Protease-associated cellular networks in malaria parasite Plasmodium falciparum

Abstract Background Malaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome) in the malaria parasite Plasmodium falciparum and its sibling species 123, providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum. Results We investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database 4, and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H) system 5, blood stage microarray experiments 678, proteomics 9101112, literature text mining, and sequence homology analysis. Seventy-seven (77) out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs). These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins), range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide processing, cell cycle progression, transcriptional regulation, and signal transduction networks. Conclusions Our network analysis of proteases from P. falciparum uses a so-called guilt-by-association approach to extract sets of proteins from the proteome that are candidates for further study. Novel protease targets and previously unrecognized members of the protease-associated sub-systems provide new insights into the mechanisms underlying parasitism, pathogenesis and virulence.</p

In Silico Identification of Specialized Secretory-Organelle Proteins in Apicomplexan Parasites and In Vivo Validation in Toxoplasma gondii

Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions

Proteases in Malaria Parasites - A Phylogenomic Perspective

Malaria continues to be one of the most devastating global health problems due to the high morbidity and mortality it causes in endemic regions. The search for new antimalarial targets is of high priority because of the increasing prevalence of drug resistance in malaria parasites. Malarial proteases constitute a class of promising therapeutic targets as they play important roles in the parasite life cycle and it is possible to design and screen for specific protease inhibitors. In this mini-review, we provide a phylogenomic overview of malarial proteases. An evolutionary perspective on the origin and divergence of these proteases will provide insights into the adaptive mechanisms of parasite growth, development, infection, and pathogenesis.

Genome Comparison of Human and Non-Human Malaria Parasites Reveals Species Subset-Specific Genes Potentially Linked to Human Disease

Genes underlying important phenotypic differences between Plasmodium species, the causative agents of malaria, are frequently found in only a subset of species and cluster at dynamically evolving subtelomeric regions of chromosomes. We hypothesized that chromosome-internal regions of Plasmodium genomes harbour additional species subset-specific genes that underlie differences in human pathogenicity, human-to-human transmissibility, and human virulence. We combined sequence similarity searches with synteny block analyses to identify species subset-specific genes in chromosome-internal regions of six published Plasmodium genomes, including Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. To improve comparative analysis, we first revised incorrectly annotated gene models using homology-based gene finders and examined putative subset-specific genes within syntenic contexts. Confirmed subset-specific genes were then analyzed for their role in biological pathways and examined for molecular functions using publicly available databases. We identified 16 genes that are well conserved in the three primate parasites but not found in rodent parasites, including three key enzymes of the thiamine (vitamin B1) biosynthesis pathway. Thirteen genes were found to be present in both human parasites but absent in the monkey parasite P. knowlesi, including genes specifically upregulated in sporozoites or gametocytes that could be linked to parasite transmission success between humans. Furthermore, we propose 15 chromosome-internal P. falciparum-specific genes as new candidate genes underlying increased human virulence and detected a currently uncharacterized cluster of P. vivax-specific genes on chromosome 6 likely involved in erythrocyte invasion. In conclusion, Plasmodium species harbour many chromosome-internal differences in the form of protein-coding genes, some of which are potentially linked to human disease and thus promising leads for future laboratory research

Exploration of factors that influence Plasmodium falciparum fitness and virulence

