A genetically hmgb2 attenuated blood stage P. berghei induces crossed-long live protection.

Due to the lack of efficiency to control malaria elicited by sub-unit vaccine preparations, vaccination with live-attenuated Plasmodium parasite as reported 70 years ago with irradiated sporozoites regained recently a significant interest. The complex life cycle of the parasite and the different stages of development between mammal host and anopheles do not help to propose an easy vaccine strategy. In order to achieve a complete long-lasting protection against Plasmodium infection and disease, we considered a genetically attenuated blood stage parasite in the hmgb2 gene coding for the high-mobility-group-box 2 (HMGB2). This Plasmodium protein belongs to the HMGB family and hold as the mammal proteins, a double life since it acts first as a nuclear factor involved in chromatin remodelling and transcription regulation and second, when secreted as an active pro-inflammatory alarmin protein. Even though the number of reports on whole living attenuated blood stage parasites is limited when compared to attenuated sporozoites, the results reported with Plasmodium KO parasites are very encouraging. In this report, we present a novel strategy based on pre-immunization with Δhmgb2PbNK65 parasitized red blood cells that confer long-lasting protection in a murine experimental cerebral malaria model against two highly pathogenic homologous and heterologous parasites.

Preparing for Transmission: Gene Regulation in Plasmodium Sporozoites.

Plasmodium sporozoites are transmitted to mammals by anopheline mosquitoes and first infect the liver, where they transform into replicative exoerythrocytic forms, which subsequently release thousands of merozoites that invade erythrocytes and initiate the malaria disease. In some species, sporozoites can transform into dormant hypnozoites in the liver, which cause malaria relapses upon reactivation. Transmission from the insect vector to a mammalian host is a critical step of the parasite life cycle, and requires tightly regulated gene expression. Sporozoites are formed inside oocysts in the mosquito midgut and become fully infectious after colonization of the insect salivary glands, where they remain quiescent until transmission. Parasite maturation into infectious sporozoites is associated with reprogramming of the sporozoite transcriptome and proteome, which depends on multiple layers of transcriptional and post-transcriptional regulatory mechanisms. An emerging scheme is that gene expression in Plasmodium sporozoites is controlled by alternating waves of transcription activity and translational repression, which shape the parasite RNA and protein repertoires for successful transition from the mosquito vector to the mammalian host.

Identification of Plasmodium falciparum nuclear proteins by mass spectrometry and proposed protein annotation.

The nuclear proteome of Plasmodium falciparum results from the continual shuttle of proteins between the cell cytoplasm-nucleus and vice versa. Using shotgun proteomics tools, we explored the nuclear proteins of mixed populations of Plasmodium falciparum extracted from infected erythrocytes. We combined GeLC-MS/MS and 2D-LC-MS/MS with a peptide ion exclusion procedure in order to increase the detection of low abundant proteins such as those involved in gene expression. We have identified 446 nuclear proteins covering all expected nuclear protein families involved in gene regulation. All structural ribosomal (40S and 60S) proteins were identified which is consistent with the nuclear localization of ribosomal biogenesis. Proteins involved in the translation machinery were also found suggesting that translational events might occur in the nucleus in P. falciparum as previously hypothesized in eukaryotes. These data were compared to the protein list established by PlasmoDB and submitted to Plasmobase a recently reported Plasmodium annotation website to propose new functional putative annotation of several unknown proteins found in the nuclear extracts.

Plasmobase: a comparative database of predicted domain architectures for Plasmodium genomes.

