CVI Accepts, published online ahead of print on 23 July 2008

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Clin. Vaccine Immunol. doi:10.1128/CVI.00093-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization of an outer membrane protein of Salmonella that confers protection against typhoid

Nowsheen Hamid and S. K. Jain^*

Department of Biotechnology, Hamdard University, New Delhi 110062, India

^* To whom correspondence should be addressed. Email: skjain{at}jamiahamdard.ac.in.

Typhoid caused by Salmonella enterica ser. Typhi remains a major health concern worldwide. The emergence of multi-drug resistant (MDR) strains of Salmonella with increased virulence, communicability and survivability leading to increased morbidity and mortality has further complicated its management. Currently available vaccines for typhoid have less than desired efficacy and certain unacceptable side effects, making it pertinent to search for new immunogens suitable for vaccine formulation. The outer membrane proteins (OMPs) of Salmonella have been implicated as possible candidates for conferring protection against typhoid. OMPs interface the cell with the environment, thus representing important virulence factors with significant role in the pathobiology of Gram-negative bacteria and enhanced bacterial adaptation. An outer membrane protein of Salmonella enterica ser. Typhimurium with an apparent molecular weight of 49kDa has been identified that is highly immunogenic, evokes humoral and cell mediated immune responses and confers 100% protection to immunized rats against challenge with very high doses (upto 100xLD₅₀) of Salmonella enterica ser. Typhimurium. Further, very efficient clearance of bacteria from the reticulo-endothelial system of immunized animals was seen. This protein is recognized by the antibodies present in serum of typhoid patients. When SDS-PAGE gel eluted protein was further analyzed by high HPLC and two-dimensional (2D) electrophoresis, two polypeptides of same molecular weight were resolved. These have different isoelectric points and gave two peaks with different retention time in reverse phase HPLC. However, only one of the two bands interacted with patient serum. The immunogenic studies (ELISA and DTH) indicated that the immunoreactive protein evoked strong immune response in rats. The N-terminal sequencing and homology analysis of this protein with sequences in protein database of Salmonella resulted in a match with the N-terminal sequences of a protein in Salmonella enterica ser. Typhi (CT18 and Ty2 strains). The homology search further revealed it to be a hypothetical protein with unidentified ORFs in Salmonella Typhi with 447 aminoacid residues corresponding to a molecular weight of 49kDa. The nucleotide sequence of the encoding gene was deduced and the gene was amplified by PCR using appropriate primers. A 1.3kb band was amplified that was purified and sequenced to confirm its identity. These OMPs provide promising targets for the development of candidate vaccine against typhoid.