Plasmodium falciparum Falcipain-2a Polymorphisms in Southeast Asia and Their Association With Artemisinin Resistance

Plasmodium falciparum Falcipain-2a Polymorphisms in Southeast Asia and Their Association With... Abstract Background Falcipain-2a ([FP2a] PF3D7_1115700) is a Plasmodium falciparum cysteine protease and hemoglobinase. Functional FP2a is required for potent activity of artemisinin, and in vitro selection for artemisinin resistance selected for an FP2a nonsense mutation. Methods To investigate associations between FP2a polymorphisms and artemisinin resistance and to characterize the diversity of the enzyme in parasites from the China-Myanmar border, we sequenced the full-length FP2a gene in 140 P falciparum isolates collected during 2004–2011. Results The isolates were grouped into 8 different haplotype groups. Haplotype group I appeared in samples obtained after 2008, coinciding with implementation of artemisinin-based combination therapy in this region. In functional studies, compared with wild-type parasites, the FP2a haplotypes demonstrated increased ring survival, and all haplotype groups exhibited significantly reduced FP2a activity, with group I showing the slowest protease kinetics and reduced parasite fitness. Conclusions These results suggest that altered hemoglobin digestion due to FP2a mutations may contribute to artemisinin resistance. ACT, artemisinin resistance, falcipain 2a, malaria, Plasmodium falciparum Evolution of drug resistance in malaria parasites is one of the biggest roadblocks to malaria control and elimination. The recent emergence of resistance in Plasmodium falciparum to frontline artemisinin-based combination therapies (ACTs) in Southeast Asia has raised international concerns [1]. The potential spread of resistant parasite strains to Africa, the heartland of malaria transmission, would be catastrophic for global malaria control and elimination. Therefore, it is essential to identify the molecular markers of resistance and vigilantly monitor the spread of resistant genotypes. Resistance against artemisinin and its derivatives in P falciparum is clinically manifested as slow parasite clearance in patients [2], and resistant parasites show increased survival when exposed to pharmacologically relevant concentrations of dihydroartemisinin at the ring stage in vitro [3]. Genomic and genetic analyses identified and confirmed that mutations in the propeller domain of the kelch protein K13 are associated with artemisinin resistance [4]. In Western Cambodia, where artemisinin resistance was first recognized, the K13 C580Y mutation has approached fixation, and resistant parasites carrying this mutation have spread to Thailand and Laos [4, 5]. Identification of artemisinin-resistant field isolates from Cambodia lacking K13 mutations suggests the involvement of additional resistance mechanisms [6]. Association studies have identified other genetic loci associated with in vivo and in vitro resistance to artemisinins [7, 8], and these results await genetic confirmation. Falcipains (FPs) are cysteine proteases that digest hemoglobin in the food vacuole to provide amino acids (AAs) for intraerythrocytic parasite development. Plasmodium falciparum encodes 4 FP genes (FP1, FP2a, FP2b, and FP3), among which FP2a (PF3D7_1115700), with peak expression in the early trophozoite stage, and FP3, with expression later in the erythrocytic cycle, appear to be the principal cysteine proteases involved in hemoglobin digestion [9]. Falcipain-2a knockout (KO) trophozoites are characterized by a swollen hemoglobin-filled food vacuole due to transiently reduced hemoglobin digestion [10, 11], which is linked to diminished parasite susceptibility to artemisinins, suggesting that hemoglobin digestion is required for artemisinin activity [12, 13]. Intriguingly, in addition to acquiring a K13 mutation (M476I), parasites selected in vitro for artemisinin resistance also acquired a nonsense mutation at codon 69 of FP2a [4]. This result and the finding that FP2a expression is required for potent artemisinin activity [12] suggest that FP2a mutations might contribute to artemisinin resistance. We previously showed that P falciparum isolates with K13 mutations collected from the China-Myanmar border were significantly associated with persistence of parasitemia on day 3 after the onset of treatment and with increased survival in the ring-stage survival assay (RSA), an in vitro marker for artemisinin resistance [14]. Given the potential contribution of FP2a polymorphisms to artemisinin resistance, we investigated their association with in vitro sensitivity to artemisinins in 140 clinical parasite isolates from the China-Myanmar border area, where artemisinins have been used to treat malaria for over 3 decades. We show that FP2a haplotypes with perturbed hemoglobin digestion and decreased fitness are common in this region and associated with decreased artemisinin sensitivity, suggesting selection of altered FP2a by the selective pressure of ACTs. MATERIALS AND METHODS Parasite Isolates and Drug Assay Plasmodium falciparum clinical isolates were collected during 2004–2011 in malaria clinics located along the China-Myanmar border, cultured, and archived [15]. Monoclonal parasite isolates were confirmed by genotyping at 3 polymorphic genes merozoite surface protein 1 (msp1), msp2, and glutamate-rich protein, as reported previously [8]. Parasites were maintained in O+ human erythrocytes as described [16, 17]. A total of 140 monoclonal isolates were analyzed, including 2 collected in 2004, 18 in 2007, 44 in 2008, 64 in 2009, 11 in 2010, and 1 in 2011. The RSA was performed as previously described [14]. Informed consent or assent was obtained from all patients or legal guardians before enrollment. All studies were approved by the ethical committees from the participating institutions and the local health department. Sequencing of Falcipain 2a Gene Parasite genomic deoxyribonucleic acid (DNA) was extracted using the Wizard Genomic DNA Purification Kit (Promega). The full-length FP2a gene was amplified and sequenced using primers listed in Supplementary Table S1. Sequences were assembled using Vector NTI (Invitrogen) with manual editing and aligned against the reference FP2a sequence of 3D7 (PF3D7_1115700 in PlasmoDB) using MEGA6.0 [18, 19]. A phylogenetic tree was constructed using the Neighbor-Joining method with 1000 pseudo-replications. We grouped the haplotypes based on their clustering on the phylogenetic tree [20]. The FP2a sequences reported in this study were deposited in GenBank (accession numbers MG768692–MG768822 and MG768823–MG768831). Molecular Modeling The three-dimensional (3D) structure of FP2a in complex with the Plasmodium berghei homolog of falstatin was retrieved from the Protein Data Bank ([PDB] 3PNR:aa245-484), and Swiss modeler was used to model identified FP2a mutations. For mapping the mutation in falstatin, the 3D structure was predicted by using the I-TASSER online protein structure prediction tool [21]. Web-based softwares PROVEAN (Protein Variation Effect Analyzer) and SIFT (Sorting Intolerant From Tolerant) were used to predict the effects of FP2a mutations. Assays for Parasite Phenotypes and Fitness To test parasite fitness under AA-deficient conditions, parasites at 1% parasitemia with 2% hematocrit were cultured in either complete media (CM) composed of Roswell Park Memorial Institute (RPMI) medium 1640 (Gibco Life Technologies, Grand Island, NY) with 25 mM NaHCO3, 25 mM HEPES (pH 7.4), 11 mM glucose, 0.367 mM hypoxanthine, and 5 µg/L gentamicin supplemented with 0.5% Albumax (Gibco Life Technologies), or AA-depleted medium ([CM-AA] identical to CM except for RPMI without AAs) (US Biological Science) following a previously described method [22]. Parasite growth was quantified from