Structural and functional studies of a 50kDa antigenic protein from Salmonella enterica serovar Typhi (original) (raw)
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A simple, rapid and early diagnostic test for typhoid fever urgently needed for clinicians. It's have been discovered 36 kDa OMP's from Makassar, South Sulawesi, Indonesia. This study was aimed to determine potency of 36 KDa OMP's to react with antibody of typhoid fever patients as candidate immunodiagnostic of typhoid fever. An OMP 's of S. Typhi with an apparent molecular mass of 36 kDa that is highly immunogenic, evokes humoral and cell-mediated immune responses, and confers 100% protection to immunized rats against challenge with very high doses of S. Typhi has been identified. Further, very efficient clearance of bacteria from the liver of immunized animals was seen. This protein is recognized by the antibodies present in serum of typhoid patients. The agglutination slide indicated that the immunoreactive protein evoked a strong immune response in rats. Serology test by dri-dot resulted OMP can react with serum of typhoid patients (n=25) with sensitivity 100%.
Characterization of the Salmonella typhi Outer Membrane Protein C
Korean Journal of Microbiology and Biotechnology, 2013
Salmonella enterica serovar typhi, a Gram-negative food-borne pathogen, causes typhoid fever in humans. OmpC is an outer membrane porin of S. typhi expressed throughout the infection period. OmpC is potentially an attractive antigen for multivalent vaccines and diagnostic kit designs. In this study we combined in silico, in vitro and in vivo approaches to analyze various aspects of OmpC's antigenic properties. The conserved region, in addition to secondary and tertiary structures, and linear B cell epitopes, were predicted. A number of results obtained from in silico analyses were validated by experimental studies. OmpC was amplified, cloned and then expressed, with the recombinant protein then being purified. BALB/c mice were immunized by purified denatured OmpC. The titer of antibody was raised. Results of challenges with the pathogen revealed that the immunity is non-protective. Most of the theoretical and experimental results were in consensus. Introduced linear B cell epitopes can be employed for the design of diagnostic kits based on antigen-antibody interactions.
Immunologic Research, 2015
Typhoid fever is a significant global health problem with highest burden on the developing world. The severity of typhoid is often underestimated, and currently available serological diagnostic assays are inadequate due to lack in requisite sensitivity and specificity. This underlines an absolute need to develop a reliable and accurate diagnostics that would benefit long-term disease control and treatment and to understand the real disease burden. Here, we have utilized flagellin protein of S. typhi that is surface accessible, abundantly expressed, and highly immunogenic, for developing immunodiagnostic tests. Flagellin monomers are composed of conserved amino-terminal and carboxy-terminal, and serovar-specific middle region. We have generated a panel of murine monoclonal antibodies (mAbs) against the middle region of flagellin, purified from large culture of S. typhi to ensure its native conformation. These mAbs showed unique specificity and very high affinity toward S. typhi flagellin without showing any cross-reactivity with other serovars. Genetic analysis of mAbs also revealed high frequency of somatic mutation due to antigenic selection process across variable region to achieve high binding affinity. These antibodies also displayed stable binding in stringent reaction conditions for antigen-antibody interactions, like DMSO, urea, KSCN, guanidinium HCl, and extremes of pH. One of the mAbs potentially reversed the TLR5-mediated immune response, in vitro by inhibiting TLR5-flagellin interaction. In our study, binding of these mAbs to flagellin, with high affinity, present on bacterial surface, as well as in soluble form, validates their potential use in developing improved diagnostics with significantly higher sensitivity and specificity.
