Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.
Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
S K
SK
Collinson
L
L
Emödy
K H
KH
Müller
T J
TJ
Trust
W W
WW
Kay
D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D004591:Electrophoresis, Polyacrylamide Gel; D010861:Fimbriae, Bacterial / Q000502:physiology / Q000648:ultrastructure*; D006801:Humans; D007150:Immunohistochemistry; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012477:Salmonella enteritidis / Q000472:pathogenicity / Q000648:ultrastructure*