How SipA, a toxin from Salmonella, increases stability of F-actin.

Abstract

Salmonella infection is a disease that is mainly limited to the intestinal tract, but can also be systemic and may be deadly for young children or people with a weakened immune system. Salmonella strains produce protein toxins that target cytoplasmic actin to promote bacterial uptake by a host cell, which is essential for pathogenicity. The SipA protein is a Salmonella effector delivered into the host cell by bacterial type III secretion system. SipA has been reported to stabilize F-actin upon binding, and to promote actin polymerization in vitro. Only very low resolution reconstructions from negative stain existed to show how SipA binds to actin. Here, we present a 3.5Å cryo-EM reconstruction of the SipA425-685/F-actin complex. Our reconstruction shows that SipA binds actin with nearest neighbor exclusion along actin's long-pitch strand. The globular SipA domain binds actin at subunit n through ionic and hydrophobic interactions with both actin strands with nanomolar affinity. The extended C-terminal tail is inserted in between the two actin strands at subunit n+2 preventing the binding of another SipA molecule at this site. Thus, SipA will be bound at n, n+1, n+4, n+5, etc. The C-terminal tail of SipA binds to the same site as two F-actin toxins, phalloidin (Pha) and jasplakinolide (Jasp). All three molecules interact with actin mainly through hydrophobic interactions, however SipA binds F-actin with higher affinity than Pha or Jasp, and outcompetes the latter two in competitive binding assay. The SipA binding involves conformational changes of actin's D-loop which provides additional intra-strand interactions. The observed stabilization of actin filaments upon SipA binding is therefore likely due to the introduction of additional inter- and intra-strand interactions within the actin filament.