title: The use of flow cytometry to detect expression of subunits encoded by 11 Salmonella enterica serotype Typhimurium fimbrial operons.
abstract: The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome contains 13 putative fimbrial operons termed agf (csg), fim, pef, lpf, 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 detected 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, lpfABCDE, 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 vivo growth conditions drastically alter the repertoire of fimbrial antigens expressed in S. Typhimurium.
authors.affiliation: Department of Medical Microbiology and Immunology, College of Medicine, Texas A and M University System Health Science Center, 407 Reynolds Medical Building, College Station 77843-1114, USA.
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Humphries
authors.forename: Manuela
authors.initials: M
authors.lastname: Raffatellu
authors.forename: Sebastian
authors.initials: S
authors.lastname: Winter
authors.forename: Eric H
authors.initials: EH
authors.lastname: Weening
authors.forename: Robert A
authors.initials: RA
authors.lastname: Kingsley
authors.forename: Robert
authors.initials: R
authors.lastname: Droleskey
authors.forename: Shuping
authors.initials: S
authors.lastname: Zhang
authors.forename: Josely
authors.initials: J
authors.lastname: Figueiredo
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Khare
authors.forename: Jairo
authors.initials: J
authors.lastname: Nunes
authors.forename: L Garry
authors.initials: LG
authors.lastname: Adams
authors.forename: Renée M
authors.initials: RM
authors.lastname: Tsolis
authors.forename: Andreas J
authors.initials: AJ
authors.lastname: Bäumler
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D007082:Ileum / Q000382:microbiology; D008026:Ligation; D016253:Microscopy, Immunoelectron; D009876:Operon; D012486:Salmonella typhimurium / Q000145:classification / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D012703:Serotyping
chemical_list: D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001426:Bacterial Proteins; D003470:Culture Media; C110675:SEF21 protein, Salmonella enteritidis; D036781:Fimbriae Proteins