Malaria is an ancient disease that still has profound impact on human population. The virulence of the most lethal malaria parasite, Plasmodium falciparum, can be attributed to several features of the parasite. P. falciparum is known for its indiscriminate red blood cell (RBC) invasion and aptitude for cytoadherence. The latter is associated with various disease pathologies. Thisthesis explores factors that influence the virulence and fitness of P. falciparum, both from the host and parasite perspective. The association between ABO blood groups and protection from severe malaria has sparked many studies, and blood group O has emerged as protective against severe disease. This protection has been attributed to the binding of uninfected RBCs (uRBC) by the parasitized RBC (pRBC), a mechanism known as rosetting. Using a robust high-throughput flow cytometric method, we characterized rosetting for six parasite strains/isolates in all four major ABO blood groups. Rosettes formed in non-O blood shielded the major parasite surface antigen (PfEMP1) from antibody recognition. As blood group A is further subdivided based on qualitative and quantitative properties of the A-antigen, we found that levels of A-antigen on RBCs were positively correlated with rosette sturdiness against disruption by heparin and antibodies. RIFINs, another large family of surface antigens, has been implicated in blood group A rosetting. Members of this family can be divided into A- and B-RIFINS, depending on cellular localization and parasite stage expression. To set the scene for future studies of RIFINS, we generated and validated antibodies for various antibody-based methods. We identified two nonrosetting RIFIN-expressing parasite lines that had not been characterized before. Their dominant rif transcripts were identified by RNA sequencing. As PfEMP1s along with RIFINs and other surface adhesins must be trafficked and inserted into the pRBC membrane to fulfil their cytoadhesive function, we hypothesized that his process might be affected by varied conditions in the host. Here, we describe the loss of pRBC’s adhesive capacities in acidified environment for rosetting and placental binding parasite stains. The reduction was associated with the loss of surface exposed PfEMP1 due to disturbances in the last steps of PfEMP1 trafficking and membrane insertion. Heparin-derivatives, including sevuparin, have sparked interest as possible adjunctive therapeutics in severe malaria treatment. Here, we investigated the mechanisms behind the invasion inhibition by clinically well-tolerated sevuparin and explored the additional antiparasitic properties of this compound. Sevuparin severely affected parasite intracellular development with delayed schizogony and reduced parasitemia after drug removal. The metabolic disturbances manifested in abnormal morphology, abundant extracellular parasites, and reduction of PfEMP1 on the pRBC surface. Inhibition by sevuparin was distinct from classical plasmodial surface anion channel (PSAC) inhibitors, suggesting the involvement of other channels or transporters. Using protein pull-downs from membranes of pRBCs and uRBCs, we identified putative sevuparin interactomes. Due to the identification of multiple human proteins linked to cation homeostasis and haemolysis, we measured cellular sodium levels. Upon treatment with sevuparin, cellular sodium levels were increased in pRBCs, whereas no differences were noted in uRBCs. In conclusion, we found that A-antigen levels on RBCs affect rosette characteristics, which should be considered in future studies investigating associations between blood group A and risk to develop severe malaria. We have validated tools for the study of RIFIN family of proteins and their possible function in disease pathogenesis. In addition, we demonstrated that PfEMP1 trafficking to the surface is pH sensitive. Finally, we showed that sevuparin has multimodal activity against malaria parasites

Characterisation of Plasmodium falciparum antigens for the development of serological assay or vaccine candidates for malaria