Giemsa-stained thin smears at 24, 42, and 48 hours postinvasion. The growth competency of parasites in CM-AA was calculated as ([no. of schizonts in CM-AA-]/[no. of schizonts in CM] × 100%) at the time when at least 15% of parasites cultured in CM had reached the schizont stage. Multiplication competency was determined by counting the average number of merozoites in mature schizonts (Navg) at the time when at least 40% of parasites cultured in CM were at the ring stage. Merozoite nuclei were stained with 0.8 × SYBR Green I and counted in a Guava easyCyte 5HT Benchtop Flow Cytometer at λex = 488 nm and λem = 525 ± 30 nm. At least 50000 total events in the green channel were collected. Green fluorescence intensity (FI) from the gated populations was normalized by dividing by the mean FI of purified rings, rounding off to the nearest integer, and plotting into histograms with bins of 4 using MS Excel. Growth in CM-AA and Navg were obtained from at least 3 technical replicates, with 2 biological replicates each. The FP2a protease activity was quantified via a modified method [23] wherein extracts from synchronized trophozoites (20 h after invasion) were tested for the ability to hydrolyze 7-amino-4-methyl coumarin from Z-Leu-Arg-AMC ([Z-LR-AMC] Bachem), assessed at λex = 355 nm and λem = 460 nm for 60 minutes. Fluorescence intensities at 100, 50, 25, 12.5, and 6.25 µM Z-AMC were plotted in GraphPad Prism 5. The first 10–15 minutes of reactions were fitted to y = mx + b, and linear slopes (m) were plotted against [Z-LR-AMC] to obtain the “Km” and “Vmax” values. Hydrolysis is attributed to FP2a, because it is maximally expressed in the early trophozoite stage and was shown to be responsible for 93% of the cysteine protease activity in extracts from early trophozoites [9, 23, 24]. Protein concentrations in trophozoite extracts were determined using the Pierce Coomassie Plus (Bradford) Assay Kit (Thermo Fisher), and 1 µg protein was used for all experiments. Hemoglobin Degradation Assays To assess the hemoglobinase activity of different field isolates, 5 µg of the trophozoite extract was added to 30-μL reactions containing 2 µg of hemoglobin in 100 mM NaOAc, pH 5.5, and 1 mM glutathione. Reactions were carried out at 37°C for 2 hours, stopped by the addition of 10 µL 4× sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and then electrophoresed on 15% SDS-PAGE gels. These were either stained with Coomassie or transferred onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were probed with rabbit antisera directed to either human hemoglobin (diluted 1:5000; Sigma) or P falciparum-aldolase (diluted 1:5000; Abcam). Statistical Analysis Statistical tests were done in MS Excel, SigmaPlot version 10, and GraphPad Prism 5. RESULTS Genetic Diversity of Falcipain 2a Sequencing of FP2a in 140 clinical isolates collected from the China-Myanmar border identified 34 nonsynonymous single-nucleotide polymorphisms (SNPs), with at least 1 SNP in 97.1% (137 of 140) of the isolates (Supplementary Table S2). Three SNPs (Y3C, D123G, and F165I) have not been described previously. The FP2a consists of a prodomain composed of a transmembrane domain, an inhibitory prodomain and a functional cysteine protease domain (Figure 1A). Six of the identified SNPs (T343P, D345G, A353T, V393I, A400P, and Q414E) are in the cysteine protease domain, 5 (M245I, E248D, E249A, K255R, and N257E) in the prodomain, and none in the hemoglobin-binding domain. A number of SNPs demonstrated high prevalence in the 140 isolates, notably, Q15H (77.3%), V51I (56.7 %), S59F (56.7%), K255R (84.4%), N257E (85.1%), T343P (71.6%), D345G (73.1%), and Q414E (74.5%) (Supplementary Figure S1A). Only 3 isolates (2 collected in 2008 and 1 in 2010) had the wild-type (WT) 3D7 sequence. The FP2a SNPs resulted in 25 distinct haplotypes. Because these were too diverse to be analyzed individually, they were studied as FP2a haplotype groups based on their phylogenetic clustering patterns (Figure 1B). For the 8 identified haplotype groups, no clear temporal frequency trends were recognized, except for group I, which appeared after 2008 (Figure 1B). It is interesting to note that our earlier study identified 8 of 140 isolates that carried a nonsynonymous mutation (T213I) in the falstatin gene [8], which encodes an endogenous P falciparum cysteine protease inhibitor. These 8 isolates were all in FP2a haplotype group I. Figure 1. View largeDownload slide Falcipain-2a (FP2a) haplotypes of 140 China-Myanmar Plasmodium falciparum isolates. (A) Domain organization of the FP2a protein showing functional cysteine protease domain ([CP] amino acids [AA], 261–482), inhibitor domain ([ID] AA, 165–222), and the transmembrane domain ([TM] AA, 35–57). (B) Twenty-five distinct haplotypes identified in the 140 isolates were categorized into 8 haplotype groups based on sequence phylogeny. The left panel shows each haplotype groups and their frequency in 140 samples. The bar graph (right) shows the proportions of haplotype groups over time. Figure 1. View largeDownload slide Falcipain-2a (FP2a) haplotypes of 140 China-Myanmar Plasmodium falciparum isolates. (A) Domain organization of the FP2a protein showing functional cysteine protease domain ([CP] amino acids [AA], 261–482), inhibitor domain ([ID] AA, 165–222), and the transmembrane domain ([TM] AA, 35–57). (B) Twenty-five distinct haplotypes identified in the 140 isolates were categorized into 8 haplotype groups based on sequence phylogeny. The left panel shows each haplotype groups and their frequency in 140 samples. The bar graph (right) shows the proportions of haplotype groups over time. Comparison of the 140 FP2a sequences with 45 published FP2a sequences revealed clear geographical separation of Asian and African parasites (Supplementary Figure S2 and Supplementary Table S3). Asian isolates had greater nucleotide and haplotype diversity, suggesting that these parasites have experienced stronger selection pressure (Supplementary Table S4). Structural Predictions To predict whether the identified FP2a SNPs interfere with enzymatic activity, we mapped the SNPs in the cysteine protease domain of FP2a in complex with the P berghei homolog of falstatin ([PbICP] inhibitor of cysteine proteases) cocrystal structure (3PNR.pdb: AA, 245–484) (Figure 2A); this structure was used because a structure of P falciparum falstatin is not available. Model predictions show A353, A400, and Q414 in the binding interface of FP2a and PbICP, whereas A353 is partially buried and A400 is fully buried into the interface and located in the center of a short helix (Figure 2B). A400P may disrupt the helix structure and make it too bulky to fit in the FP2a-PbICP interface. Q414E may disrupt the hydrogen bond with the inhibitor at Q308 (Figure 2C). In silico web-based PROVEAN and SIFT programs predict that T343P and D345G may alter the protein structure. Because none of these SNPs occur singly in any of the isolates, we performed phenotypic assays based on the haplotype phylogeny (Figure 1B). Figure 2. View largeDownload slide Three-dimensional modeling of Pf3D7-falcipain-2a (FP2a) (amino acids, 245–484) using 3PNR.pdb. (A) Single-nucleotide polymorphisms in the protease domain (T343P, D345G, A353T, V393I, A400P, and Q414E) and the prodomain (M245I, E248D, E249A, K255R, and N257E) are highlighted. (B) A353, A400, and Q414 lie in the binding interface of FP2a and its inhibitor. A353 is partially buried, whereas A400 is fully buried in the interface and located in the middle of a short helix. (C) Q414 forms a hydrogen bond with the inhibitor. Abbreviation: ICP, Plasmodium berghei homolog of falstatin (inhibitor of cysteine proteases). Figure 2. View largeDownload slide Three-dimensional modeling of Pf3D7-falcipain-2a (FP2a) (amino acids, 245–484) using 3PNR.pdb. (A) Single-nucleotide