Search

Chloroquine resistance in Plasmodium flaciparum was first reported around 40 years ago, although the molecular mechanism of resistance has remained unknown. It has been found that chloroquine resistance can be partly reversed by verapamil. The mapping of the chloroquine-resistant locus to a 36 kb segment is reported, and the candidate gene required for the resistance mechanism, is identified.

The var gene family of Plasmodium falciparum codes for the PfEMP1 proteins, which are responsible for antigenic variation and the adherence of erythrocytes infected by Plasmodium falciparum to the endothelium. There are 50 to 150 var genes on multiple chromosomes. The gene product is a 200 to 350 kDa protein that has a membrane anchor, a transmembrane sequence and an extracellular domain that is similar to that of other cell-cell adhesion proteins such as the cadherins.

The CG2 protein of Plasmodium falciparum has been postulated to be involved in chloroquine resistance in the malaria-causing parasite. A recent study has even claimed that this protein resembles a sodium-hydrogen exchanger (NHE). Sequence analysis of this protein reveals that it is not similar to NHE. The hydrophobic domains of CG2 are not characteristic of NHEs and the transport domain is not homologous to that of NHEs. Further, immunoelectronmicroscopy revealed that CG2 is not a membrane protein.

Summary Invading pathogens trigger specific host responses, an understanding of which might identify genes that function in pathogen recognition and elimination. In this study, we performed trans-species expression quantitative trait locus (ts-eQTL) analysis using genotypes of the Plasmodium yoelii malaria parasite and phenotypes of mouse gene expression. We significantly linked 1,054 host genes to parasite genetic loci (LOD score ≥ 3.0). Using LOD score patterns, which produced results that differed from direct expression-level clustering, we grouped host genes that function in related pathways, allowing functional prediction of unknown genes. As a proof of principle, 14 of 15 randomly selected genes predicted to function in type I interferon (IFN-I) responses were experimentally validated using overexpression, small hairpin RNA knockdown, viral infection, and/or infection of knockout mice. This study demonstrates an effective strategy for studying gene function, establishes a functional gene database, and identifies regulators in IFN-I pathways. [ABSTRACT FROM AUTHOR]

The debate over whether the human malaria parasite Plasmodium falciparum underwent a recent severe population bottleneck (“Malaria''s Eve” hypothesis) has attracted great attention recently. Understanding the genetic diversity and evolutionary history of the parasite has practical implications for developing disease control measures. [Copyright &y& Elsevier]

Artemisinin and its derivatives (ART) are crucial first-line antimalarial drugs that rapidly clear parasitemia, but recrudescences of the infection frequently follow ART monotherapy. For this reason, ART must be used in combination with one or more partner drugs that ensure complete cure. The ability of malaria parasites to survive ART monotherapy may relate to an innate growth bistability phenomenon whereby a fraction of the drug-exposed population enters into metabolic quiescence (dormancy) as persister forms. Characterization of the events that underlie entry and waking from persistence may lead to lasting breakthroughs in malaria chemotherapy that can prevent recrudescences and protect the future of ART-based combination therapies. Artemisinin treatment clears malaria parasitemia rapidly but is frequently followed by recrudescent infection unless the artemisinin (or artemisinin derivative) is combined with an effective partner drug. Recrudescence was identified as RI type artemisinin resistance in 1984, when artemisinin-based combination therapy (ACT) was also proposed. RI type artemisinin resistance is innate to Plasmodium falciparum and has not progressed to higher levels (RII or RIII), despite decades of ACT distributions and partner drug failures. Recrudescences from persisters are not associated with parasite clearance times after artemisinin treatment. Persistence as a survival mechanism is distinct from drug resistance acquired by genetic mutations, a distinction that is at risk of being lost when persisters are incorrectly distinguished as 'the drug-resistant parasites'. Understanding and targeting persisters may suggest strategies for lasting breakthroughs in malaria chemotherapy. [ABSTRACT FROM AUTHOR]

