Submicroscopic malaria infections in pregnant women from six departments in Haiti.

OBJECTIVES: To describe the epidemiology of malaria in pregnancy in Haiti. METHODS: Cross-sectional study among pregnant women in six departments of Haiti. After obtaining informed consent, whole blood samples and demographic surveys were collected to investigate malaria prevalence, anaemia and socio-behavioural risk factors for infection, respectively. A total of 311 pregnant women were screened for Plasmodium falciparum infection using a rapid diagnostic test (RDT), microscopy and a novel, quantitative reverse transcriptase polymerase chain reaction method (qRT-PCR). RESULTS: Overall, 1.2% (4/311) of pregnant women were tested positive for malaria infection by both microscopy and RDT. However, using the qRT-PCR, 16.4% (51/311) of pregnant women were positive. The prevalence of malaria infection varied with geographical locations ranging between 0% and 46.4%. Additionally, 53% of pregnant women had some form of anaemia; however, no significant association was found between anaemia and submicroscopic malaria infection. The socio-behavioural risk factors identified to be protective of malaria infection were marital status (P < 0.05) and travel within one month prior to screening (P < 0.05). CONCLUSION: This study is the first to document the high prevalence of submicroscopic malaria infections among pregnant women in Haiti and identify social and behavioural risk factors for disease transmission.

High prevalence of asymptomatic malaria infections: a cross-sectional study in rural areas in six departments in Haiti.

BACKGROUND: Public health measures are poised for transition from malaria control to malaria elimination on the island of Hispaniola. Assessment of the reservoir of asymptomatic infections from which acute malaria cases may derive is critical to plan and evaluate elimination efforts. Current field technology is ill suited for detecting sub-microscopic infections, thus highly sensitive survey methods capable of detecting virtually all infections are needed. In this study the prevalence of infection with Plasmodium falciparum was determined in patients seeking medical care primarily for non-febrile conditions in six departments in Haiti using a newly designed qRT-PCR-based assay. METHODS: Three different methods of parasite detection were compared to assess their utility in approximating the prevalence of P. falciparum infections in the population: malaria rapid diagnostic test (RDT) designed to detect histidine-rich protein 2 (HRP2), thick smear microscopy, and a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based upon the small sub-unit ribosomal RNA. The limit of detection of the qRT-PCR assay utilized was 0.0003 parasite/µL of blood. Venous blood was obtained from a total of 563 subjects from six departments in Haiti, all of whom were seeking medical attention without complaints consistent with malaria. Each subject was questioned for knowledge and behaviour using demographic and epidemiological survey to identify risk factors for disease transmission. RESULTS: Among the 563 samples tested, ten and 16 were found positive for malaria by RDT and microscopy, respectively. Using the qRT-PCR test to assess the infection status of these subjects, an additional 92 were identified for a total of 108. Based upon the qRT-PCR assay results, a wide variation in prevalence of infection in asymptomatic subjects was seen between geographic locations ranging from 4-41%. The prevalence of infection was highest in the Grand Anse, Nord and Sud-Est Departments, and demographic data from questionnaires provide evidence for focal disease transmission. CONCLUSIONS: The qRT-PCR assay is sufficiently sensitive to identify an unexpectedly large number of asymptomatic, submicroscopic infections. Identifying and clearing these infections presents a significant challenge to both control and elimination efforts, but the qRT-PCR assay offers a reliable method to identify them.

Malaria elimination in Haiti by the year 2020: an achievable goal?

