Identification Of Antigenic Proteins Of Salmonella enterica subspecies enterica serovar Typhi In Sera Of Patients With Acute Typhoid Fever
Loading...
Date
2016-06
Authors
Chin, Kai Ling
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Typhoid fever is caused by a Gram-negative bacteria, Salmonella enterica subspecies enterica serovar Typhi (S. Typhi). It is a worldwide disease infecting mostly the under-developed and developing countries. Due to a lack of sensitive and specific biomarkers available in the market, diagnosis of typhoid fever remains a problem for the clinician. This may be due to the fact that these biomarkers were identified using pure microbiological cultures grown in in vitro conditions. Infection is a dynamic process and the expression of antigens depend on the environmental challenges faced by the bacteria. This study used serum as a source of in vivo antigens as it contains physiological signatures from both host and bacterial origin that are produced in vivo during infection. However, the presence of high abundance proteins (HAPs) in human serum masks the detection of low abundance proteins (LAPs) and low molecular weight proteins, which contain candidate protein biomarkers relevant to a particular disease state. Pooled Typhoid Fever Sera (PTFS) and Pooled Normal Human Sera (PNHS) were subjected to 2 different serum separation methods, i.e. 1) affinity chromatography to remove albumin and immunoglobulin G (IgG), and 2) membrane ultrafiltration to remove proteins with molecular weights greater than 100 kDa. These protein preparations were then subjected to 2 protein analysis methods, i.e. 1) SDS-PAGE-Western blot, and 2) 2D-PAGE-Western blot. The results showed that different separation methods gave different biomarker profiles. Typhoid-related antigens found in PTFS were
characterised using mass spectrometry analysis and 11 S. Typhi proteins were successfully identified using LC-ESI-MS/MS. Bioinformatic analyses were used to select S. Typhi proteins as potential biomarkers for diagnosis of typhoid fever. Based on high antigenicity prediction scores and low percentage sequence similarity with Escherichia coli (E. coli), 6 S. Typhi proteins, namely, blue copper oxidase (CueO), outer membrane protein C (OmpC), transaldolase B (TalB), cell invasion protein A (SipA), flagellar hook-associated protein 1 (FlgK), and hemolysin E (HlyE) were selected for further studies. Recombinant proteins (rCueO, rOmpC, rTalB, rSipA, rFlgK, and rHlyE) were produced using DNA cloning technique to obtain sufficient antigens for development of Western blot and indirect ELISAs to evaluate their diagnostic potential. Western blot analysis using a panel of test sera consisting of 4 typhoid fever, 2 non-typhoid fever and 2 normal human subjects showed that only antibodies from typhoid fever sera reacted with rCueO, rOmpC, rFlgK, and rHlyE, but not from non-typhoid fever and normal human subjects. Indirect ELISAs were used to test a panel of sera consisting of 30 typhoid fever, 30 non-typhoid fever, and 30 normal human subjects. The results showed that all 4 biomarkers were specific for diagnosis of typhoid fever with specificity of more than 90%. However, rHlyE had the highest diagnostic sensitivity of 90% compared to rCueO (50%), rOmpC (50%), and rFlgK (50%). Thus, rHlyE can serve as an invaluable biomarker for the diagnosis of typhoid fever.
Description
Keywords
Typhoid fever is caused by a Gram-negative bacteria , Salmonella enterica subspecies enterica serovar Typhi.