Galectins is a family of multifunctional lectins. Fifteen galectins have been identified from a variety of cells and tissues of vertebrates and invertebrates. Galectins have been shown to play pivotal roles in host–pathogen interaction such as adhesion of pathogens to host cells and activation of host innate and adaptive immunity. In recent years, the roles of galectins during parasite infections have gained increasing attention. Galectins produced by different hosts can act as pattern recognition receptors detecting conserved pathogen-associated molecular patterns of parasites, while galectins produced by parasites can modulate host responses. This review summarizes some recent studies on the roles of galectins produced by parasitic protozoa, nematodes, and trematodes and their hosts. Understanding the roles of galectins in host–parasite interactions may provide targets for immune intervention and therapies of parasitic infections. [ABSTRACT FROM AUTHOR]

Highlights: [•] This is the first study that identified gene-related microsatellite of Biomphalaria glabrata using data mining. [•] Genetic linkage map construction based on F2 progenies between resistant and susceptible snails coding B. glabrata markers. [Copyright &y& Elsevier]

• Australian tea tree oil display a range of biological and pharmacological properties. • Tea tree oil and its 15 major constituents have effects on parasitic infections. • The parasites include protozoa, nematodes, cestodes, and monogenean parasites. • Prolonged survival or curative effect on protozoan infections in treated animals. • Helminthic burden was reduced or controlled in treated animals. Australian tea tree (Melaleuca alternifolia) oil (TTO) and its monoterpene constituents such as terpinen-4-ol (T4O), 1,8-cineole, limonene, p-cymene, and α-terpinene have been shown to be effective in controlling a wide range of parasitic infections. The anti-parasitic effects of these compounds are mainly due to their anti-histamine and anti-acetylcholinesterase activities as well as their ability to modulate host inflammatory responses. This review attempts to summarize recent advances in the uses of TTO and its 15 major monoterpene constituents in treating parasitic infections in both humans and animals. Activities against parasitic protozoans (Plasmodium falciparum, Leishmania spp., Trypanosoma spp., Acanthamoeba castellanii, Trichomonas vaginalis, Eimeria, and Ichthyophthirius multifiliis), nematodes (Haemonchus contortus and Anisakis simplex), cestode (Echinococcus ortleppi), and monogeneans (Gasterosteus spp. and Dactylogyrus minutus) have been reported, showing good potentials in treating parasitic infections. Further studies are necessary for developing anti-parasite therapies using TTO or its monoterpenes constituents. [ABSTRACT FROM AUTHOR]

• Plasmodium yoelii can repair CRISPR/Cas9-generated DNA breaks by microhomology-mediated end joining. • Size mutants of the central repeat in circumsporozoite surface protein (CSP) were successfully generated. • A minimum size of the CSP central repeat region is required for mosquito stage development. CRISPR/Cas9 is a powerful genome editing method that has greatly facilitated functional studies in many eukaryotic organisms including malaria parasites. Due to the lack of genes encoding enzymes necessary for the non-homologous end joining DNA repair pathway, genetic manipulation of malaria parasite genomes is generally accomplished through homologous recombination requiring the presence of DNA templates. Recently, an alternative double-strand break repair pathway, microhomology-mediated end joining, was found in the Plasmodium falciparum parasite. Taking advantage of the MMEJ pathway, we developed a MMEJ-based CRISPR/Cas9 (mCRISPR) strategy to efficiently generate multiple mutant parasites simultaneously in genes with repetitive sequences. As a proof of principle, we successfully produced various size mutants in the central repeat region of the Plasmodium yoelii circumsporozoite surface protein without the use of template DNA. Monitoring mixed parasite populations and individual parasites with different sizes of CSP-CRR showed that the CSP-CRR plays a role in the development of mosquito stages, with severe developmental defects in parasites with large deletions in the repeat region. However, the majority of the csp mutant parasite clones grew similarly to the wild type P. yoelii 17XL parasite in mice. This study develops a useful technique to efficiently generate mutant parasites with deletions or insertions, and shows that the CSP-CRR plays a role in parasite development in mosquito. [ABSTRACT FROM AUTHOR]

