N-terminal conservation of putative type III secreted effectors of Salmonella typhimurium.

Abstract

Sir, Salmonella typhimurium is a facultative intracellular pathogen that can cause gastroenteritis in humans and a systemic disease in susceptible mice. Essential for the full virulence of this pathogen are two type III secretion systems and their effectors, which are translocated into the host cell. The first system, encoded by Salmonella Pathogenicity Island (SPI) −1 is required for efficient colonization of the intestinal epithelium, while the SPI-2 encoded system is essential for survival and replication in macrophages during systemic phases of disease (Groisman et al., 1999, Pathogenicity islands and the evolution of salmonella virulence. In Pathogenicity Islands and Other Mobile Virulence Elements. Kaper and Hacker (eds). Washington, DC: American Society for Microbiology Press, pp. 127–150). While these SPIs appear to constitute complete virulence packages, recent evidence has demonstrated that some translocated effectors of these systems are encoded outside the two pathogenicity islands. A recent paper by Miao et al. (Miao et al., 1999, Mol Microbiol34: 850–864) characterizes two novel effectors of S. typhimurium, SspH 1 and SspH 2. These effectors are not encoded within either SPI and contain leucine-rich repeats. The authors provide strong evidence that SspH 1 and SspH 2 are type III secreted effectors: fusions to the catalytic domain of adenylate cyclase from Bordetella pertussis (CyaA) are delivered into host cells in a type III secretion-dependent manner. Importantly, the N-terminal 208 codons of sspH 1 and 211 codons of sspH 2 (not including the Leucine-rich repeats) are sufficient for translocation of the CyaA moiety. This suggests that the N-termini of each effector contains a translocation signal for delivery into the host cell. Based on the similarity of SspH 1 and SspH 2 with SlrP, another Leucine-rich repeat-containing protein required for S. typhimurium virulence in mice (Tsolis et al., 1999, Infect Immun67: 6385–6393), Miao et al. suggest it is likely that SlrP is also a type III secreted effector protein. We found a region of similarity shared between these proteins (Fig. 1) that was also present in SifA, a virulence factor previously described for its role in the formation of Salmonellainduced filaments (Sifs) in epithelial cells (Stein et al., 1996, Mol Microbiol20: 151–164). These elongated filaments were identified by immunostaining with antibodies to lysosomal glycoproteins and are formed only by intracellular S. typhimurium (Garcia-del Portillo et al., 1993, Proc Natl Acad Sci USA90: 10544–10548). Mutants of sifA, encoded within the potABCD operon at ≈ 27 cs on the bacterial chromosome (outside both SPIs), do not form Sifs and are attenuated for systemic virulence in mice. Partial multiple alignment of putative effectors of S. typhimurium type III secretion systems. Identical residues are in black, well conserved residues in dark grey and least conserved residues in light grey. Alignments were performed with Clustal W and coloured with Boxshade through the Biology Workbench 3.2 server (http://workbench.sdsc.edu/CGI/BW. cgi). While the function of SifA remains unknown, its similarity to SspH 1 and SspH 2 within their N-terminal translocation domain (but not the remainder of the protein) suggests that SifA is also a type III secreted effector of S. typhimurium. The highest similarity was found between SifA and SspH 2, which are 36% identical within their first 50 amino acids. In their paper, Miao et al. demonstrate that translocation of SspH 2 is dependent on the SPI-2-encoded type III secretion system, implicating SPI-2 in the delivery of SifA into host cells. This possibility is consistent with the fact that Sif formation is witnessed 6 h after infection, when SPI-2 genes are maximally induced (Cirillo et al., 1998, Mol Microbiol, 30: 175–188). Further evidence suggesting a role for SPI-2 in SifA translocation comes from the previous observation that the OmpR–EnvZ two component regulatory system is necessary for Sif formation (Mills et al., 1998, Infect Immun66: 1806–1811) and the recent finding that SPI-2 is regulated, in part, by this system (Lee et al., 2000, J Bacteriol, 182: 771–781). Finally, we have recently determined that S. typhimurium mutants with a deletion in ssaR, a putative component of the SPI-2 type III secretion apparatus, do not form Sifs in infected HeLa cells (data not shown). Together, these findings suggest that SifA is a translocated effector of the SPI-2 type III secretion system. We performed a search of the S. typhi and S. typhimurium genome projects for proteins with similarity to SifA. S. typhi encodes a SifA homologue 90% identical to S. typhimurium SifA, consistent with the observation that intracellular S. typhi can form Sifs in epithelial cells (Mills and Finlay, 1994, Microb Pathog, 17: 409–423). Translated sequences that encode two putative SifA-related proteins were also identified. SifA-related 1 was identified on contig 1370 of S. typhimurium LT2 and its S. typhi homologue found on contig 418. A second putative SifA-related protein was found in the LT2 database on contig 1495. Of note, similarity was limited to the N-termini of these proteins, suggesting that each may perform a unique function. Our analysis suggests the existence of two families of type III secreted effectors in S. typhimurium which share considerable N-terminal similarity: a Leucine-rich repeat family (SspH 1, SspH 2 and SlrP) and a family of SifA-related proteins. Whether the conserved N-terminal region constitutes, in whole or in part, a secretion/translocation signal and how this region might confer targeting to either of the type III secretion apparati in S. typhimurium remains to be determined. Because the putative effectors encoded within SPI-2 lack this conserved N-terminal region, its role as a translocation signal may be limited to the delivery of SPI-2 effectors encoded outside of the second pathogenicity island. Genomic sequence data of S. typhi were obtained through the Sanger Centre (http://www.sanger.ac.uk) and the sequence data of S. typhimurium LT2 obtained from Washington University School of Medicine (http://genome.wustl.edu/gsc). This work was supported by the Medical Research Council of Canada.