polymorphisms in the protease domain (T343P, D345G, A353T, V393I, A400P, and Q414E) and the prodomain (M245I, E248D, E249A, K255R, and N257E) are highlighted. (B) A353, A400, and Q414 lie in the binding interface of FP2a and its inhibitor. A353 is partially buried, whereas A400 is fully buried in the interface and located in the middle of a short helix. (C) Q414 forms a hydrogen bond with the inhibitor. Abbreviation: ICP, Plasmodium berghei homolog of falstatin (inhibitor of cysteine proteases). Association of Falcipain 2a Haplotypes and K13 Mutations With Decreased Artemisinin Sensitivity As a proxy for artemisinin resistance, we determined RSA values for the 140 parasite strains. A survival rate above 1% in the RSA predicts clinical resistance [3]. Mean RSA values for haplotype groups I (2.0%) and IV (6.6%) were significantly higher than for WT parasites (3D7 and 3 field isolates with the same sequences) (0.43%) (Figure 3A). In addition, the mean RSA value (2.5%) for all 137 isolates with FP2a SNPs was significantly higher than that for the WT group. Considering specific SNPs, group I is predominantly composed of isolates with the F446I K13 mutation (9 of 10 isolates), 8 of which also contain the falstatin T213I mutation, whereas group IV has 5 of 9 isolates with the K13 P574L mutation and 1 with F446I (Supplementary Figure S3). Figure 3. View largeDownload slide Phenotypic characterization of representative isolates. (A) Mean survival rates from ring-stage survival assay of 140 isolates grouped according to falcipain-2a (FP2a) haplotype groups (I–VIII). Wild type (WT) = 3D7 and 3 field isolates with WT K13 and FP2a sequences. ALL = mean for all 137 isolates with FP2a single-nucleotide polymorphisms (SNPs). (B) Mean growth in amino acid-depleted complete medium (CM-AA) of 29 representative isolates. ALL = mean growth in CM-AA for all 27 isolates with FP2a SNPs. (C) Average number of merozoites (Navg) in the mature schizonts of 29 representative isolates. ALL = mean Navg for all 27 isolates with FP2a SNPs. *, P < .05 (t test) vs WT in A–C. (D) Michealis-Menten curve-fits of Z-LR-AMC hydrolysis by trophozoite extracts from WT and parasites in haplotype groups I–VIII. Figure 3. View largeDownload slide Phenotypic characterization of representative isolates. (A) Mean survival rates from ring-stage survival assay of 140 isolates grouped according to falcipain-2a (FP2a) haplotype groups (I–VIII). Wild type (WT) = 3D7 and 3 field isolates with WT K13 and FP2a sequences. ALL = mean for all 137 isolates with FP2a single-nucleotide polymorphisms (SNPs). (B) Mean growth in amino acid-depleted complete medium (CM-AA) of 29 representative isolates. ALL = mean growth in CM-AA for all 27 isolates with FP2a SNPs. (C) Average number of merozoites (Navg) in the mature schizonts of 29 representative isolates. ALL = mean Navg for all 27 isolates with FP2a SNPs. *, P < .05 (t test) vs WT in A–C. (D) Michealis-Menten curve-fits of Z-LR-AMC hydrolysis by trophozoite extracts from WT and parasites in haplotype groups I–VIII. Phenotypic Analysis of Representative Falcipain 2a Haplotypes Drug resistance may engender a fitness cost, including altered hemoglobin digestion and growth [25, 26]. In particular, hemoglobin digestion was found to be crucial for enabling artemisinin activity [12, 13]. The fitness of 29 representative isolates from different FP2a haplotype groups was compared after growth in CM versus CM-AA (Supplementary Figure S4). The growth competencies of 27 isolates from different haplotype groups (I = 31.6%, II = 22.2%, III = 29.2%, IV = 46.1%, V = 16.9%, VI = 17.0%, VII = 11.1%, VIII = 5.9%; all groups = 32.5%) were significantly lower than those of WT parasites (105.7%; t test; P < .05) (Figure 3B), indicating that field isolates with FP2a SNPs have suboptimal growth, likely due to altered hemoglobin catabolism. Moreover, multiplication competency (Navg) was lower for most of the isolates with altered FP2a compared with that of the WT parasites (Navg = 16.2) (Supplementary Figure S4). However, only group I (Navg = 8.7) and IV (Navg = 9.8) parasites had significantly fewer merozoites compared with WT parasites (Figure 3C). We next investigated the effect of FP2a SNPs on enzyme activity [9, 27]. We measured cysteine protease activity of trophozoite extracts (predominantly from FP2a) against the substrate Z-LR-AMC and extrapolated Km and Vmax values from Michealis-Menten curves (Figure 3D). A range of Vmax values was obtained, with the highest value for WT parasites (50.2 pM/s) (Table 1), and significantly lower values for all haplotype groups, indicating that altered FP2a digests the substrate less rapidly than the WT enzyme. Km values had a relatively wide range, with group I exhibiting the highest value (35.4 µM) (Table 1), implying that this FP2a haplotype has lower enzyme to substrate affinity or lower rate of substrate conversion to product. Z-LR-AMC hydrolysis by all haplotype groups was significantly different from that of the WT parasites (analysis of variance, P < .0001) (Figure 3D). We also looked for alterations in the morphologies of these isolates, because trophozoites treated with cysteine protease inhibitors and FP2a KO parasites show a swollen and dark-staining food vacuole, consistent with accumulation of undigested hemoglobin [10]. Mutant isolates belonging to groups I and IV developed enlarged food vacuoles, although the abnormality was not as prominent as in WT parasites treated with E-64 (Supplementary Figure S6). In addition, analysis of representative parasite isolates demonstrated that parasites belonging to groups I and IV showed reduced hemoglobin hydrolysis activity compared with the WT parasites (Supplementary Figure S7). Table 1. FP2a Protease Kineticsa Haplotype Groups  Vmax (pM/s)  Km (μM)  WT  50.2 ± 4.4  9.6 ± 3.1  I  14.9 ± 2.9  35.4 ± 15.6  II  9.2 ± 1.4  17.7 ± 7.5  III  8.1 ± 1.8  4.7 ± 5.4  IV  10.8 ± 8.2  14.5 ± 3.7  V  7.0 ± 6.6  11.3 ± 3.4  VI  9.4 ± 7.4  6.6 ± 2.2  VII  14.2 ± 1.3  13.1 ± 3.7  VIII  15.1 ± 2.6  13.91 ± 7.4  Haplotype Groups  Vmax (pM/s)  Km (μM)  WT  50.2 ± 4.4  9.6 ± 3.1  I  14.9 ± 2.9  35.4 ± 15.6  II  9.2 ± 1.4  17.7 ± 7.5  III  8.1 ± 1.8  4.7 ± 5.4  IV  10.8 ± 8.2  14.5 ± 3.7  V  7.0 ± 6.6  11.3 ± 3.4  VI  9.4 ± 7.4  6.6 ± 2.2  VII  14.2 ± 1.3  13.1 ± 3.7  VIII  15.1 ± 2.6  13.91 ± 7.4  Abbreviations: ANOVA, analysis of variance; FP2a, falcipain-2a; WT, wild type. aVmax and Km were extrapolated from Z-LR-AMC vs initial rates of AMC production. I–VIII vs WT (P < .0001, ANOVA). View Large Table 1. FP2a Protease Kineticsa Haplotype Groups  Vmax (pM/s)  Km (μM)  WT  50.2 ± 4.4  9.6 ± 3.1  I  14.9 ± 2.9  35.4 ± 15.6  II  9.2 ± 1.4  17.7 ± 7.5  III  8.1 ± 1.8  4.7 ± 5.4  IV  10.8 ± 8.2  14.5 ± 3.7  V  7.0 ± 6.6  11.3 ± 3.4  VI  9.4 ± 7.4  6.6 ± 2.2  VII  14.2 ± 1.3  13.1 ± 3.7  VIII  15.1 ± 2.6  13.91 ± 7.4  Haplotype Groups  Vmax (pM/s)  Km (μM)  WT  50.2 ± 4.4  9.6 ± 3.1  I  14.9 ± 2.9  35.4 ± 15.6  II  9.2 ± 1.4  17.7 ± 7.5  III  8.1 ± 1.8  4.7 ± 5.4  IV  10.8 ± 8.2  14.5 ± 3.7  V  7.0 ± 6.6  11.3 ± 3.4  VI  9.4 ± 7.4  6.6 ± 2.2  VII  14.2 ± 1.3  13.1 ± 3.7  VIII  15.1 ± 2.6  13.91 ± 7.4  Abbreviations: ANOVA, analysis of variance; FP2a, falcipain-2a; WT, wild type. aVmax and Km were extrapolated from Z-LR-AMC vs initial rates of AMC production. I–VIII vs WT (P < .0001, ANOVA). View Large DISCUSSION We evaluated the sequences of FP2a genes from 140 clinical P falciparum isolates from the China-Myanmar border. Remarkably, 97% of studied strains had SNPs in FP2a compared with the reference 3D7 strain, with a great deal of diversity compared with African strains [28]. Parasites with FP2a SNPs demonstrated decreased FP2a activity and decreased artemisinin sensitivity. Given that hemoglobin degradation by FP2a is required for artemisinin activity, the observed