The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium . However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii , we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite ( PyCas9ki ) of the rodent malaria parasite P. yoelii . The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii . [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 has been successfully adapted for gene editing in malaria parasites including Plasmodium falciparum and Plasmodium yoelii . However, the reported methods were limited to editing one gene at a time. In practice, it is often desired to modify multiple genetic loci in a parasite genome. Here we describe a CRISPR/Cas9 mediated genome editing method that allows successive modification of more than one gene in the genome of P. yoelii using an improved single-vector system (pYCm) we developed previously. Drug resistant genes encoding human dihydrofolate reductase (hDHFR) and a yeast bifunctional protein (yFCU), with cytosine deaminase (CD) and uridyl phosphoribosyl transferase (UPRT) activities in the plasmid, allowed sequential positive (pyrimethamine, Pyr) and negative (5-fluorocytosine, 5FC) selections and generation of transgenic parasites free of the episomal plasmid after genetic modification. Using this system, we were able to efficiently tag a gene of interest ( Pyp28 ) and subsequently disrupted two genes ( Pyctrp and Pycdpk3 ) that are individually critical for ookinete motility. Disruption of the genes either eliminated ( Pyctrp ) or greatly reduced ( Pycdpk3 ) ookinete forward motility in matrigel in vitro and completely blocked oocyst development in mosquito midgut. The method will greatly facilitate studies of parasite gene function, development, and disease pathogenesis. [ABSTRACT FROM AUTHOR]

Highlights: [•] Genome sequencing of Plasmodium yoelii parasite strains. [•] Identification of large numbers of single nucleotide polymorphism (SNP). [•] Development of a high throughput and reliable genotyping microarray. [•] Detection of SNPs among P. yoelli strains and genetic cross progeny. [Copyright &y& Elsevier]

Highlights: [•] Quantitative high-throughput screen (qHTS) of small molecules (SMs). [•] SMs as a tool for studying phenotypes and gene function. [•] Combination of SM screening and genome-wide approaches. [•] Target identification and pathway/network building. [ABSTRACT FROM AUTHOR]

Summary: Background: Artemisinin-resistant Plasmodium falciparum has been reported in Pailin, western Cambodia, detected as a slow parasite clearance rate in vivo. Emergence of this phenotype in western Thailand and possibly elsewhere threatens to compromise the effectiveness of all artemisinin-based combination therapies. Parasite genetics is associated with parasite clearance rate but does not account for all variation. We investigated contributions of both parasite genetics and host factors to the artemisinin-resistance phenotype in Pursat, western Cambodia. Methods: Between June 19 and Nov 28, 2009, and June 26 and Dec 6, 2010, we enrolled patients aged 10 years or older with uncomplicated falciparum malaria, a density of asexual parasites of at least 10 000 per μL of whole blood, no symptoms or signs of severe malaria, no other cause of febrile illness, and no chronic illness. We gave participants 4 mg/kg artesunate at 0, 24, and 48 h, 15 mg/kg mefloquine at 72 h, and 10 mg/kg mefloquine at 96 h. We assessed parasite density on thick blood films every 6 h until undetectable. The parasite clearance half-life was calculated from the parasite clearance curve. We genotyped parasites with 18 microsatellite markers and patients for haemoglobin E, α-thalassaemia, and a mutation of G6PD, which encodes glucose-6-phosphate dehydrogenase. To account for the possible effects of acquired immunity on half-life, we used three surrogates for increased likelihood of exposure to P falciparum: age, sex, and place of residence. This study is registered with ClinicalTrials.gov, number NCT00341003. Findings: We assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. We enrolled 168 patients with falciparum malaria who met inclusion criteria. The geometric mean half-life was 5·85 h (95% CI 5·54–6·18) in Pursat, similar to that reported in Pailin (p=0·109). We identified two genetically different parasite clone groups: parasite group 1 (PG1) and parasite group 2 (PG2). Non-significant increases in parasite clearance half-life were seen in patients with haemoglobin E (0·55 h; p=0·078), those of male sex (0·96 h; p=0·064), and in 2010 (0·68 h; p=0·068); PG1 was associated with a significant increase (0·79 h; p=0·033). The mean parasite heritability of half-life was 0·40 (SD 0·17). Interpretation: Heritable artemisinin resistance is established in a second Cambodian province. To accurately identify parasites that are intrinsically susceptible or resistant to artemisinins, future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation. Funding: Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. [Copyright &y& Elsevier]

