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.</p

Effects of proteases on CLAG3 and PSAC-mediated transport.

<p>(A) Immunoblots showing CLAG3 hydrolysis in HB3 and Dd2 parasites by chymotrypsin (“Ch”), trypsin (“Tr”), or pronase E (“PrE”); mouse anti-CLAG3 generated using a recombinant C-terminal fragment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093759#pone.0093759-Nguitragool1" target="_blank">[23]</a>. The band at ∼160 kDa reflects uncleaved CLAG3; a C-terminal proteolysis fragment at ∼35 kDa is visible upon protease treatment. Addition of 2 mM PMSF abolishes cleavage by chymotrypsin. (B) Osmotic lysis kinetics for HB3- and Dd2-infected cells in sorbitol. Control traces represent matched samples not exposed to proteases or PMSF (black traces). While pronase E retards PSAC-mediated osmotic lysis and trypsin is without effect in both parasites (green and blue traces, respectively), chymotrypsin inhibits transport in HB3- but not Dd2-infected cells (red solid traces). (C) Mean ± S.E.M. PSAC inhibition determined from osmotic lysis experiments, normalized to 0% for no protease controls. (D) Mean ± S.E.M. inhibition resulting from chymotrypsin treatment of erythrocytes infected with indicated parasites.</p

Schematic showing models for protease action on PSAC.

<p>(A) Functional channel with a variable extracellular loop and permeating solute (red circle and arrow). (B) Trypsin digestion of extracellular loop, yielding a positively charged end. Solute transport is preserved. (C-E) Possible models of chymotrypsin inhibition in sensitive clones. Panel C shows a collapsed pore due to proteolysis at a critical site in the extracellular loop. Panel D shows steric hindrance of the channel pore, which may prevent solute permeation. Panel E shows cleavage at additional site(s) exposed after cleavage of the extracellular loop.</p

<i>clag3</i> genes accounts for the differing sensitivities to chymotrypsin.

<p>(A) Mean ± S.E.M. block of sorbitol uptake by chymotrypsin treatment on indicated parental lines and progeny clones (black and gray bars, respectively). (B) Logarithm of odds (LOD) scores from a primary scan of QTL associated with PSAC inhibition. The peak at the 5′ end of chromosome 3 contains the two <i>clag3</i> genes. The <i>P</i> = 0.05 significance threshold (dashed horizontal line) was calculated from 1000 permutations. Inset shows results from a secondary scan for additional QTL after controlling for the <i>clag3</i> locus. No other loci reached the <i>P</i> = 0.05 threshold (dashed horizontal line). (C) Osmotic lysis kinetics for indicated parasites after selection for expression of a specific <i>clag3</i> gene. Black and red traces represent no protease control and chymotrypsin-treated cells, respectively. The ribbon schematic at the top of each panel shows the gene structure for the two <i>clag3</i> genes in each parasite with active transcription indicated by a bent arrow. The <i>clag3</i> gene resulting from allelic exchange in HB3<i>^3rec</i> has a gray shaded 3′ end to indicate the fragment derived from Dd2 (“<i>chimera</i>”). For each parasite, relative expression of the two paralogs is shown with an ethidium-stained gel at the bottom right of each panel. (D) Mean ± S.E.M. chymotrypsin-induced inhibition for each selected parasite.</p

Genetic differentiation (<i>Fst</i>) of <i>P</i>. <i>vivax</i> populations.

<p>Genetic differentiation (<i>Fst</i>) of <i>P</i>. <i>vivax</i> populations.</p

Substantial population structure of <i>Plasmodium vivax</i> in Thailand facilitates identification of the sources of residual transmission

<div><p>Background</p><p><i>Plasmodium vivax</i> transmission in Thailand has been substantially reduced over the past 10 years, yet it remains highly endemic along international borders. Understanding the genetic relationship of residual parasite populations can help track the origins of the parasites that are reintroduced into malaria-free regions within the country.</p><p>Methodology/Results</p><p>A total of 127 <i>P</i>. <i>vivax</i> isolates were genotyped from two western provinces (Tak and Kanchanaburi) and one eastern province (Ubon Ratchathani) of Thailand using 10 microsatellite markers. Genetic diversity was high, but recent clonal expansion was detected in all three provinces. Substantial population structure and genetic differentiation of parasites among provinces suggest limited gene flow among these sites. There was no haplotype sharing among the three sites, and a reduced panel of four microsatellite markers was sufficient to assign the parasites to their provincial origins.</p><p>Conclusion/Significance</p><p>Significant parasite genetic differentiation between provinces shows successful interruption of parasite spread within Thailand, but high diversity along international borders implies a substantial parasite population size in these regions. The provincial origin of <i>P</i>. <i>vivax</i> cases can be reliably determined by genotyping four microsatellite markers, which should be useful for monitoring parasite reintroduction after malaria elimination.</p></div

Genetic differentiation (<i>Fst</i>) of <i>P</i>. <i>vivax</i> populations.

<p>Genetic differentiation (<i>Fst</i>) of <i>P</i>. <i>vivax</i> populations.</p

Optimal panel of MS markers and determination of haplotype loss during the removal of each MS.

<p>The removal of MS was prioritized from lower to higher <i>H</i>_<i>E</i> according to the X-axis. The remaining haplotypes after removal were shown by the percentages of all haplotypes.</p

Principal coordinate analysis of <i>P</i>. <i>vivax</i> haplotypes from three provinces.

<p>Principal coordinate analysis of <i>P</i>. <i>vivax</i> haplotypes from three provinces.</p

Population genetic structure of <i>P</i>. <i>vivax</i> in three provinces (K = 2–5).

<p>The structure was plotted by using 10 (A) and (B) 4 MS markers (MS2, MS6, MS10 and MS12).</p