nucleotide and haplotype diversity in the Asian isolates could be attributed to selection of parasites with decreased FP2a activity due to extensive artemisinin and ACT usage in Southeast Asia. Although the data could not allow robust examination of the evolution of FP2a over time due to limitation in sample size in certain years, haplotype group I emerged after 2008, coinciding with the implementation of extensive ACT use. Taken together, our results suggest that use of ACTs has selected for alterations in FP2a in Southeast Asia and that decreased hemoglobin digestion due to the FP2a alterations contributes to artemisinin resistance. Among the plethora of SNPs identified in FP2a, 6 were mapped to the protease domain and potentially interfere with enzyme activity. It is noteworthy that none of the mutations were present in the hemoglobin-binding domain. Modeling predicts that A353T, A400P, and Q414E lie in the binding interface of FP2a and the endogenous cysteine protease inhibitor falstatin, and it may therefore affect binding of the inhibitor with FP2a. In particular, A400 lies in a helix, and Q414 forms a hydrogen bond with the inhibitor. It remains to be tested whether other SNPs outside of the catalytic domain could be compensatory [29–33]. Falcipain 2a has a much larger prodomain than those of most papain-family proteases. The upstream globular portion of the prodomain is largely responsible for inhibition, and a downstream portion is responsible for folding of the catalytic domain [30, 31]. N-terminal portions of the FP2a prodomain mediate trafficking of the mature enzyme to the food vacuole, its principal site of action. It was shown that a 20-AA stretch of the luminal portion and a 10-AA stretch of the cytoplasmic portion of the prodomain were required for efficient trafficking to the food vacuole [34]. None of the prodomain SNPs described in this study have been shown to be required in the minimum inhibitory domain [35] or for food vacuole trafficking [34]. However, we cannot rule out deleterious effects of these mutations. Other SNPs outside of the catalytic domain are not known to be required for catalytic activity or binding with hemoglobin [29–33], although the specific impacts of identified SNPs and haplotypes have not been tested. Protease activities of different FP2 isoforms have been tested previously [29, 30, 36], but none of these isoforms conformed to the haplotype groups documented in this study. Future studies may use a similar approach to determine the enzyme activities of the different haplotype groups identified here. The use of isogenic parasite lines with different FP2a haplotypes would clarify the impacts of these SNPs on enzyme activity and artemisinin resistance. The group I haplotype stood out as the only haplotype with a mutation in falstatin. Falstatin is a reversible, active site-binding inhibitor of FP2 and FP3, and it is present in all asexual stages except trophozoites, the stage at which the cysteine protease activity of P falciparum is maximal [37]. Treatment of schizonts with antifalstatin antibodies inhibited invasion of erythrocytes by merozoites, suggesting a crucial role for falstatin during erythrocyte invasion [37]. It is noteworthy that the T213I mutation in falstatin is upstream of the predicted functional inhibitory domain (Supplementary Figure S5), and according to sequence alignment this residue (T213) is not conserved in the falstatin homologs in P berghie and Plasmodium vivax. Because falstatin is not expressed at the trophozoite stage, when hemoglobin degradation is maximal, the presence of mutated falstatin, along with its distinct FP2a haplotype “partner,” merits future investigation to determine impacts on FP2a activity and drug resistance [38]. Fitness of the clinical isolates, measured as ability to grow and multiply in AA-deficient medium, showed slower growth of all haplotype groups with mutant FP2a, compared with that of WT parasites, implying altered hemoglobin catabolism in these isolates. Because FP2a KO did not appear to affect parasite growth rates [10], it is surprising that our group I and IV isolates produced a lower number of merozoites, potentially leading to decreased virulence or infectivity. It is interesting to note that reduced hemoglobin degradation activity was also observed in representative isolates belonging to groups I and IV compared with WT parasites. When tested for Z-LR-AMC hydrolysis, we saw a clear differentiation of FP2a haplotypes, with perturbed hemoglobin digestion in all isolates with mutant FP2a. Group I FP2a exhibited the highest Km, indicating higher substrate concentration requirement for maximum proteolytic velocity and a lower than WT reaction velocity (Vmax), supporting an association between abnormal FP2a activity and reduced growth in AA-deficient medium. In addition, group I and IV mutant parasites developed enlarged food vacuoles (Supplementary Figure S6), similar to what was reported in FP2a KO parasites [10]. The identification in FP2a mutant parasites of reduced FP2a activity, decreased hemoglobin digestion, enlarged food vacuoles, and enhanced ring stage survival after treatment with dihydroartemisinin all support the hypothesis that altered FP2a activity contributes to artemisinin resistance [12]. CONCLUSIONS Mutations in the propeller domain of the P falciparum K13 protein are central to artemisinin resistance, but identification of other genetic loci associated with resistance [7, 8] and reports of artemisinin-resistant field isolates lacking K13 mutations suggest the involvement of additional resistance mechanisms. Selection of an FP2a loss-of-function mutation in parasites selected in vitro for artemisinin resistance [4] and resistance to artemisinins in FP2a KO parasites [13] emphasize an important role of this protease in artemisinin resistance. Because the hemoglobin degradation pathway is a common target of antimalarial drugs and multiple enzymes are involved in this pathway, it seems logical that mutations in the genes encoding hemoglobinases have emerged to contribute to drug resistance. Recently, amplification of genes encoding the aspartic proteases plasmepsin 2 and 3 has emerged in Cambodian parasite populations and is associated with piperaquine resistance [39, 40]. Similarly, our results showing association of altered FP2a with decreased artemisinin sensitivity in field isolates provide evidence for a role of FP2a in this phenomenon. It is important to note that piperaquine resistance (plasmepsin 2/3 amplification) has evolved in the genetic background of artemisinin resistance (K13 mutations) [41, 42]. Analogously, our study also found a link of several FP2a haplotypes with K13 mutations. All but 1 parasite in group IV had K13 mutations and was associated with the highest ring survival rates. In contrast, 14 of 16 group III parasites had WT K13 and the lowest ring survival rates. Thus, the association of FP2a and K13 haplotypes deserves future population studies to elucidate the evolutionary history of artemisinin resistance. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Financial support. This study was funded by grants (U19AI089672 and R01AI128940) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health and a grant (81761128017) from National Natural Science Foundation of China. Potential conflicts of interest. All authors: No reported conflicts of interest. 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Google Scholar CrossRef Search ADS PubMed  42. Duru V, Khim N, Leang Ret al.   Plasmodium falciparum dihydroartemisinin-piperaquine failures in Cambodia are associated with mutant K13 parasites presenting high survival rates in novel piperaquine in vitro assays: retrospective and prospective investigations. BMC Med  2015; 13: 305. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Plasmodium falciparum Falcipain-2a Polymorphisms in Southeast Asia and Their Association With Artemisinin Resistance