Abstract: Despite significant improvement in the malaria situation of the Greater Mekong Subregion (GMS), malaria control for the region continues to face a multitude of challenges. The extremely patchy malaria distribution, especially along international borders, makes disease surveillance and targeted control difficult. The vector systems are also diverse with dramatic differences in habitat ecology, biting behavior, and vectorial capacity, and there is a lack of effective transmission surveillance and control tools. Finally, in an era of heavy deployment of artemisinin-based combination therapies, the region acts as an epicenter of drug resistance, with the emergence of artemisinin resistant Plasmodium falciparum posing a threat to both regional and global malaria elimination campaigns. This problem is further exacerbated by the circulation of counterfeit and substandard artemisinin drugs. Accordingly, this Southeast Asian Malaria Research Center, consisting of a consortium of US and regional research institutions, has proposed four interlinked projects to address these most urgent problems in malaria control. The aims of these projects will help to substantially improve our understanding of malaria epidemiology, vector systems and their roles in malaria transmission, as well as the mechanisms of drug resistance in parasites. Through the training of next-generation scientists in malaria research, this program will help build up and strengthen regional research infrastructure and capacities, which are essential for sustained malaria control in this region. [Copyright &y& Elsevier]

Abstract: The Greater Mekong Subregion (GMS), comprised of six countries including Cambodia, China''s Yunnan Province, Lao PDR, Myanmar (Burma), Thailand and Vietnam, is one of the most threatening foci of malaria. Since the initiation of the WHO''s Mekong Malaria Program a decade ago, malaria situation in the GMS has greatly improved, reflected in the continuous decline in annual malaria incidence and deaths. However, as many nations are moving towards malaria elimination, the GMS nations still face great challenges. Malaria epidemiology in this region exhibits enormous geographical heterogeneity with Myanmar and Cambodia remaining high-burden countries. Within each country, malaria distribution is also patchy, exemplified by ‘border malaria’ and ‘forest malaria’ with high transmission occurring along international borders and in forests or forest fringes, respectively. ‘Border malaria’ is extremely difficult to monitor, and frequent malaria introductions by migratory human populations constitute a major threat to neighboring, malaria-eliminating countries. Therefore, coordination between neighboring countries is essential for malaria elimination from the entire region. In addition to these operational difficulties, malaria control in the GMS also encounters several technological challenges. Contemporary malaria control measures rely heavily on effective chemotherapy and insecticide control of vector mosquitoes. However, the spread of multidrug resistance and potential emergence of artemisinin resistance in Plasmodium falciparum make resistance management a high priority in the GMS. This situation is further worsened by the circulation of counterfeit and substandard artemisinin-related drugs. In most endemic areas of the GMS, P. falciparum and Plasmodium vivax coexist, and in recent malaria control history, P. vivax has demonstrated remarkable resilience to control measures. Deployment of the only registered drug (primaquine) for the radical cure of vivax malaria is severely undermined due to high prevalence of glucose-6-phosphate dehydrogenase deficiency in target human populations. In the GMS, the dramatically different ecologies, diverse vector systems, and insecticide resistance render traditional mosquito control less efficient. Here we attempt to review the changing malaria epidemiology in the GMS, analyze the vector systems and patterns of malaria transmission, and identify the major challenges the malaria control community faces on its way to malaria elimination. [Copyright &y& Elsevier]

Abstract: The DNA amplification process can be a source of bias and artifacts, especially when amplifying genomic areas with extreme AT or GC content. The human malaria parasite Plasmodium falciparum has an AT-rich genome, and some of its highly AT-rich regions have been shown to be refractory to polymerase chain reaction (PCR) amplification. Biased amplification may lead to erroneous conclusions for studies investigating genome-wide gene expression, nucleosome position, and copy number variation. Here we compare genome-wide nucleosome coverage in libraries amplified at three different extension temperatures and show that reduction in PCR extension temperature from 70°C to 60°C can greatly increase the fraction of coverage at AT-rich regions of the P. falciparum genome. Our method will improve the efficiency and coverage in sequencing an AT-rich genome. [ABSTRACT FROM AUTHOR]

