Salmonella enteritidisRck‐mediated invasion requires activation of Rac1, which is dependent on the class I PI 3‐kinases‐Akt signaling pathway

Abstract

The Salmonella outer membrane protein Rck mediates a Zipper-like entry mechanism controlled by Rac, the Arp2/3 complex, and actin polymerization. However, little is known about the early steps leading to Rac activation and Rck-mediated internalization. The use of pharmacological inhibitors or PI 3-kinase dominant-negative mutant induced more than 80% less invasion without affecting attachment. Moreover, Rck-mediated internalization caused an increase in the association of p85 with at least one tyrosine-phosphorylated protein, indicating that class I PI 3-kinase activity was stimulated. We also report that this PI 3-kinase activity is essential for Rac1 activation. However, Rac recruitment at the Rck-mediated entry site was independent of its activation. Using a pharmacological approach or Akt-knockout cells, we also demonstrated that Akt was phosphorylated in response to Rck-mediated internalization as demonstrated by immunoblotting analysis and that all three Akt isoforms were required during this process. Overall, our results describe a signaling pathway involving tyrosine phosphorylation, class I PI 3-kinase, Akt activation, and Rac activation, leading to Rck-dependent Zipper entry. The specificity of this signaling pathway with regard to that of the type 3 secretion system, which is the other invasion process of Salmonella, is discussed.

Involvement of c-Src Tyrosine Kinase Upstream of Class I Phosphatidylinositol (PI) 3-Kinases in Salmonella Enteritidis Rck Protein-mediated Invasion

“…GST and GST-113-159 Rck were purified as described previously (4,6). Bacteria were resuspended in lysis buffer (50 mM Tris pH 8, 40 mM EDTA, 25% sucrose, 100 mM MgCl 2 , 0.2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and cOmplete protease inhibitor mixture (Roche Applied Science)) and sonicated.…”

“…After clearing, fusion proteins were affinity-purified from the soluble fraction on glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer's instructions. Purified GST alone or GST-113-159 Rck proteins were coated on latex beads (Sigma), as described previously (4,6).…”

“…Viable bacteria (intra-and extracellular) were counted after plating serial dilutions on tryptic soy agar. Invasion was quantified by the gentamicin protection assay to kill any remaining extracellular bacteria, as described previously (4,6). After a 90-min incubation with 100 g/ml gentamicin (Invitrogen), cells were washed and then lysed by adding 1 ml of cold distilled water.…”

“…After a 10-min incubation, cells were rinsed with cold PBS and solubilized by the addition of 500 l of ice-cold lysis buffer (1% Nonidet P-40, 50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, and protease inhibitor mix- ture). Immunoprecipitation was performed using anti-phosphotyrosine (4G10; Millipore) as described previously (6). In brief, after preclearance, protein concentrations of the lysates were determined, and equal quantities of total protein were used for immunoprecipitation.…”

“…Rck induces the activation of host cell signal transduction pathways, leading to the reorganization of the host actin cytoskeleton, the formation of a weak membrane rearrangement at the site of bacteria-cell contact, and the entry of the bacteria into the host cell (4). The study of Rck-mediated entry has recently enabled a model of the signaling cascade to be constructed, where interaction of Rck with its unknown cell receptor induces (p85-p110) PI 3-kinase activation through tyrosine phosphorylation of one or more unknown proteins (6). Until now, the mechanism induced by Rck, leading to tyrosine phosphorylation and class I PI 3-kinase activation, has not been characterized.…”