Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis

Abstract

Novel fimbriae were isolated and purified from the human enteropathogen SalmoneUa 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% 13-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.

Comparative Genetics of the rdar Morphotype inSalmonella

“…Conservation of Tafi production and rdar morphotype formation in these other subgroups suggests an important role in the life cycles of diverse Salmonella isolates. Due to characteristic growth conditions (growth under 30°C, nutrient limitation, low osmolarity) (16,25) and increased resistance to desiccation (26,68) and antimicrobial agents (2,55,56,68), we hypothesize that the rdar morphotype has a fundamental role for Salmonella survival outside host environments. This may contribute to contamination of different food products (4,30) and passage between susceptible hosts.…”

“…In Salmonella, Tafi are produced together with cellulose (70), capsular and extracellular polysaccharides (17,26,67), and BapA (38), all of which come together to form a recalcitrant extracellular matrix that links individual cells together. Extracellular matrix production is associated with multicellular properties of Salmonella, both in the formation of the rdar morphotype (16,52) and in the formation of pellicles at the air-liquid interface in standing culture (51,56). These phenotypes enhance the resistance of Salmonella to antimicrobial stresses (2,55) and contribute to long-term survival (68).…”

Sequence Determinants of Bacterial Amyloid Formation

“…18 For example, CsgA produced by wild-type cells is whole cell-associated and SDS insoluble. 19 Brief treatment with FA liberates CsgA monomers from curli fibers produced by wild-type strain MC4100. 6 Similar to the wild-type strain, CsgA produced by csgA/pLR5 was SDS insoluble and required brief pretreatment with FA to migrate into the gel (Figure 2(a), lanes 1 and 2).…”

AgfC and AgfE facilitate extracellular thin aggregative fimbriae synthesis in Salmonella Enteritidis

“…Thin aggregative fimbriae (Tafi) are the only fimbriae dependent on the ENP pathway. Tafi were identified in Salmonella Enteritidis by Collinson and co-workers, and the operon was termed agf (Collinson et al, 1991), whereas in Escherichia coli, the homologue discovered was named curli, and the operon termed csg (Collinson et al, 1992;Arnqvist et al, 1992). Tafi were later renamed curli using the csg nomenclature in Salmonella (Romling et al, 1998), but will be referred to by the original Salmonella nomenclature of agf here.…”