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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0022-1899
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1537-6613
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10.1093/infdis/jiy188
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Abstract

Abstract Background Falcipain-2a ([FP2a] PF3D7_1115700) is a Plasmodium falciparum cysteine protease and hemoglobinase. Functional FP2a is required for potent activity of artemisinin, and in vitro selection for artemisinin resistance selected for an FP2a nonsense mutation. Methods To investigate associations between FP2a polymorphisms and artemisinin resistance and to characterize the diversity of the enzyme in parasites from the China-Myanmar border, we sequenced the full-length FP2a gene in 140 P falciparum isolates collected during 2004–2011. Results The isolates were grouped into 8 different haplotype groups. Haplotype group I appeared in samples obtained after 2008, coinciding with implementation of artemisinin-based combination therapy in this region. In functional studies, compared with wild-type parasites, the FP2a haplotypes demonstrated increased ring survival, and all haplotype groups exhibited significantly reduced FP2a activity, with group I showing the slowest protease kinetics and reduced parasite fitness. Conclusions These results suggest that altered hemoglobin digestion due to FP2a mutations may contribute to artemisinin resistance. ACT, artemisinin resistance, falcipain 2a, malaria, Plasmodium falciparum Evolution of drug resistance in malaria parasites is one of the biggest roadblocks to malaria control and elimination. The recent emergence of resistance in Plasmodium falciparum to frontline artemisinin-based combination therapies (ACTs) in Southeast Asia has raised international concerns [1]. The potential spread of resistant parasite strains to Africa, the heartland of malaria transmission, would be catastrophic for global malaria control and elimination. Therefore, it is essential to identify the molecular markers of resistance and vigilantly monitor the spread of resistant genotypes. Resistance against artemisinin and its derivatives in P falciparum is clinically manifested as slow parasite clearance in patients [2], and resistant parasites show increased survival when exposed to pharmacologically relevant concentrations of dihydroartemisinin at the ring stage in vitro [3]. Genomic and genetic analyses identified and confirmed that mutations in the propeller domain of the kelch protein K13 are associated with artemisinin resistance [4]. In Western Cambodia, where artemisinin resistance was first recognized, the K13 C580Y mutation has approached fixation, and resistant parasites carrying this mutation have spread to Thailand and Laos [4, 5]. Identification of artemisinin-resistant field isolates from Cambodia lacking K13 mutations suggests the involvement of additional resistance mechanisms [6]. Association studies have identified other genetic loci associated with in vivo and in vitro resistance to artemisinins [7, 8], and these results await genetic confirmation. Falcipains (FPs) are cysteine proteases that digest hemoglobin in the food vacuole to provide amino acids (AAs) for intraerythrocytic parasite development. Plasmodium falciparum encodes 4 FP genes (FP1, FP2a, FP2b, and FP3), among which FP2a (PF3D7_1115700), with peak expression in the early trophozoite stage, and FP3, with expression later in the erythrocytic cycle, appear to be the principal cysteine proteases involved in hemoglobin digestion [9]. Falcipain-2a knockout (KO) trophozoites are characterized by a swollen hemoglobin-filled food vacuole due to transiently reduced hemoglobin digestion [10, 11], which is linked to diminished parasite susceptibility to artemisinins, suggesting that hemoglobin digestion is required for artemisinin activity [12, 13]. Intriguingly, in addition to acquiring a K13 mutation (M476I), parasites selected in vitro for artemisinin resistance also acquired a nonsense mutation at codon 69 of FP2a [4]. This result and the finding that FP2a expression is required for potent artemisinin activity [12] suggest that FP2a mutations might contribute to artemisinin resistance. We previously showed that P falciparum isolates with K13 mutations collected from the China-Myanmar border were significantly associated with persistence of parasitemia on day 3 after the onset of treatment and with increased survival in the ring-stage survival assay (RSA), an in vitro marker for artemisinin resistance [14]. Given the potential contribution of FP2a polymorphisms to artemisinin resistance, we investigated their association with in vitro sensitivity to artemisinins in 140 clinical parasite isolates from the China-Myanmar border area, where artemisinins have been used to treat malaria for over 3 decades. We show that FP2a haplotypes with perturbed hemoglobin digestion and decreased fitness are common in this region and associated with decreased artemisinin sensitivity, suggesting selection of altered FP2a by the selective pressure of ACTs. MATERIALS AND METHODS Parasite Isolates and Drug Assay Plasmodium falciparum clinical isolates were collected during 2004–2011 in malaria clinics located along the China-Myanmar border, cultured, and archived [15]. Monoclonal parasite isolates were confirmed by genotyping at 3 polymorphic genes merozoite surface protein 1 (msp1), msp2, and glutamate-rich protein, as reported previously [8]. Parasites were maintained in O+ human erythrocytes as described [16, 17]. A total of 140 monoclonal isolates were analyzed, including 2 collected in 2004, 18 in 2007, 44 in 2008, 64 in 2009, 11 in 2010, and 1 in 2011. The RSA was performed as previously described [14]. Informed consent or assent was obtained from all patients or legal guardians before enrollment. All studies were approved by the ethical committees from the participating institutions and the local health department. Sequencing of Falcipain 2a Gene Parasite genomic deoxyribonucleic acid (DNA) was extracted using the Wizard Genomic DNA Purification Kit (Promega). The full-length FP2a gene was amplified and sequenced using primers listed in Supplementary Table S1. Sequences were assembled using Vector NTI (Invitrogen) with manual editing and aligned against the reference FP2a sequence of 3D7 (PF3D7_1115700 in PlasmoDB) using MEGA6.0 [18, 19]. A phylogenetic tree was constructed using the Neighbor-Joining method with 1000 pseudo-replications. We grouped the haplotypes based on their clustering on the phylogenetic tree [20]. The FP2a sequences reported in this study were deposited in GenBank (accession numbers MG768692–MG768822 and MG768823–MG768831). Molecular Modeling The three-dimensional (3D) structure of FP2a in complex with the Plasmodium berghei homolog of falstatin was retrieved from the Protein Data Bank ([PDB] 3PNR:aa245-484), and Swiss modeler was used to model identified FP2a mutations. For mapping the mutation in falstatin, the 3D structure was predicted by using the I-TASSER online protein structure prediction tool [21]. Web-based softwares PROVEAN (Protein Variation Effect Analyzer) and SIFT (Sorting Intolerant From Tolerant) were used to predict the effects of FP2a mutations. Assays for Parasite Phenotypes and Fitness To test parasite fitness under AA-deficient conditions, parasites at 1% parasitemia with 2% hematocrit were cultured in either complete media (CM) composed of Roswell Park Memorial Institute (RPMI) medium 1640 (Gibco Life Technologies, Grand Island, NY) with 25 mM NaHCO3, 25 mM HEPES (pH 7.4), 11 mM glucose, 0.367 mM hypoxanthine, and 5 µg/L gentamicin supplemented