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
Journal Title
Journal ISSN
Volume Title
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.
Description
Keywords
The potential infection markers , from liver aspirates and serum samples.