Secreted effector proteins of Salmonella dublin act in concert to induce enteritis

Abstract

The ability of enteropathogenic salmonellae to recruit inflammatory cells and induce secretory responses in the infected ileum is considered to be a main feature in Salmonella-induced enteritis. Interactions between the pathogen and intestinal epithelial cells result in a variety of cellular responses mediating inflammation and fluid secretion. It is becoming apparent that proteins secreted by the Inv-Spa type III secretion system of Salmonella spp. play a key role in the induction of these responses. We have recently demonstrated that the SopB effector protein is translocated into eukaryotic cells via a Sip-dependent pathway and mediates inflammation and fluid secretion in infected ileal mucosa. However, SopB did not appear to be the only effector involved, as inactivation of the sopB gene only partially impaired enteropathogenicity. We suggested that at least some of such protein effectors are likely to be proteins of the same class as SopB, i.e., secreted effector proteins translocated into eukaroyotic cells via a Sip-dependent pathway. In this work, we identify SopD, another secreted protein belonging to the family of Sop effectors of Salmonella dublin. Using the cya reporter system we showed that SopD is translocated into eukaroyotic cells. We assessed the potential involvement of SopD in enteropathogenicity and found that inactivation of sopD has an additive effect in relation to the sopB mutation.

FIG. 1

Sequence alignment of the N-terminal amino acid sequences of S. dublin SopD and S. typhimurium ORF4 (a hypothetical 36.1-kDa protein encoded by a gene located downstream of the cysJIH operon [16]). The SopD protein was isolated from filaments produced by S. dublin (28) and was purified by SDS-PAGE. The N-terminal amino acid sequence was determined by Edman degradation.

FIG. 2

SDS-PAGE analysis of the proteins secreted by different S. dublin strains. The secreted proteins deposited into filaments were isolated as described earlier (28). The samples were separated by SDS–12% PAGE and stained with Coomassie blue.

FIG. 3

Southern blot analysis showing conservation of sopD in different Salmonella serotypes. Total cell DNA was isolated from S. choleraisuis A57 (lane 1), S. dublin 2229 (lane 2), S. enteritidis 149 (lane 3), S. gallinarum 287/91 (lane 4), S. pullorum 449/87 (lane 5), and S. typhimurium 12/75 (lane 6) and digested with HpaI. A DNA fragment including the full-length sopD was amplified by PCR, labelled, and used as a probe. The positions of size markers (in kilobases) are indicated on the left. The sopD gene of S. dublin is known to contain one HpaI site.

FIG. 4

Accumulation of cAMP in HeLa cells over a period of time (in minutes) as a measure of the translocation of the SopD-Cya fusion protein mediated by S. dublin 2229 (squares) and S. dublin 2229(pMJ600) (circles). Filled symbols represent data from HeLa cells incubated in the presence of cytochalasin D (1 μg/ml). Each point is the mean result of samples from three wells. Error bars indicate standard deviations.

FIG. 5

Accumulation of cAMP within J774.2 cells in relation to total cell protein at 1 h (open bars) and 2 h (solid bars) after infection with different S. dublin strains harboring plasmid pMJ600. Each bar represents the mean result of samples from four wells. Error bars indicate standard deviations. cAMP values are given as nanomoles per microgram of protein.

FIG. 6

Effects of sopD, sopB, and double sopD-sopB mutations on the ability of S. dublin to induce fluid secretion and PMN influx. S. dublin 2229 (wild type), SB2 (sopB), SD1 (sopD), and SB2SD1 (sopB sopD) were used in ligated loop assays. The secretory response is defined as the volume of fluid within a loop per length of loop. The PMN influx is defined as radioactive counts of PMNs within test loops per radioactive count of PMNs in negative-control loops. Values are the means of three loops and are representative of results from three independent experiments. Error bars indicate standard deviations.