>pcrmp</it>3 and Δ<it>pcrmp</it>4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine.</p> <p>Results</p> <p>Disruption of <it>pcrmp3 </it>and <it>pcrmp4 </it>in <it>P. berghei </it>revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to <it>pbcrmp1 </it>and <it>2</it>. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Δ<it>crmp3 </it>and Δ<it>crmp4 </it>sporozoites does not confer protective immunity upon subsequent challenge.</p> <p>Conclusions</p> <p>PCRMP3 and 4 play multiple roles during the <it>Plasmodium </it>life cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes. However, although Δ<it>pcrmp3 </it>and Δ<it>pcrmp4 </it>parasites are completely growth-impaired in the liver, immunization with live sporozoites does not induce the protective immune responses that have been shown for other genetically-attenuated parasites.</p>
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