Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells

Abstract

Type III secreted Sop protein effectors are delivered into target eukaryotic cells and elicit cellular responses underlying Salmonella pathogenicity. In this work, we have identified another secreted protein, SopE2, and showed that SopE2 is an important invasion-associated effector. SopE2 is encoded by the sopE2 gene which is present and conserved in pathogenic strains of Salmonella. SopE2 is highly homologous to SopE, a protein encoded by a gene within a temperate bacteriophage and present in only some pathogenic strains.

FIG. 1

Analysis of the proteins secreted by different Salmonella strains (sipB mutants). The secreted proteins deposited by each strain into proteinaceous filaments were isolated as described earlier (17). The protein samples from serovar Dublin B1 (lane 1), Salmonella enterica serovar Gallinarum S9.B1 (lane 2), Salmonella enterica serovar Enteritidis S13.B1 (lane 3), serovar Typhimurium F98.B1 (lane 4), serovar Typhimurium ST4/74.B1 (lane 5), serovar Typhimurium ST5306.B1 (lane 6), and Salmonella enterica serovar Choleraesuis A57.B1 (lane 7) were separated by SDS–12% PAGE and were stained with Coomassie brilliant blue (A) or were transferred onto nitrocellulose membrane and probed with an anti-SopE monoclonal antibody (B). Molecular mass is shown on the left.

FIG. 2

Sequence alignment of serovars Dublin SopE and Typhimurium F98 SopE2. Lines indicate identical amino acids, and colons and periods indicate conservative amino acid substitutions.

FIG. 3

Western blot analysis of the secreted proteins of the wild-type serovar Typhimurium F98 (lane 2), serovar Typhimurium SE2.1 sopE2 mutant (lane 3), and serovar Typhimurium SE2.1/pSopE2st transcomplemented sopE2 mutant (lane 4) strains. Culture supernatant proteins from different Salmonella strains were prepared by precipitation with 10% trichloroacetic acid as previously described (17), were separated by SDS–PAGE, were transferred to nitrocellulose membrane, and were probed with anti-SopE2 monoclonal antibody.

FIG. 4

Invasion of HeLa cells by the wild-type serovar Typhimurium F98, serovar Typhimurium SE2.1 sopE2 mutant, and serovar Typhimurium SE2.1/ pSopE2st transcomplemented sopE2 mutant strains. All strains were grown overnight at 26°C, then diluted 10-fold and grown at 37°C to an optical density at 600 nm of 0.5. The cells were infected with bacteria at an approximate ratio of 1:10. HeLa cells were infected for 1 h and then washed and exposed to gentamicin (100 μg/ml) treatment for 1 h. The cells were lysed with 1% Triton X-100, and the numbers of bacteria surviving gentamicin treatment were calculated by viable count. The experiments were carried out in triplicate. Each bar is derived from the mean of three samples per strain. The error bars indicate standard errors of the means calculated from triplicate plating.