Identification of cognate host targets and specific ubiquitylation sites on the Salmonella SPI-1 effector SopB/SigD.
Salmonella enterica is a bacterial pathogen responsible for enteritis and typhoid fever. Virulence is linked to two Salmonella pathogenicity islands (SPI-1 and SPI-2) on the bacterial chromosome, each of which encodes a type III secretion system. While both the SPI-1 and SPI-2 systems secrete an array of effectors into the host, relatively few host proteins have been identified as targets for their effects. Here we use stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry-based proteomics to identify the host targets of the SPI-1 effector, SopB/SigD. The only host protein found to bind immunoprecipitated SopB was the small G-protein Cdc42. The interaction was confirmed by reciprocal immunoprecipitation, and Cdc42 also bound glutathione S-transferase-fused SopB and SopB delivered through infection by the bacteria, confirming the interaction by an orthogonal method and in a more physiological context. The region of SopB responsible for the interaction was mapped to residues 117-168, and SopB is ubiquitylated at both K19 and K541, likely as monoubiquitylation. SopB colocalizes with activated Cdc42 near the plasmalemma, but we found no evidence that SopB alone can alter Cdc42 activity. This approach is also widely applicable to identify binding partners to other bacterial effectors.
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada.
Lindsay D
LD
Rogers
Anders R
AR
Kristensen
Erin C
EC
Boyle
Dale P
DP
Robinson
Richard T
RT
Ly
B Brett
BB
Finlay
Leonard J
LJ
Foster
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D019779:Salmonella enterica / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion; D054875:Ubiquitination; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; C419942:Spi1 protein, Salmonella; C489224:SopB protein, Salmonella; D020764:cdc42 GTP-Binding Protein