Haiti and the Dominican Republic, which share the island of Hispaniola, are the last locations in the Caribbean where malaria still persists. Malaria is an important public health concern in Haiti with 17,094 reported cases in 2014. Further, on January 12, 2010, a record earthquake devastated densely populated areas in Haiti including many healthcare and laboratory facilities. Weakened infrastructure provided fertile reservoirs for uncontrolled transmission of infectious pathogens. This situation results in unique challenges for malaria epidemiology and elimination efforts. To help Haiti achieve its malaria elimination goals by year 2020, the Laboratoire National de Santé Publique and Henry Ford Health System, in close collaboration with the Direction d'Épidémiologie, de Laboratoire et de Recherches and the Programme National de Contrôle de la Malaria, hosted a scientific meeting on "Elimination Strategies for Malaria in Haiti" on January 29-30, 2015 at the National Laboratory in Port-au-Prince, Haiti. The meeting brought together laboratory personnel, researchers, clinicians, academics, public health professionals, and other stakeholders to discuss main stakes and perspectives on malaria elimination. Several themes and recommendations emerged during discussions at this meeting. First, more information and research on malaria transmission in Haiti are needed including information from active surveillance of cases and vectors. Second, many healthcare personnel need additional training and critical resources on how to properly identify malaria cases so as to improve accurate and timely case reporting. Third, it is necessary to continue studies genotyping strains of Plasmodium falciparum in different sites with active transmission to evaluate for drug resistance and impacts on health. Fourth, elimination strategies outlined in this report will continue to incorporate use of primaquine in addition to chloroquine and active surveillance of cases. Elimination of malaria in Haiti will require collaborative multidisciplinary approaches, sound strategic planning, and strong ownership of strategies by the Haiti Ministère de la Santé Publique et de la Population.

Survey of Plasmodium falciparum multidrug resistance-1 and chloroquine resistance transporter alleles in Haiti.

BACKGROUND: In Haiti where chloroquine (CQ) is widely used for malaria treatment, reports of resistance are scarce. However, recent identification of CQ resistance genotypes in one site is suggestive of an emerging problem. Additional studies are needed to evaluate genetic mutations associated with CQ resistance, especially in the Plasmodium falciparum multi-drug resistance-1 gene (pfmdr1) while expanding the already available information on P. falciparum CQ transporter gene (pfcrt) in Haiti. METHODS: Blood samples were collected on Whatman filter cards (FTA) from eight clinics spread across Haiti. Following the confirmation of P. falciparum in the samples, PCR protocols were used to amplify regions of pfmdr1and pfcrt codons of interest, (86, 184, 1034, 1042, and 1246) and (72-76), respectively. Sequencing and site-specific restriction enzyme digestions were used to analyse these DNA fragments for the presence of single nucleotide polymorphisms (SNPs) known to confer resistance to anti-malarial drugs. RESULTS: P. falciparum infection was confirmed in160 samples by amplifying a segment of the P. falciparum 18S small subunit ribosomal RNA gene (pfssurrna). The sequence of pfmdr1 in 54 of these samples was determined between codons 86,184 codons 1034, 1042 and 1246. No sequence differences from that of the NF54 clone 3D7 were found among the 54 samples except at codon 184, where a non-silent mutation was found in all samples predicted to alter the amino acid sequence replacing tyrosine with phenylalanine (Y184F). This altered sequence was also confirmed by restriction enzyme digestion. The sequence of pfmdr1 at codons 86, 184, 1034 and 1042 encoded the NFSN haplotype. The sequence of pfcrt codons 72-76 from 79 samples was determined and found to encode CVMNK, consistent with a CQ sensitive genotype. CONCLUSION: The presence of the Y184F mutation in pfmdr1 of P. falciparum parasites in Haiti may have implications for resistance to antimalarial drugs. The absence of mutation in pfcrt at codon 76 among 79 isolates tested suggests that sensitivity to CQ in Haiti remains common. Wide-spread screening of the pfmdr1 and pfcrt especially among patients experiencing treatment failure may be a useful tool in early detection of the emergence of antimalarial drug resistance in Haiti.

Apical surface expression of aspartic protease Plasmepsin 4, a potential transmission-blocking target of the plasmodium ookinete.

To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target.

Plasmepsin 4-deficient Plasmodium berghei are virulence attenuated and induce protective immunity against experimental malaria.

