The role of the cell surface proteins of Burkholderia pseudomallei in the serodiagnosis of melioidosis and comparative serological proteomic analysis of the humoral immune response in melioidosis
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Date
2006
Authors
Sheik Abdul Kader, Zainoodin
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Abstract
Melioidosis refers to an infection by the bacterium, B. pseudomallei, affecting man and
animals. Clinical manifestations are protean, ranging from mild and self-limited
diseases to progressive multi-organ infection with fatal consequences, if untreated.
Treatment with prolonged course of antibiotics is required for the acute infection and
the prevention of development of persistence of infection and relapse. The diagnosis
based on clinical evidence is a challenging task, as the presenting symptoms mimic
many other common infectious diseases. Direct isolation remains the mainstay in the
laboratory diagnosis for melioidosis although it is less sensitive and time consuming.
Over the last two decades, many experimental approaches towards the development of a
reliable serological assay for rapid serodiagnosis of melioidosis have been reported.
However, these attempts were often met with limited success.
In the present study, a standardized protocol of dot enzyme immunoassay (dot EIA test)
with two optimized concentrations of the cell surface proteins (CSP) antigens from B.
pseudomallei was established for serological diagnosis of melioidosis by detecting the
IgM, IgG and IgA antibody isotypes as markers for current infection. The sensitivity of
the dot EIA test for the detection of IgM, IgG and IgA antibody isotypes individually
were 47.7%, 89.2% and 60% respectively. However, the overall combined detection of
the IgM, IgG and IgA antibody isotypes improved the sensitivity and specificity of the
dot EIA test to 95.5% and 92.5%, respectively. In addition, the dot EIA performed
satisfactorily in the prospective study for the diagnosis of melioidosis by detecting the
IgM, IgG and IgA antibody isotypes in the sera tested from clinically suspected cases of
melioidosis. The preliminary finding from this study demonstrated for the first time the
potential use of the CSP of the B. pseudomallei as a source of antigens in the dot EIA
test for early and accurate diagnosis of melioidosis by detecting the IgM, IgG and IgA
antibody isotypes in the patient’s serum.
Characterization of the SDS-PAGE separated CSP antigens by conventional Western
blot (WB) analysis revealed 25 antigenic bands recognized by melioidosis sera with
variable frequency and intensity. When the patterns of the antigenic bands were
compared between the 55 sera from melioidosis patients, no single pattern was found to
be diagnostically adequate. Great diversity in the humoral immune response against the
CSP antigens was observed in each of the melioidosis patient during the time course of
the infection. The heterogeneous antigenic protein profiles observed in the WB assay
were categorized into three clusters of antigens. These included broadly diffused
antigenic bands with the molecular weights ranging from 43 kDa to 36.8 kDa (cluster
1), regularly spaced antigenic bands with molecular weights ranging from 35.6 kDa to
25 kDa (cluster 2), and low molecular weights antigenic bands with molecular weights
ranging from 22 kDa to 13.2 kDa (cluster 3). Some degree of comparable cross-reaction
was observed between normal sera and non-melioidosis patients sera from endemic
regions against the antigens in cluster 1. However, the endemic sera showed very
limited cross-reactions with the antigens in the clusters 2 and 3. These data suggested
that healthy individuals and non-melioidosis patients in endemic regions possessed
antibodies specific to the CSP reflecting significant degree of background immunity to
melioidosis. Although the reactivity of the non-melioidosis and melioidosis sera were
not distinguishable in the WB analysis especially against the antigens in the cluster 1,
whether the non-melioidosis and melioidosis sera recognized similar and different
epitopes present in the same antigens remains to be elucidated.
In serological proteomic study, the CSP was fractionated based on molecular charge in
preparative Rotofor isoelectric focusing (IEF) and polyacrylamide gel IEF (PAGE IEF).
Fractionation in the preparative Rotofor IEF produced 20 rotofor fractions. A new
protocol to perform immunoblotting of the proteins separated by PAGE IEF gel was
developed. The IEF immunoblot analysis of the 20 rotofor fractions with pooled
melioidosis sera demonstrated presence of several strongly reacting bands located at the
pI values approximately 4 to 6.5. Preliminary analysis was performed to determine the
antigenic composition in the selected reactive rotofor fractions number F16 and F7 by
PAGE IEF method. The fractionated proteins in the gel matrix were extracted in
adequate quantity by sonication for subsequent analysis. Preliminary analysis by SDSPAGE
of the extracted proteins revealed the existence of the isoforms protein antigens
and successful enrichment of the hidden low abundance antigenic proteins present in the
CSP that otherwise may not be possible to detect by the conventional WB analysis. The
purified proteins extracted from the selected fractions from IEF study showed strong
and specific reaction with pooled melioidosis sera. These antigens can be used along
with the antigens identified by the conventional WB assay of the CSP separated by
SDS-PAGE to develop an improved multi-antigens based diagnostic assay for
melioidosis.
In summary, this work would clearly be relevant for the development of newer serologic
tests for diagnosis for melioidosis. Elucidation of the pathological mechanism in
melioidosis disease progression and recovery, exposition of more antigenic components
for diagnostic application and development of vaccine for melioidosis would be other
spin offs.
Description
PhD
Keywords
Biological science , Burkholderia pseudomallei , Serodiagnosis , Melioidosis