Flp/<i>FRT</i>-mediated disruption of <i>ptex150</i> and <i>exp2</i> in <i>Plasmodium falciparum</i> sporozoites inhibits liver-stage development.

Abstract

<i>Plasmodium falciparum</i> causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during <i>P. falciparum</i> liver infection remains unexplored. Here, we use the FlpL/<i>FRT</i> system to conditionally excise genes in <i>P. falciparum</i> sporozoites for functional liver-stage studies. Disruption of PTEX members <i>ptex150</i> and <i>exp2</i> did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage <i>Plasmodium</i> export element (PEXEL) proteins. <i>P. falciparum</i> liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a <i>Plasmodium berghei</i> LISP2 reporter all localized to the periphery of <i>P. falciparum</i> liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in <i>ptex150-FRT</i> and <i>exp2-FRT</i> liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for <i>P. falciparum</i> development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking.