Plasmodium vivax has accounted for an enormous share of the global malaria burden in recent years, along with Plasmodium falciparum. The wide distribution of P. vivax and recent evidences of severe ...and complicated vivax malaria across several endemic regions of the world suggest that this disease may have been more overlooked than benign. While P. falciparum has been extensively studied, P. vivax has received limited research attention owing to its complex nature and absence of a continuous culture system.
This review briefly describes the epidemiology of vivax malaria, analyzes challenges towards effective control and summarizes major insights provided by genomics and transcriptomics research in the area. Subsequently, the review provides a detailed description of the applications of proteomics in vivax malaria research, focusing on both host responses and parasite proteomics studies to understand P. vivax biology. Expert commentary: In recent years, proteomics technologies are being used effectively to understand P. vivax biology and the underlying pathogenesis. Technological advances in mass spectrometry configurations, multiomics investigations and emerging strategies such as targeted proteomics may also immensely aid in studying disease severity, improving existing diagnosis and identifying new drug and vaccine targets.
Glutathione peroxidase‐like thioredoxin peroxidase (PfTPxGl) is an antioxidant enzyme trafficked to the apicoplast, a secondary endosymbiotic organelle, in Plasmodium falciparum. Apicoplast ...trafficking signals usually consist of N‐terminal signal and transit peptides, but the trafficking signal of PfTPxGl appears to exhibit important differences. As transfection is a protracted process in P. falciparum, we expressed the N terminus of PfTPxGl as a GFP fusion protein in a related apicomplexan, Toxoplasma gondii, in order to dissect its trafficking signals. We show that PfTPxGl possesses an N‐terminal signal anchor that takes the protein to the endoplasmic reticulum in Toxoplasma—this is the first step in the apicoplast targeting pathway. We dissected the residues important for endomembrane system uptake, membrane anchorage, orientation, spacing, and cleavage. Protease protection assays and fluorescence complementation revealed that the C terminus of the protein lies in the ER lumen, a topology that is proposed to be retained in the apicoplast. Additionally, we examined one mutant, responsible for altered PfTPxGl targeting in Toxoplasma, in Plasmodium. This study has demonstrated that PfTPxGl belongs to an emergent class of proteins that possess signal anchors, unlike the canonical bipartite targeting signals employed for the trafficking of luminal apicoplast proteins. This work adds to the mounting evidence that the signals involved in the targeting of apicoplast membrane proteins may not be as straightforward as those of luminal proteins, and also highlights the usefulness of T. gondii as a heterologous system in certain aspects of this study, such as reducing screening time and facilitating the verification of membrane topology.
Glutathione peroxidase‐like thioredoxin peroxidase (PfTPxGl) is an antioxidant enzyme trafficked to the apicoplast in Plasmodium falciparum. This study demonstrates that PfTPxGl belongs to an emergent class of proteins that possess signal anchors, unlike the canonical bipartite targeting signals employed for the trafficking of luminal apicoplast proteins, while highlighting the usefulness of Toxoplasma in the study of membrane‐bound Plasmodium apicoplast proteins.
Vivax malaria is the most widely distributed human malaria resulting in 80–300million clinical cases every year. It causes severe infection and mortality but is generally regarded as a benign disease ...and has not been investigated in detail. The present study aimed to perform human serum proteome analysis in a malaria endemic area in India to identify potential serum biomarkers for vivax malaria and understand host response. The proteomic analysis was performed on 16 age and gender matched subjects (vivax patients and control) in duplicate. Protein extraction protocols were optimized for large coverage of the serum proteome and to obtain high-resolution data. Identification of 67 differentially expressed and statistically significant (Student's t-test; p<0.05) protein spots was established by MALDI-TOF/TOF mass spectrometry. Many of the identified proteins such as apolipoprotein A and E, serum amyloid A and P, haptoglobin, ceruloplasmin, and hemopexin are interesting from a diagnostic point of view and could further be studied as potential serum biomarkers. The differentially expressed serum proteins in vivax malaria identified in this study were subjected to functional pathway analysis using multiple software, including Ingenuity Pathway Analysis (IPA), Protein ANalysis THrough Evolutionary Relationships (PANTHER) and Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation tool for better understanding of the biological context of the identified proteins, their involvement in various physiological pathways and association with disease pathogenesis. Functional pathway analysis of the differentially expressed proteins suggested the modulation of multiple vital physiological pathways, including acute phase response signaling, complement and coagulation cascades, hemostasis and vitamin D metabolism pathway due to this parasitic infection. This article is part of a Special Issue entitled: Proteomics: The clinical link.
