Plasmodium knowlesi provides a rapid in vitro and in vivo transfection system that enables double-crossover gene knockout studies

Abstract

Transfection technology for malaria parasites provides a valuable tool for analyzing gene function and correlating genotype with phenotype. Transfection models are even more valuable when appropriate animal models are available in addition to complete in vitro systems to be able to fully analyze parasite-host interactions. Here we describe the development of such a model by using the nonhuman primate malaria Plasmodium knowlesi. Blood-stage parasites were adapted to long-term in vitro culture. In vitro-adapted parasites could re-adapt to in vivo growth and regain wild-type characteristics after a single passage through an intact rhesus monkey. P. knowlesi parasites, either in vitro adapted or in vivo derived, were successfully transfected to generate circumsporozoite protein (CSP) knockout parasites by double-crossover mechanisms. In vitro-transfected and cloned CSP knockout parasites were derived in a time span of only 18 days. Microscopic evaluation of developing oocysts from mosquitoes that had fed on CSP knockout parasites confirmed the impairment of sporozoite formation observed in P. berghei CSP knockout parasites. The P. knowlesi model currently is the only malaria system that combines rapid and precise double-crossover genetic manipulation procedures with complete in vitro as well as in vivo possibilities. This allows for full analysis of P. knowlesi genotype-phenotype relationships and host-parasite interactions in a system closely related to humans.

FIG. 1.

DNA constructs and analysis of integration into P. knowlesi CSP locus after transfection. (A) Linear DNA construct designed for integration into the P. knowlesi CSP locus. The 5-kb selectable marker cassette (TgDHFR cassette) contains P. berghei dhfr/ts flanking regions controlling expression of mutagenized T. gondii dhfr/ts. Relevant restriction sites are indicated: R, EcoRI; P, PstI; H, HindIII; B, BamHI. (C) The location of the PCR primers A and B and the specific probe used for Southern blotting are shown. ORF-containing sequences are marked with an open arrow, and sequences used for targeted integration are indicated. (B) PCR analysis of transfected parasites. Lanes 1 to 3 show PCR with CSP integration-specific primers A and B. Lanes: 1, P. knowlesi H-strain DNA; 2, PkCSPko clone DNA; 3, pD_B.D_Tm.D_B/CSPko vector DNA. PkCSPko and the transfection construct were all positive for PCR with the two T. gondii dhfr/ts-specific primers (not shown). (C) Southern blot analysis of transfected parasites. PstI-digested DNA from P. knowlesi H strain (lane 2), PkCSPko clone (lane 3), and the transfection vector pD_B.D_Tm.D_B/CSPko (lane 1) was used to prepare a Southern blot. The blot was probed with a P. knowlesi CSP-specific probe (see panel A).

FIG. 2.

In vitro growth characteristics of P. knowlesi H strain. Parasitemia development over a 15-day period. Parasites were cultured as described in Materials and Methods, and after 3 months the parasitemia level was determined daily and expressed as a percentage of infected red blood cells. Arrows indicate the days on which the culture was diluted.

FIG. 3.

Microscopic evaluation of wild-type and CSP knockout P. knowlesi oocyst development in A. stephensi midguts. (A) Intact wild-type oocyst 9 days postfeeding. (B) Intact CSP knockout oocyst 11 days postfeeding. (C) Wild-type oocyst releasing sporozoites (marked with arrows) 8 days postfeeding. (D) Force-induced rupture of CSP knockout oocyst 11 days postfeeding. Sporozoite release was never observed in CSP knockout oocysts.