| Salmonella enterica serovar Paratyphi A (SPA), a Gram-negative bacteria, is an emerging food and water-borne pathogen causing paratyphoid fever and is host-adapted to human only. The World Health Organization (WHO) has estimated that typhoid fever (including paratyphoid fever) causes approximately22million illnesses and220,000deaths per year. SPA is the most prevalent cause of paratyphoid fever. SPA infection rates have been rising, particularly in South East Asia including south of China, where this serovar is now responsible for30-50%of enteric fever cases. This increase has been associated with rises in antibiotic resistance among paratyphoid infections. It may also be associated with vaccination against Typhi, which unfortunately provides little cross-protection against Paratyphi A. Meanwhile, the present use of inactivated whole-cell paratyphoid A vaccines is restricted by their high rates of side effect. It is necessary to research and develop new protective vaccine of paratyphoid A fever. Therefore, the scholars of enteric pathogens field pay more attention to the study of SPA.At present, the rapid development of molecular biology, genomics, proteomics, bioinformatics and other subjects can provide the revolutionary methods for vaccine research and development. Reverse Vaccinology, an analysis based on the genomic information to screen candidate protein antigens, includes the following main steps: first, after obtaining the pathogen genome sequence, microarray analysis were used to predict encoding secreted proteins, surface-associated antigen and virulence factor; secondly, utility of proteomics to analyze the target proteins, such as outer membrane proteins, or DNA microarrays to identify highly expressed genes and genes regulation in vivo; thirdly, summarize the above results, the candidate protein antigens were defined and were high-throughput cloned and expressed in certain expression system; finally, candidate vaccine were selected by immunological experiments in vivo or in vitro. So, following the rapid development of genomics and bioinformatics analysis, gene annotation is no longer limited to the number of total proteins and function divided into several groups according to the forecast, but can forecast all proteins of cell including more information, such as localization and function. In addition, the development of proteomics plays an active role on vaccine research. Proteomics technology is a complement to genomics technology, also provides effective technical support on vaccine development. Comprehensive application of genomics, bioinformatics, DNA microarray and proteomics technology, can determine different components of the pathogens qualitatively and quantitatively. It is helpful for developing subunit vaccine.In this study, a reference strain of Salmonella enterica serovar Paratyphi A, CMCC50973, obtained from National Center for Medical Culture Collection (CMCC, Beijing, China), was used. This strain is a stronger virulence isolates from a SPA-infected patient and was planned for the study of paratyphoid fever vaccine. The genomic DNA of CMCC50973strain was extracted and entrusted to BGI-Shenzhen to determine the complete genome sequence. The genome of CMCC50973consists of a single, circular chromosome of4,608,961bp with an average G+C content of52.24%, and including a total of4,618complete open reading frames (ORFs),7rRNA clusters,100tRNAs,56sRNAs. All4,618genes were annotated according to different database of KEGG, COG, SwissProt, TrEMBL, or NR. Comparing with reference NC006511sequence of SPA strain ATCC9150, there are143single nucleotide polymorphisms (SNPs) and12small insertion or deletion events (Indels). Of the143SNPs,112were located in open reading frames and31were in the intergenic regions. Out of12Indels, insertion events and deletion events have six respectively. In summary, the complete genome sequence of CMCC50973will lay the foundation to explore more detailed microbial genetics and biological information, such as the interaction between genes, the new regulatory factors, prediction and screening of new, more specific protective antigen gene, facilitate additional bioinformation and enable in-depth studies of SPA sequence variations.Expression proteomics research is the basis of proteomics research. To establish the protein reference map and database of whole-cell proteins under normal physiological conditions, first of all, can help us to have an overall understanding of the expression of protein; second, can provide a good foundation for subsequent comparative proteomics studies, such as comparative analysis the protein expression changes of different conditions of cell survival, post-translational processing and modification, protein changes at the subcellular level, also help for discovery and identification of the proteins with specific function and their genes; third, can help us to find some new vaccine targets, screen immune response antigens, and proceed future pathogenic studies. Therefore, we used the selected virulent SPA strain CMCC50973, performed the whole-cell protein expression profiling studies. In this study, the proteome reference maps of the end of cells growth log phase were thoroughly analyzed by the use of multiple overlapping narrow pH range (pH4.0-5.0, pH4.5-5.5, pH5.0-6.0, pH5.5-6.7, and pH6-11) two-dimensional gel electrophoresis. Altogether,705spots representing519different protein entries were identified by MALDI-TOF/TOF MS. Some gene products were found in multiple spots indicating isoforms. Calculated values of molecular weight and isoelectric point of identified proteins were similar to the predicted values of genome annotation, but there are also some differences. The most identified proteins contain which related to energy production and conversion, transport and metabolism of the three major nutrients, amino acid transport and translation, and so on. We also analyzed the abundance of identified proteins at different stages and confirmed the expression of67hypothetical proteins. CMCC50973whole-cell proteome map can help us understand the genomic expression information from the overall level, and provide a basis for looking for new vaccine targets or screening of the immune response antigen.Outer membrane proteins (OMPs) of Gram-negative bacteria play an essential role in bacterial pathogenesis and are directly involved in the interaction with host environments. Thus they represent important virulence factors and can induce a good immune response in host, which are usually facilitated the targets of antimicrobial drugs and vaccines. Salmonella OMPs also have been investigated as potential vaccine candidates and diagnostic antigens. Our former test showed that the outer membrane protein BtuB of SPA had a strong immune response with convalescent patient serum of paratyphoid fever. In order to further study the immunogenicity and immune protection of BtuB, the protein was cloned and expressed in Escherichia coli. About90%purity rBtuB protein was obtained after purification. Animal trials have shown that the recombinant protein had good immunogenicity. The lethal challenged experiments showed60%immunized mice with rBtuB were protected. The rBtuB protein showed good immunogenicity but unsatisfactory immune protection, it might be considered for conjugate vaccine carrier and used with other antigens.
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