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► Alteration in serum proteome in vivax malaria was investigated. ► Classical 2DE and 2D-DIGE were implicated for serum proteome profiling. ► 31 differentially expressed serum proteins were identified using MALDI TOF/TOF MS. ► Identified proteins are involved in vital physiological pathways. ► Few identified proteins could further be studied as potential serum biomarkers.
The global burden of dengue continues to worsen, specifically in tropical and subtropical countries, and has evolved as a major public health problem. We investigated the changes in serum proteome in ...dengue fever (DF) patients from a dengue-endemic area of India to obtain mechanistic insights about the disease pathogenesis, the host immune response, and identification of potential serum protein biomarkers of this infectious disease. In this study, serum samples from DF patients, healthy subjects, and patients with falciparum malaria (an infectious disease control) were investigated by 2D-DIGE in combination with MALDI-TOF/TOF MS. The findings were validated with Western blotting. Functional clustering of the identified proteins was performed using PANTHER and DAVID tools. Compared to the healthy controls, we found significant changes in the expression levels of 48 protein spots corresponding to 18 unique proteins (7 downregulated and 11 upregulated) in DF patients (p<0.05). Among these differentially-expressed proteins, 11 candidates exhibited different trends in dengue fever compared to falciparum malaria. Importantly, our results suggest that dengue virus infection leads to alterations in expression levels of multiple serum proteins involved in diverse and vital physiological pathways, including acute phase response signaling, complement cascades, hemostasis, and blood coagulation. For the first time we report here that the serum levels of hemopexin, haptoglobin, serum amyloid P, and kininogen precursor, are altered in DF. This study informs the pathogenesis and host immune response to dengue virus infection, as well as the current search for new diagnostic and molecular drug targets.
The existing armament of drugs for the treatment and prevention of malaria is inadequate due to development of resistance. In addition to this due to lack of economic enticement the rate of new drug ...development and new drug discovery in the segment of parasitic diseases is very low as compared to the other segments. This has necessitated the better deployment and usage of existing antimalarial drugs as well as discovery of antimalarial activity of drugs which are well characterized for other diseases; these approaches help to reduce the time and cost required for new drug discovery. The present study evaluated the antimalarial activity of antituberculosis drugs rifampicin, isoniazide, and ethambutol in monotherapy and combination in Plasmodium berghei-infected mice. Animals were observed for mortality, parasite progression, and toxicity for a period of 1 month. Rifampicin + isoniazide and rifampicin + isoniazide + ethambutol treatment resulted in an overall survival rate of 60% compared to 0% in vehicle-fed animals by 4 weeks after post-infection without showing any toxicity.
