Disruption of Plasmodium falciparum chitinase markedly impairs parasite invasion of mosquito midgut

Abstract

To initiate invasion of the mosquito midgut, Plasmodium ookinetes secrete chitinolytic activity to penetrate the peritrophic matrix surrounding the blood meal. While ookinetes of the avian malaria parasite Plasmodium gallinaceum appear to secrete products of two chitinase genes, to date only one chitinase gene, PfCHT1, has been identified in the nearly completed Plasmodium falciparum strain 3D7 genome database. To test the hypothesis that the single identified chitinase of P. falciparum is necessary for ookinete invasion, the PfCHT1 gene was disrupted 39 bp upstream of the stop codon. PfCHT1-disrupted parasites had normal gametocytogenesis, exflagellation, and ookinete formation but were markedly impaired in their ability to form oocysts in Anopheles freeborni midguts. Confocal microscopy demonstrated that the truncated PfCHT1 protein was present in mutant ookinetes but that the concentration of mutant PfCHT1 within the apical end of the ookinetes was substantially reduced. These data suggest that full-length PfCHT1 is essential for intracellular trafficking and secretion and that the PfCHT1 gene product is necessary for ookinetes to invade the mosquito midgut.

FIG. 1

Design and experimental verification of PfCHT1 gene disruption in P. falciparum strain 3D7. (A) A PCR-amplified partial coding sequence corresponding to nucleotides 153 to 1095 of PfCHT1 (stippled box labeled pfcht1) was inserted into plasmid pHDWT, which contains the human dihydrofolate reductase gene as a selectable marker under the control of the 5′ untranslated sequence of Pfhrp3 and the 3′ untranslated sequence of Pfhrp2. The 6.8-kb disruption plasmid is indicated as pPfCHT1KO1. Primers 1 and 4 are from the 5′ and 3′ ends, respectively, of the PfCHT1 coding region not included in the disruption construct. Primer 2 is from the 3′ Pfhrp2 untranslated region, and primer 3 is from the pBluescript plasmid backbone. The wild-type 3D7 PfCHT1 locus on chromosome 12 is diagrammed before and after (labeled as 19.1) the predicted integration event. The asterisk indicates the chitinase enzymatic active site. (B) Southern blot analysis of the PfCHT1 locus in wild-type (WT) 3D7 and mutant 19.1. Genomic DNA was digested with either SpeI or BglII and probed with the digoxigenin-labeled PfCHT1 coding sequence; chemiluminescence was used for development of the blot. ORF, open reading frame. (C) PCR analysis of wild-type 3D7 (wt) and mutant 19.1 with pairs of oligonucleotide primers schematically depicted in panel A. PCR of the Pfs25 gene encoding the 25-kDa P. falciparum zygote-ookinete surface protein was performed as a positive control to demonstrate the presence of amplifiable DNA. Std, molecular size standards. (D) Reverse transcriptase PCR was performed using RNA extracted from wild-type 3D7 or 19.1 gametocytes. RT+ and RT− indicate the presence and absence of reverse transcriptase in the reaction mixture, respectively. Primers to amplify Pfs25 were used as a positive control to demonstrate equivalent amounts of Pfs25 RNA in the wild-type 3D7 and 19.1 RNA samples.

FIG. 2

Confocal microscopy of wild-type 3D7 and 19.1 ookinetes. The presence of the P. falciparum zygote-ookinete surface proteins and PfCHT1 was simultaneously assessed in ookinetes found in A. freeborni midguts 30 h after ingestion of a blood meal. MAb, monoclonal antibody. The arrow indicates the apical end of an ookinete.