A malária causada por Plasmodium falciparum continua a ser um grande problema de saúde nas regiões tropicais e subtropicais em todo o mundo. Várias proteínas do parasita expressas durante o ciclo replicativo no hospedeiro vertebrado, foram descritas nos últimos anos e estão sendo consideradas como potenciais candidatos à vacina anti-malária; no entanto ainda existem algumas proteínas que não foram identificadas como potenciais alvos de diagnostico, terapêuticos e/ou candidatos a vacina. Este estudo retrospetivo descreve a identificação e caracterização de antigénios imunogénicos de P. falciparum como possíveis candidatos para o desenvolvimento de novos testes serológicos ou candidatos a vacina para malaria. Os métodos: Um extrato total de P. falciparum (PfET) do clone 3D7, foi selecionado como o nosso alvo de estudo. A antigenicidade do PfET foi avaliada usando soro humano de indivíduos anteriormente expostos à malária. Foram analisadas 436 amostras de soro, das quais 419 soros de indivíduos, com história clínica de malária, oriundos de alguns países de Africa, América e Asia, previamente diagnosticados na Unidade de Clínica Tropical do IHMT no 2012; e 17 soros de indivíduos sem história clínica de malária foram usados como controle negativo. Foram determinados anticorpos Totais, IgM, IgG, IgG1, IgG2, IgG3, IgG4 anti-P. falciparum por ELISA. Foi feita a caracterização imunoquímica de antigénios de P. falciparum responsáveis por tais reatividades serológicas por immunoblotting. Foi efetuada uma análise in silico de frações proteicas de P. falciparum previamente identificadas por espectrometria de massa. Resultados: Anticorpos anti-P. falciparum reconheceram os antigénios do PfET, com maior contribuição de anticorpos tipo IgG (72%), IgG1 (43%) e IgG3 (64%). O immunoblotting revelou frações proteicas de P. falciparum com massa molecular entre 20 -190 kDa, das quais bandas com massa molecular de 25, 35, 40, 45, 75, 80, 100 e 150 kDa apresentaram maior reatividade antigénica. A análise in silico identificou 29 epitopes correspondentes a seis proteínas de P. falciparum previamente identificadas: Elongation factor 1 alpha; Protein disulphide isomerase (PDI), Phosphoglycerate kinase; Glucose regulated protein homologue; Rhoptry associated protein 2 (RAP-2); Rhoptry-associated protein 3 (RAP-3); a análise in silico sugere que RAP-2, RAP-3, PDI, apresentam características imunogénicas essenciais como epitopes consensos, signal peptidica, domínio transmembrana. Conclusão: Este estudo mostra a caracterização de antigénios do PfET, tendo em conta que algumas das suas proteínas apresentaram várias características imunogénicas importantes, como aqui se mostra, estas proteínas podem ser consideradas como um bom candidato à vacina. São assim recomendados estudos complementares destinados a avaliar a sua capacidade de imunogenicidade e indução de proteção contra a malaria. Futuramente espera-se potencializar a ação destas proteínas e contribuir na caracterização de potenciais novos alvos antigénicos de P. falciparum importantes para o diagnóstico sorológico ou candidatos a vacina para malaria.Plasmodium falciparum malaria remains a major health problem in tropical and subtropical regions worldwide. Several parasite proteins expressed during the replicative cycle in the vertebrate host have been described in recent years, are being considered as potential candidates for anti-malaria vaccine; there are still some proteins that have not been identified as potential diagnostic, therapeutic and/or vaccine candidates. This retrospective study describes the identification and characterization of immunogenic P. falciparum antigens as possible candidates for the development of new serological tests or candidates for the malaria vaccine. Methods: A of P. falciparum total extract 3D7 (PfTE) was selected as our study target. The antigenicity of PfTE was evaluated using human sera from individuals previously exposed to malaria. We analyzed 436 serum samples, of which 419 sera from individuals with a clinical history of malaria, from some countries of Africa, America and Asia, previously diagnosed at the Tropical Clinic Unit of the IHMT in 2012; and 17 sera from healthy Portuguese individuals, who had never been in malaria-endemic area, were used as negative control. Total antibodies, IgM, IgG, IgG1, IgG2, IgG3, IgG4 anti-P. falciparum were determined by ELISA. Immunochemical characterization of P. falciparum antigens responsible for such serological reactivities was performed by immunoblotting. An in silico analysis of protein fractions of P. falciparum previously identified by mass spectrometry was performed. Results: Anti-P. falciparum antibodies recognised antigens of PfET, with a higher contribution of IgG (72%), IgG1 (43%) and IgG3 (64%) antibodies. Immunoblotting revealed protein fractions of P. falciparum with molecular mass between 20 -190 kDa, of which bands with molecular mass of 25, 35, 40, 45, 75, 80, 100 and 150 kDa showed higher antigenic reactivity. In silico analysis identified 29 epitopes corresponding to six P. falciparum proteins previously identified: Elongation factor 1 alpha; Protein disulphide isomerase (PDI), Phosphoglycerate kinase; Glucose regulated protein homologue; Rhoptry associated protein 2 (RAP-2); Rhoptry-associated protein 3 (RAP-3); in silico analysis suggests that RAP-2, RAP-3, PDI, present essential immunogenic characteristics as consensus epitopes, signal peptide, transmembrane domain. Conclusion: This study shows the characterization of PfTE antigens, taking into account that some of its proteins presented several important immunogenic characteristics, as shown here, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability against malaria are thus recommended. It is expected to potentiate the action of these proteins and contribute to the characterization of potential new antigenic targets of P. falciparum important for the serological diagnosis or vaccine candidates for malari

Juxtamembrane Shedding of Plasmodium falciparum AMA1 Is Sequence Independent and Essential, and Helps Evade Invasion-Inhibitory Antibodies

The malarial life cycle involves repeated rounds of intraerythrocytic replication interspersed by host cell rupture which releases merozoites that rapidly invade fresh erythrocytes. Apical membrane antigen-1 (AMA1) is a merozoite protein that plays a critical role in invasion. Antibodies against AMA1 prevent invasion and can protect against malaria in vivo, so AMA1 is of interest as a malaria vaccine candidate. AMA1 is efficiently shed from the invading parasite surface, predominantly through juxtamembrane cleavage by a membrane-bound protease called SUB2, but also by limited intramembrane cleavage. We have investigated the structural requirements for shedding of Plasmodium falciparum AMA1 (PfAMA1), and the consequences of its inhibition. Mutagenesis of the intramembrane cleavage site by targeted homologous recombination abolished intramembrane cleavage with no effect on parasite viability in vitro. Examination of PfSUB2-mediated shedding of episomally-expressed PfAMA1 revealed that the position of cleavage is determined primarily by its distance from the parasite membrane. Certain mutations at the PfSUB2 cleavage site block shedding, and parasites expressing these non-cleavable forms of PfAMA1 on a background of expression of the wild type gene invade and replicate normally in vitro. The non-cleavable PfAMA1 is also functional in invasion. However – in contrast to the intramembrane cleavage site - mutations that block PfSUB2-mediated shedding could not be stably introduced into the genomic pfama1 locus, indicating that some shedding of PfAMA1 by PfSUB2 is essential. Remarkably, parasites expressing shedding-resistant forms of PfAMA1 exhibit enhanced sensitivity to antibody-mediated inhibition of invasion. Drugs that inhibit PfSUB2 activity should block parasite replication and may also enhance the efficacy of vaccines based on AMA1 and other merozoite surface proteins