Elsevier

Journal of Proteomics

Volume 75, Issue 10, 6 June 2012, Pages 3063-3080
Journal of Proteomics

Serum proteome analysis of vivax malaria: An insight into the disease pathogenesis and host immune response

https://doi.org/10.1016/j.jprot.2011.10.018Get rights and content

Abstract

Vivax malaria is the most widely distributed human malaria resulting in 80–300 million 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.

Graphical abstract

Highlights

► 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.

Introduction

Malaria remains a leading global health problem, especially in tropical and subtropical areas, with a worldwide incidence of 300–500 million clinical cases per year leading to at least one million deaths annually. According to the recent WHO statistics about 100 countries in the world are considered malarious and 3.3 billion people, nearly half of the world's population are at the risk of malaria [1]. Plasmodium falciparum and Plasmodium vivax account for over 90% of the total malaria cases worldwide [2]. The estimated number of annual clinical cases of vivax malaria ranges from 70 to 390 million, and outside of the African continents vivax malaria accounts for more than 50% of all malaria cases, yet the morbidity associated with this infection and its spectrum of disease is less studied [3]. India has the largest population in the world at the risk of malaria with 85% of the total Indian population living in malarious zones [4]. In spite of the overall global burden, economic impact and severity of the disease, P. vivax is less studied since the parasite cannot be propagated continuously in the laboratory except in non-human primates [5]. Increasing resistance to chloroquine and commonly used antimalarials in P. vivax parasites and the recent reports of involvement of this species in complicated malaria suggests the need for further research in vivax malaria [6].

Completion of the genome sequencing of different species of Plasmodium and its insect and vertebrate hosts have propelled the growth of proteomics into different areas of malaria research, including the understanding of host–pathogen interactions [7], disease pathogenesis and host immune response [8], characterization of global and stage specific parasite proteome [9], [10], analysis of parasite secretome [11] and elucidation of mechanisms of drug action [12]. Proteomic techniques pose tremendous potential to provide a wealth of new information to accelerate malaria research and aid identification of new targets of vaccines and drugs. Serum is an easily accessible body fluid, which is considered as an attractive sample for clinical research since it contains different types of proteins released by various diseased tissues and provides an inclusive representation of the pathophysiological condition of a patient [13]. Diagnostic applications of serum proteomics have steadily been growing over the last decade and it has been used for sensitive detection of surrogate proteins markers for cancers and different types of other human diseases [13]. However, very few studies have been conducted to identify surrogate protein markers for malaria in serum, plasma or other types of biological fluids. A few studies have identified protein markers such as SOD-1 [14], haptoglobin and serum amyloid A [15], IP-10, sTNF-R2 sFas and VEGF [16], apoptotic factors and neuroprotective angiogenic growth factors [17]. These findings open up new opportunities for the comprehensive screening of both uncomplicated and complicated malaria for better understanding of disease pathogenesis.

The aim of the present study was to identify the potential serum protein markers using proteomic techniques and enhance our understanding of host immune response in vivax malaria. Two dimensional electrophoresis (2DE) and two dimensional fluorescence difference gel electrophoresis (2D-DIGE) were used for serum protein separation. The 92 protein spots, which were significantly (p < 0.05) modulated as compared to the controls, were analyzed using MALDI-TOF/TOF and identity of 67 protein spots was established. Functional pathway analysis of the differentially expressed proteins revealed alterations in multiple physiological pathways including acute phase response signaling, complement and coagulation cascades, hemostasis and vitamin D metabolism pathway due to P. vivax infection. The significance of identified proteins such as apolipoprotein A-1 and E, serum amyloid A and P, haptoglobin, ceruloplasmin and hemopexin as well as various physiological pathways is discussed in the context of vivax malaria. We anticipate that this proteomic analysis would contribute to better understanding of the disease pathogenesis and establishment of diagnostic surrogates to provide new molecular targeted therapies.

Section snippets

Subject selection and sample collection

This study was undertaken with the approval of the Institutional Ethics Committee of Seth GS Medical College and KEM Hospital and written informed consent was obtained from each participant before screening. Patients presenting to the malaria outpatient department with a history suggestive of uncomplicated malaria were screened. If the microscopic examination of a thick peripheral blood smear by a trained technician revealed P. vivax infection with asexual parasite count more than 1000 per μL

Comparative evaluation of serum protein extraction and sample processing protocols

In order to circumvent the challenges associated with the serum sample such as extremely broad dynamic range of protein concentration, presence of high-abundance proteins and salts, nucleic acids and other contaminants, and to obtain a comprehensive coverage of the serum proteome on gels, multiple processing steps are required prior to the proteomic analysis. In this study, firstly we evaluated different protein extraction protocols from published papers to obtain maximum coverage of the serum