BACKGROUND: With the availability of complete genome sequences of both human and non-human Plasmodium parasites, it is now possible to use comparative genomics to look for orthology across Plasmodium species and for species specific genes. This comparative analyses could provide important clues for the development of new strategies to prevent and treat malaria in humans, however, the number of functionally annotated proteins is still low for all Plasmodium species. In the context of genomes that are hard to annotate because of sequence divergence, such as Plasmodium, domain co-occurrence becomes particularly important to trust predictions. In particular, domain architecture prediction can be used to improve the performance of existing annotation methods since homologous proteins might share their architectural context. RESULTS: Plasmobase is a unique database designed for the comparative study of Plasmodium genomes. Domain architecture reconstruction in Plasmobase relies on DAMA, the state-of-the-art method in architecture prediction, while domain annotation is realised with CLADE, a novel annotation tool based on a multi-source strategy. Plasmobase significantly increases the Pfam domain coverage of all Plasmodium genomes, it proposes new domain architectures as well as new domain families that have never been reported before for these genomes. It proposes a visualization of domain architectures and allows for an easy comparison among architectures within Plasmodium species and with other species, described in UniProt. CONCLUSIONS: Plasmobase is a valuable new resource for domain annotation in Plasmodium genomes. Its graphical presentation of protein sequences, based on domain architectures, will hopefully be of interest for comparative genomic studies. It should help to discover species-specific genes, possibly underlying important phenotypic differences between parasites, and orthologous gene families for deciphering the biology of these complex and important Apicomplexan organisms. In conclusion, Plasmobase is a flexible and rich site where any biologist can find something of his/her own interest. AVAILABILITY: Plasmobase is accessible at http://genome.lcqb.upmc.fr/plasmobase/ .

Plasmodium falciparum proteins involved in cytoadherence of infected erythrocytes to chemokine CX3CL1.

Malaria caused by Plasmodium falciparum is associated with cytoadherence of infected red blood cells (iRBC) to endothelial cells. Numerous host molecules have been involved in cytoadherence, including the adhesive chemokine CX3CL1. Most of the identified parasite ligands are from the multigenic and hypervariable Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family which makes them poor targets for the development of a broadly protective vaccine. Using proteomics, we have identified two 25-kDa parasite proteins with adhesive properties for CX3CL1, called CBP for CX3CL1 Binding Proteins. CBPs are coded by single-copy genes with little polymorphic variation and no homology with other P. falciparum gene products. Specific antibodies raised against epitopes from the predicted extracellular domains of each CBP efficiently stain the surface of RBC infected with trophozoites or schizonts, which is a strong indication of CBP expression at the surface of iRBC. These anti-CBP antibodies partially neutralize iRBC adherence to CX3CL1. This adherence is similarly inhibited in the presence of peptides from the CBP extracellular domains, while irrelevant peptides had no such effect. CBP1 and CBP2 are new P. falciparum ligands for the human chemokine CX3CL1. The identification of this non-polymorphic P. falciparum factors provides a new avenue for innovative vaccination approaches.

Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence.

Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of ancient domain duplications, the reconstruction of the history of protein architectures, and the estimation of protein domain age. Website and software: http://www.lcqb.upmc.fr/CLADE.

Disruption of Parasite hmgb2 Gene Attenuates Plasmodium berghei ANKA Pathogenicity.

Eukaryotic high-mobility-group-box (HMGB) proteins are nuclear factors involved in chromatin remodeling and transcription regulation. When released into the extracellular milieu, HMGB1 acts as a proinflammatory cytokine that plays a central role in the pathogenesis of several immune-mediated inflammatory diseases. We found that the Plasmodium genome encodes two genuine HMGB factors, Plasmodium HMGB1 and HMGB2, that encompass, like their human counterparts, a proinflammatory domain. Given that these proteins are released from parasitized red blood cells, we then hypothesized that Plasmodium HMGB might contribute to the pathogenesis of experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that develops in C57BL/6 (susceptible) mice infected with Plasmodium berghei ANKA and that in many aspects resembles human cerebral malaria elicited by P. falciparum infection. The pathogenesis of experimental cerebral malaria was suppressed in C57BL/6 mice infected with P. berghei ANKA lacking the hmgb2 gene (Δhmgb2 ANKA), an effect associated with a reduction of histological brain lesions and with lower expression levels of several proinflammatory genes. The incidence of ECM in pbhmgb2-deficient mice was restored by the administration of recombinant PbHMGB2. Protection from experimental cerebral malaria in Δhmgb2 ANKA-infected mice was associated with reduced sequestration in the brain of CD4(+) and CD8(+) T cells, including CD8(+) granzyme B(+) and CD8(+) IFN-γ(+) cells, and, to some extent, neutrophils. This was consistent with a reduced parasite sequestration in the brain, lungs, and spleen, though to a lesser extent than in wild-type P. berghei ANKA-infected mice. In summary, Plasmodium HMGB2 acts as an alarmin that contributes to the pathogenesis of cerebral malaria.

