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.

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Salmonella enterica, like many gram-negative pathogens, uses type three secretion systems (TTSS) to infect its hosts. The three TTSS of Salmonella, namely, TTSS-1, TTSS-2, and flagella, play a major role in the virulence of this bacterium, allowing it to cross the intestinal barrier and to disseminate systemically. Previous data from our laboratory have demonstrated the involvement of the chromosomal region harboring the yfgL, engA, and yfgJ open reading frames in S. enterica serovar Enteritidis virulence. Using microarray analysis and real-time reverse transcription-PCR after growth of bacterial cultures favorable for either TTSS-1 or TTSS-2 expression, we show in this study that the deletion in S. enterica serovar Enteritidis of yfgL, encoding an outer membrane lipoprotein, led to the transcriptional down-regulation of most Salmonella pathogenicity island 1 (SPI-1), SPI-2, and flagellar genes encoding the TTSS structural proteins and effector proteins secreted by these TTSS. In line with these results, the virulence of the ⌬yfgL mutant was greatly attenuated in mice. Moreover, even if YfgL is involved in the assembly of outer membrane proteins, the regulation of TTSS expression observed was not due to an inability of the ⌬yfgL mutant to assemble TTSS in its membrane. Indeed, when we forced the transcription of SPI-1 genes by constitutively expressing HilA, the secretion of the TTSS-1 effector protein SipA was restored in the culture supernatant of the mutant. These results highlight the crucial role of the outer membrane lipoprotein YfgL in the expression of all Salmonella TTSS and, thus, in the virulence of Salmonella. Therefore, this outer membrane protein seems to be a privileged target for fighting Salmonella.Salmonella enterica infections are an important worldwide health problem. Salmonella serovars are responsible for diseases ranging from mild gastroenteritis to life-threatening systemic infections. During the course of infection, these serovars use many virulence factors, among which the type III secretion systems (TTSS) play a major role. TTSS-1, encoded by Salmonella pathogenicity island 1 (SPI-1), mainly allows intestinal epithelial cell invasion (57), thereby allowing the bacteria to cross the intestinal barrier. TTSS-2, encoded by SPI-2, is required for intracellular survival and multiplication (54) and, consequently, is important for systemic dissemination of the bacteria. The virulence phenotypes associated with SPI-1 and SPI-2 are dependent on the ability of the TTSS to deliver effector proteins into the host cell cytosol. Thus, bacteria hijack the eukaryotic cellular machinery for their own profit. The flagella, which share a common architectural design with TTSS, are involved in the motility of the bacteria and favor the interaction with the intestinal epithelium (31, 47). However, their role in Salmonella virulence remains controversial (26,27,47).We previously characterized a Salmonella enterica subsp. enterica serovar Enteritidis mutant which was altered in motility and in invasion of Cac...