Cloning of Salmonella enterica serovar Enteritidis fimbrial protein SefA as a surface protein in Escherichia coli confers the ability to attach to eukaryotic cell lines

Abstract

The gene for the Salmonella enterica serovar Enteritidis fimbrial protein SefA was cloned into an Escherichia coli surface expression vector and confirmed by Western blot assay. E. coli clones expressing SefA attached to avian ovary granulosa cells and HEp-2 cells, providing evidence for the involvement of SefA in the ability of Salmonella to attach to eukaryotic cells.

FIG. 1.

SDS-PAGE and Western blot of SefA samples using SefA monoclonal antibodies. (A) SDS-PAGE of total membrane fractions collected from cells grown at 37°C. (B) Western blot of SDS-PAGE gel shown in panel A using monoclonal mouse antibodies 69/25 as an immunoprobe for SefA. (C) SDS-PAGE of outer membrane fractions collected from cells grown at 25°C. Lanes: 1, molecular mass standards (99.4, 66.2, 45, 31, 21.5, and 14.4 kDa); 2, crude SefA; 3, purified SefA; 4, E. coli JM109; 5, E. coli JM109(pTX101); 6, E. coli JM109(pDUG3A). Arrows indicate Lpp-OmpA-SefA fusion protein at ∼31 kDa.

FIG. 2.

Attachment of indicated bacterial cells to avian ovary granulosa cells: S. Enteritidis CDC9 (A), E. coli JM109(pTX101) (B), and E. coli JM109(pDUG3A) (C). Cells were stained with 10% Giemsa stain and photographed with a light microscope at ×400 magnification.

FIG. 3.

Attachment of indicated bacterial cells to HEp-2 cells: S. Enteritidis CDC9 (A), E. coli JM109(pTX101) (B), and E. coli JM109(pDUG3A) (C). Cells were stained with 10% Giemsa stain and photographed with a light microscope at ×400 magnification.