In silico and biological survey of transcription-associated proteins implicated in the transcriptional machinery during the erythrocytic development of Plasmodium falciparum.

BACKGROUND: Malaria is the most important parasitic disease in the world with approximately two million people dying every year, mostly due to Plasmodium falciparum infection. During its complex life cycle in the Anopheles vector and human host, the parasite requires the coordinated and modulated expression of diverse sets of genes involved in epigenetic, transcriptional and post-transcriptional regulation. However, despite the availability of the complete sequence of the Plasmodium falciparum genome, we are still quite ignorant about Plasmodium mechanisms of transcriptional gene regulation. This is due to the poor prediction of nuclear proteins, cognate DNA motifs and structures involved in transcription. RESULTS: A comprehensive directory of proteins reported to be potentially involved in Plasmodium transcriptional machinery was built from all in silico reports and databanks. The transcription-associated proteins were clustered in three main sets of factors: general transcription factors, chromatin-related proteins (structuring, remodelling and histone modifying enzymes), and specific transcription factors. Only a few of these factors have been molecularly analysed. Furthermore, from transcriptome and proteome data we modelled expression patterns of transcripts and corresponding proteins during the intra-erythrocytic cycle. Finally, an interactome of these proteins based either on in silico or on 2-yeast-hybrid experimental approaches is discussed. CONCLUSION: This is the first attempt to build a comprehensive directory of potential transcription-associated proteins in Plasmodium. In addition, all complete transcriptome, proteome and interactome raw data were re-analysed, compared and discussed for a better comprehension of the complex biological processes of Plasmodium falciparum transcriptional regulation during the erythrocytic development.

Temperature shift and host cell contact up-regulate sporozoite expression of Plasmodium falciparum genes involved in hepatocyte infection.

Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria.

Whole-transcriptome analysis of Plasmodium falciparum field isolates: identification of new pathogenicity factors.

BACKGROUND: Severe malaria and one of its most important pathogenic processes, cerebral malaria, involves the sequestration of parasitized red blood cells (pRBCs) in brain postcapillary venules. Although the pathogenic mechanisms underlying malaria remain poorly characterized, it has been established that adhesion of pRBCs to endothelial cells (ECs) can result in cell apoptosis, which in turn may lead to disruption of the blood-brain barrier. The nature of the parasite molecules involved in the pathogenesis of severe malaria remains elusive. METHODS: Whole-transcriptome profiling of nonapoptogenic versus apoptogenic parasite field isolates obtained from Gabonese children was performed with pan-genomic Plasmodium falciparum DNA microarrays; radiolabeled instead of fluorescent cDNAs were used to improve the sensitivity of signal detection. RESULTS: Our methods allowed the identification of 59 genes putatively associated with the induction of EC apoptosis. Silencing of Plasmodium gene expression with specific double-stranded RNA was performed on 8 selected genes; 5 of these, named "Plasmodium apoptosis-linked pathogenicity factors" (PALPFs), were found to be linked to parasite apoptogenicity. Of these genes, 2 might act via parasite cytoadherence. CONCLUSION: This is the first attempt to identify genes involved in parasite pathogenic mechanisms against human ECs. The finding of PALPFs illuminates perspectives for novel therapeutic strategies against cerebral complications of malaria.

Detection, characterization and regulation of antisense transcripts in HIV-1.