with 0.5% Albumax (Gibco Life Technologies), or AA-depleted medium ([CM-AA] identical to CM except for RPMI without AAs) (US Biological Science) following a previously described method [22]. Parasite growth was quantified from Giemsa-stained thin smears at 24, 42, and 48 hours postinvasion. The growth competency of parasites in CM-AA was calculated as ([no. of schizonts in CM-AA-]/[no. of schizonts in CM] × 100%) at the time when at least 15% of parasites cultured in CM had reached the schizont stage. Multiplication competency was determined by counting the average number of merozoites in mature schizonts (Navg) at the time when at least 40% of parasites cultured in CM were at the ring stage. Merozoite nuclei were stained with 0.8 × SYBR Green I and counted in a Guava easyCyte 5HT Benchtop Flow Cytometer at λex = 488 nm and λem = 525 ± 30 nm. At least 50000 total events in the green channel were collected. Green fluorescence intensity (FI) from the gated populations was normalized by dividing by the mean FI of purified rings, rounding off to the nearest integer, and plotting into histograms with bins of 4 using MS Excel. Growth in CM-AA and Navg were obtained from at least 3 technical replicates, with 2 biological replicates each. The FP2a protease activity was quantified via a modified method [23] wherein extracts from synchronized trophozoites (20 h after invasion) were tested for the ability to hydrolyze 7-amino-4-methyl coumarin from Z-Leu-Arg-AMC ([Z-LR-AMC] Bachem), assessed at λex = 355 nm and λem = 460 nm for 60 minutes. Fluorescence intensities at 100, 50, 25, 12.5, and 6.25 µM Z-AMC were plotted in GraphPad Prism 5. The first 10–15 minutes of reactions were fitted to y = mx + b, and linear slopes (m) were plotted against [Z-LR-AMC] to obtain the “Km” and “Vmax” values. Hydrolysis is attributed to FP2a, because it is maximally expressed in the early trophozoite stage and was shown to be responsible for 93% of the cysteine protease activity in extracts from early trophozoites [9, 23, 24]. Protein concentrations in trophozoite extracts were determined using the Pierce Coomassie Plus (Bradford) Assay Kit (Thermo Fisher), and 1 µg protein was used for all experiments. Hemoglobin Degradation Assays To assess the hemoglobinase activity of different field isolates, 5 µg of the trophozoite extract was added to 30-μL reactions containing 2 µg of hemoglobin in 100 mM NaOAc, pH 5.5, and 1 mM glutathione. Reactions were carried out at 37°C for 2 hours, stopped by the addition of 10 µL 4× sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and then electrophoresed on 15% SDS-PAGE gels. These were either stained with Coomassie or transferred onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were probed with rabbit antisera directed to either human hemoglobin (diluted 1:5000; Sigma) or P falciparum-aldolase (diluted 1:5000; Abcam). Statistical Analysis Statistical tests were done in MS Excel, SigmaPlot version 10, and GraphPad Prism 5. RESULTS Genetic Diversity of Falcipain 2a Sequencing of FP2a in 140 clinical isolates collected from the China-Myanmar border identified 34 nonsynonymous single-nucleotide polymorphisms (SNPs), with at least 1 SNP in 97.1% (137 of 140) of the isolates (Supplementary Table S2). Three SNPs (Y3C, D123G, and F165I) have not been described previously. The FP2a consists of a prodomain composed of a transmembrane domain, an inhibitory prodomain and a functional cysteine protease domain (Figure 1A). Six of the identified SNPs (T343P, D345G, A353T, V393I, A400P, and Q414E) are in the cysteine protease domain, 5 (M245I, E248D, E249A, K255R, and N257E) in the prodomain, and none in the hemoglobin-binding domain. A number of SNPs demonstrated high prevalence in the 140 isolates, notably, Q15H (77.3%), V51I (56.7 %), S59F (56.7%), K255R (84.4%), N257E (85.1%), T343P (71.6%), D345G (73.1%), and Q414E (74.5%) (Supplementary Figure S1A). Only 3 isolates (2 collected in 2008 and 1 in 2010) had the wild-type (WT) 3D7 sequence. The FP2a SNPs resulted in 25 distinct haplotypes. Because these were too diverse to be analyzed individually, they were studied as FP2a haplotype groups based on their phylogenetic clustering patterns (Figure 1B). For the 8 identified haplotype groups, no clear temporal frequency trends were recognized, except for group I, which appeared after 2008 (Figure 1B). It is interesting to note that our earlier study identified 8 of 140 isolates that carried a nonsynonymous mutation (T213I) in the falstatin gene [8], which encodes an endogenous P falciparum cysteine protease inhibitor. These 8 isolates were all in FP2a haplotype group I. Figure 1. View largeDownload slide Falcipain-2a (FP2a) haplotypes of 140 China-Myanmar Plasmodium falciparum isolates. (A) Domain organization of the FP2a protein showing functional cysteine protease domain ([CP] amino acids [AA], 261–482), inhibitor domain ([ID] AA, 165–222), and the transmembrane domain ([TM] AA, 35–57). (B) Twenty-five distinct haplotypes identified in the 140 isolates were categorized into 8 haplotype groups based on sequence phylogeny. The left panel shows each haplotype groups and their frequency in 140 samples. The bar graph (right) shows the proportions of haplotype groups over time. Figure 1. View largeDownload slide Falcipain-2a (FP2a) haplotypes of 140 China-Myanmar Plasmodium falciparum isolates. (A) Domain organization of the FP2a protein showing functional cysteine protease domain ([CP] amino acids [AA], 261–482), inhibitor domain ([ID] AA, 165–222), and the transmembrane domain ([TM] AA, 35–57). (B) Twenty-five distinct haplotypes identified in the 140 isolates were categorized into 8 haplotype groups based on sequence phylogeny. The left panel shows each haplotype groups and their frequency in 140 samples. The bar graph (right) shows the proportions of haplotype groups over time. Comparison of the 140 FP2a sequences with 45 published FP2a sequences revealed clear geographical separation of Asian and African parasites (Supplementary Figure S2 and Supplementary Table S3). Asian isolates had greater nucleotide and haplotype diversity, suggesting that these parasites have experienced stronger selection pressure (Supplementary Table S4). Structural Predictions To predict whether the identified FP2a SNPs interfere with enzymatic activity, we mapped the SNPs in the cysteine protease domain of FP2a in complex with the P berghei homolog of falstatin ([PbICP] inhibitor of cysteine proteases) cocrystal structure (3PNR.pdb: AA, 245–484) (Figure 2A); this structure was used because a structure of P falciparum falstatin is not available. Model predictions show A353, A400, and Q414 in the binding interface of FP2a and PbICP, whereas A353 is partially buried and A400 is fully buried into the interface and located in the center of a short helix (Figure 2B). A400P may disrupt the helix structure and make it too bulky to fit in the FP2a-PbICP interface. Q414E may disrupt the hydrogen bond with the inhibitor at Q308 (Figure 2C). In silico web-based PROVEAN and SIFT programs predict that T343P and D345G may alter the protein structure. Because none of these SNPs occur singly in any of the isolates, we performed phenotypic assays based on the haplotype phylogeny (Figure 1B). Figure 2. View largeDownload slide Three-dimensional modeling of Pf3D7-falcipain-2a (FP2a) (amino acids, 245–484) using 3PNR.pdb. (A) Single-nucleotide polymorphisms in the protease domain (T343P, D345G, A353T, V393I, A400P, and Q414E) and the prodomain (M245I, E248D, E249A, K255R, and N257E) are highlighted. (B) A353, A400, and Q414 lie in the binding interface of FP2a and its inhibitor. A353 is partially buried, whereas A400 is fully buried in the interface and located in the middle of a short helix. (C) Q414 forms a hydrogen bond with the inhibitor. Abbreviation: ICP, Plasmodium berghei homolog of falstatin (inhibitor of cysteine proteases). Figure 2. View largeDownload slide Three-dimensional modeling of Pf3D7-falcipain-2a (FP2a) (amino acids, 245–484) using 3PNR.pdb. (A) Single-nucleotide polymorphisms in the protease domain (T343P, D345G, A353T, V393I, A400P, and Q414E) and the prodomain (M245I, E248D, E249A, K255R, and N257E) are highlighted. (B) A353, A400, and Q414 lie in the binding interface of FP2a and its inhibitor. A353 is partially buried, whereas A400 is fully buried in the interface and located in the middle of a short helix. (C) Q414 forms a hydrogen bond with the inhibitor. Abbreviation: ICP, Plasmodium berghei homolog of falstatin (inhibitor of cysteine proteases). Association of Falcipain 2a Haplotypes and K13 Mutations With Decreased Artemisinin Sensitivity As a proxy for artemisinin resistance, we determined RSA values for the 140 parasite strains. A survival rate above 1% in the RSA predicts clinical resistance [3]. Mean RSA values for haplotype groups I (2.0%) and IV (6.6%) were significantly higher than for WT parasites (3D7 and 3 field isolates with the same sequences) (0.43%) (Figure 3A). In addition, the mean RSA value (2.5%) for all 137 isolates with FP2a SNPs was significantly higher than that for the WT group. Considering specific SNPs, group I is predominantly composed of isolates with the F446I K13 mutation (9 of 10 isolates), 8 of which also contain the falstatin T213I mutation, whereas group IV has 5 of 9 isolates with the K13 P574L mutation and 1 with F446I (Supplementary Figure S3). Figure 3. View largeDownload slide Phenotypic characterization of representative isolates. (A) Mean survival rates from ring-stage survival assay of 140 isolates grouped according to falcipain-2a (FP2a) haplotype groups (I–VIII). Wild type (WT) = 3D7 and 3 field isolates with WT K13 and FP2a sequences. ALL = mean for all 137 isolates with FP2a single-nucleotide polymorphisms (SNPs). (B) Mean growth in amino acid-depleted complete medium (CM-AA) of 29 representative isolates. ALL = mean growth in CM-AA for all 27 isolates with FP2a SNPs. (C) Average number of merozoites (Navg) in the mature schizonts of 29 representative isolates. ALL = mean Navg for all 27 isolates with FP2a SNPs. *, P < .05 (t test) vs WT in A–C. (D) Michealis-Menten curve-fits of Z-LR-AMC hydrolysis by trophozoite extracts from WT and parasites in haplotype groups I–VIII. Figure 3. View largeDownload slide Phenotypic characterization of representative isolates. (A) Mean survival rates from ring-stage survival assay of 140 isolates grouped according to falcipain-2a (FP2a) haplotype groups (I–VIII). Wild type (WT) = 3D7 and 3 field isolates with WT K13 and FP2a sequences. ALL = mean for all 137 isolates with FP2a single-nucleotide polymorphisms (SNPs). (B) Mean growth in amino acid-depleted complete medium (CM-AA) of 29 representative isolates. ALL = mean growth in CM-AA for all 27 isolates with FP2a SNPs. (C) Average number of merozoites (Navg) in the mature schizonts of 29 representative isolates. ALL = mean Navg for all 27 isolates with FP2a SNPs. *, P < .05 (t test) vs WT in A–C. (D) Michealis-Menten curve-fits of Z-LR-AMC hydrolysis by trophozoite extracts from WT and parasites in haplotype groups I–VIII. Phenotypic Analysis of Representative Falcipain 2a Haplotypes Drug resistance may engender a fitness cost, including altered hemoglobin digestion and growth [25, 26]. In particular, hemoglobin digestion was found to be crucial for enabling artemisinin activity [12, 13]. The fitness of 29 representative isolates from different FP2a haplotype groups was compared after growth in CM versus CM-AA (Supplementary Figure S4). The growth competencies of 27 isolates from different haplotype groups (I = 31.6%, II = 22.2%, III = 29.2%, IV = 46.1%, V = 16.9%, VI = 17.0%, VII = 11.1%, VIII = 5.9%; all groups = 32.5%) were significantly lower than those of WT parasites (105.7%; t test; P < .05) (Figure 3B), indicating that field isolates with FP2a SNPs have suboptimal growth, likely due to altered hemoglobin catabolism. Moreover, multiplication competency (Navg) was lower for most of the isolates with altered FP2a compared with that of the WT parasites (Navg = 16.2) (Supplementary Figure S4). However, only group I (Navg = 8.7) and IV (Navg = 9.8) parasites had significantly fewer merozoites compared with WT parasites (Figure 3C). We next investigated the effect of FP2a SNPs on enzyme activity [9, 27]. We measured cysteine protease activity of trophozoite extracts (predominantly from FP2a) against the substrate Z-LR-AMC and extrapolated Km and Vmax values from Michealis-Menten curves (Figure 3D). A range of Vmax values was obtained, with the highest value for WT parasites (50.2 pM/s) (Table 1), and significantly lower values for all haplotype groups, indicating that altered FP2a digests the substrate less rapidly than the WT enzyme. Km values had a relatively wide range, with group I exhibiting the highest value (35.4 µM) (Table 1), implying that this FP2a haplotype has lower enzyme to substrate affinity or lower rate of substrate conversion to product. Z-LR-AMC hydrolysis by all haplotype groups was significantly different from that of the WT parasites (analysis of variance, P < .0001) (Figure 3D). We also looked for alterations in the morphologies of these isolates, because trophozoites treated with cysteine protease inhibitors and FP2a KO parasites show a swollen and dark-staining food vacuole, consistent with accumulation of undigested hemoglobin [10]. Mutant isolates belonging to groups I and IV developed enlarged food vacuoles, although the abnormality was not as prominent as in WT parasites treated with E-64 (Supplementary Figure S6). In addition, analysis of representative parasite isolates demonstrated that parasites belonging to groups I and IV showed reduced hemoglobin hydrolysis activity compared with the WT parasites (Supplementary Figure S7). Table 1. FP2a Protease Kineticsa Haplotype Groups  Vmax (pM/s)  Km (μM)  WT  50.2 ± 4.4  9.6 ± 3.1  I  14.9 ± 2.9  35.4 ± 15.6  II  9.2 ± 1.4  17.7 ± 7.5  III  8.1 ± 1.8  4.7 ± 5.4  IV  10.8 ± 8.2  14.5 ± 3.7  V  7.0 ± 6.6  11.3 ± 3.4  VI  9.4 ± 7.4  6.6 ± 2.2  VII  14.2 ± 1.3  13.1 ± 3.7  VIII  15.1 ± 2.6  13.91 ± 7.4  Haplotype Groups  Vmax (pM/s)  Km (μM)  WT  50.2 ± 4.4  9.6 ± 3.1  I  14.9 ± 2.9  35.4 ± 15.6  II  9.2 ± 1.4  17.7 ± 7.5  III  8.1 ± 1.8  4.7 ± 5.4  IV  10.8 ± 8.2  14.5 ± 3.7  V  7.0 ± 6.6  11.3 ± 3.4  VI  9.4 ± 7.4  6.6 ± 2.2  VII  14.2 ± 1.3  13.1 ± 3.7  VIII  15.1 ± 2.6  13.91 ± 7.4  Abbreviations: ANOVA, analysis of variance; FP2a, falcipain-2a; WT, wild type. aVmax and Km were extrapolated from Z-LR-AMC vs initial rates of AMC production. I–VIII vs WT (P < .0001, ANOVA). View Large Table 1. FP2a Protease Kineticsa Haplotype Groups  Vmax (pM/s)  Km (μM)  WT  50.2 ± 4.4  9.6 ± 3.1  I  14.9 ± 2.9  35.4 ± 15.6  II  9.2 ± 1.4  17.7 ± 7.5  III  8.1 ± 1.8  4.7 ± 5.4  IV  10.8 ± 8.2  14.5 ± 3.7  V  7.0 ± 6.6  11.3 ± 3.4  VI  9.4 ± 7.4  6.6 ± 2.2  VII  14.2 ± 1.3  13.1 ± 3.7  VIII  15.1 ± 2.6  13.91 ± 7.4  Haplotype Groups  Vmax (pM/s)  Km (μM)  WT  50.2 ± 4.4  9.6 ± 3.1  I  14.9 ± 2.9  35.4 ± 15.6  II  9.2 ± 1.4  17.7 ± 7.5  III  8.1 ± 1.8  4.7 ± 5.4  IV  10.8 ± 8.2  14.5 ± 3.7  V  7.0 ± 6.6  11.3 ± 3.4  VI  9.4 ± 7.4  6.6 ± 2.2  VII  14.2 ± 1.3  13.1 ± 3.7  VIII  15.1 ± 2.6  13.91 ± 7.4  Abbreviations: ANOVA, analysis of variance; FP2a, falcipain-2a; WT, wild type. aVmax and Km were extrapolated from Z-LR-AMC vs initial rates of AMC production. I–VIII vs WT (P < .0001, ANOVA). View Large DISCUSSION We evaluated the sequences of FP2a genes from 140 clinical P falciparum isolates from the China-Myanmar border. Remarkably, 97% of studied strains had SNPs in FP2a compared with the reference 3D7 strain, with a great deal of diversity