A Proteomics Study On Potential Parasite And Host Markers Of Infection In Serum And/Or Pus Of Patients With Amoebic Liver Abscess

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Date
2012-08
Authors
Othman, Nurulhasanah
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Publisher
Universiti Sains Malaysia
Abstract
Entamoeba histolytica, a protozoan parasite that causes amoebiasis, is still a health burden in many underdeveloped and developing countries. The most common clinical manifestation of extraintestinal amoebiasis is amoebic liver abscess (ALA). Research on finding infection markers for ALA is important in order to improve its diagnosis. Proteomic approach has been widely utilized to discover infection markers for various diseases. In this study, this approach was performed to identifY the potential infection markers from liver aspirates and serum samples from ALA patients. First, real-time PCR assay was developed to detect the presence of E. histolytica DNA in the pus aspirates and the corresponding serum samples were tested with ELISA to detect the presence of anti-amoebic antibodies. These pus aspirates and serum samples were then used for proteomic analysis. Proteins from pus aspirates were extracted and serum samples were depleted before being subjected to two-dimensional electrophoresis (2-DE), Western blot and mass-spectrometry to identify the antigenic proteins. In addition differential human serum protein expression profiles between ALA and normal individuals (control) were also performed using 2-DE and mass-spectrometry. Detection limits of the real-time PCR were found to be 10 copies/reaction and 1 o-2 cells/ml using pTEh1 plasmid and DNA from known number of trophozoites, respectively. Standard curves constructed from serial dilutions of pTEh1 plasmid and DNA from known number of trophozoites showed R2=0.97, with 88% efficiency and R2=0.98 with 98% efficiency, respectively. The real-time PCR assay detected fifteen "clear-cut" ALA pus aspirate samples, their corresponding serum samples gave positive result when tested with ELISA. These pus aspirates and sera sets were used in the proteomic experiments. Western blot analyses of the electrophoresed proteins from three pooled pus aspirates, and pooled serum samples showed the presence of antigenic 14 kDa and 42 kDa proteins, using pooled serum of hamster with ALA and IgG purified from pooled serum samples from patients with ALA as primary antibodies. Massspectrometry analysis identified five E. histolytica proteins in the 14 kDa protein band from pus aspirates namely kinetochore protein Spc25 domain-containing protein, Gal/GalNAc lectin heavy subunit, chromodomain-helicase-DNA-binding protein, tyrosine kinase and a conserved hypothetical protein. Meanwhile, the 42 kDa protein identified from the serum samples showed presence of E. histolytica BspA, leucine rich repeat family from pooled serum samples and cullin family proteins from two of three individual samples. Subsequent analysis of the mass spectra, the tyrosine kinase protein from 14 kDa band and BspA, leucine rich repeat family protein from the 42 kDa band were the most probable identifications. Meanwhile, the presence of cullin family protein in two out of three serum samples should not be ignored and further study is needed to verify the presence of this protein in individual samples using MS targeted approach.
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The potential infection markers , from liver aspirates and serum samples.
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