Plasmodium parasites lacking plasmepsin 4 (PM4), an aspartic protease that functions in the lysosomal compartment and contributes to hemoglobin digestion, have only a modest decrease in the asexual blood-stage growth rate; however, PM4 deficiency in the rodent malaria parasite Plasmodium berghei results in significantly less virulence than that for the parental parasite. P. berghei Deltapm4 parasites failed to induce experimental cerebral malaria (ECM) in ECM-susceptible mice, and ECM-resistant mice were able to clear infections. Furthermore, after a single infection, all convalescent mice were protected against subsequent parasite challenge for at least 1 year. Real-time in vivo parasite imaging and splenectomy experiments demonstrated that protective immunity acted through antibody-mediated parasite clearance in the spleen. This work demonstrates, for the first time, that a single Plasmodium gene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild-type parasites. Parasite blood-stage attenuation should help identify protective immune responses against malaria, unravel parasite-derived factors involved in malarial pathologies, such as cerebral malaria, and potentially pave the way for blood-stage whole organism vaccines.

Genetic Markers of Adaptation of Plasmodium falciparum to Transmission by American Vectors Identified in the Genomes of Parasites from Haiti and South America.

The malaria parasite, Plasmodium falciparum, was introduced into Hispaniola and other regions of the Americas through the slave trade spanning the 16th through the 19th centuries. During this period, more than 12 million Africans were brought across the Atlantic to the Caribbean and other regions of the Americas. Since malaria is holoendemic in West Africa, a substantial percentage of these individuals carried the parasite. St. Domingue on Hispaniola, now modern-day Haiti, was a major port of disembarkation, and malaria is still actively transmitted there. We undertook a detailed study of the phylogenetics of the Haitian parasites and those from Colombia and Peru utilizing whole-genome sequencing. Principal-component and phylogenetic analyses, based upon single nucleotide polymorphisms (SNPs) in protein coding regions, indicate that, despite the potential for millions of introductions from Africa, the Haitian parasites share an ancestral relationship within a well-supported monophyletic clade with parasites from South America, while belonging to a distinct lineage. This result, in stark contrast to the historical record of parasite introductions, is best explained by a severe population bottleneck experienced by the parasites introduced into the Americas. Here, evidence is presented for targeted selection of rare African alleles in genes which are expressed in the mosquito stages of the parasite's life cycle. These genetic markers support the hypothesis that the severe population bottleneck was caused by the required adaptation of the parasite to transmission by new definitive hosts among the Anopheles (Nyssorhynchus) spp. found in the Caribbean and South America.IMPORTANCE Historical data suggest that millions of P. falciparum parasite lineages were introduced into the Americas during the trans-Atlantic slave trade, which would suggest a paraphyletic origin of the extant isolates in the Western Hemisphere. Our analyses of whole-genome variants show that the American parasites belong to a well-supported monophyletic clade. We hypothesize that the required adaptation to American vectors created a severe bottleneck, reducing the effective introduction to a few lineages. In support of this hypothesis, we discovered genes expressed in the mosquito stages of the life cycle that have alleles with multiple, high-frequency or fixed, nonsynonymous mutations in the American populations which are rarely found in African isolates. These alleles appear to be in gene products critical for transmission through the anopheline vector. Thus, these results may inform efforts to develop novel transmission-blocking vaccines by identifying parasite proteins functionally interacting with the vector that are important for successful transmission. Further, to the best of our knowledge, these are the first whole-genome data available from Haitian P. falciparum isolates. Defining the genome of these parasites provides genetic markers useful for mapping parasite populations and monitoring parasite movements/introductions.

Recombinant plasmepsin 1 from the human malaria parasite plasmodium falciparum: enzymatic characterization, active site inhibitor design, and structural analysis.

A mutated form of truncated proplasmepsin 1 (proPfPM1) from the human malaria parasite Plasmodium falciparum, proPfPM1 K110pN, was generated and overexpressed in Escherichia coli. The automaturation process was carried out at pH 4.0 and 4.5, and the optimal catalytic pH of the resulting mature PfPM1 was determined to be pH 5.5. This mature PfPM1 showed comparable binding affinity to peptide substrates and inhibitors with the naturally occurring form isolated from parasites. The S3-S3' subsite preferences of the recombinant mature PfPM1 were explored using combinatorial chemistry based peptide libraries. On the basis of the results, a peptidomimetic inhibitor (compound 1) was designed and yielded 5-fold selectivity for binding to PfPM1 versus the homologous human cathepsin D (hcatD). The 2.8 A structure of the PfPM2-compound 1 complex is reported. Modeling studies were conducted using a series of peptidomimetic inhibitors (compounds 1-6, Table 3) and three plasmepsins: the crystal structure of PfPM2, and homology derived models of PfPM1 and PfPM4.

Role of Plasmodium falciparum digestive vacuole plasmepsins in the specificity and antimalarial mode of action of cysteine and aspartic protease inhibitors.

Hemoglobin (Hb) degradation is essential for the growth of the intraerythrocytic stages of malarial parasites. This process, which occurs inside an acidic digestive vacuole (DV), is thought to involve the action of four aspartic proteases, termed plasmepsins (PMs). These enzymes have received considerable attention as potential antimalarial drug targets. Leveraging the availability of a set of PM-knockout lines generated in Plasmodium falciparum, we report here that a wide range of previously characterized or novel aspartic protease inhibitors exert their antimalarial activities independently of their effect on the DV PMs. We also assayed compounds previously shown to inhibit cysteine proteases residing in the DV. The most striking observation was a ninefold increase in the potency of the calpain inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) against parasites lacking all four DV PMs. Genetic ablation of PM III or PM IV also decreased the level of parasite resistance to the beta-hematin binding antimalarial chloroquine. On the basis of the findings of drug susceptibility and isobologram assays, as well as the findings of studies of the inhibition of Hb degradation, morphological analyses, and stage specificity, we conclude that the DV PMs and falcipain cysteine proteases act cooperatively in Hb hydrolysis. We also identify several aspartic protease inhibitors, designed to target DV PMs, which appear to act on alternative targets early in the intraerythrocytic life cycle. These include the potent diphenylurea compound GB-III-32, which was found to be fourfold less potent against a P. falciparum line overexpressing plasmepsin X than against the parental nontransformed parasite line. The identification of the mode of action of these inhibitors will be important for future antimalarial drug discovery efforts focusing on aspartic proteases.

Elimination or more accurate estimation? Investigation of trends in malaria diagnoses in the Ouest Department of Haiti from 2008 to 2017.

BACKGROUND: According to the 2016 World Malaria Report, the malaria incidence in Haiti declined by > 40% between 2010 and 2015. Though elimination efforts have likely contributed, this time period also corresponded to a national change in diagnostic methods. METHODS: Monthly reports of aggregated patient data were acquired from five clinics in the Ouest Department of Haiti. Generalized linear models were used to compare the number of febrile patients tested, the number of positive tests, and the proportion of tests that were positive (TRP) before and after the national adoption of rapid diagnostic tests (RDTs). RESULTS: Prior to the earthquake when microcopy was used for diagnosis, a total of 1,727 patients with 557 (32.3%) positive; post-earthquake testing was reduced and the TPR was variable; during the post recovery period when RDTs were used exclusivly, a total of 5,132 patients were tested using RDTs, only 83 (1.62%) were positive. Compared to the pre-earthquake period, there was a 69% increase in the number of patients tested (IRR: 1.69; 95% CI IRR 1.59, 2.79), and a 97.0% decrease in the proportion of patients with a positive test result (IRR: 0.03; 95% CI IRR 0.02, 0.04) in the post-recovery period. CONCLUSIONS: While the decline in malaria indicators between 2010 and 2015 has been cited as evidence of progress towards elimination, these reports derived estimates of the malaria burden in Haiti using two different diagnostic tests. Thus, comparison of these periods in the context of malaria elimination should be made with caution.

Effects on growth, hemoglobin metabolism and paralogous gene expression resulting from disruption of genes encoding the digestive vacuole plasmepsins of Plasmodium falciparum.

Four of the plasmepsins of Plasmodium falciparum are localised in the digestive vacuole (DV) of the asexual blood stage parasite (PfPM1, PfPM2, PfPM4 and PfHAP), and each of these aspartic proteinases has been successfully targeted by gene disruption. This study describes further characterisation of the single-plasmepsin knockout mutants, and the creation and characterisation of double-plasmepsin knockout mutants lacking complete copies of pfpm2 and pfpm1 or pfhap and pfpm2. Double-plasmepsin knockout mutants were created by transfecting pre-existing knockout mutants with a second plasmid knockout construct. PCR and Southern blot analysis demonstrate the integration of a large concatamer of each plasmid construct into the targeted gene. All mutants have been characterised to assess the involvement of the DV plasmepsins in sustaining growth during the asexual blood stage. Analyses reaffirmed that knockout mutants Deltapfpm1 and Deltapfpm4 had lower replication rates in the asexual erythrocytic stage than the parental line (Dd2), but double-plasmepsin knockout mutants lacking intact copies of either pfpm2 and pfpm1, or pfpm2 and pfhap, had normal growth rates compared with Dd2. The amount of crystalline hemozoin produced per parasite during the asexual cycle was measured in each single-plasmepsin knockout to estimate the effect of each DV plasmepsin on hemoglobin digestion. Only Deltapfpm4 had a statistically significant reduction in hemozoin accumulation, indicating that hemoglobin digestion was impaired in this mutant. In the single-plasmepsin knockouts, no statistically significant differences were found in the steady state levels of mRNA from the remaining intact DV plasmepsin genes. Disruption of a DV plasmepsin gene does not affect the accumulation of mRNA encoding the remaining paralogous plasmepsins, and Western blot analysis confirmed that the accumulation of the paralogous plasmepsins in each knockout mutant was similar among all clones examined.

Expression and characterization of the ATP-binding domain of a malarial Plasmodium vivax gene homologous to the B-subunit of the bacterial topoisomerase DNA gyrase.

We have previously reported the presence of a DNA gyrase-like topoisomerase activity associated with the 35kb apicoplast DNA in the malarial parasite Plasmodium falciparum [Weissig V, Vetro-Widenhouse TS, Rowe TC. Topoisomerase II inhibitors induce cleavage of nuclear and 35kb plastid DNAs in the malarial parasite Plasmodium falciparum. DNA Cell Biol 1997;16:1483]. Sequences encoding polypeptides homologous to both the A and B subunits of bacterial DNA gyrase have been identified in the genome sequence of P. falciparum among data produced by the Malaria Genome Consortium and the University of Florida Malaria Gene Sequence Tag Project. Based on these findings, we have cloned and expressed a region of the Plasmodium vivax GyrB gene encoding a 43kDa polypeptide homologous to the ATP-binding domain of Escherichia coli DNA gyrase. The 43kDa PvGyrB polypeptide was found to have intrinsic ATPase activity with a K(m) of 0.27mM and a k(cat) of 0.051s(-1). The PvGyrB ATPase was also sensitive to the bacterial DNA gyrase inhibitor coumermycin. The implications of these findings are discussed.

Structural insights into the activation of P. vivax plasmepsin.

The malarial aspartic proteinases (plasmepsins) have been discovered in several species of Plasmodium, including all four of the human malarial pathogens. In P.falciparum, plasmepsins I, II, IV and HAP have been directly implicated in hemoglobin degradation during malaria infection, and are now considered targets for anti-malarial drug design. The plasmepsins are produced from inactive zymogens, proplasmepsins, having unusually long N-terminal prosegments of more than 120 amino acids. Structural and biochemical evidence suggests that the conversion process of proplasmepsins to plasmepsins differs substantially from the gastric and plant aspartic proteinases. Instead of blocking substrate access to a pre-formed active site, the prosegment enforces a conformation in which proplasmepsin cannot form a functional active site. We have determined crystal structures of plasmepsin and proplasmepsin from P.vivax. The three-dimensional structure of P.vivax plasmepsin is typical of the monomeric aspartic proteinases, and the structure of P.vivax proplasmepsin is similar to that of P.falciparum proplasmepsin II. A dramatic refolding of the mature N terminus and a large (18 degrees ) reorientation of the N-domain between P.vivax proplasmepsin and plasmepsin results in a severe distortion of the active site region of the zymogen relative to that of the mature enzyme. The present structures confirm that the mode of inactivation observed originally in P.falciparum proplasmepsin II, i.e. an incompletely formed active site, is a true structural feature and likely represents the general mode of inactivation of the related proplasmepsins.

