Crystal Structure of the Salmonella enterica Serovar Typhimurium Virulence Factor SrfJ,a Glycoside Hydrolase Family Enzyme

To cause infection, Salmonella enterica serovar Typhimurium uses type III secretion systems, which are encoded on two Salmonella pathogenicity islands, SPI-1 and SPI-2, the latter of which is thought to play a crucial role in bacterial proliferation in Salmonella-containing vacuoles (SCVs) after invading cells. S. Typhimurium SrfJ, located outside SPI-2, is also known to be involved in Salmonella pathogenicity and has high amino acid sequence homology with human lysosomal glucosylceramidase (GlcCerase). We present the first crystal structure of SrfJ at a resolution of 1.8 Å. The overall fold of SrfJ shares high structure similarities with that of human GlcCerase, comprising two distinctive domains: a (β/α)₈-barrel catalytic domain and a β-sandwich domain. As in human GlcCerase, the pocket-shaped active site of SrfJ is located on the C-terminal side of the barrel, and two conserved glutamic acid residues are used for the enzyme catalysis. Moreover, a glycerol-bound form of SrfJ reveals that the glucose ring moiety of the substrate might similarly bind to the enzyme as to human GlcCerase, suggesting that SrfJ might function as a glycoside hydrolase. Although some structural differences are observed between SrfJ and human GlcCerase in the substrate entrance of the active site, we speculate that, based on the high structural similarities to human GlcCerase in the overall fold and the active-site environment, SrfJ might have a GlcCerase activity and use the activity to enhance Salmonella virulence by modifying SCV membrane lipids.Gram-negative bacterial pathogens deliver effector proteins into host cells through type III secretion systems (TTSS). The TTSS apparatus is a molecular syringe which spans the inner and outer membranes of pathogens and secretes translocon and effector proteins. Translocon proteins locate at the tip of the needle structure and are involved in the translocation of effector proteins by forming pores in the host cell membrane (3). The translocated effector proteins function to manipulate diverse host cellular processes such as cytoskeleton assembly, vesicle transport, and signal transduction, thereby promoting bacterial virulence (9).Salmonella enteric serovar Typhimurium (S. Typhimurium) causes a systemic infection in mice and is an intensively studied model of typhoid fever. This gram-negative bacterium can invade host cells and then survive by replicating within a membrane-bound compartment known as the Salmonella-containing vacuole (SCV) (16). Both invasion and intracellular survival are mediated by numerous virulence genes, which are clustered within the pathogenicity islands, SPI-1 and SPI-2 (18). The regulation of virulence proteins encoded by each pathogenicity island depends on the different stages of infection. While most of the genes within SPI-1 are required for the invading host cells and the early stages of SCV development (8), the genes in SPI-2 play a crucial role in bacterial proliferation in SCVs after cell invasion (23). The SsrA-SsrB two-component regulatory system is known to regulate the expression of genes within SPI-2 for bacterial virulence (4). Recent works have shown that several genes located outside of SPI-2 are under the control of the SsrA-SsrB regulator as well, and these have been proposed as putative virulence factors (10, 25).S. Typhimurium SrfJ was initially identified as a gene that is strongly activated by SsrB outside SPI-2 (25). Furthermore, a mutation on srfJ leads to mild attenuation of virulence in mice (22). Interestingly, SrfJ shares high amino acid sequence similarity with human lysosomal glucosylceramidase (GlcCerase) (25), which is a peripheral membrane protein catalyzing the hydrolysis of glucosylceramide (GlcCer) to β-glucose and ceramide in the presence of the modulator protein saposin C and lipid (11). Inherited defects in GlcCerase result in lysosomal GlcCer accumulation and, as a consequence, Gaucher disease, the most common lysosomal storage disease (19). Both human GlcCerase and SrfJ have been grouped into glycoside hydrolase (GH) family 30 containing GlcCerase (EC 3.2.1.45), β-1,6-glucanse (EC 3.2.1.75), and β-xylosidase (EC 3.2.1.37) of the GH-A clan in the CAZy database (http://afmb.cnrs-mrs.fr/CAZY). Among the members of GH family 30, structural information is available only on the human enzyme. The biochemical function of SrfJ and its role in Salmonella virulence remain to be elucidated. In order to better understand the function of SrfJ, we have determined the crystal structure of SrfJ from S. Typhimurium at a resolution of 1.8 Å.