Abstract: Genetic crosses have been employed to study various traits of rodent malaria parasites and to locate loci that contribute to drug resistance, immune protection, and disease virulence. Compared with human malaria parasites, genetic crossing of rodent malaria parasites is more easily performed; however, genotyping methods using microsatellites (MSs) or large-scale single nucleotide polymorphisms (SNPs) that have been widely used in typing Plasmodium falciparum are not available for rodent malaria species. Here we report a genome-wide search of the Plasmodium yoelii yoelii (P. yoelii) genome for simple sequence repeats (SSRs) and the identification of nearly 600 polymorphic MS markers for typing the genomes of P. yoelii and Plasmodium berghei. The MS markers are randomly distributed across the 14 physical chromosomes assembled from genome sequences of three rodent malaria species, although some variations in the numbers of MS expected according to chromosome size exist. The majority of the MS markers are AT-rich repeats, similar to those found in the P. falciparum genome. The MS markers provide an important resource for genotyping, lay a foundation for developing linkage maps, and will greatly facilitate genetic studies of P. yoelii. [Copyright &y& Elsevier]

Abstract: A complex of high-molecular-mass proteins (PfRhopH) of the human malaria parasite Plasmodium falciparum induces host protective immunity and therefore is a candidate for vaccine development. Understanding the level of polymorphism and the evolutionary processes is important for advancements in both vaccine design and knowledge of the evolution of cell invasion in this parasite. In the present study, we sequenced the entire open reading frames of seven genes encoding the proteins of the PfRhopH complex (rhoph2, rhoph3, and five rhoph1/clag gene paralogs). We found that four rhoph1/clag genes (clag2, 3.1, 3.2, and 8) were highly polymorphic. Amino acid substitutions and indels are predominantly clustered around amino acid positions 1000–1200 of these four rhoph1/clag genes. An excess of nonsynonymous substitutions over synonymous substitutions was detected for clag8 and 9, indicating positive selection. The McDonald–Kreitman test with a Plasmodium reichenowi orthologous sequence also supports positive selection on clag8. Based on the ratio of interspecific genetic distance to intraspecific distance, the time to the most recent common ancestor of the clag2 and 8 polymorphisms was estimated to be 1.89 and 0.87 million years ago, respectively, assuming divergence of P. falciparum and P. reichenowi 6 million years ago. In addition to a copy number polymorphism, gene conversion events were detected for the rhoph1/clag genes on chromosome 3, which likely play a role in increasing the diversity of each locus. Our results indicate that a high diversity of the PfRhopH1/Clag multigene family is maintained by diversifying selection forces over a considerably long period. [Copyright &y& Elsevier]

Abstract: VAR2CSA is the main candidate for a pregnancy malaria vaccine, but vaccine development may be complicated by sequence polymorphism. Here, we obtained partial or full-length var2CSA sequences from 106 parasites and applied novel computational methods and three-dimensional modeling to investigate VAR2CSA geographic variation and selection pressure. Our analysis reveals structural patterns of VAR2CSA sequence variation in which polymorphic sites group into segments of limited diversity. Within these segments, two or three basic types characterize a substantial majority of the parasite samples. Comparison to the primate malaria Plasmodium reichenowi shows that these basic types have ancient origins. Globally, var2CSA genes are comprised of a mosaic of these ancestral polymorphic segments that have recombined extensively between var2CSA alleles. Three-dimensional modeling reveals that polymorphic segments concentrate in flexible loops at characteristic locations in the six VAR2CSA Duffy binding-like (DBL) adhesion domains. Individual DBL domain surfaces have distinct patterns of diversifying selection, suggesting that limited and differing portions of each DBL domain are targeted by host antibody. Since standard phylogenetic tree analysis is inadequate for highly recombining genes like var2CSA, we developed a novel phylogenetic approach that incorporates recombination and tracks new mutations in segment types. In the resulting tree, P. reichenowi is confirmed as an outlier and African and Asian P. falciparum isolates have slightly diverged. These findings validate a new approach to modeling protein evolution in the presence of frequent recombination and provide a clearer understanding of how var gene products function as immunoevasive binding ligands. [Copyright &y& Elsevier]