Crystallization and preliminary X-ray analysis of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae.

Plasmepsin 4 from the malarial parasite Plasmodium malariae (PmPM4) is a member of the plasmepsins (Plasmodium pepsins), a subfamily of the pepsin-like aspartic proteases whose ortholog in the malarial parasite P. falciparum is involved in hemoglobin digestion in its digestive vacuole. Crystals of PmPM4 in complex with the small-molecule inhibitor AG1776 have been grown from a precipitant of 15% PEG 4000 and 200 mM ammonium sulfate in 100 mM sodium acetate pH 4.5. X-ray diffraction data were collected on a Rigaku rotating-anode generator from a single crystal under cryoconditions, with a maximal useful diffraction pattern to 3.3 A resolution. The crystals are shown to be orthorhombic and have been assigned to space group P2(1)2(1)2, with unit-cell parameters a = 95.88, b = 112.58, c = 90.40 A and a scaling Rsym of 0.104 for 14,334 unique reflections. Packing consideration and self-rotation function results indicate that there are two molecules per asymmetric unit. It is expected that in the near future the structure of PmPM4 will be obtained using molecular-replacement methods, obtaining phases from previously determined plasmepsin structures. Elucidation of the structure of PmPM4 in complex with inhibitors may be paramount to producing new antimalarial therapeutic agents.

Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei.

The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1', S2' and S3'. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design.

Plasmepsin 4, the food vacuole aspartic proteinase found in all Plasmodium spp. infecting man.

Plasmepsins are aspartic proteinases of the malaria parasite, and seven groups of plasmepsins have been identified by comparing genomic sequence data available for the genes encoding these enzymes from Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium berghei, and Plasmodium yoelii. The food vacuole plasmepsins typified by plasmepsin 4 from P. falciparum (PfPM4) constitute one of these groups. Genes encoding the ortholog of PfPM4 have been cloned from Plasmodium ovale, Plasmodium malariae, and P. vivax. In addition, P. falciparum contains three paralagous food vacuole plasmepsins or plasmepsin-like enzymes that appear to have arisen by gene duplication, plasmepsins 1 (PfPM1), 2 (PfPM2) and HAP, and all four were localized to purified food vacuole preparations by two-dimensional gel electrophoresis and mass spectroscopic analysis. The three paralogs of PfPM4 do not have counterparts in the six other Plasmodium spp. examined by genomic DNA blot analysis and by review of available genomic sequence data. The presence of these paralogs among the food vacuole plasmepsins in P. falciparum as compared with the other three species causing malaria in man will impact efforts to rationally design antimalarials targeting the food vacuole plasmepsins.

Recombinant expression and enzymatic subsite characterization of plasmepsin 4 from the four Plasmodium species infecting man.

Plasmepsin 4 from Plasmodium falciparum and orthologs from Plasmodium malariae, Plasmodium ovale and Plasmodium vivax have been expressed in recombinant form, and properties of the active site of each enzyme characterized by kinetic analysis. A panel of chromogenic peptide substrates systematically substituted at the P3, P2, P2' and P3' positions was used to estimate enzyme/ligand interactions in the corresponding enzyme subsites based upon kinetic data. The kinetic parameters kcat, Km and kcat/Km were measured to identify optimal substrates for each enzyme and also sequences that were readily cleaved by the plasmepsins but poorly by host aspartic peptidases. Computer generated models were utilized to compare enzyme structures and interpret kinetic results. The orthologous plasmepsins share highly similar subsite specificities. In the S3 and S2 subsites, the plasmepsin 4 orthologs all preferred hydrophobic amino acid residues, Phe or Ile, but rejected charged residues such as Lys or Asp. In S2' and S3' subsites, these plasmepsins tolerated both hydrophobic and hydrophilic residues. Subsite specificities of the plasmepsin 4 family of orthologs are similar to those of human cathepsins D and E, except in S3' where the plasmepsins accept substrates containing Ser significantly better than either of these human aspartic proteases. Peptidomimetic methyleneamino reduced-peptide inhibitors, which have inhibition constants in the picomolar range, were prepared for each plasmepsin 4 ortholog based upon substrate preferences. A peptidomimetic inhibitor designed for plasmepsin 4 from P. falciparum having Ser in P3' had the lowest Ki of the series of inhibitors prepared, but did not significantly improve the selectivity of the inhibitor for plasmepsin 4 versus human cathepsin D.

