A host‐specific virulence protein of Erwinia herbicola pv. gypsophilae is translocated into human epithelial cells by the Type III secretion system of enteropathogenic Escherichia coli

Abstract

summary HsvG is a virulence factor that determines the host specificity of Erwinia herbicola pathovars gypsophilae and betae on gypsophila. We used the calmodulin adenylate cyclase reporter (CyaA) to demonstrate that HsvG is secreted and translocated into HeLa cells by the type III secretion system (TTSS) of the enteropathogenic Escherichia coli (EPEC). A fusion of HsvG-CyaA containing 271 amino acids of the N-terminus of HsvG were introduced into a wild-type EPEC, espB mutant deficient in translocation and an escV mutant deficient in secretion. A significant secretion was detected in EPEC/HsvG-CyaA and its espB mutant, but not with the escV mutant. Translocation was only observed with the wild-type EPEC, and not with the other two mutants. To localize the secretion and translocation signals of HsvG, fusions containing 39, 11 and 3 amino acids of the N-terminus of HsvG were constructed and expressed in EPEC. A fusion containing the first 39 N-terminal amino acids of HsvG was secreted and translocated at significant level (31-35%) as compared to the original fusion. In contrast, fusions containing the 3 and 11 amino acids failed to be secreted and translocated.

Analysis of the Expression, Secretion and Translocation of the Salmonella enterica Type III Secretion System Effector SteA

“…Plasmids expressing CyaA' fusions were derivatives of pIZ1673, a modification of pSIF003-R1 [81] (a gift from I. Rosenshine, the Hebrew University of Jerusalem) that was constructed by deletion of the lacI gene using enzymes Hpa I and Sph I. Therefore, expression of CyaA fusions from derivatives of pIZ1673 is no longer dependent on IPTG.…”

The Salmonella SpiC protein targets the mammalian Hook3 protein function to alter cellular trafficking

“…The resulting product was digested with Bam HI and cloned into the Bam HI site of plasmid pSIF003‐R1 (a gift from Ilan Rosenshine). Plasmid pSIF003‐RI was designed for constructing gene fusions encoding chimeras between a protein of interest and CyaA (Valinsky et al ., 2002; Tu et al ., 2003). This plasmid was derived from pEX‐CyaA1‐412 (Wolff et al ., 1998) by deleting the His‐tag and the ribosomal binding site (RBS), but preserving the unique Bam HI site for cloning.…”

“…The cloning of a DNA insert containing an RBS sequence into the Bam HI site in‐frame with cyaA creates an active CyaA fusion. In addition, the lacI gene was cloned into pSIF003‐RI in order to ensure the tight regulation of CyaA expression from the p tac promoter (Valinsky et al ., 2002; Tu et al ., 2003).…”

pthG from Pantoea agglomerans pv. gypsophilae encodes an avirulence effector that determines incompatibility in multiple beet species

“…A direct translocation of PthG into beet or gypsophila cells has not yet been demonstrated. However, as reported for the HsvG effector located on the pPATH Pag (Valinsky et al ., 2002), it has been shown that PthG can be translocated into human HeLa cells via the TTSS system of enteropathogenic Escherichia coli (Ezra et al . 2002).…”

“…A direct translocation of PthG into beet or gypsophila cells has not yet been demonstrated. However, as reported for the HsvG effector located on the pPATH Pag (Valinsky et al, 2002), it has been shown that PthG can be translocated into human HeLa cells via the TTSS system of enteropathogenic Escherichia coli (Ezra et al 2002). Bombardment of intact pthG ( pGreen-sGFP-pthG) into host leaves (beet) resulted in a massive cell death as evidenced by the drastic reduction in the number of points of GFP co-expression when compared to cells bombarded with a construct containing a deletion of the C-terminus of the pthG (pGreen-sGFP-pthG∆C).…”