Protective Properties and Surface Localization of Plasmodium falciparum Enolase

Department of Biological Sciences, TIFR, Homi Babha Road, Colaba, Mumbai 400 005, India.
Infection and Immunity (Impact Factor: 3.73). 12/2007; 75(11):5500-8. DOI: 10.1128/IAI.00551-07
Source: PubMed


The enolase protein of the human malarial parasite Plasmodium falciparum has recently been characterized. Apart from its glycolytic function, enolase has also been shown to possess antigenic properties
and to be present on the cell wall of certain invasive organisms, such as Candida albicans. In order to assess whether enolase of P. falciparum is also antigenic, sera from residents of a region of Eastern India where malaria is endemic were tested against the recombinant
P. falciparum enolase (r-Pfen) protein. About 96% of immune adult sera samples reacted with r-Pfen over and above the seronegative controls.
Rabbit anti-r-Pfen antibodies inhibited the growth of in vitro cultures of P. falciparum. Mice immunized with r-Pfen showed protection against a challenge with the 17XL lethal strain of the mouse malarial parasite
Plasmodium yoelii. The antibodies raised against r-Pfen were specific for Plasmodium and did not react to the host tissues. Immunofluorescence as well as electron microscopic examinations revealed localization
of the enolase protein on the merozoite cell surface. These observations establish malaria enolase to be a potential protective

