scholarly journals Systematic Genetic Analysis of the Plasmodium falciparum MSP7-Like Family Reveals Differences in Protein Expression, Location, and Importance in Asexual Growth of the Blood-Stage Parasite

2010 ◽  
Vol 9 (7) ◽  
pp. 1064-1074 ◽  
Author(s):  
Madhusudan Kadekoppala ◽  
Solabomi A. Ogun ◽  
Steven Howell ◽  
Ruwani S. Gunaratne ◽  
Anthony A. Holder
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Expression ◽  
Surface Protein ◽  
Gene Families ◽  
Merozoite Surface Protein ◽  
Blood Stage ◽  
Asexual Blood Stage ◽  
Content Type ◽  
Specific Proteins ◽  
Proteolytic Cleavages

ABSTRACT Proteins located on Plasmodium falciparum merozoites, the invasive form of the parasite's asexual blood stage, are of considerable interest in vaccine research. Merozoite surface protein 7 (MSP7) forms a complex with MSP1 and is encoded by a member of a multigene family located on chromosome 13. The family codes for MSP7 and five MSP7-related proteins (MSRPs). In the present study, we have investigated the expression and the effect of msrp gene deletion at the asexual blood stage. In addition to msp7, msrp2, msrp3, and msrp5 are transcribed, and mRNA was easily detected by hybridization analysis, whereas mRNA for msrp1 and msrp4 could be detected only by reverse transcription (RT)-PCR. Notwithstanding evidence of transcription, antibodies to recombinant MSRPs failed to detect specific proteins, except for antibodies to MSRP2. Sequential proteolytic cleavages of MSRP2 resulted in 28- and 25-kDa forms. However, MSRP2 was absent from merozoites; the 25-kDa MSRP2 protein (MSRP225) was soluble and secreted upon merozoite egress. The msrp genes were deleted by targeted disruption in the 3D7 line, leading to ablation of full-length transcripts. MSRP deletion mutants had no detectable phenotype, with growth and invasion characteristics comparable to those of the parental parasite; only the deletion of MSP7 led to a detectable growth phenotype. Thus, within this family some of the genes are transcribed at a significant level in asexual blood stages, but the corresponding proteins may or may not be detectable. Interactions of the expressed proteins with the merozoite also differ. These results highlight the potential for unexpected differences of protein expression levels within gene families.

scholarly journals A Chimeric Plasmodium falciparum Merozoite Surface Protein Vaccine Induces High Titers of Parasite Growth Inhibitory Antibodies

2013 ◽  
Vol 81 (10) ◽  
pp. 3843-3854 ◽  
Author(s):  
James R. Alaro ◽  
Andrea Partridge ◽  
Kazutoyo Miura ◽  
Ababacar Diouf ◽  
Ana M. Lopez ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
T Cell ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Plasmodium Yoelii ◽  
T Cell Response ◽  
Blood Stage ◽  
Fusion Partner ◽  
Content Type ◽  
Cpg Oligodeoxynucleotide

ABSTRACTThe C-terminal 19-kDa domain ofPlasmodium falciparummerozoite surface protein 1 (PfMSP119) is an established target of protective antibodies. However, clinical trials ofPfMSP142, a leading blood-stage vaccine candidate which contains the protective epitopes ofPfMSP119, revealed suboptimal immunogenicity and efficacy. Based on proof-of-concept studies in thePlasmodium yoeliimurine model, we produced a chimeric vaccine antigen containing recombinantPfMSP119(rPfMSP119) fused to the N terminus ofP. falciparummerozoite surface protein 8 that lacked its low-complexity Asn/Asp-rich domain, rPfMSP8 (ΔAsn/Asp). Immunization of mice with the chimeric rPfMSP1/8 vaccine elicited strong T cell responses to conserved epitopes associated with the rPfMSP8 (ΔAsn/Asp) fusion partner. While specific forPfMSP8, this T cell response was adequate to provide help for the production of high titers of antibodies to bothPfMSP119and rPfMSP8 (ΔAsn/Asp) components. This occurred with formulations adjuvanted with either Quil A or with Montanide ISA 720 plus CpG oligodeoxynucleotide (ODN) and was observed in both inbred and outbred strains of mice.PfMSP1/8-induced antibodies were highly reactive with two major alleles ofPfMSP119(FVO and 3D7). Of particular interest, immunization withPfMSP1/8 elicited higher titers ofPfMSP119-specific antibodies than a combined formulation of rPfMSP142and rPfMSP8 (ΔAsn/Asp). As a measure of functionality,PfMSP1/8-specific rabbit IgG was shown to potently inhibit thein vitrogrowth of blood-stage parasites of the FVO and 3D7 strains ofP. falciparum. These data support the further testing and evaluation of this chimericPfMSP1/8 antigen as a component of a multivalent vaccine forP. falciparummalaria.

