Characterization of actin nucleation activity of SipC from Salmonella typhimurium

SipC is one of Salmonella SPI-1 effector proteins which are secreted from bacteria and translocated into host cells to engage actin dynamics leading to bacterial uptake. SipC possesses dual effector translocation and actin-modulating functions. SipC was identified as the first example of direct pathogen-encoded actin nucleator. In order to demonstrate the biological significance of actin nucleation activity of SipC during Salmonella invasion, we have delineated the actin nucleation activity from its effector translocation activity. Our data show that the central amino acid 201-220 region is essential for its actin nucleation activity and the amino acid 321-409 region is required for its effector translocation activity. A SipC nucleation deficient mutant, which maintained its effector translocation activity, had significantly reduced ability to induce actin cytoskeleton rearrangements resulting in lower bacterial invasion into HeLa cells. Furthermore, we investigated how SipC nucleates actin filaments. Our study shows that SipC199-409 promotes actin assembly from the barbed-end and remains associated at the pointed-end of actin filaments. We found that SipC199-409 forms transient multimers and stabilized dimers are active in actin assembly both in vitro and in vivo. Biochemical cross-link analysis suggested that it might form parallel dimers in an extended conformation. Previously identified amino acid residues 201-220 are essential for efficient dimer formation and required for causing mouse colitis in a mouse model. Our results allowed us to present a model in which SipC forms transient multimers to promote actin nucleation. We then tried to assess how SipC and other Salmonella actin-modulators cooperate during Salmonella invasion. We show that nucleation activity of SipC functionally cooperates with SipA in vivo and contributes to Salmonella entry in a ruffling independent manner. We also demonstrate that nucleation activity of SipC is involved in the initial cell adhesion process. Profuse membrane ruffles which engulf bacteria are then induced by SopB, SopE or SopE2. These data represent a more detailed view of Salmonella entry process which involves dynamic actin cytoskeletons and allow us to propose a novel mechanism by which two bacterial actin-binding proteins contribute to Salmonella invasion through SipC-mediated cell adhesion