Molecular characterisation of a major 29 kDa surface antigen of Sarcocystis neurona.

A gene encoding a major 29 kDa surface antigen from Sarcocystis neurona, the primary causative agent of equine protozoal myeloencephalitis (EPM), was cloned, sequenced, and expressed as a recombinant protein. A cDNA library was prepared in the expression vector lambda ZAP from polyA+mRNA isolated from S. neurona merozoites cultivated in vitro. Random sequencing of 96 clones identified a clone of an abundant transcript having a translated amino acid sequence with 30% identity to the 31-kDa surface antigen of Sarcocystis muris cyst merozoites. Southern blot analysis indicated that the corresponding gene exists in low copy number within the S. neurona genome, but RNA blot analysis and other data indicated that the gene transcript is highly abundant. The sequence of the cDNA clone encoded an open reading frame specifying a polypeptide of 276 amino acids with a predicted size of 28.7 kDa. The deduced amino acid sequence displayed a hypothetical N-terminal signal peptide sequence followed by a polypeptide containing 12 cysteines. The coding region of the cDNA insert was subcloned into the expression vector pET14b, and a fusion protein expressed. The recombinant polypeptide was recognised by mAb 2A7 and mAb 1631, directed against a 29 kDa native protein found on the surface of cultured merozoites. Antibodies in serum and cerebrospinal fluid from a horse with EPM recognised a 29 kDa native protein of S. neurona merozoites and the 29 kDa recombinant protein. This S. neurona surface antigen is named SnSAG1.

Depletion of natural killer cells does not result in neurologic disease due to Sarcocystis neurona in mice with severe combined immunodeficiency.

Sarcocystis neurona is an apicomplexan parasite that is the primary etiologic agent of equine protozoal myeloencephalitis in horses. Protective immune responses in horses have not been determined, but interferon-gamma (IFN-gamma) is considered critical for protection from neurologic disease in mice. The role of adaptive and innate immune responses in control of parasites was explored by infecting BALB/c, IFN-gamma knockout (GKO), and severe combined immune deficient (SCID) mice with S. neurona (10(4) sporocysts/mouse). Immune competent BALB/c mice eliminated parasites within 30 days, with no sign of neurologic disease, whereas GKO mice developed fulminant neurologic disease. In contrast, SCID mice remained healthy throughout the experimental period despite the persistence of parasite at low levels in some mice. Treatment with anti-IFN-gamma antibody resulted in neurologic disease in infected SCID mice. Although SCID mice lack adaptive immune responses, they have natural killer (NK) cells capable of producing significant quantities of IFN-gamma. Therefore, SCID mice were infected with sporocysts of S. neurona and treated with anti-asialo GM1. Depletion of NK cells, confirmed by flow cytometry, did not result in neurologic disease in SCID mice. These results indicate that IFN-gamma mediates protection from neurologic disease in SCID mice. Protective levels of IFN-gamma may originate from a low number of nondepleted NK cells or from a non-T cell, non-NK cell population.

Sarcocystis meningoencephalitis in a northern gannet (Morus bassanus).

Sarcocystis sp. schizonts were found in the cerebellum of a northern gannet (Morus bassanus), exhibiting neurologic signs, found on the Florida (USA) east coast. Based upon molecular characterization of DNA isolated from the brain of the gannet, this Sarcocystis sp. appeared to be closely related, if not identical, to an unnamed Sarcocystis sp. typified by isolates 1085 and 1086 collected from feces of a Virginia opossum (Didelphis virginiana) on the east coast of Florida. Because the life cycle of this parasite appears to be land based, urban waste discharge to marine/estuarine environments may be a source of infection for marine species.