A variant extracellular residue accounts for PSAC resistance to chymotrypsin in Dd2 parasites.

<p>(A) Multiple sequence alignment of an extracellular loop on indicated CLAG3.1 and CLAG3.2 sequences, representing geographically divergent parasites (Dd2, from Indochina; 3D7, probably Africa; 7G8 Brazil; HB3, Honduras). A variable segment is apparent in gray shading (Consensus); residues susceptible to chymotrypsin cleavage are shown in blue. Two sites refractory to cleavage in Dd2 CLAG3.1 are highlighted in red. (B) Schematic showing the allelic exchange strategy to introduce a single mutation in the Dd2 <i>clag3.1</i> gene. Plasmid carrying the mutation is shown at the top; single homologous recombination into the Dd2 genome produces an intact full-length gene with a single site mutation and unchanged UTR sequences. (C) Southern blot showing integration of plasmid into the Dd2^L1115F genome. Indicated DNA samples were digested and probed with an hDHFR-specific probe. Dd2 is not recognized by this probe, but Dd2^L1115F yields a single band whose size differs from that of the plasmid. (D) Osmotic lysis kinetics for Dd2^L1115F without and with 1h chymotrypsin treatment (black and red traces, respectively). Inset shows preferential expression of the mutated <i>clag3.1</i> gene in this clone. (E) Mean ± S.E.M. chymotrypsin-induced inhibition for indicated parasites. (F) Dose responses for inhibition of sorbitol permeability (<i>P</i>) by ISPA-28. Black and red symbols represent mean ± S.E.M. inhibition for Dd2 and Dd2^L1115F parasites, respectively. Solid lines represent best fits to the sum of two Langmuir isotherms.