|Droleskey, Robert - Bob|
Submitted to: Molecular Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/7/2003
Publication Date: 9/20/2003
Citation: Humphries, A.D., Raffatellu, M., Winter, S., Kingsley, R.A., Droleskey, R.E., Zhang, S., Figueiredo, J., Khare, S., Nunes, J., Adams, L.G. 2003. The use of flow cytometry to detect expression of subunits encoded by 11 Salmonella enterica serotype typhimurium fimbrial operona. Molecular Microbiology. 48:1357-1376. Interpretive Summary: The bacteria Salmonella is one of the main causes of food poisoning in humans in the United States. One of the many ways that the bacteria causes people to get sick is by attaching itself to the inside of the gut using long fibers called fimbriae. Our research has identified some of the different kinds of fibers that Salmonella can produce and under what growth conditions they are made. Specifically, we have identified different fibers that are produced only when these bacteria grow inside the gut and not when they are grown under laboratory conditions. These newly identified fibers may play an important role in the attachment of the bacteria to the wall of the gut resulting in food poisoning. This work is important since all previous work on attachment fibers is based on laboratory grown bacteria. By identifying these new fibers, researchers will be able to investigate new ways to prevent the attachment of Salmonella to the lining of the gut and increase the chances for the prevention of food poisoning caused by Salmonella.
Technical Abstract: The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome contains 13 putative fimbrial operons termed agf (csg), fim, pef, Ipf, bcf, saf, stb, stc, std, stf, sth, sti, and stj. Evidence for in vitro expression of fimbrial proteins encoded by these operons is currently only available for agf, fim, and pef. We raised antisera against putative major fimbrial subunits of S. Typhimurium, including AgfA, FimA, PefA, LpfA, BcfA, StbA, StcA, StdA, StfA, SthA and StiA. Elaboration of StcA on the bacterial surface could be setected by flow cytometry and immunoelectron microscopy after expression of the cloned stcABCD operon from a heterologous T7 promoter in Escherichia coli. To study the expression of fimbrial antigens in S. Typhimurium by flow cytometry, we constructed strains carrying deletions of agfAB, pefBACDI, IpfABCDE, bcfABCDEFG, stbABCD, stcABC, stdAB, stfACDEFG, sthABCDE or stiABCDE. Using these deletion mutants for gating, expression of fimbrial antigens was measured by flow cytometry in cultures grown in vitro or in samples recovered 8 h after infection of bovine ligated ileal loops with S. Typhimurium. FimA was the only fimbrial antigen expressed by S. Typhimurium after static growth in Luria-Bertani (LB) broth. Injection of static LB broth cultures of S. Typhimurium into bovine ligated ileal loops resulted in the expression of BcfA, FimA, LpfA, PefA, StbA, StcA, StdA, StfA and StiA. These data show that in vitro growth conditions drastically alter the repertoire of fimbrial antigens expressed in S. Typhimurium.