Special Feature

doi:10.1038/nindia.2015.120 Published online 27 August 2015

Proteomics in malaria research

[Nature India Special Issue: Proteomics Research in India]

Pushkar Sharma1, Inderjeet Kaur2, Pawan Malhotra2 & Sanjeeva Srivastava3



© Havard Davis /Alamy
Genome sequence and proteome analysis of many parasitic organisms have provided new hope for the identification of new vaccine/drug targets and their corresponding inhibitors or drugs. Among these, the most significant progress has been made with the malaria parasite.

Malaria is the most prevalent tropical parasitic disease killing at least a million people annually. Genome sequences of different species of Plasmodium and its insect as well as vertebrate hosts have propelled the growth of integrated omics approaches in different spheres of malaria research, including understanding of the host-pathogen interactions, disease etiology and pathogenic mechanism, characterisation of stage-specific parasite proteome as well as post-translational modifications and elucidation of mechanisms of antimalarial drug action.

Proteomics is an effective tool for the identification of next-generation biomarkers and potential drug/vaccine targets. Nonetheless, this emerging field is also fraught with various challenges, such as in vivo proteomic profiling of human malaria parasites, expression and purification of proteins in large quantities, difficulties in targeting low-abundance target analytes within complex biological samples, and analysis and interpretation of huge multi-omics datasets.

Research leads

Plasmodium genomes encode about 5,300 proteins, more than half of which are hypothetical proteins since they do not show sufficient similarity with proteins from other organisms1. In the last decade, a series of proteomics studies have tried to understand the expression of Plasmodium proteins at different parasite stages2, 3 and also illustrated post-translational modifications that many of these proteins undergo4, 5, 6, 7, 8.

These modifications have been crucial for protein functions such as haemoglobin degradation, host invasion and merozoite egress9. Proteome analyses have further led to the identification of a library of cell surface and secreted proteins that probably are responsible for host cell invasion and immune modulations10, organelle specific proteins and drug sensitive proteins11. A significant number of proteomics studies have also been performed to understand the development, pathogenesis and drug resistance in apicomplexan parasites12–19. Additionally, chemical proteomics approaches have provided new chemistries to develop new anti-malarials20. A number of novel Plasmodium secretory proteins at asexual blood stages have been identified alongside a haemoglobin degradation-hemozoin formation complex21, 22.

Proteome analysis studies have led to the identification of potential new targets such as haemoglobin degradation enzymes22, enzymes/proteins of purine salvage pathway and of protein and polyamine metabolism23, proteins associated with parasite specific trafficking/transport pathways24, 25, GPI anchored proteins26, proteins associated with proteasome machinery and proteins linked with spread of drug resistance27. These global proteomic studies have provided researchers enough arsenal to develop novel anti-parasitic strategies both for new drugs and vaccine development.

Signaling studies

Researchers have also noted the presence of several putative effectors of cell signaling in the parasite genome28.

The post-genome era saw a flurry of activity in the use of reverse genetics to understand the function of enzymes like protein kinases and phosphatases4, 29, 30, 31 – major modulators of protein phosphorylation. While these and other efforts revealed that signaling may regulate most stages of parasite development, the underlying mechanisms remained unclear and the signaling map of the parasite remains ambiguous.

Efforts to understand the role of second messengers like calcium and phosphoinositides (PIPs) in the parasite have resulted in explaining novel signaling and trafficking pathways. For instance, researchers have shown that phospholipase C-mediated calcium release may regulate protein kinases like PfCDPKs and PfPKB, which in turn may regulate key processes like host erythrocyte invasion and sexual differentiation32.

Using mass spectrometry, several regulatory phosphorylation sites on PfCDPK133 and its substrates like PfGAP4534 have been identified. Indian teams have also identified several novel substrates for Plasmodium kinases. Some of these substrates have been validated by performing in vitro kinase assays with recombinant substrate proteins and LC-MS/MS analysis.

A combination of traditional approaches with the omics approach will help understand the mechanism through which signaling pathways regulate the development of malaria parasite.

Identifying diagnostic and prognostic markers

Most of the attention on infectious diseases of the developing world has focused on the development of rapid diagnostic tests and novel therapeutics to ensure timely treatment and improved survival rates. Existing diagnostic tests are either too expensive or time consuming or difficult to implement in developing countries due to the lack of resources and expertise. The inability to predict disease severity is also a major challenge to effective clinical management and prevention of long-term malaria complications. These limitations have spurred the search for better diagnostic and prognostic markers in malaria that can be easily measured in body fluids.

