Identification of heat shock factor binding protein in Plasmodium falciparum

Background: Heat shock factor binding protein (HSBP) was originally discovered in a yeast two-hybrid screen as an interacting partner of heat shock factor (HSF). It appears to be conserved in all eukaryotes studied so far, with yeast being the only exception. Cell biological analysis of HSBP in mammals suggests its role as a negative regulator of heat shock response as it appears to interact with HSF only during the recovery phase following exposure to heat stress. While the identification of HSF in the malaria parasite is still eluding biologists, this study for the first time, reports the presence of a homologue of HSBP in Plasmodium falciparum. Methods: PfHSBP was cloned and purified as his-tag fusion protein. CD (Circular dichroism) spectroscopy was performed to predict the secondary structure. Immunoblots and immunofluorescence approaches were used to study expression and localization of HSBP in P. falciparum. Cellular fractionation was performed to examine subcellular distribution of PfHSBP. Immunoprecipitation was carried out to identify HSBP interacting partner in P. falciparum. Results: PfHSBP is a conserved protein with a high helical content and has a propensity to form homo-oligomers. PfHSBP was cloned, expressed and purified. The in vivo protein expression profile shows maximal expression in trophozoites. The protein was found to exist in oligomeric form as trimer and hexamer. PfHSBP is predominantly localized in the parasite cytosol, however, upon heat shock, it translocates to the nucleus. This study also reports the interaction of PfHSBP with PfHSP70-1 in the cytoplasm of the parasite. Conclusions: This study emphasizes the structural and biochemical conservation of PfHSBP with its mammalian counterpart and highlights its potential role in regulation of heat shock response in the malaria parasite. Analysis of HSBP may be an important step towards identification of the transcription factor regulating the heat shock response in P. falciparum.

Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.

Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the x-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the x-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA- M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.

A novel method to count the red blood cells in thin blood films

This paper describes a novel idea to identify the total number of red blood cells (RBCs) as well as their location in a Giemsa stained thin blood film image. This work is being undertaken as a part of developing an automated malaria parasite detection system by scanning a photograph of thin blood film in order to evaluate the parasitemia of the blood. Not only will this method eliminates the segmentation procedures that are normally used to segment the cells in the microscopic image, but also avoids any image pre-processing to deal with non uniform illumination prior to cell detection. The method utilizes basic knowledge on cell structure and brightness of the components due to Giemsa staining of the sample and detects and locates the RBCs in the image.

Genetic diversity and selection in three plasmodium vivax merozoite surface protein 7 (pvmsp-7) genes in a colombian population

A completely effective vaccine for malaria (one of the major infectious diseases worldwide) is not yet available; different membrane proteins involved in parasite-host interactions have been proposed as candidates for designing it. It has been found that proteins encoded by the merozoite surface protein (msp)-7 multigene family are antibody targets in natural infection; the nucleotide diversity of three Pvmsp-7 genes was thus analyzed in a Colombian parasite population. By contrast with P. falciparum msp-7 loci and ancestral P. vivax msp-7 genes, specie-specific duplicates of the latter specie display high genetic variability, generated by single nucleotide polymorphisms, repeat regions, and recombination. At least three major allele types are present in Pvmsp-7C, Pvmsp-7H and Pvmsp-7I and positive selection seems to be operating on the central region of these msp-7 genes. Although this region has high genetic polymorphism, the C-terminus (Pfam domain ID: PF12948) is conserved and could be an important candidate when designing a subunit-based antimalarial vaccine.

Búsqueda de selección en el polimorfismo 677C>T (c.665C>T) del gen de la metilentetrahidrofolato reductasa (MTHFR) en una población Colombiana

Se realizó un estudio genético – poblacional en dos grupos etarios de población colombiana con la finalidad de evaluar las diferencias genéticas relacionadas con el polimorfismo MTHFR 677CT en busca de eventos genéticos que soporten la persistencia de este polimorfismo en la especie humana debido que este ha sido asociado con múltiples enfermedades. De esta manera se genotipificaron los individuos, se analizaron los genotipos, frecuencias alélicas y se realizaron diferentes pruebas genéticas-poblacionales. Contrario a lo observado en poblaciones Colombianas revisadas se identificó la ausencia del Equilibrio Hardy-Weinberg en el grupo de los niños y estructuras poblacionales entre los adultos lo que sugiere diferentes historias demográficas y culturales entre estos dos grupos poblacionales al tiempo, lo que soporta la hipótesis de un evento de selección sobre el polimorfismo en nuestra población. De igual manera nuestros datos fueron analizados junto con estudios previos a nivel nacional y mundial lo cual sustenta que el posible evento selectivo es debido a que el aporte de ácido fólico se ha incrementado durante las últimas dos décadas como consecuencia de las campañas de fortificación de las harinas y suplementación a las embarazadas con ácido fólico, por lo tanto aquí se propone un modelo de selección que se ajusta a los datos encontrados en este trabajo se establece una relación entre los patrones nutricionales de la especie humana a través de la historia que explica las diferencias en frecuencias de este polimorfismo a nivel espacial y temporal.  