Scientific Reports
Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typhoid fever. Thus, mapping of B-cell epitopes of Salmonella enterica serovar Typhi (S. Typhi) is critical to identify key immunogenic regions of HlyE. A random 20-mer peptide library was used for biopanning with enriched anti-HlyE polyclonal antibodies from typhoid patient sera. Bioinformatic tools were used to refine, analyze and map the enriched peptide sequences against the protein to identify the epitopes. The analysis identified both linear and conformational epitopes on the HlyE protein. The predicted linear GAAAGIVAG and conformational epitope PYSQESVLSADSQNQK were further validated against the pooled sera. The identified epitopes were then used to isolate epitope specific monoclonal antibodies by antibody phage display. Monoclonal scFv antibodies were enriched for both linear and conformational epitopes. Molecular docking was performed to elucidate the antigen-antibody interaction of the monoclonal antibodies against the epitopes on the HlyE monomer and oligomer structure. An in-depth view of the mechanistic and positional characteristics of the antibodies and epitope for HlyE was successfully accomplished by a combination of phage display and bioinformatic analysis. The predicted function and structure of the antibodies highlights the possibility of utilizing the antibodies as neutralizing agents for typhoid fever. Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin with hemolytic and cytotoxic activities which is also known as cytolysin A (ClyA) and silent hemolysin A (SheA). Serological data shows that substantial amounts of HlyE are produced by S. Typhi and Paratyphi A but not for non typhoidal strains of Salmonella such as serovar Paratyphi B and Paratyphi C during human infection 1. Recent serodiagnostic studies conducted with HlyE under denatured and native conditions showed both forms of HlyE to be antigenic and specific to detect typhoid fever (TF) 2, 3. Therefore there is a strong indication that HlyE could pose as an important virulence determinant for Salmonella Typhi and Paratyphi A pathogenesis. To date, there is no crystal structure available for Salmonella enterica serovar Typhi (S. Typhi) HlyE protein in the protein database. Nevertheless, protein sequence analysis reveals that both E. coli HlyE and S. Typhi HlyE share high similarities at amino acid sequence level. This makes it difficult to isolate antibodies that are specific to S. Typhi HlyE. As a basis of comparison, the crystal structure of E. coli HlyE can be used for homology modelling. E. coli HlyE demonstrates a long rod shape which is a new architecture design for toxin family structures. Based on electron microscopy observation, HlyE molecule is predicted to oligomerize to form a channel that projects out from the membrane, subsequently allowing the formation of a pore 4. Although S. Typhi HlyE is an immunogenic antigen with the potential for diagnostic applications, there is still a gap in knowledge with regards to the antigenic epitopes of S. Typhi HlyE that allows the human antibody response to distinguish it from other bacterial HlyE. The ability of typhoid sera to exhibit specificity against S. Typhi HlyE suggest the presence of S. Typhi HlyE specific epitopes that is identified by typhoid patient sera 3. Therefore, epitope mapping of the patient sera will provide important insight to these epitopes and the inherent response by the immune system against S. Typhi HlyE.
Serodiagnostic evaluation of recombinant CdtB of S. Typhi as a potential candidate for acute typhoid
Immunologic research, 2018
Typhoid fever caused by human restricted Salmonella typhi presents a considerable health burden on developing South-Asian nations like India. The suboptimal sensitivity and specificity associated with culture-based isolation of etiological agent and the extensively used surface antigen-based serological assays often lead to misdiagnosis and inappropriate antimicrobial treatment. The increasing reports of the emergence of resistant strains and undefined disease burden signify the critical need for an inexpensive, reliable, easy-to-use, and highly sensitive diagnostic test for typhoid fever. Utilizing S. typhi-specific and immunogenic antigens in sero-diagnostic assays could lead to precise diagnosis of acute typhoid and prompt treatment. In this study, we report cloning, expression, and purification of recombinant Cytolethal distending toxin subunit B (CdtB) of S. typhi, which is reported to be highly specific, immunogenic, and expressed only upon S. typhi infection. We further evalu...