For over a decade now, several attempts to discover novel biomarkers in human bio-fluids such as serum, plasma and urine have been made by various research groups. Such studies have involved the use of proteomic technologies to profile host responses to infectious diseases35, 36, 37, 38, 39. The high-throughput proteomic technology platforms not only investigate the systemic alterations of protein expression in response to diseases but also enable visualisation of the underlying interconnecting protein networks and signaling pathways, facilitating the discovery of unique markers of infection40. Proteomic technologies have also been used to discover biomarkers that demonstrate the presence of the infecting organisms41. One of the first attempts to unravel the proteome of the malaria parasite, Plasmodium vivax from clinical samples provided new leads towards the identification of diagnostic markers, novel therapeutic targets and an enhanced understanding of malaria pathogenesis42.

Efforts to decipher host responses to malaria infection have revealed a panel of proteins with a distinct pattern of differential abundance that can discriminate malaria patients from healthy subjects and patients with other infectious diseases43.

Similarly, analysis of serum proteome of dengue and leptospirosis patients has led to the identification of unique protein signatures and molecular targets44, 45, 46. A comparative serum proteomic analysis of severe and non-severe malaria in search of prognostic markers using quantitative proteomics has highlighted the presence of muscular, cytoskeletal and anti-oxidant proteins in patient sera revealing extensive oxidative stress and cellular damage in severe malaria. These findings are currently being validated in a larger cohort of patients using immunoassays.

The application of proteomic technologies has shown promising leads. However, early disease detection, measurement of therapeutic efficacy, prediction of disease severity and tailored patient therapy are still some distance away.


1National Institute of Immunology, New Delhi, India (pushkar@nii.ac.in). 2International Centre for Genetic Engineering & Biotechnology, New Delhi, India (pawanm@icgeb.res.in; inderjeet@icgeb.res.in). 3Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India (sanjeeva@iitb.ac.in).


References

1. Carlton, J. M, et al. Comparative genomics of the neglected human malaria parasite Plasmodium vivax. Nature. 455, 757-763 (2008).

2. Florens, L. et al. A proteomic view of the Plasmodium falciparum life cycle. Nature 419, 520-526 (2002).

3. Lasonder, E. et al. Nature 419, 537-542 (2002).

4. Solyakov, L. et al. Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum. Nat. Commun. 2, 565 (2011).

5. Treeck, M. et al. The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites’ boundaries. Cell Host Microbe. 10, 410-419 (2011).

6. Jones, M. L. Getting stuck in: protein palmitoylation in Plasmodium. Trends Parasitol. 28, 496-503 (2012).

7.  Pease, B. N. et al. Global analysis of protein expression and phosphorylation of three stages of Plasmodium falciparum intraerythrocytic development. J. Proteome Res. 12, 4028-4045 (2013).

8. Lasonder, E. et al. Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites. Proteomics. (2015) doi: 10.1002/pmic.201400508. [Epub ahead of print]

9. Foth, B. J. et al. Quantitative time-course profiling of parasite and host cell proteins in the human malaria parasite Plasmodium falciparum. Mol. Cell Proteomics. 10, M110.006411 (2011).

10.Crosnier, C. et al. A library of functional recombinant cell-surface and secreted P. falciparum merozoite proteins. Mol. Cell Proteomics. 12, 3976-3986 (2013).

11. Siddiki, A. M. A. M. Z. et al. A review on subcellular or organellar proteomics with special reference to apicomplexan parasites. Bangl. J. Vet. Med. 5, 01- 07 (2007).

12.Belli, S. I. et al. Global protein expression analysis in apicomplexan parasites: current status. Proteomics. 5, 918-924 (2005).

13. Gunasekera, K. et al. Proteome remodelling during development from blood to insect-form Trypanosoma brucei quantified by SILAC and mass spectrometry. BMC Genomics. 13, 556 (2012).

14. Walker J. et al. Discovery of factors linked to antimony resistance in Leishmania panamensis through differential proteome analysis. Mol. Biochem Parasitol. 183, 166- 176 (2012).

15. Che, F. Y. et al. Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii. Mol. Cell Proteomics. 10, M110.000745 (2011).

16.Yang, P. Y. et al. Parasite-based screening and proteome profiling reveal orlistat, an FDA-approved drug, as a potential anti Trypanosoma brucei agent. Chemistry. 18, 8403-8413 (2012).

17. Matrangolo, F. S. et al. Comparative proteomic analysis of antimony-resistant and -susceptible Leishmania braziliensis and Leishmania infantum chagasi lines. Mol. Biochem Parasitol. 190, 63-75 (2013).

18. Queiroz, R. M. et al. Cell surface proteome analysis of human-hosted Trypanosoma cruzi life stages. J. Proteome Res. 13, 3530-3541 (2014).

19. Zhou, H. et al. Differential proteomic profiles from distinct Toxoplasma gondii strains revealed by 2D-difference gel electrophoresis. Exp. Parasitol. 133, 376-382 (2013).

