Characterization of an Outer Membrane Protein ofSalmonella entericaSerovar Typhimurium That Confers Protection against Typhoid

Abstract

Typhoid caused by Salmonella enterica serovar Typhi remains a major health concern worldwide. The emergence of multidrug-resistant 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 considered possible candidates for conferring protection against typhoid. OMPs interface the cell with the environment, thus representing important virulence factors with a significant role in the pathobiology of gram-negative bacteria and bacterial adaptation. An OMP of Salmonella enterica serovar Typhimurium with an apparent molecular mass of 49 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 (up to 100 times the 50% lethal dose) of Salmonella enterica serovar Typhimurium has been identified. Further, very efficient clearance of bacteria from the reticuloendothelial systems of immunized animals was seen. This protein is recognized by the antibodies present in serum of typhoid patients. When sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel-eluted protein was further analyzed by highperformance liquid chromatography (HPLC) and two-dimensional electrophoresis, two polypeptides with the same molecular weight were resolved. These have different isoelectric points and gave two peaks with different retention times in reverse-phase HPLC. However, only one of the two bands interacted with patient serum. The immunogenicity studies (enzyme-linked immunosorbent assay and delayed-type hypersensitivity [DTH]) indicated that the immunoreactive protein evoked a strong immune response in rats. The N-terminal sequencing and analysis of the homology of this protein with sequences in the protein database of Salmonella resulted in a match with the N-terminal sequences of a protein in Salmonella enterica serovar Typhi (CT18 and Ty2 strains). The homology search further revealed it to be a hypothetical protein, whose gene had unidentified open reading frames in Salmonella serovar Typhi encoding 447 amino acid residues, corresponding to a molecular mass of 49 kDa. The nucleotide sequence of the encoding gene was deduced, and the gene was amplified by PCR using appropriate primers. An amplified 1.3-kb band was purified and sequenced to confirm its identity. These OMPs provide promising targets for the development of a candidate vaccine against typhoid.Typhoid fever is an acute systemic illness caused by Salmonella enterica serovar Typhi. The organisms are noncapsulated, nonsporulating, facultative anaerobic bacilli, which have characteristic flagellar, somatic, and outer coat antigens (32). Salmonella enterica serovar Typhimurium is its murine/rodent counterpart...

Immune response in Lutjanus erythropterus induced by the major outer membrane protein (OmpU) of Vibrio alginolyticus

“…Isolation of OMPs was carried out using the method of Hamid & Jain (2008). The bacteria were harvested and washed 3 times with 10 mM Tris-HCl (pH 7.5), and 1.0 g (wet weight) of bacteria was extracted with 20 ml extraction buffer (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 6 M urea) for 1 h at 4°C.…”

Cloning, Sequencing and In Silico Characterization of OmpF Protein of Salmonella typhimurium for its Immune-Potential

“…Vaccination along with other control measures is currently being used in poultry industry to reduce the zoonotic risk (Desin et al, 2013). In last few years, vaccine control strategic programmes were in search of a suitable strong immunogenic vaccine candidate, this lead to an extensive study on outer membrane proteins of Salmonella (Hamid and Jain, 2008;Ghosh et al, 2011). Outer membrane proteins are good surface immunogens (Nandakumar et al, 1993), and are used in several studies to investigate the capability as a vaccine candidate in poultry (Meenakshi et al, 1999;Khan et al, 2003;Okamura et al, 2012;Prejit et al, 2013).…”

Immune response to Vi polysaccharide, heat-killed whole cells, and outer membrane protein of Salmonella typhi

“…This was carried out using two different extraction buffers; the first one followed the method of Foulaki et al [16] modified by Hamid and Jain [17], using Tris extraction buffer (10 mM Tris-HCl [pH 7.5], 10 mM EDTA, and 6 M urea). In brief, the harvested bacterial cells were washed using 10 mM Tris-HCl (pH 7.5), then extracted with the extraction buffer (20 mL/1 g wet bacteria shaken using a Bellco roller drum, Bellco Glass Inc., Vineland, USA) for one hour at 4°C.…”

“…The test was repeated in another group but using lithium-extracted OMP [17] The test group for the heat-killed whole cells was IP injected with two doses of 10 8 heat-killed cells (in 200 µL PBS) one week apart. For the different tested antigens, a control group was included for comparison under similar experimental conditions with PBS.…”