The sigA gene which is borne on the she pathogenicity island of Shigella flexneri 2a encodes an exported cytopathic protease involved in intestinal fluid accumulation

Abstract

In this study, the sigA gene situated on the she pathogenicity island of Shigella flexneri 2a was cloned and characterized. Sequence analysis showed that sigA encodes a 139.6-kDa protein which belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) subfamily of autotransporter proteins. The demonstration that SigA is autonomously secreted from the cell to yield a 103-kDa processed form and possesses a conserved C-terminal domain for export from the cell were consistent with the autotransporter pathway of secretion. Functional analysis showed that SigA is a secreted temperature-regulated serine protease capable of degrading casein. SigA was cytopathic for HEp-2 cells, suggesting that it may be a cell-altering toxin with a role in the pathogenesis of Shigella infections. SigA was at least partly responsible for the ability of S. flexneri to stimulate fluid accumulation in ligated rabbit ileal loops.

FIG. 1

Analysis of the expression and secretion of SigA in E. coli carrying the sigA gene. Concentrated culture supernatants were subjected to SDS-PAGE, and the gel was stained with Coomassie blue. Lanes: A, molecular mass markers (indicated in kilodaltons at the left); B, supernatant of DH5α/pPBA1100 (vector only); C, supernatant of DH5α/pSBA479 (sigA). The arrowhead indicates the 103-kDa secreted protein encoded by sigA.

FIG. 2

Temperature regulation of SigA. Supernatants from S. flexneri SBA1356 grown at 37°C (lane A) or 21°C (lane B) were immunoblotted and probed with rabbit antiserum raised against SigA. The arrowhead indicates the 103-kDa SigA protein expressed at 37°C.

FIG. 3

Protease activity of SigA. Proteolytic activity of concentrated supernatants derived from E. coli and S. flexneri 2a was measured as described in Materials and Methods. Columns: A, E. coli DH5α/pSBA479 (sigA); B, DH5α/pPBA1100 (vector only); C, S. flexneri 2a YSH6000T (parent); D, SBA1361 (sigA); E, SBA1351 (sigA::kan). Error bars indicate standard errors in assays performed at least in duplicate.

FIG. 4

Effect of SigA protein on epithelial cells. SigA (B) and Pet (A) proteins were added to cell cultures at a final concentration of 25 μg/ml per well and incubated for 3 h. Release from the glass substratum of the cellular focal adhesion points and rounding of cells is indicated by arrows. (C) HEp-2 cells incubated with PMSF (25 μg/ml)-treated SigA preparations. No cell rounding or detachment of focal adhesion contacts is apparent. Appropriate vector control preparations were also added (D; see text).