scholarly journals Comparative Immunogenicities of Full-Length Plasmodium falciparum Merozoite Surface Protein 3 and a 24-Kilodalton N-Terminal Fragment

2011 ◽  
Vol 18 (8) ◽  
pp. 1221-1228 ◽  
Author(s):  
Maryam Imam ◽  
Yengkhom Sangeeta Devi ◽  
Akhilesh K. Verma ◽  
Virander Singh Chauhan
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Terminal Region ◽  
Content Type ◽  
Conserved Domain ◽  
Terminal Fragment ◽  
Falciparum Merozoite ◽  
Form Part ◽  
Parasite Lysate

ABSTRACTRecombinantPlasmodium falciparummerozoite surface protein 3 (PfMSP3F) and a 24-kDa fragment from its N terminus (MSP3N) that includes the essential conserved domain, which elicits the maximum antibody (Ab)-dependent cellular inhibition (ADCI), were expressed as soluble proteins inEscherichia coli. Both proteins were found to be stable in both soluble and lyophilized forms. Immunization with MSP3F and MSP3N formulated separately with two human-compatible adjuvants, aluminum hydroxide (Alhydrogel) and Montanide ISA 720, produced significant antibody responses in mice and rabbits. Polyclonal Abs against both antigens recognized native MSP3 in the parasite lysate. These two Abs also recognized two synthetic peptides, previously characterized to possess B cell epitopes from the N-terminal region. Antibody depletion assay showed that most of the IgG response is directed toward the N-terminal region of the full protein. Anti-MSP3F and anti-MSP3N rabbit antibodies did not inhibit merozoite invasion or intraerythrocytic development but significantly reduced parasitemia in the presence of human monocytes. The ADCI demonstrated by anti-MSP3N antibodies was comparable to that exhibited by anti-MSP3F antibodies (both generated in rabbit). These results suggest that the N-terminal fragment of MSP3 can be considered a vaccine candidate that can form part of a multigenic vaccine against malaria.

scholarly journals Identification of MMV Malaria Box Inhibitors of Plasmodium falciparum Early-Stage Gametocytes Using a Luciferase-Based High-Throughput Assay

2013 ◽  
Vol 57 (12) ◽  
pp. 6050-6062 ◽  
Author(s):  
Leonardo Lucantoni ◽  
Sandra Duffy ◽  
Sophie H. Adjalley ◽  
David A. Fidock ◽  
Vicky M. Avery
Keyword(s):  
Plasmodium Falciparum ◽  
High Throughput ◽  
Early Stage ◽  
Blood Stage ◽  
Stage I ◽  
Mosquito Vector ◽  
Asexual Blood Stage ◽  
Content Type ◽  
Malaria Box ◽  
Gametocytocidal Activity

ABSTRACTThe design of new antimalarial combinations to treatPlasmodium falciparuminfections requires drugs that, in addition to resolving disease symptoms caused by asexual blood stage parasites, can also interrupt transmission to the mosquito vector. Gametocytes, which are essential for transmission, develop as sexual blood stage parasites in the human host over 8 to 12 days and are the most accessible developmental stage for transmission-blocking drugs. Considerable effort is currently being devoted to identifying compounds active against mature gametocytes. However, investigations on the drug sensitivity of developing gametocytes, as well as screening methods for identifying inhibitors of early gametocytogenesis, remain scarce. We have developed a luciferase-based high-throughput screening (HTS) assay using tightly synchronous stage I to III gametocytes from a recombinantP. falciparumline expressing green fluorescent protein (GFP)-luciferase. The assay has been used to evaluate the early-stage gametocytocidal activity of the MMV Malaria Box, a collection of 400 compounds with known antimalarial (asexual blood stage) activity. Screening this collection against early-stage (I to III) gametocytes yielded 64 gametocytocidal compounds with 50% inhibitory concentrations (IC50s) below 2.5 μM. This assay is reproducible and suitable for the screening of large compound libraries, with an average percent coefficient of variance (%CV) of ≤5%, an average signal-to-noise ratio (S:N) of >30, and a Z′ of ∼0.8. Our findings highlight the need for screening efforts directed specifically against early gametocytogenesis and indicate the importance of experimental verification of early-stage gametocytocidal activity in the development of new antimalarial candidates for combination therapy.

