Disruption of Plasmodium falciparumChitinase Markedly Impairs Parasite Invasion of Mosquito Midgut

Design and experimental verification ofPfCHT1 gene disruption in P. falciparumstrain 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 ofPfhrp3 and the 3′ untranslated sequence ofPfhrp2. 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′ Pfhrp2untranslated 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 PfCHT1locus 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 amplifyPfs25 were used as a positive control to demonstrate equivalent amounts of Pfs25 RNA in the wild-type 3D7 and 19.1 RNA samples.