In my preliminary study, a big-scale gene screen was conducted in Cambridge using 1,440 transposon mutants of Salmonella Typhimurium (S. Typhimurium) wild-type SL1344 to infect HEp-2 cells in vitro and Transposon Directed Insertion-site Sequencing (TraDIS) was subsequently applied for detection of the attenuated transposon mutants in the conditioned output pools. A novel Salmonella virulence gene speG, not located within the Salmonella Pathogenicity Islands, was discovered to be responsible for bacterial intracellular replication in human epithelial cells. Little is known regarding the role of speG in Salmonella and its infected host cells although most current knowledge in its encoded protein (spermidine- /spermine-N1-acetyltransferase) and its involvement in polyamine metabolism derived from studies of E. coli and Shigella. Therefore, speG in Salmonella is worthwhile being further characterized for its phenotype and clinical significance. Intestinal M cells in Peyer’s patches, the specialized antigen-sampling cells of the mucosal immune system, are exploited by Salmonella and other pathogens as a route of invasion. M cells are characterized morphologically having sparse, irregular microvilli on their apical surface, with a basolateral cytoplasmic pocket harboring immune cells, and functionally having transcytotic capacity of particles to the invaginated immune cells. So far an in vitro M cell model has been developed using Transwells to culture polarized Caco-2 cell monolayers for 14 days and subsequently co-culture with basolateral Raji B lymphocytes for another 4-6 days. Hence, this model is feasible for further studies in the interactions between Salmonella and human M cells/B cells. Microarray is a powerful tool to study transcriptome of all genes in diverse cells. Microarray can broaden the horizon in characterization of speG by thorough understanding of the other involved gene expression/metabolic pathways in the bacteria and the infected host cells, rather than its own phenotype in S. Typhimurium. To date, no systemic transcriptome analysis has been done simultaneously in the pathogen (S. Typhimurium) and the infected host cells (M cells/B cells) to investigate crosstalks between them. Taken together, this study is designed using a Caco-2/Raji B cells co-culture model for global investigation in the expression of speG-regulated genes in S. Typhimurium, the infected in vitro M cells, and the responsive immune B cells beneath the in vitro M cells. This provides detailed information in this “adhesive, invasive, but relatively non-proliferative” attenuated strain to serve as an oral vaccine or targeted therapy vector. Based on the obtained information in the involved metabolic pathways of this gene, computer-aided drug design (CADD) could be performed to develop new anti-microbial drugs for suppression of Salmonella intracellular proliferation and specific antagonists for inhibition of bacterial pathogenesis in host cells. Moreover, this research offers comprehensive genomic surveys in characterization of in vitro M cells, which assist in better understanding of the important role of M cells in human innate and adaptive immunity. Supposing M cells are a good target for oral vaccines or immunotherapy, successful establishment of in vitro M cell may be developed as a platform to test new vaccines or drugs before in vivo animal studies and clinical trials are practicable.
|Effective start/end date||4/1/13 → 7/31/14|