A Salmonella typhimurium Effector Protein SifA Is Modified by Host Cell Prenylation and S-Acylation Machinery

Anthony I Magee,Adrian P Kelly,John Trowsdale,Piero Mastroeni,James L Hutchinson,Anna T Reinicke

doi:10.1074/jbc.m500076200

Abstract

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria. It associates with the Salmonella-containing vacuole but how it interacts with the membrane is unknown. Here we show by immunofluorescence, S100 fractionation and Triton X-114 partitioning that the membrane association and targeting properties of SifA are influenced by a motif encoded within the C-terminal six amino acids. This sequence shares homology with both CAAX and Rab geranylgeranyl transferase prenylation motifs. We characterized the post-translational processing of SifA and showed that the cysteine residue within the CAAX motif is modified by isoprenoid addition through the action of protein geranylgeranyl transferase I. SifA was additionally modified by S-acylation of an adjacent cysteine residue. Similar modifications to host cell proteins regulate numerous functions including protein targeting, membrane association, protein-protein interaction, and signal transduction. This is the only known example of a bacterial effector protein that is modified both by mammalian cell S-acylation and prenylation machinery.

Highlights

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria
We characterized the post-translational processing of SifA and showed that the cysteine residue within the CAAX motif is modified by isoprenoid addition through the action of protein geranylgeranyl transferase I
We show by sequence analysis that the motif aligns with consensus motifs utilized by all three known prenyltransferases, protein geranylgeranyl transferase I (PGGT), protein farnesyl transferase (PFT), and Rab geranylgeranyl transferase (RGGT)

Summary

Introduction

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria. It associates with the Salmonella-containing vacuole but how it interacts with the membrane is unknown. Similar modifications to host cell proteins regulate numerous functions including protein targeting, membrane association, protein-protein interaction, and signal transduction This is the only known example of a bacterial effector protein that is modified both by mammalian cell S-acylation and prenylation machinery. In non-phagocytic cells it triggers its own invasion by inducing membrane ruffling and macropinocytosis [3] This is directed by a type 3 secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI-1), which translocates effector proteins into the host cell cytosol [4]. Their secretion can be induced in vitro by acidic conditions suggesting that reductions in pH, usually associated with phagosome maturation [15], may trigger SPI-2 translocation in vivo [16]

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Results

Conclusion