Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker

Kin, Wong Weng; Tan, Zi Ning; Othman, Nurulhasanah; Lim, Boon Huat; Mohamed, Zeehaida; García, Alfonso Olivos; Noordin, Rahmah

doi:10.1128/cvi.05356-11

Cited by 13 publications

(14 citation statements)

References 18 publications

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“…In addition, some of the proteins identified in this study will help to develop diagnostic tools capable of detecting both cyst and trophozoite forms of the parasite (Tables S4, S5, S6). A recent study shows that sera of amebic liver abscess patients can recognize a 110 kDa protein (annotated as pyruvate phosphate dikinase, EHI_009530) [53]. This protein was identified in all 5 cyst samples in our study (see Table 2 or Table S3), supporting the notion that proteins identified in this study have promise as candidate diagnostic targets.…”

Section: Discussionsupporting

confidence: 87%

Proteomic Analysis of the Cyst Stage of Entamoeba histolytica

et al. 2012

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BackgroundThe category B agent of bioterrorism, Entamoeba histolytica has a two-stage life cycle: an infective cyst stage, and an invasive trophozoite stage. Due to our inability to effectively induce encystation in vitro, our knowledge about the cyst form remains limited. This also hampers our ability to develop cyst-specific diagnostic tools.AimsThree main aims were (i) to identify E. histolytica proteins in cyst samples, (ii) to enrich our knowledge about the cyst stage, and (iii) to identify candidate proteins to develop cyst-specific diagnostic tools.MethodsCysts were purified from the stool of infected individuals using Percoll (gradient) purification. A highly sensitive LC-MS/MS mass spectrometer (Orbitrap) was used to identify cyst proteins.ResultsA total of 417 non-redundant E. histolytica proteins were identified including 195 proteins that were never detected in trophozoite-derived proteomes or expressed sequence tag (EST) datasets, consistent with cyst specificity. Cyst-wall specific glycoproteins Jacob, Jessie and chitinase were positively identified. Antibodies produced against Jacob identified cysts in fecal specimens and have potential utility as a diagnostic reagent. Several protein kinases, small GTPase signaling molecules, DNA repair proteins, epigenetic regulators, and surface associated proteins were also identified. Proteins we identified are likely to be among the most abundant in excreted cysts, and therefore show promise as diagnostic targets.Major ConclusionsThe proteome data generated here are a first for naturally-occurring E. histolytica cysts, and they provide important insights into the infectious cyst form. Additionally, numerous unique candidate proteins were identified which will aid the development of new diagnostic tools for identification of E. histolytica cysts.

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Section: Discussionsupporting

confidence: 87%

Proteomic Analysis of the Cyst Stage of Entamoeba histolytica

et al. 2012

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“…They contribute to the virulence of the parasite by their adherence to the host tissue, and cause contact-dependent cytolysis of target cells; they are also involved in phagocytosis and contribute to resistance to cell lysis inflicted by the host [36]. The GalNAc lectin protein was also reported by Wong et al [15] as one of the components of excretory-secretory antigens of E. histolytica . In addition, the abundance of the Gal/GalNAc lectin protein on the surface of the parasite and its antigenic property have allowed it to be used for antigen detection in the TechLab Entamoeba histolytica II ELISA kit (TechLAB Inc.) [37].…”

Section: Resultsmentioning

confidence: 99%

“…The E. histolytica Gal/Gal-NAc lectin antigen is being utilized in commercial antigen detection tests such as the TechLab E. histolytica II ELISA (TechLab Inc). A study on ES proteins showed the diagnostic potential of pyruvate phosphate dikinase (PPDK), and its recombinant form has been used to develop a lateral flow dipstick test [15, 16]. In terms of treatment, auronofin has been identified as an effective drug which targets thioredoxin reductase, an ES protein of E. histolytica [17].…”

