Salmonella causes severe gastroenteritis in humans, entering non‐phagocytic cells to initiate intracellular replication. Bacterial engulfment occurs by macropinocytosis, which is dependent upon nucleation of host cell actin polymerization and condensation (‘bundling’) of actin filaments into cables. This is stimulated by contact‐induced delivery of an array of bacterial effector proteins, including the four Sips ( Salmonella invasion proteins). Here we show in vitro that SipC bundles actin filaments independently of host cell components, a previously unknown pathogen activity. Bundling is directed by the SipC N‐terminal domain, while additionally the C‐terminal domain nucleates actin polymerization, an activity so far known only in eukaryotic proteins. The ability of SipC to cause actin condensation and cytoskeletal rearrangements was confirmed in vivo by microinjection into cultured cells, although as SipC associates with lipid bilayers it is possible that these activities are normally directed from the host cell membrane. The data suggest a novel mechanism by which a pathogen directly modulates the cytoskeletal architecture of mammalian target cells.

Pathogen‐induced remodelling of the host cell actin cytoskeleton drives internalization of invasive Salmonella by non‐phagocytic intestinal epithelial cells. Two Salmonella actin‐binding proteins are involved in internalization: SipC is essential for the process, while SipA enhances its efficiency. Using purified SipC and SipA proteins in in vitro assays of actin dynamics and F‐actin bundling, we demonstrate that SipA stimulates substantially SipC‐mediated nucleation of actin polymerization. SipA additionally enhances SipC‐ mediated F‐actin bundling, and SipC–SipA collaboration generates stable networks of F‐actin bundles. The data show that bacterial SipC and SipA cooperate to direct efficient modulation of actin dynamics, independently of host cell proteins. The ability of SipA to enhance SipC‐induced reorganization of the actin cytoskeleton in vivo was confirmed using semi‐ permeabilized cultured mammalian cells.

Salmonellae employ two type III secretion systems (T3SSs), SPI1 and SPI2, to deliver virulence effectors into mammalian cells. SPI1 effectors, including actin-binding SipA, trigger initial bacterial uptake, whereas SPI2 effectors promote subsequent replication within customized Salmonella-containing vacuoles (SCVs). SCVs sequester actin filaments and subvert microtubule-dependent motors to migrate to the perinuclear region. We demonstrate that SipA delivery continues after Salmonella internalization, with dosage being restricted by host-mediated degradation. SipA is exposed on the cytoplasmic face of the SCV, from where it stimulates bacterial replication in both nonphagocytic cells and macrophages. Although SipA is sufficient to target and redistribute late endosomes, during infection it cooperates with the SPI2 effector SifA to modulate SCV morphology and ensure perinuclear positioning. Our findings define an unexpected additional function for SipA postentry and reveal precise intracellular communication between effectors deployed by distinct T3SSs underlying SCV biogenesis.

Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform–ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking ‘pump-action’ conformational changes that underpin effector injection.