Abstract: The rodent malaria parasite Plasmodium yoelii has been an important animal model for studying malaria pathology and host–parasite interactions. Compared with other rodent malaria parasites such as Plasmodium chabaudi, however, genetic mapping studies on P. yoelii have been limited, partly due to the absence of genetic markers and the lack of well characterized phenotypes. Taking advantage of the available genome sequence, we initiated a project to develop a high-resolution microsatellite (MS) map for P. yoelii to study malaria disease phenotypes. Here we report screening the P. yoelii genome for simple sequence repeats and development of an inexpensive method (modified from a previously reported procedure) for typing malaria parasite MS: instead of labeling individual polymerase chain reaction primers, a single fluorescently labeled primer was used to type the MS markers. We evaluated various polymerase chain reaction cycling conditions and M13-tailed/labeled M13 primer ratios to establish a simple and robust procedure for typing P. yoelii MS markers. We also compared typing efficiencies between individually labeled primers and the M13-tailed single labeled primer method and found that the two approaches were comparable. Preliminary analyses of seven P. yoelii isolates deposited at MR4 with 77 MS showed that the markers were highly polymorphic and that the isolates belonged to two groups, suggesting potential common ancestry or laboratory contaminations among the isolates. The MS markers and the typing method provide important tools for genetic studies of P. yoelii. There is a good possibility that this method can be applied to type MS from other malaria parasites including important human pathogens Plasmodium falciparum and Plasmodium vivax. [Copyright &y& Elsevier]

Abstract: In Plasmodium falciparum, var genes encode adhesive proteins that are transported to the surface of infected erythrocytes and act as major virulence determinants for infected erythrocyte binding and immune evasion. Var genes are highly diverse and can be classified into five major groups (UpsA, B, C, D, and E). Previous serological studies have suggested that the UpsA var group may contain common antigenic types that have important roles in severe childhood malaria. Here, our analysis found that UpsA vars are highly diverse between 22 world-wide parasite isolates, although they could be grouped into two broad clusters that may be separately recombining. By comparison, orthologs of the UpsA-linked Type 3 var and UpsE-linked var2csa were detected in nearly all parasite isolates, and a var2csa ortholog was also present in a chimpanzee malaria P. reichenowi that diverged from P. falciparum ∼5–7 million years ago. Although the specific function of Type 3 var genes is unknown, var2csa is a leading candidate for a pregnancy associated malaria vaccine. Compared to typical var genes, var2csa is unusually conserved but still had only 54–94% amino acid identity in extracellular binding regions. However, var2csa alleles have extensive gene mosaicism within polymorphic blocks that are shared between world-wide parasite isolates and recognizable in P. rechenowi suggesting a high rate of self–self recombination and an ancient and globally-related pool of var2csa polymorphism. These studies aid our understanding of the evolutionary mechanisms that shape var diversity and will be important to the development of vaccines against pregnancy associated malaria and severe malaria. [Copyright &y& Elsevier]

Abstract: The Plasmodium falciparum clone, Dd2, that requires sialic acid for invasion can switch to a sialic acid independent pathway, Dd2(NM). To elucidate the molecular basis of the switch in invasion phenotype of Dd2 to Dd2(NM), we performed expression profiling of the parasites using an oligonucleotide microarray and real-time RT-PCR. We found that four genes were upregulated in Dd2(NM) by microarray analysis, only two of which could be confirmed by real time RT-PCR. One gene, PfRH4, is a member of the reticulocyte homology family and the other, PEBL, is a pseudogene of the Duffy binding-like family. The two genes are contiguous but transcribed in opposite directions. The DNA sequence of these ORFs, their 5′-intergenic region and a 1.1kb region 3′ to each ORF are identical between Dd2 and Dd2(NM), suggesting that their transcription upregulation relates to transactivating factors. The transcription upregulation of PfRH4 was reflected at the protein level as PfRH4 protein expression was detected in Dd2(NM) and not in Dd2. Other sialic acid independent and dependent clones of P. falciparum showed variable transcript levels of PfRH4 and PEBL, unrelated to their dependence on sialic acid for invasion, suggesting that different P. falciparum clones use different receptors for sialic acid independent invasion. As Dd2(NM) is a selected subclone of Dd2, the marked upregulation of PfRH4 expression in Dd2(NM) suggests its role in erythrocyte invasion through the sialic acid independent pathway of Dd2(NM). [Copyright &y& Elsevier]