BACKGROUND: We and others have recently demonstrated that the human retrovirus HTLV-I was producing a spliced antisense transcript, which led to the synthesis of the HBZ protein. The objective of the present study was to demonstrate the existence of antisense transcription in HIV-1 and to provide a better characterization of the transcript and its regulation. RESULTS: Initial experiments conducted by standard RT-PCR analysis in latently infected J1.1 cell line and pNL4.3-transfected 293T cells confirmed the existence of antisense transcription in HIV-1. A more adapted RT-PCR protocol with limited RT-PCR artefacts also led to a successful detection of antisense transcripts in several infected cell lines. RACE analyses demonstrated the existence of several transcription initiation sites mapping near the 5' border of the 3'LTR (in the antisense strand). Interestingly, a new polyA signal was identified on the antisense strand and harboured the polyA signal consensus sequence. Transfection experiments in 293T and Jurkat cells with an antisense luciferase-expressing NL4.3 proviral DNA showed luciferase reporter gene expression, which was further induced by various T-cell activators. In addition, the viral Tat protein was found to be a positive modulator of antisense transcription by transient and stable transfections of this proviral DNA construct. RT-PCR analyses in 293T cells stably transfected with a pNL4.3-derived construct further confirmed these results. Infection of 293T, Jurkat, SupT1, U937 and CEMT4 cells with pseudotyped virions produced from the antisense luciferase-expressing NL4.3 DNA clone led to the production of an AZT-sensitive luciferase signal, which was however less pronounced than the signal from NL4.3Luc-infected cells. CONCLUSION: These results demonstrate for the first time that antisense transcription exists in HIV-1 in the context of infection. Possible translation of the predicted antisense ORF in this transcript should thus be re-examined.

High-mobility-group box nuclear factors of Plasmodium falciparum.

In eukaryotes, the high-mobility-group (HMG) nuclear factors are highly conserved throughout evolution and are divided into three families, including HGMB, characterized by an HMG box domain. Some HMGB factors are DNA structure specific and preferentially interact with distorted DNA sequences, trigger DNA bending, and hence facilitate the binding of nucleoprotein complexes that in turn activate or repress transcription. In Plasmodium falciparum, two HMGB factors were predicted: PfHMGB1 and PfHMGB2. They are small proteins, under 100 amino acids long, encompassing a characteristic HMG box domain closely related to box B of metazoan factors, which comprises two HMG box domains, A and B, in tandem. Computational analyses supported the conclusion that the Plasmodium proteins were genuine architectural HMGB factors, and in vitro analyses performed with both recombinant proteins established that they were able to interact with distorted DNA structures and bend linear DNA with different affinities. These proteins were detected in both asexual- and gametocyte-stage cells in Western blotting experiments and mainly in the parasite nuclei. PfHMGB1 is preferentially expressed in asexual erythrocytic stages and PfHMGB2 in gametocytes, in good correlation with transcript levels of expression. Finally, immunofluorescence studies revealed differential subcellular localizations: both factors were observed in the nucleus of asexual- and sexual-stage cells, and PfHMGB2 was also detected in the cytoplasm of gametocytes. In conclusion, in light of differences in their levels of expression, subcellular localizations, and capacities for binding and bending DNA, these factors are likely to play nonredundant roles in transcriptional regulation of Plasmodium development in erythrocytes.

PfMyb1, a Plasmodium falciparum transcription factor, is required for intra-erythrocytic growth and controls key genes for cell cycle regulation.

During the complex life cycle of Plasmodium falciparum, divided between mosquito and human hosts, the regulation of morphologic changes implies a fine control of transcriptional regulation. Transcriptional control, however, and in particular its molecular actors, transcription factors and regulatory motifs, are as yet poorly described in Plasmodium. In order to decipher the molecular mechanisms implicated in transcriptional regulation, a transcription factor belonging to the tryptophan cluster family was studied. In a previous work, the PfMyb1 protein, contained in nuclear extracts, was shown to have DNA binding activity and to interact specifically with myb regulatory elements. We used long pfmyb1 double-stranded RNA (dsRNA) to interfere with the cognate messenger expression. Parasite cultures treated with pfmyb1 dsRNA exhibited a 40% growth inhibition when compared with either untreated cultures or cultures treated with unrelated dsRNA, and parasite mortality occurred during trophozoite to schizont transition. In addition, the pfmyb1 transcript and protein decreased by as much as 80% in treated trophozoite cultures at the time of their maximum expression. The global effect of this partial loss of transcript and protein was investigated using a thematic DNA microarray encompassing genes involved in signal transduction, cell cycle and transcriptional regulation. SAM software enabled us to identify several genes that were differentially expressed and probably directly or indirectly under the control of PfMyb1. Using chromatin immuno-precipitation, we demonstrated that PfMyb1 binds, within the parasite nuclei, to several promoters and therefore participates directly in the transcriptional regulation of the corresponding genes. This study provides the first evidence of a regulation network involving a Plasmodium transcription factor.

