title: Salmonella enteritidis fimbriae displaying a heterologous epitope reveal a uniquely flexible structure and assembly mechanism.
abstract: Two distinct Salmonella fimbrins, AgfA and SefA, comprising thin aggregative fimbriae SEF17 and SEF14, respectively, were each genetically engineered to carry PT3, an alpha-helical 16-amino acid Leishmania T-cell epitope derived from the metalloprotease gp63. To identify regions within AgfA and SefA fimbrins amenable to replacement with this epitope, PCR-generated chimeric fimbrin genes were constructed and used to replace the native chromosomal agfA and sefA genes in Salmonella enteritidis. Immunoblot analysis using anti-SEF17 and anti-PT3 sera demonstrated that all ten AgfA chimeric fimbrin proteins were expressed by S. enteritidis under normal growth conditions. Immunoelectron microscopy confirmed that eight of the AgfA::PT3 proteins were effectively assembled into cell surface-exposed fimbriae. The PT3 replacements in AgfA altered Congo red (CR) binding, cell-cell adhesion and cell surface properties of S. enteritidis to varying degrees. However, these chimeric fimbriae were still highly stable, being resistant to proteinase K digestion and requiring harsh formic acid treatment for depolymerization. In marked contrast to AgfA, none of the chimeric SefA proteins were expressed or assembled into fimbriae. Since each PT3 replacement constituted over 10% of the AgfA amino acid sequence and all ten replacements collectively represented greater than 75% of the entire AgfA primary sequence, the ability of AgfA to accept large sequence substitutions and still assemble into fibers is unique among fimbriae and other structural proteins. This structural flexibility may be related to the novel fivefold repeating sequence of AgfA and its recently proposed structure Proper formation of chimeric fimbrial fibers suggests an unusual assembly mechanism for thin aggregative fimbriae which tolerates aberrant structures. This study opens a range of possibilities for Salmonella thin aggregative fimbriae as a carrier of heterologous epitopes and as an experimental model for studies of protein structure.
authors.affiliation: Department of Biochemistry, University of Victoria, Victoria, Canada.
authors.forename: A P
authors.initials: AP
authors.lastname: White
authors.forename: S K
authors.initials: SK
authors.lastname: Collinson
authors.forename: P A
authors.initials: PA
authors.lastname: Banser
authors.forename: D J
authors.initials: DJ
authors.lastname: Dolhaine
authors.forename: W W
authors.initials: WW
authors.lastname: Kay
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001704:Biopolymers / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D003224:Congo Red / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology*; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005561:Formates / Q000378:metabolism; D007891:Leishmania / Q000235:genetics / Q000276:immunology; D008666:Metalloendopeptidases / Q000235:genetics / Q000276:immunology; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D018583:Pliability; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012477:Salmonella enteritidis / Q000166:cytology* / Q000235:genetics / Q000254:growth & development / Q000502:physiology
chemical_list: C107306:AgfA protein, Salmonella enterica; D001426:Bacterial Proteins; D001704:Biopolymers; D018984:Epitopes, T-Lymphocyte; D005561:Formates; D011993:Recombinant Fusion Proteins; C080931:sefA protein, Salmonella enteritidis; C030544:formic acid; D036781:Fimbriae Proteins; D003224:Congo Red; D019286:Endopeptidase K; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania