Mutational analysis of Salmonella translocated effector members SifA and SopD2 reveals domains implicated in translocation, subcellular localization and function

• Published in: Microbiology (Society for General Microbiology) Vol. 152; no. 8; pp. 2323 - 2343
• Main Authors: Brown, Nat F, Szeto, Jason, Jiang, Xiuju, Coombes, Brian K, Finlay, B. Brett, Brumell, John H
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01-08-2006; Society for General Microbiology
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Bacterial Proteins - analysis; Bacterial Proteins - chemistry; Bacterial Proteins - physiology; Bacteriology; Base Sequence; Biological and medical sciences; Cell Line; Fundamental and applied biological sciences. Psychology; Genetics; Glycoproteins - analysis; Glycoproteins - chemistry; Glycoproteins - physiology; Golgi Apparatus - metabolism; Humans; Mice; Microbiology; Molecular Sequence Data; Protein Structure, Tertiary; Protein Transport; Salmonella enterica; Structure-Activity Relationship; Translocation; Salmonella; Mutagenesis; Pathogenic; Bacteria; Recombinant protein; Localization; Fluorescence microscopy; Enterobacteriaceae
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.28995-0

1 Michael Smith Laboratories and Departments of Biochemistry and Molecular Biology, Microbiology, and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
2 Infection, Immunity, Injury, and Repair Program, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
3 Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada
4 Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada
Correspondence John H. Brumell john.brumell{at}sickkids.ca
Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen causing disease in several hosts. These bacteria use two distinct type III secretion systems that inject effector proteins into the host cell for invasion and to alter maturation of the Salmonella -containing vacuole. Members of the Salmonella translocated effector (STE) family contain a conserved N-terminal translocation signal of approximately 140 aa. In this study, the STE family member SifA was examined using deletion strategies. Small deletions (approx. 20 residues long) throughout SifA were sufficient to block its secretion and/or translocation into host cells. Transfection of HeLa cells with a GFP-SifA fusion was previously shown to be sufficient to induce formation of Sif-like tubules resembling structures present in Salmonella -infected cells. The present study showed that both N- and C-terminal domains of SifA are required for this phenotype. Furthermore, both domains could induce aggregation of Lamp1-positive compartments, provided they were coupled to the minimal C-terminal membrane-anchoring motif of SifA. Mutation or deletion of the conserved STE N-terminal WEK(I/M)xxFF translocation motif of SopD2 disrupted its association with Lamp1-positive compartments, implicating these residues in both effector translocation and subcellular localization. Interestingly, one GFP-SifA deletion mutant lacking residues 42–101, but retaining the WEK(I/M)xxFF motif, targeted the Golgi apparatus. In addition, short peptides containing the signature WEK(I/M)xxFF motif derived from the N-termini of Salmonella effectors SopD2, SseJ and SspH2 were sufficient to localize GFP to the Golgi. These studies suggest that Salmonella effectors contain multifunctional motifs or domains that regulate several effector traits, including protein secretion/translocation, localization and subversion of host cell systems. Conditions that perturb the tertiary structure of effectors can influence their localization in host cells by liberating cryptic intracellular targeting motifs.
Abbreviations: BFA, brefeldin A; Lamp1, lysosome-associated membrane protein 1; SCV, Salmonella -containing vacuole; Sif, Salmonella -induced filament; SPI, Salmonella pathogenicity island; STE, Salmonella translocated effector; T3SS, type III secretion system; TAPAS-1, tryptophan anchoring phosphatidic acid selective binding domain 1
These authors contributed equally to the work.