20. Penarete-Vargas, D. M. et al. A chemical proteomics approach for the search of pharmacological targets of the antimalarial clinical candidate albitiazolium in Plasmodium falciparum using photo crosslinking and click chemistry. PLoS One. 9, e113918 (2014).

21. Singh, M. et al. Proteome analysis of Plasmodium falciparum extracellular secretory antigens at asexual blood stages reveals a cohort of proteins with possible roles in immune modulation and signaling. Mol. Cell Proteomics. 8, 2102-2118 (2009).

22. Chugh, M. et al. Protein complex directs hemoglobin-to-hemozoin formation in Plasmodium falciparum. Proc. Natl. Acad. Sci. USA. 110, 5392-53977 (2013 )

23. Donaldson, T. & Kim, K. Targeting Plasmodium falciparum purine salvage enzymes: a look at structure-based drug development. Infect. Disord. Drug Targets. 10, 191-199 (2010).

24. Kirk, K. et al. Plasmodium permeomics: membrane transport proteins in the malaria parasite. Curr. Top. Microbiol. Immunol. 295, 325-356 (2005).

25. Desai, S. A. & Miller, L. H. Malaria: Protein-export pathway illuminated. Nature 511, 541-542 (2014).

26. Gilson, P. R. et al. Identification and stoichiometry of glycosylphosphatidylinositol-anchored membrane proteins of the human malaria parasite Plasmodium falciparum. Mol. Cell Proteomics. 5, 1286-1299 (2006).

27. Chung, D. W. & Le Roch, K. G. Targeting the Plasmodium ubiquitin/proteasome system with anti-malarial compounds: promises for the future. Infect. Disord. Drug Targets. 10, 158-164 (2010).

28. Gardner, M. J. et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498-511 (2002).

29. Ward, P. et al. Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote. BMC. Genomics 5, 79 (2004).

30. Guttery, D. S. et al. Genome-wide functional analysis of Plasmodium protein phosphatases reveals key regulators of parasite development and differentiation. Cell Host Microbe 16, 128-140 (2014).

31. Tewari, R. et al. The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell Host Microbe 8, 377- 387 (2010).

32. Sharma, P. & Chitnis, C. E. Key molecular events during host cell invasion by Apicomplexan pathogens. Curr. Opin. Microbiol. 16, 432-437 (2013).

33. Ahmed, A. et al. Novel insights into the regulation of malarial calcium-dependent protein kinase 1. FASEB J. (2012).

34. Thomas, D. C. et al. Regulation of Plasmodium falciparum glideosome associated protein 45 (PfGAP45) phosphorylation. PLoS One. 7, e35855 (2012).

35. Taneja, S. et al. Plasma and urine biomarkers in acute viral hepatitis E. Proteome Sci. 7, 39 (2009).

36. Sahu, A. et al. Host response profile of human brain proteome in toxoplasma encephalitis co-infected with HIV. Clin. Proteomics 11, 39 (2014).

37. Gouthamchandra, K. et al. Serum proteomics of hepatitis C virus infection reveals retinol-binding protein 4 as a novel regulator. J. Gen. Virol. 95, 1654–1667 (2014).

38. Puttamallesh, V. N. et al. Proteomic profiling of serum samples from chikungunya-infected patients provides insights into host response. Clin. Proteomics 10, 14 (2013).

39. Anuradha, R. et al. Circulating microbial products and acute phase proteins as markers of pathogenesis in lymphatic filarial disease. PLoS Pathog. 8, e1002749 (2012).

40. Petricoin, E. F. et al. Clinical proteomics: translating benchside promise into bedside reality. Nat. Rev. Drug Discov. 1, 683–695 (2002).

41. Kashyap, R. S. et al. Diagnostic markers for tuberculosis ascites: A preliminary study. Biomark. Insights 5, 87–94 (2010).

42. Acharya, P. et al. Clinical proteomics of the neglected human malarial parasite Plasmodium vivax. PLoS ONE 6, (2011).

43. Ray, S. et al. Proteomic investigation of falciparum and vivax malaria for identification of surrogate protein markers. PloS One 7, e41751 (2012).

44. Ray, S. et al. Serum proteome changes in dengue virus-infected patients from a dengue-endemic area of India: towards new molecular targets? Omics J. Integr. Biol. 16, 527–536 (2012).

45. Srivastava, R. et al. Serum profiling of leptospirosis patients to investigate proteomic alterations. J. Proteomics 76 Spec No., 56–68 (2012).

46. Ray, S. et al. Proteomic analysis of Plasmodium falciparum induced alterations in humans from different endemic regions of India to decipher malaria pathogenesis and identify surrogate markers of severity. J Proteomics (in press) (2015).