Rck of Salmonella enterica, subspecies enterica serovar Enteritidis, mediates Zipper-like internalization

Abstract

Salmonella can invade non-phagocytic cells through its type III secretion system (T3SS-1), which induces a Trigger entry process. This study showed that Salmonella enterica, subspecies enterica serovar Enteritidis can also invade cells via the Rck outer membrane protein. Rck was necessary and sufficient to enable non-invasive E. coli and Rckcoated beads to adhere to and invade different cells. Internalization analysis of latex beads coated with different Rck peptides showed that the peptide containing amino acids 140-150 promoted adhesion, whereas amino acids between 150 and 159 modulated invasion. Expression of dominant-negative derivatives and use of specific inhibitors demonstrated the crucial role of small GTPases Rac1 and Cdc42 in activating the Arp2/3 complex to trigger formation of actin-rich accumulation, leading to Rck-dependent internalization. Finally, scanning and transmission electron microscopy with Rck-coated beads and E. coli expressing Rck revealed microvillus-like extensions that formed a Zipper-like structure, engulfing the adherent beads and bacteria. Overall, our results provide new insights into the Salmonella T3SS-independent invasion mechanisms and strongly suggest that Rck induces a Zipper-like entry mechanism. Consequently, Salmonella seems to be the first bacterium found to be able to induce both Zipper and Trigger mechanisms to invade host cells.

Heterogeneity of type III secretion system (T3SS)-1-independent entry mechanisms used by Salmonella Enteritidis to invade different cell types

“…3b). Moreover, some actin remodelling observed with the invA mutant was similar to that described for the Zipper entry process mediated by Rck (Rosselin et al, 2010). Local actin accumulation was best visualized with scattered bacteria.…”

“…The S. Enteritidis LA5 wild-type strain (Nal R ) is a field isolate from infected chickens (AllenVercoe et al, 1997). Construction of the Drck mutant of strain LA5 (Nal R ), the Drck mutant complemented with a pUC19 vector carrying rck (Drck-rck) (Cb R ) and the invA mutant has been previously described (Fardini et al, 2007;Rosselin et al, 2010). The invADrck mutant (Km R ) was obtained by transduction of the Drck : : Cm mutation from strain LA5 Drck : : Cm into the LA5 invA : : aphDT mutant using P22HT105int, as described by Schmieger (1999).…”

“…Rck is known to be involved in Salmonella invasion in a T3SS-1-independent manner, and is poorly expressed at the different growth phases (exponential, stationary, with Salmonella T3SS-1-independent invasion mechanisms and without shaking) in standard in vitro culture conditions (Kim & Surette, 2006;Rosselin et al, 2010). To investigate whether Rck was involved in the in vitro invasion assays described above, we analysed Rck expression under the conditions used for those experiments.…”

“…In contrast to the Trigger mechanism, Salmonella can also invade cells by a Zipper entry process through the Rck outermembrane protein, which induces a local accumulation of actin, leading to a discrete membrane alteration (Cossart & Sansonetti, 2004;Rosselin et al, 2010). Rck is sufficient to promote entry in a T3SS-1-independent manner.…”

“…However, its contribution to Salmonella virulence remains unclear. It has been shown in vitro that rck expression is induced by SdiA (Ahmer et al, 1998;Rosselin et al, 2010), a quorumsensing regulator, but the conditions of in vivo Rck expression have not yet been characterized. In addition to Rck, PagN is another invasin whose entry mechanism remains uncharacterized (Lambert & Smith, 2008).…”