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Available from: Monika Mehta, Aug 18, 2014
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    • "Enolase has been reported as vaccine candidate in Plasmodium sp [19] and other microorganisms such as Candida albicans [20], [21], [22], Chlamydia pneumonia [23] and Streptococcus sp [24], [25]. There have been reports regarding the presence of anti-enolase antibodies among malaria patients [26] and prophylactic potential of recombinant P. falciparum enolase in mice against a challenge with a lethal strain of P. yoelii suggested that enolase is a potential protective antigen [19]. Since there has been no report of enolase as well as aldolase as vaccine candidates in case of Leishmania, it was pertinent to assess their immunogenic and prophylactic potential against VL- a fatal infection. "
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    ABSTRACT: Th1 immune responses play an important role in controlling Visceral Leishmaniasis (VL) hence, Leishmania proteins stimulating T-cell responses in host, are thought to be good vaccine targets. Search of such antigens eliciting cellular responses in Peripheral blood mononuclear cells (PBMCs) from cured/exposed/Leishmania patients and hamsters led to the identification of two enzymes of glycolytic pathway in the soluble lysate of a clinical isolate of Leishmania donovani - Enolase (LdEno) and aldolase (LdAld) as potential Th1 stimulatory proteins. The present study deals with the molecular and immunological characterizations of LdEno and LdAld. The successfully cloned and purified recombinant proteins displayed strong ability to proliferate lymphocytes of cured hamsters' along with significant nitric-oxide production and generation of Th1-type cytokines (IFN-γ and IL-12) from stimulated PBMCs of cured/endemic VL patients. Assessment of their prophylactic potentials revealed ∼90% decrease in parasitic burden in rLdEno vaccinated hamsters against Leishmania challenge, strongly supported by an increase in mRNA expression levels of iNOS, IFN-γ, TNF-α and IL-12 transcripts along with extreme down-regulation of TGF-β, IL-4 and IL-10. However, animals vaccinated with rLdAld showed comparatively lesser prophylactic efficacy (∼65%) with inferior immunological response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of both the proteins was done using computational approach. Additionally, in-silico 3-D modelling of the proteins was done in order to explore the possibility of exploiting them as potential drug targets. The comparative molecular and immunological characterizations strongly suggest rLdEno as potential vaccine candidate against VL and supports the notion of its being effective T-cell stimulatory protein.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "In this study, we reported the cloning, expression and in silico immunological characterization of a T. cruzi cDNA enolase by molecular techniques, affinity chromatography, SDS-PAGE and immunodetection (western blot). The results showed that purified His-tag recombinant protein (rTcENO) encodes a 46.1 kDa protein and can be specifically recognized by anti-histidine monoclonal antibody (Fig. 6, lane 1), this molecular weight is corresponding with the previously reported size for several recombinant purified enolases of P. falciparum (49 kDa), C. albicans (46 kDa), Anisakis simplex (47 kDa), Sarcocystis neurona (47 kDa), Echinococcus granulosus (49.92 kDa) and Clonorchis sinensis (50 kDa) (Li et al. 2011; Pal-Bhowmick et al. 2007; Rodriguez et al. 2006; Wilson et al. 2004; Gan et al. 2010; Wang et al. 2011). Furthermore, based on multiple sequence alignment of enolases, the predicted amino acid sequence of the TcENO had an identity of 81.03 % to the enolase of T. brucei and 72–73 % with the enolases of Leishmania genus (Fig. 2). "
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    ABSTRACT: Nowadays, Chagas disease is a major health problem in Latin America that has been disseminated also into non-endemic countries. Currently, a vaccine against Chagas disease does not exist. In the present study, the gene encoding Trypanosoma cruzi enolase (TcENO) was amplified, cloned, and sequenced and the recombinant protein was purified. We used in silico and an experimental assay to investigate the immunological role of TcENO. The in silico assays showed that TcENO sequence contains characteristic motifs of enolase; additionally, a transmembranal region was identified, and this could indicate the potential membrane localization of TcENO. Moreover, both B lymphocyte and cytotoxic T lymphocytes (CTL) predicted epitopes were localized; these results suggest the possibility that TcENO can develop both humoral and cellular immune responses. Furthermore, the presence of antibodies was verified by western blot assays, showing that the purified recombinant protein was detected by sera from experimentally infected mice and sera of patients with Chagas disease. These results indicate that TcENO is immunogenic and could be used as a vaccine candidate.
    Full-text · Article · Jan 2014 · Parasitology Research
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    • "In Plasmodium falciparum, immuno-gold electron microscopic imaging to determine the sub-cellular distribution of enolase revealed its association with the food vacuole, in addition to it being present in the nucleus, cytosol and the cell surface [5]. Similar to enolases from several other organisms, parasite enolase also binds to plasminogen [5] and was suggested to have a role in invasion of RBCs [21]. Adaptive changes in the proteome of P. falciparum merozoites on switching the invasion dependence from sialated to non-sialated receptor on erythrocytes, showed up-regulation of enolase [22]. "
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    ABSTRACT: Plasmodium enolase localizes to several sub-cellular compartments viz. cytosol, nucleus, cell membrane, food vacuole (FV) and cytoskeleton, without having any organelle targeting signal sequences. This enzyme has been shown to undergo multiple post-translational modifications (PTMs) giving rise to several variants that show organelle specific localization. It is likely that these PTMs may be responsible for its diverse distribution and moonlighting functions. While most variants have a MW of ~50 kDa and are likely to arise due to changes in pI, food vacuole (FV) associated enolase showed three forms with MW~50, 65 and 75 kDa. Evidence from immuno-precipitation and western analysis indicates that the 65 and 75 kDa forms of FV associated enolase are ubiquitinated. Using mass spectrometry (MS), definitive evidence is obtained for the nature of PTMs in FV associated variants of enolase. Results showed several modifications, viz. ubiquitination at K147, phosphorylation at Y148 and acetylation at K142 and K384. MS data also revealed the conjugation of three ubiquitin (Ub) molecules to enolase through K147. Trimeric ubiquitin has a linear peptide linkage between the NH2-terminal methionine of the first ubiquitin (Ub1) and the C-terminal G76 of the second (Ub2). Ub2 and third ubiquitin (Ub3) were linked through an atypical isopeptide linkage between K6 of Ub2 and G76 of Ub3, respectively. Further, the tri-ubiquitinated form was found to be largely associated with hemozoin while the 50 and 65 kDa forms were present in the NP-40 soluble fraction of FV. Mass spectrometry results also showed phosphorylation of S42 in the cytosolic enolase from P. falciparum and T337 in the cytoskeleton associated enolase from P. yoelii. The composition of food vacuolar proteome and likely interactors of enolase are also being reported.
    Preview · Article · Aug 2013 · PLoS ONE
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