compared with African strains [28]. Parasites with FP2a SNPs demonstrated decreased FP2a activity and decreased artemisinin sensitivity. Given that hemoglobin degradation by FP2a is required for artemisinin activity, the observed nucleotide and haplotype diversity in the Asian isolates could be attributed to selection of parasites with decreased FP2a activity due to extensive artemisinin and ACT usage in Southeast Asia. Although the data could not allow robust examination of the evolution of FP2a over time due to limitation in sample size in certain years, haplotype group I emerged after 2008, coinciding with the implementation of extensive ACT use. Taken together, our results suggest that use of ACTs has selected for alterations in FP2a in Southeast Asia and that decreased hemoglobin digestion due to the FP2a alterations contributes to artemisinin resistance. Among the plethora of SNPs identified in FP2a, 6 were mapped to the protease domain and potentially interfere with enzyme activity. It is noteworthy that none of the mutations were present in the hemoglobin-binding domain. Modeling predicts that A353T, A400P, and Q414E lie in the binding interface of FP2a and the endogenous cysteine protease inhibitor falstatin, and it may therefore affect binding of the inhibitor with FP2a. In particular, A400 lies in a helix, and Q414 forms a hydrogen bond with the inhibitor. It remains to be tested whether other SNPs outside of the catalytic domain could be compensatory [29–33]. Falcipain 2a has a much larger prodomain than those of most papain-family proteases. The upstream globular portion of the prodomain is largely responsible for inhibition, and a downstream portion is responsible for folding of the catalytic domain [30, 31]. N-terminal portions of the FP2a prodomain mediate trafficking of the mature enzyme to the food vacuole, its principal site of action. It was shown that a 20-AA stretch of the luminal portion and a 10-AA stretch of the cytoplasmic portion of the prodomain were required for efficient trafficking to the food vacuole [34]. None of the prodomain SNPs described in this study have been shown to be required in the minimum inhibitory domain [35] or for food vacuole trafficking [34]. However, we cannot rule out deleterious effects of these mutations. Other SNPs outside of the catalytic domain are not known to be required for catalytic activity or binding with hemoglobin [29–33], although the specific impacts of identified SNPs and haplotypes have not been tested. Protease activities of different FP2 isoforms have been tested previously [29, 30, 36], but none of these isoforms conformed to the haplotype groups documented in this study. Future studies may use a similar approach to determine the enzyme activities of the different haplotype groups identified here. The use of isogenic parasite lines with different FP2a haplotypes would clarify the impacts of these SNPs on enzyme activity and artemisinin resistance. The group I haplotype stood out as the only haplotype with a mutation in falstatin. Falstatin is a reversible, active site-binding inhibitor of FP2 and FP3, and it is present in all asexual stages except trophozoites, the stage at which the cysteine protease activity of P falciparum is maximal [37]. Treatment of schizonts with antifalstatin antibodies inhibited invasion of erythrocytes by merozoites, suggesting a crucial role for falstatin during erythrocyte invasion [37]. It is noteworthy that the T213I mutation in falstatin is upstream of the predicted functional inhibitory domain (Supplementary Figure S5), and according to sequence alignment this residue (T213) is not conserved in the falstatin homologs in P berghie and Plasmodium vivax. Because falstatin is not expressed at the trophozoite stage, when hemoglobin degradation is maximal, the presence of mutated falstatin, along with its distinct FP2a haplotype “partner,” merits future investigation to determine impacts on FP2a activity and drug resistance [38]. Fitness of the clinical isolates, measured as ability to grow and multiply in AA-deficient medium, showed slower growth of all haplotype groups with mutant FP2a, compared with that of WT parasites, implying altered hemoglobin catabolism in these isolates. Because FP2a KO did not appear to affect parasite growth rates [10], it is surprising that our group I and IV isolates produced a lower number of merozoites, potentially leading to decreased virulence or infectivity. It is interesting to note that reduced hemoglobin degradation activity was also observed in representative isolates belonging to groups I and IV compared with WT parasites. When tested for Z-LR-AMC hydrolysis, we saw a clear differentiation of FP2a haplotypes, with perturbed hemoglobin digestion in all isolates with mutant FP2a. Group I FP2a exhibited the highest Km, indicating higher substrate concentration requirement for maximum proteolytic velocity and a lower than WT reaction velocity (Vmax), supporting an association between abnormal FP2a activity and reduced growth in AA-deficient medium. In addition, group I and IV mutant parasites developed enlarged food vacuoles (Supplementary Figure S6), similar to what was reported in FP2a KO parasites [10]. The identification in FP2a mutant parasites of reduced FP2a activity, decreased hemoglobin digestion, enlarged food vacuoles, and enhanced ring stage survival after treatment with dihydroartemisinin all support the hypothesis that altered FP2a activity contributes to artemisinin resistance [12]. CONCLUSIONS Mutations in the propeller domain of the P falciparum K13 protein are central to artemisinin resistance, but identification of other genetic loci associated with resistance [7, 8] and reports of artemisinin-resistant field isolates lacking K13 mutations suggest the involvement of additional resistance mechanisms. Selection of an FP2a loss-of-function mutation in parasites selected in vitro for artemisinin resistance [4] and resistance to artemisinins in FP2a KO parasites [13] emphasize an important role of this protease in artemisinin resistance. Because the hemoglobin degradation pathway is a common target of antimalarial drugs and multiple enzymes are involved in this pathway, it seems logical that mutations in the genes encoding hemoglobinases have emerged to contribute to drug resistance. Recently, amplification of genes encoding the aspartic proteases plasmepsin 2 and 3 has emerged in Cambodian parasite populations and is associated with piperaquine resistance [39, 40]. Similarly, our results showing association of altered FP2a with decreased artemisinin sensitivity in field isolates provide evidence for a role of FP2a in this phenomenon. It is important to note that piperaquine resistance (plasmepsin 2/3 amplification) has evolved in the genetic background of artemisinin resistance (K13 mutations) [41, 42]. Analogously, our study also found a link of several FP2a haplotypes with K13 mutations. All but 1 parasite in group IV had K13 mutations and was associated with the highest ring survival rates. In contrast, 14 of 16 group III parasites had WT K13 and the lowest ring survival rates. Thus, the association of FP2a and K13 haplotypes deserves future population studies to elucidate the evolutionary history of artemisinin resistance. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Financial support. This study was funded by grants (U19AI089672 and R01AI128940) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health and a grant (81761128017) from National Natural Science Foundation of China. Potential conflicts of interest. All authors: No reported conflicts of interest. 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The Journal of Infectious DiseasesOxford University Press

Published: Apr 12, 2018

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