scholarly journals Age-Dependent IgG Subclass Responses to Plasmodium falciparum EBA-175 Are Differentially Associated with Incidence of Malaria in Mozambican Children

2011 ◽  
Vol 19 (2) ◽  
pp. 157-166 ◽  
Author(s):  
Carlota Dobaño ◽  
Diana Quelhas ◽  
Llorenç Quintó ◽  
Laura Puyol ◽  
Elisa Serra-Casas ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Incidence ◽  
Controlled Trial ◽  
Intermittent Preventive Treatment ◽  
Antibody Test ◽  
Merozoite Surface Protein ◽  
Blood Stage ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Subclass ◽  
Content Type

ABSTRACTPlasmodium falciparumblood-stage antigens such as merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and the 175-kDa erythrocyte binding antigen (EBA-175) are considered important targets of naturally acquired immunity to malaria. However, it is not clear whether antibodies to these antigens are effectors in protection against clinical disease or mere markers of exposure. In the context of a randomized, placebo-controlled trial of intermittent preventive treatment in infants conducted between 2002 and 2004, antibody responses toPlasmodium falciparumblood-stage antigens in a cohort of 302 Mozambican children were evaluated by immunofluorescence antibody test and enzyme-linked immunosorbent assay at 5, 9, 12, and 24 months of age. We found that IgG subclass responses to EBA-175 were differentially associated with the incidence of malaria in the follow-up period. A double amount of cytophilic IgG1 or IgG3 was associated with a significant decrease in the incidence of malaria (incidence rate ratio [IRR] = 0.49, 95% confidence interval [CI] = 0.25 to 0.97, andP= 0.026 and IRR = 0.44, CI = 0.19 to 0.98, andP= 0.037, respectively), while a double amount of noncytophilic IgG4 was significantly correlated with an increased incidence of malaria (IRR = 3.07, CI = 1.08 to 8.78,P= 0.020). No significant associations between antibodies to the 19-kDa fragment of MSP-1 (MSP-119) or AMA-1 and incidence of malaria were found. Age, previous episodes of malaria, present infection, and neighborhood of residence were the main factors influencing levels of antibodies to all merozoite antigens. Deeper understanding of the acquisition of antibodies against vaccine target antigens in early infancy is crucial for the rational development and deployment of malaria control tools in this vulnerable population.

scholarly journals Plasmodium falciparum Apicomplexan-Specific Glucosamine-6-Phosphate N-Acetyltransferase Is Key for Amino Sugar Metabolism and Asexual Blood Stage Development

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Jordi Chi ◽  
Marta Cova ◽  
Matilde de las Rivas ◽  
Ana Medina ◽  
Rafael Junqueira Borges ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Apicomplexan Parasite ◽  
Blood Stage ◽  
Host Cells ◽  
Asexual Blood Stage ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Hexosamine Pathway ◽  
Depth Analysis ◽  
Stage Development

ABSTRACT UDP-N-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and N- and O-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate N-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual Plasmodium falciparum parasites. Furthermore, we present the 1.95 Å resolution structure of the GNA1 ortholog from Cryptosporidium parvum, an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for P. falciparum GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria. IMPORTANCE Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite’s ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate N-acetyltransferase (GNA1), required for the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), is essential for P. falciparum asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite C. parvum, used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.

scholarly journals Antigenic Characterization of an Intrinsically Unstructured Protein, Plasmodium falciparum Merozoite Surface Protein 2

