Salmonella-directed Recruitment of Membrane to Invasion Foci and the Role of FAK during Lipopolysaccharide-induced Inflammatory Signaling

Two central features of Salmonella pathogenesis include the invasion of host cells and the activation of an inflammatory response. Our data supports novel roles for bacterial and host proteins which contribute to each of these aspects of infection. Salmonella attachment to the intestinal epithelium triggers delivery of bacterial effector proteins into the host cytosol through a Type III Secretion System (T3SS), leading to pronounced membrane ruffling and macropinocytic uptake of the attached bacteria. SipC acts as the tip of the T3SS and is inserted into the host plasma membrane with its termini exposed in the host cytosol. We identified a direct interaction between SipC and Exo70, a component of the exocyst complex, which mediates docking and fusion of exocytic vesicles with the plasma membrane. Exocyst components precipitate with SipC and accumulate at sites of Salmonella invasion. The small GTPase, RalA, regulates assembly of the exocyst complex. We have demonstrated that RalA becomes activated during infection and that this activation is primarily due to the translocated Salmonella effector protein, SopE. Knockdown of RalA or Sec5 results in reduced membrane ruffling at sites of attachment and impairs bacterial entry into host cells. These findings suggest that Salmonella enhance invasion efficiency by promoting localized membrane expansion, directly through SipC-dependent recruitment of the exocyst and indirectly via SopE-dependent activation of RalA. These data establish a novel mechanism for Salmonella-directed recruitment of membrane to invasion foci. ii Toll-like receptor 4 (TLR4) is a host pattern recognition receptor that recognizes lipopolysaccharide (LPS), a main component of the outer membrane of Gram-negative bacteria, including Salmonella. LPS-induced activation of TLR4 initiates a signaling cascade leading to the production of inflammatory cytokines. We have shown that depletion of focal adhesion kinase (FAK) from RAW264.7 macrophages significantly impairs LPS-induced release of TNF and IL-6, and this phenotype correlates with an inhibition in the activation of the transcription factor NFB. In response to LPS, FAKdepleted macrophages also exhibit reduced RANTES gene expression, and this correlates with an inhibition in the activation of the transcription factor IRF3. These data establish a novel role for FAK during LPS-induced inflammatory cytokine production in macrophages.

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