Being and goodness

The old scholastic principle of the "convertibility" of being and goodness strikes nearly all moderns as either barely comprehensible or plain false. "Convertible" is a term of art meaning "interchangeable" in respect of predication, where the predicates can be exchanged salva veritate albeit not salva sensu: their referents are, as the maxim goes, really the same albeit conceptually different. The principle seems, at first blush, absurd. Did the scholastics literally mean that every being is good? Is that supposed to include a cancer, a malaria parasite, an earthquake that kills millions? If every being is good, then no being is bad—but how can that be? To the contemporary philosophical mind, such bafflement is understandable. It derives from the systematic dismantling of the great scholastic edifice that took place over half a millennium. With the loss of the basic concepts out of which that edifice was built, the space created by those concepts faded out of existence as well. The convertibility principle, like virtually all the other scholastic principles (not all, since some do survive and thrive in analytic philosophy), could not persist in a post-scholastic space wholly alien to it.

Fitness of transgenic Anopheles stephensi mosquitoes expressing the SM1 peptide under the control of a vitellogenin promoter

Three transgenic Anopheles stephensi lines were established that strongly inhibit transmission of the mouse malaria parasite Plasmodium berghei. Fitness of the transgenic mosquitoes was assessed based on life table analysis and competition experiments between transgenic and wild-type mosquitoes. Life table analysis indicated low fitness load for the 2 single-insertion transgenic mosquito lines VD35 and VD26 and no load for the double-insertion transgenic mosquito line VD9. However, in cage experiments, where each of the 3 homozygous transgenic mosquitoes was mixed with nontransgenic mosquitoes, transgene frequency of all 3 lines decreased with time. Further experiments suggested that reduction of transgene frequency is a consequence of reduced mating success, reduced reproductive capacity, and/or insertional mutagenesis, rather than expression of the transgene itself. Thus, for transgenic mosquitoes released in the field to be effective in reducing malaria transmission, a driving mechanism will be required.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Plasmodium falciparum Accompanied the Human Expansion out of Africa

Plasmodium falciparum is distributed throughout the tropics and is responsible for an estimated 230 million cases of malaria every year, with a further 1.4 billion people at risk of infection [1-3]. Little is known about the genetic makeup of P. falciparum populations, despite variation in genetic diversity being a key factor in morbidity, mortality, and the success of malaria control initiatives. Here we analyze a worldwide sample of 519 P. falciparum isolates sequenced for two housekeeping genes (63 single nucleotide polymorphisms from around 5000 nucleotides per isolate). We observe a strong negative correlation between within-population genetic diversity and geographic distance from sub-Saharan Africa (R(2) = 0.95) over Africa, Asia, and Oceania. In contrast, regional variation in transmission intensity seems to have had a negligible impact on the distribution of genetic diversity. The striking geographic patterns of isolation by distance observed in P. falciparum mirror the ones previously documented in humans [4-7] and point to a joint sub-Saharan African origin between the parasite and its host. Age estimates for the expansion of P. falciparum further support that anatomically modern humans were infected prior to their exit out of Africa and carried the parasite along during their colonization of the world.

Transcriptional memory and switching in the Plasmodium falciparum rif gene family

The human malaria parasite Plasmodium falciparum expresses erythrocyte-surface directed variant antigens which are important virulence factors Many are transcribed from multigene families and presumably their mode of expression is strictly controlled to guarantee immune evasion in the human host. In order to elucidate the dynamics of rif transcription and to investigate if rif switching is comparable to var switching we monitored rif variant gene expression in parasites with different cytoadhesive properties as well as after a number of reinvasions. We found identical transcripts in parasite lines with different adhesive phenotypes suggesting that rif genes do not have a critical role in determining the cytoadhesion specificity of infected erythrocytes. We show for the first time that rif genes may show a conserved mode of transcription, maintaining the previously dominant rif transcript in subsequent reinvasions, but also observed rapid switching at rates up to 45% per generation, much higher than for the var gene family. (C) 2009 Elsevier B.V. All rights reserved.