The availability of the complete genome sequence of Plasmodium falciparum has facilitated high-throughput profiling of its complex life cycle, following the application of micro-array, proteomic, and ...serial analysis of gene expression (SAGE) technologies in this system. These, in turn, have yielded unprecedented insight into global gene expression, including the foremost demonstration of antisense transcription in the parasite. For example, owing to its inherent ability to sample novel ORFs and to predict transcript orientation, SAGE analysis in asexual forms led to the initial discovery of highly abundant antisense RNAs. To determine the extent of this phenomenon in P. falciparum, we have surveyed the distribution of both sense and antisense transcripts across the asexual transcriptome for the first time. To this end, a relational database integrating SAGE expression data with genome annotation information was constructed. This allowed the comprehensive annotation of a total of 17245 SAGE tags, extending over a 350-fold expression range. Transcripts from approximately 30% of the estimated 3D7 gene loci were present at detectable levels in mixed asexual stages, where loci involved in invasion and immune evasion; and carbohydrate metabolism were highly represented in the sense transcriptome. Approximately 12% of SAGE tags, however, were derived from the non-coding strand of nuclear-encoded ORFs, indicating that endogenous antisense RNAs are widespread in this system. Notably, these antisense transcripts were absent from the mitochondrial genome. Interestingly, we note that sense and antisense tag counts from single loci across the transcriptome were inversely related. Taken together, this data may provide first hints as to the possible function of antisense transcription in this system.
Regulation of gene expression in the malaria parasite
Plasmodium falciparum
is tightly controlled and little is known about the many steps involved. One step i.e. translation initiation is also ...poorly understood and in
P. falciparum
, choice of the translation initiation site (TIS) is a critical decision largely due to the high frequency of AUGs in the relatively long 5′ untranslated regions of parasite mRNAs. The sequences surrounding the TIS have a major role to play in translation initiation and this report evaluates these sequences by mutational analysis of the heat shock protein 86 gene, transient transfection and reporter assays in the parasite. We find that purines at the −3 and +4 positions are essential for efficient translation in
P. falciparum
, similar to other eukaryotes. Interestingly, a U at the −1 position results in 2.5-fold higher reporter activity compared to wild type. Certain classes of protein biosynthetic genes show higher frequencies of U at the −1 position, suggesting that these genes may exhibit higher levels of translation. This work defines the optimal sequences for TIS choice and has implications for the design of efficient expression vectors in an important human pathogen.
Enhanced green fluorescent protein (eGFP) is a variant of wild-type GFP humanized for optimal expression in mammalian cell lines. A computational approach comparing wtGFP and eGFP showed the ...occurrence of rare proline codons within the eGFP gene that could interfere with and hamper protein production in prokaryotic expression systems. The eGFP gene excised from mammalian plasmid pEGFP N3 was used for construction of two inducible promoter-reporter fusions, T7-eGFP and PₚᵣₒU-eGFP, through directional cloning. The T7-eGFP fusion confirmed expression of eGFP protein within the bacterial strain, showing a fluorescent green cell pellet and overexpression of the ~29 kDa eGFP protein upon induction with IPTG. The proU operon aids in osmoadaptation by encoding a transport system for uptake of various compatible solutes, including glycine-betaine and proline. Expression of the proU operon is induced upon growth of bacteria in media of elevated osmolarity. When coupled to an eGFP reporter, a time course study using fluorometry demonstrated that induction of PₚᵣₒUin Escherichia coli occurred rapidly. The PₚᵣₒUinduction and recombinant eGFP production depends on time and concentration of solute (NaCl) in the medium. Cells containing the PₚᵣₒU-eGFP fusion showed maximum promoter activity at 500 mM concentration of NaCl with a sensitivity of the PₚᵣₒUpromoter being 50 mM. The relative fluorescence reflected the amount of protein synthesized proportional to the activity of induced promoter and effect of NaCl on growth was also taken into consideration. Thus, such environmentally regulated highly sensitive promoters with enhanced reporters could possibly be used as whole-cell biosensors.
With the ability to adopt an assortment of forms throughout its life cycle, and to thrive in host environments so diverse and challenging, the malaria parasite Plasmodium falciparum may well serve as ...the epitome of the regulation of gene expression. The parasite is replete with mechanisms of control, many of them unique and intriguing, permitting it to transit seamlessly from one defined ecological niche to the next. This review is an attempt to capture the essence of our current understanding of transcriptional, post-transcriptional and translational regulation in P. falciparum, and how this works for us in drug development.
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