Discussion

Although several studies have been executed to investigate malaria pathogenesis and host immune response against the different species of the parasite, various aspects remain to be elucidated further. In this study we used a proteomic approach to analyze the alterations in human serum proteome due to P. vivax infection and identified a number of differentially expressed serum proteins involved in acute phase response signaling, interleukin signaling, complement and coagulation cascades and

Conclusions

In summary, we have performed a proteomic analysis of the vivax malaria patients' serum to understand the host response and identify potential candidate biomarkers or unique proteomic pattern for P. vivax infection. In seeking towards sensitive diagnostic approach in vivax malaria, apart from clinical symptoms and microscopic examination of a blood smear, serum protein markers can be the potential candidates. Some of the differentially regulated serum proteins identified in our study such as

Funding

This research was supported by Board of Research in Nuclear Sciences (BRNS) DAE young scientist award (2009/20/37/4/BRNS) and a start-up grant 09IRCC007 from the IIT Bombay to SS. SR was supported by the IIT Bombay fellowship.

Competing interests

The authors have declared that no competing interests exist.

Acknowledgments

We thank all the vivax malaria patients and healthy volunteers who participated in our study. The active support from Tulip Jhaveri and Snehal Kamble of Department of Clinical Pharmacology, Seth GS Medical College and KEM Hospital in clinical sample collection process is gratefully acknowledged. We would also like to thank Dr. Shantanu Sengupta and Gaurav Garg, Institute of Genomics and Integrative Biology (IGIB), Delhi for the help in performing immunoturbidimetric assays and Dr. Geetanjali

References (63)

  • J.R. Delanghe et al.

    Hemopexin: a review of biological aspects and the role in laboratory medicine

    Clin Chim Acta

    (2001)
  • O. Erel et al.

    Oxidative stress and antioxidative status of plasma and erythrocytes in patients with vivax malaria

    Clin Biochem

    (1997)
  • W. McGuire et al.

    C-reactive protein and haptoglobin in the evaluation of a community–based malaria control programme

    Trans R Soc Trop Med Hyg

    (1996)
  • H. Baumann et al.

    The acute phase response

    Immunol Today

    (1994)
  • A. Rougemont et al.

    Hypohaptoglobinaemia as an epidemiological and clinical indicator for malaria. Results of two studies in a hyperendemic region in West Africa

    Lancet

    (1988)
  • S. Yerly et al.

    Development of a haptoglobin ELISA. Its use as an indicator for malaria

    Acta Trop

    (1990)
  • B.S. Das et al.

    Plasma a-tocopherol, retinol and carotenoids in children with falciparum malaria

    Am J Clin Nutr

    (1996)
  • C. Wenisch et al.

    Complement activation in severe Plasmodium falciparum malaria

    Clin Immunol Immunopathol

    (1997)
  • H.J. Vial et al.

    Inhibition of the in vitro growth of Plasmodium falciparum by D vitamins and vitamin D-3 derivatives

    Mol Biochem Parasitol

    (1982)
  • S. Muller et al.

    Vitamin and co-factor biosynthesis pathways in Plasmodium and other apicomplexan parasites

    Trends Parasitol

    (2007)
  • World Malaria Report

    World Health Organization

    (2008)
  • J.W. Park

    Changing transmission pattern of Plasmodium vivax malaria in the Republic of Korea: relationship with climate change

    Environ Health Toxicol

    (2011)
  • R.N. Price et al.

    Vivax malaria: neglected and not benign

    Am J Trop Med Hyg

    (2007)
  • S.K. Prajapati et al.

    Molecular epidemiology of Plasmodium vivax anti-folate resistance in India

    Malar J

    (2011)
  • T. Lefevre et al.

    Malaria Plasmodium agent induces alteration in the head proteome of their Anopheles mosquito host

    Proteomics

    (2007)
  • D.L. Doolan et al.

    Profiling humoral immune responses to P. falciparum infection with protein microarrays

    Proteomics

    (2008)
  • E. Lasonder et al.

    Proteomic profiling of plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

    PLoS Pathog

    (2008)
  • L. Florens et al.

    A proteomic view of the Plasmodium falciparum life cycle

    Nature

    (2002)
  • J.H. Prieto et al.

    Large scale differential proteome analysis in Plasmodium falciparum under drug treatment

    PLoS One

    (2008)
  • S. Ray et al.

    Proteomic technologies for the identification of disease biomarkers in serum: advances and challenges ahead

    Proteomics

    (2011)
  • B.B. Andrade et al.

    Plasma Superoxide Dismutase-1 as a surrogate marker of vivax malaria severity

    PLoS Negl Trop Dis

    (2010)
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