Biocatalysis and Agricultural Biotechnology, 2019
Typhoid fever is a world health problem, with 200,000 recorded death annually in developing countries. The discovery of new drug discovery and detection methods for typhoid is still continuing. In previous research, the 31 kilo Dalton (kDa) recombinant protein Fim-C-S typhi was successfully expressed. It was also reported that the recombinant protein Fim-C-S. typhi could induce the occurrence of antibodies. This study aims to further develop anti-Fim-C-S. typhi antibodies as a detection tool. The sensitivity evaluated by western immunoblotting analysis indicated that anti-Fim-C-S. typhi antibodies can significantly recognize its antigen at a minimum level of 0.125 μg. The specificity evaluation of anti-FimC-S. typhi antibodies against S. typhi bacteria extract protein showed that anti-Fim-C-S. typhi antibodies could recognize S. typhi extract protein at ± 29 KDa and ± 60 kDa. In addition, anti-Fim-C-S. typhi antibody did not recognize healthy blood extract proteins. Simulation in healthy blood samples containing bacterial antigen S. typhi and recombinant antigen Fim-C-S. typhi produce bands of 29 kDa, 31 kDa and 60 kDa have been also studied. It can be concluded that anti-Fim-C-S. typhi antibodies can be used in the development of prototype detection tool. The results from this study are expected to provide a foundation to the development of a detection methods for S. typhi that are sensitive, specific, safe and simple.
Molecular characterization of a 42 kDa subunit pili protein of Salmonella typhi causes typhoid fever
Biodiversitas Journal of Biological Diversity, 2022
Blood culture is the gold standard for diagnosing typhoid fever, but it has limitations such as media and laboratory equipment, specimen volume, and examination time. However, the Academy of Pediatrics does not recommend serology due to its low sensitivity. The purpose of this study was to determine the molecular properties of the protein pilli of Salmonella typhi (S. typhi) that the findings can be used to develop a typhoid fever diagnostic reagent. The SDS-PAGE method was used, as well sequence analysis with ProtParam, ProtScale, and PSIPRED. The SDS-PAGE profile reveals one major protein (42 kDa) and fourteen minor proteins. The pili protein subunit 42 kDa had an amino acid (AA) sequence with a length of 390 AA, according to bioinformatics analysis. According to the ProtParam results, the pili protein subunit 42 kDa has good stability with a value of 40 and is a hydrophilic protein with an average GRAVY value of-0.950. PSIPRED results show that among the secondary structural elements, coil strand predominates, followed by-helix and-strand. It is concluded that this protein is immunogenic and that it can be used to develop a more specific and sensitive diagnostic reagent for typhoid fever.
Application of Salmonella typhi's outer membrane (OMP)in diagnosis of Typhoid
In this study, a modern technique for the diagnosis of established. To reduce the identification time of the infection by typhoid fever; protein was extracted from Partially purified outer membrane protein was immobilized on a nitrocellulose membrane and tested against serum of typhoid patients to detect the antigenicity of the protein isolated using immunoblotting technique. The molecular weight of the highly antige antigenisity against the sera of Typhoid fever patients using Western blotting technique. So, this can be an initial step towards the rapid immunochromatographic strips and ELISA d antibodies against
IJERT-Screening of New Potential Drug Target for Salmonella typhi Using Biostatistics Analysis
International Journal of Engineering Research and Technology (IJERT), 2013
https://www.ijert.org/screening-of-new-potential-drug-target-for-salmonella-typhi-using-biostatistics-analysis https://www.ijert.org/research/screening-of-new-potential-drug-target-for-salmonella-typhi-using-biostatistics-analysis-IJERTV2IS90662.pdf The sequenced genome of Human and various pathogens has provided access to huge amount of data related to their genome as well as their proteome. This immense assembly of information proves to be very useful in the identification of the novel targets in pathogens. Such drug targets are then used to control the dreadful actions of various fatal diseases. In this study, the disease chosen was typhoid. It was found that typhoid is mainly caused due to the bacteria Salmonella typhi. The whole proteome of Salmonella typhi was retrieved from both UniProt and NCBI. The filtered essential proteins of pathogen, non homologous to human, represents potential drug targets. The complete proteome set of Salmonella typhi contains 19732 proteins (Uniprot). 6292 proteins were retrieved as non-redundant by CD-HIT program at 60%(identical) threshold.MSA analysis was perform to get the conserved region within these proteins. Sub-cellular localization was predicted using PA-SUB server to locate the outer membrane proteins which could be probable vaccine candidates. These represent a vast number of potential therapeutic drug targets because of their involvement in major biological processes in cell.