Worldwide sequence conservation of transmission-blocking vaccine candidate Pvs230 in Plasmodium vivax

Pfs230, surface protein of gametocyte/gamete of the human malaria parasite, Plasmodium falciparum, is a prime candidate of malaria transmission-blocking vaccine. Plasmodium vivax has an ortholog of Pfs230 (Pvs230), however, there has been no study in any aspects on Pvs230 to date. To investigate whether Pvs230 can be a vivax malaria transmission-blocking vaccine, we performed evolutionary and population genetic analysis of the Pvs230 gene (pvs230: PVX_003905). Our analysis of Pvs230 and its orthologs in eight Plasmodium species revealed two distinctive parts: an interspecies variable part (IVP) containing species-specific oligopeptide repeats at the N-terminus and a 7.5 kb interspecies conserved part (ICP) containing 14 cysteine-rich domains. Pvs230 was closely related to its orthologs, Pks230 and Pcys230, in monkey malaria parasites. Analysis of 113 pvs230 sequences obtained from worldwide, showed that nucleotide diversity is remarkably low in the non-repeat 8-kb region of pvs230 (theta pi = 0.00118) with 77 polymorphic nucleotide sites, 40 of which results in amino acid replacements. A signature of purifying selection but not of balancing selection was seen on pvs230. Functional and/or structural constraints may limit the level of polymorphism in pvs230. The observed limited polymorphism in pvs230 should ground for utilization of Pvs230 as an effective transmission-blocking vaccine. (C) 2011 Elsevier Ltd. All rights reserved.

Evidence for a common role for the serine-type Plasmodium falciparum serine repeat antigen proteases : implications for vaccine and drug design

Serine repeat antigens (SERAs) are a family of secreted “cysteine-like” proteases of Plasmodium parasites. Several SERAs possess an atypical active-site serine residue in place of the canonical cysteine. The human malaria parasite Plasmodium falciparum possesses six “serine-type” (SERA1 to SERA5 and SERA9) and three “cysteine-type” (SERA6 to SERA8) SERAs. Here, we investigate the importance of the serine-type SERAs to blood-stage parasite development and examine the extent of functional redundancy among this group. We attempted to knock out the four P. falciparum serine-type SERA genes that have not been disrupted previously. SERA1, SERA4, and SERA9 knockout lines were generated, while only SERA5, the most strongly expressed member of the SERA family, remained refractory to genetic deletion. Interestingly, we discovered that while SERA4-null parasites completed the blood-stage cycle normally, they exhibited a twofold increase in the level of SERA5 mRNA. The inability to disrupt SERA5 and the apparent compensatory increase in SERA5 expression in response to the deletion of SERA4 provides evidence for an important blood-stage function for the serine-type SERAs and supports the notion of functional redundancy among this group. Such redundancy is consistent with our phylogenetic analysis, which reveals a monophyletic grouping of the serine-type SERAs across the genus Plasmodium and a predominance of postspeciation expansion. While SERA5 is to some extent further validated as a target for vaccine and drug development, our data suggest that the expression level of other serine-type SERAs is the only barrier to escape from anti-SERA5-specific interventions.

Plasmodium falciparum induces reorganization of host membrane proteins during intraerythrocytic growth

The virulence of the malaria parasite, Plasmodium falciparum, is due in large part to the way in which it modifies the membrane of its erythrocyte host. In this work we have used confocal microscopy and fluorescence recovery after photo-bleaching to examine the lateral mobility of host membrane proteins in erythrocytes infected with P falciparum at different stages of parasite growth. The erythrocyte membrane proteins band 3 and glycophorin show a marked decrease in mobility during the trophozoite stage of growth. Erythrocytes infected with a parasite strain that does not express the knob-associated histidine-rich protein show similar effects, indicating that this parasite protein does not contribute to the immobilization of the host proteins. Erythrocytes infected with ring-stage parasites exhibit intermediate mobility indicating that the parasite is able to modify its host prior to its active feeding stage.

A new rodent model to assess blood stage immunity to the plasmodium falciparum antigen merozoite surface protein 119 reveals a protective role for invasion inhibitory antibodies

Antibodies capable of inhibiting the invasion of Plasmodium merozoites into erythrocytes are present in individuals that are clinically immune to the malaria parasite. Those targeting the 19-kD COOH-terminal domain of the major merozoite surface protein (MSP)-119 are a major component of this inhibitory activity. However, it has been difficult to assess the overall relevance of such antibodies to antiparasite immunity. Here we use an allelic replacement approach to generate a rodent malaria parasite (Plasmodium berghei) that expresses a human malaria (Plasmodium falciparum) form of MSP-119. We show that mice made semi-immune to this parasite line generate high levels of merozoite inhibitory antibodies that are specific for P. falciparum MSP-119. Importantly, protection from homologous blood stage challenge in these mice correlated with levels of P. falciparum MSP-119–specific inhibitory antibodies, but not with titres of total MSP-119–specific immunoglobulins. We conclude that merozoite inhibitory antibodies generated in response to infection can play a significant role in suppressing parasitemia in vivo. This study provides a strong impetus for the development of blood stage vaccines designed to generate invasion inhibitory antibodies and offers a new animal model to trial P. falciparum MSP-119 vaccines.