Transcriptome of 3D7 and its gametocyte-less derivative F12 Plasmodium falciparum clones during erythrocytic development using a gene-specific microarray assigned to gene regulation, cell cycle and transcription factors.

During the complex life cycle of Plasmodium falciparum, through mosquito and human, the erythrocytic cycle is responsible for malarial disease and transmission. The regulation of events that occur during parasite development, such as proliferation and differentiation, implies a fine control of transcriptional activities that in turn governs the expression profiles of sets of genes. Pathways that underline gametocyte commitment are yet poorly understood even though kinases and transcription factors have been assumed to play a crucial role in this event. In order to understand the molecular mechanisms controlling the variation of gene expression profiles that might participate in early gametocytogenesis, the transcriptome of two clones, 3D7 and its gametocyte-less derivative F12, was compared at five time points of the erythrocytic asexual development. We have used a thematic DNA microarray containing 150 PCR fragments, representative of P. falciparum genes involved in signal transduction, cell cycle and transcriptional regulation. We identified several genes eliciting different expression profiles among which some implicated in gene regulation or encoding putative transcription factors. The differential expression of transcription factor and kinase transcripts observed in the two clones may enlighten genes that might have a role in impairment of the early gametocytogenesis of the F12 clone.

The TAR RNA-binding protein, TRBP, stimulates the expression of TAR-containing RNAs in vitro and in vivo independently of its ability to inhibit the dsRNA-dependent kinase PKR.

TRBP (HIV-1 transactivating response (TAR) RNA-binding protein) and PKR, the interferon-induced dsRNA-regulated protein kinase, contain two dsRNA binding domains. They both bind to HIV-1 TAR RNAs through different sites. Binding to dsRNA activates PKR that phosphorylates the eukaryotic initiation factor eIF-2alpha leading to protein synthesis inhibition. TRBP and PKR can heterodimerize, which inhibits the kinase function of PKR and has a positive effect on HIV-1 expression. In this study, an in vitro reticulocyte assay revealed the poor expression of TAR containing CAT RNAs compared with CAT RNAs. Addition of TRBP restored translation efficiency of TAR-CAT RNA and decreased the phosphorylation status of eIF-2alpha, confirming its role as a PKR inhibitor. Unexpectedly, eIF-2alpha was phosphorylated in the presence of TAR-CAT as well as CAT RNA devoid of the TAR structure. TRBP inhibited eIF-2alpha phosphorylation in both cases, suggesting that it restores the translation of TAR-CAT RNA independently and in addition to its ability to inhibit PKR. TRBP activity on gene expression was then analyzed in a PKR-free environment using PKR-deficient murine embryo fibroblasts. In a transient reporter gene assay, TRBP stimulated the expression of a TAR-containing luciferase 3.8-fold whereas the reporter gene with mutated TAR structures or devoid of TAR was stimulated 1.5- to 2.4-fold. Overall, the activity of TRBP2 was higher when the 5'-end of the mRNA was structured and was mediated independently by each dsRBD in TRBP. Increasing concentrations of TRBP showed no significant modification of the luciferase RNA levels, suggesting that TRBP stimulates translation of TAR-containing RNAs. Therefore, TRBP is an important cellular factor for efficient translation of dsRNA containing transcripts, both by inhibiting PKR and in a PKR-independent pathway.

Immunolocalization studies of an antisense protein in HIV-1-infected cells and viral particles.

The minus DNA strand of HIV-1 presents an open reading frame that is complementary to the HIV-1 envelope messenger, is highly conserved among HIV-1 isolates, and may encode a hydrophobic protein. In previous studies, the antisense transcript has been identified both in various HIV-infected cell lines and in leukocytes of HIV-1(+) patients. The expression of the corresponding antisense protein (ASP) during natural HIV-1 infection has been indirectly evidenced by the identification of anti-ASP antibodies in the sera of HIV(+) patients. We have used immunoelectron microscopy procedures (ultra-small gold particles coupled to silver enhancement) to establish direct evidence of ASP production. ASP has then been detected in chronically and acutely HIV-1-infected cell lines. The protein, found mostly associated with various cellular membranes as well as with the virions released from the cells, indicated that ASP is a bona fide component for the virions and may participate in the particle formation.