2012 ◽  
Vol 80 (12) ◽  
pp. 4177-4185 ◽  
Author(s):  
Christopher G. Adda ◽  
Christopher A. MacRaild ◽  
Linda Reiling ◽  
Kaye Wycherley ◽  
Michelle J. Boyle ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Protein ◽  
Variable Region ◽  
Intrinsic Property ◽  
Merozoite Surface Protein ◽  
Surface Proteins ◽  
Structural Basis ◽  
Content Type ◽  
Intrinsically Unstructured Protein ◽  
Unstructured Protein

ABSTRACTMerozoite surface protein 2 (MSP2) is an abundant glycosylphosphatidylinositol (GPI)-anchored protein ofPlasmodium falciparum, which is a potential component of a malaria vaccine. As all forms of MSP2 can be categorized into two allelic families, a vaccine containing two representative forms of MSP2 may overcome the problem of diversity in this highly polymorphic protein. Monomeric recombinant MSP2 is an intrinsically unstructured protein, but its conformational properties on the merozoite surface are unknown. This question is addressed here by analyzing the 3D7 and FC27 forms of recombinant and parasite MSP2 using a panel of monoclonal antibodies raised against recombinant MSP2. The epitopes of all antibodies, mapped using both a peptide array and by nuclear magnetic resonance (NMR) spectroscopy on full-length recombinant MSP2, were shown to be linear. The antibodies revealed antigenic differences, which indicate that the conserved N- and C-terminal regions, but not the central variable region, are less accessible in the parasite antigen. This appears to be an intrinsic property of parasite MSP2 and is not dependent on interactions with other merozoite surface proteins as the loss of some conserved-region epitopes seen using the immunofluorescence assay (IFA) on parasite smears was also seen on Western blot analyses of parasite lysates. Further studies of the structural basis of these antigenic differences are required in order to optimize recombinant MSP2 constructs being evaluated as potential vaccine components.

scholarly journals Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

2013 ◽  
Vol 13 (5) ◽  
pp. 550-559 ◽  
Author(s):  
Ben C. L. van Schaijk ◽  
T. R. Santha Kumar ◽  
Martijn W. Vos ◽  
Adam Richman ◽  
Geert-Jan van Gemert ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Plasmodium Falciparum ◽  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Blood Stage ◽  
Acid Biosynthesis ◽  
Asexual Blood Stage ◽  
Liver Stage ◽  
Content Type ◽  
Fas Ii

ABSTRACT The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum , the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F . Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii , where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.

scholarly journals Plasmodium falciparum Malaria in the Peruvian Amazon, a Region of Low Transmission, Is Associated with Immunologic Memory

2012 ◽  
Vol 80 (4) ◽  
pp. 1583-1592 ◽  
Author(s):  
Eva H. Clark ◽  
Claudia J. Silva ◽  
Greta E. Weiss ◽  
Shanping Li ◽  
Carlos Padilla ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Protein ◽  
Plasmodium Falciparum Malaria ◽  
Merozoite Surface Protein ◽  
Content Type ◽  
Low Transmission ◽  
Apical Membrane Antigen 1 ◽  
Specific Memory ◽  
Erythrocyte Binding ◽  
Transmission Region

ABSTRACTThe development of clinical immunity toPlasmodium falciparummalaria is thought to require years of parasite exposure, a delay often attributed to difficulties in developing protective antibody levels. In this study, we evaluated severalP. falciparumvaccine candidate antigens, including apical membrane antigen 1 (AMA-1), circumsporozoite protein (CSP), erythrocyte binding antigen 175 (EBA-175), and the 19-kDa region of merozoite surface protein 1 (MSP119). After observing a more robust antibody response to MSP119, we evaluated the magnitude and longevity of IgG responses specific to this antigen in Peruvian adults and children before, during, and afterP. falciparuminfection. In this low-transmission region, even one reported prior infection was sufficient to produce a positive anti-MSP119IgG response for >5 months in the absence of reinfection. We also observed an expansion of the total plasmablast (CD19+CD27+CD38high) population in the majority of individuals shortly after infection and detected MSP1-specific memory B cells in a subset of individuals at various postinfection time points. This evidence supports our hypothesis that effective antimalaria humoral immunity can develop in low-transmission regions.

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