Section: Introductionmentioning

confidence: 99%

Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC–ESI–MS/MS and LC–MALDI–TOF/TOF

et al. 2016

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BackgroundExcretory-secretory (ES) proteins of E. histolytica are thought to play important roles in the host invasion, metabolism, and defence. Elucidation of the types and functions of E. histolytica ES proteins can further our understanding of the disease pathogenesis. Thus, the aim of this study is to use proteomics approach to better understand the complex ES proteins of the protozoa.Methods E. histolytica ES proteins were prepared by culturing the trophozoites in protein-free medium. The ES proteins were identified using two mass spectrometry tools, namely, LC–ESI–MS/MS and LC–MALDI–TOF/TOF. The identified proteins were then classified according to their biological processes, molecular functions, and cellular components using the Panther classification system (PantherDB).ResultsA complementary list of 219 proteins was identified; this comprised 201 proteins detected by LC–ESI–MS/MS and 107 proteins by LC–MALDI–TOF/TOF. Of the 219 proteins, 89 were identified by both mass-spectrometry systems, while 112 and 18 proteins were detected exclusively by LC–ESI–MS/MS and LC–MALDI–TOF/TOF respectively. Biological protein functional analysis using PantherDB showed that 27% of the proteins were involved in metabolic processes. Using molecular functional and cellular component analyses, 35% of the proteins were found to be involved in catalytic activity, and 21% were associated with the cell parts.ConclusionThis study showed that complementary use of LC–ESI–MS/MS and LC–MALDI–TOF/TOF has improved the identification of ES proteins. The results have increased our understanding of the types of proteins excreted/secreted by the amoeba and provided further evidence of the involvement of ES proteins in intestinal colonisation and evasion of the host immune system, as well as in encystation and excystation of the parasite.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-016-9135-8) contains supplementary material, which is available to authorized users.

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“…Thus similar observation may be expected ESA. Western blot analysis using patients serum samples revealed the high diagnostic value of native and recombinant forms of PPDK for detection of ALA [16,17]. Since PPDK is a component of E. histolytica ESA, it is conceivable that it may be also useful as a target for detection of this parasite in stool sample.…”

Section: Discussionmentioning

confidence: 99%

“…Meanwhile E. histolytica excretory-secretory antigens (EhESA) was produced using the method we have described earlier [17]. New Zealand white rabbits (Oryctolagus cuniculus; female, 2.8-3.0 kg, 11-13 weeks old) were used for immunizations, with electro-eluted rPPDK, or E. histolytica ESA.…”

Section: Production and Purification Of Polyclonal Antibodiesmentioning

confidence: 99%

Development and initial evaluation of a lateral flow dipstick test for antigen detection ofEntamoeba histolyticain stool sample

Saidin

Yunus

Othman

et al. 2017

Pathogens and Global Health

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Entamoeba histolytica infection remains a public health concern in developing countries. Early diagnosis of amoebiasis can avoid disease complications, thus this study was aimed at developing a test that can rapidly detect the parasite antigens in stool samples. Rabbits were individually immunized with recombinant pyruvate phosphate dikinase (rPPDK) and E. histolytica excretorysecretory antigens to produce polyclonal antibodies. A rapid dipstick test was produced using anti-rPPDK PAb lined on the dipstick as capture reagent and anti-EhESA PAb conjugated to colloidal gold as the detector reagent. Using E. histolytica-spiked in stool sample of a healthy individual, the detection limit of the dipstick test was found to be 1000 cells ml −1. Meanwhile when rPPDK was spiked in the stool sample, the minimum concentration detected by the dipstick test was 0.1 μg ml −1. The performances of the dipstick, commercial Techlab E. histolytica II enzyme-linked immunosorbent assays (ELISA) and real-time PCR were compared using 70 stool samples from patients infected with Entamoeba species (n = 45) and other intestinal pathogens (n = 25). When compared to real-time PCR, the diagnostic sensitivity of the dipstick for detection of E. histolytica was 65.4% (n = 17/26); while the diagnostic specificity when tested with stool samples containing other intestinal pathogens was 92% (23/25). In contrast, Techlab E. histolytica II ELISA detected 19.2% (5/26) of the E. histolytica-positive samples as compared to real-time PCR. The lateral flow dipstick test produced in this study enabled rapid detection of E. histolytica, thus it showed good potential to be further developed into a diagnostic tool for intestinal amoebiasis.

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Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker

Cited by 13 publications

References 18 publications

Proteomic Analysis of the Cyst Stage of Entamoeba histolytica

Proteomic Analysis of the Cyst Stage of Entamoeba histolytica

Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC–ESI–MS/MS and LC–MALDI–TOF/TOF

Development and initial evaluation of a lateral flow dipstick test for antigen detection ofEntamoeba histolyticain stool sample

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