Functional dissection of SiiE, a giant non-fimbrial adhesin of Salmonella enterica

Abstract

SummarySalmonella enterica deploys the giant non-fimbrial adhesin SiiE to adhere to the apical side of polarized epithelial cells. The establishment of close contact is a prerequisite for subsequent invasion mediated by translocation of effector proteins of the Salmonella Pathogenicity Island 1 (SPI1)-encoded type III secretion system (T3SS). Although SiiE is secreted into the culture medium, the adhesin is retained on the bacterial envelope in the phase of highest bacterial invasiveness. To dissect the structural requirements for secretion, retention and adhesive properties, comprehensive deletional and functional analyses of various domains of SiiE were performed. We observed that b-sheet and coiled-coil domains in the N-terminal moiety of SiiE are required for the control of SiiE retention on the surface and co-ordinated release. These results indicate a novel molecular mechanism for the control of surface display of a T1SS-secreted adhesin that acts cooperatively with the SPI1-T3SS.

Gaussia princeps Luciferase as a Reporter for Transcriptional Activity, Protein Secretion, and Protein-Protein Interactions in Salmonella enterica Serovar Typhimurium

“…3C). Deletion of the first 15 N-terminal amino acids of Ig53 abolished the ability to mediate T1S of Gluc, which is consistent with previous results from SiiE mutational analysis (28). As a control, we did Western blots to detect the secreted fusion proteins in precipitated supernatants.…”

“…To test whether Gluc is suitable monitoring type I secretion (T1S), we set out to characterize the C-terminal signal sequence of the T1SS substrate SiiE. Previously, mutational analyses in conjunction with Western blots and a customized SiiE-specific enzyme-linked immunosorbent assay (ELISA) led to the identification of a C-terminal portion including Ig domain 53 to be essential for mediating SiiE transport via the siiCDF-encoded T1SS (11,28). In a complementary setup, we constructed fusion proteins consisting of an N-terminal GlucM43I and different C-terminal moieties of SiiE to define the minimal secretion signal.…”

“…We used this improved Gluc variant in fusion proteins together with domains of the SPI-4-encoded adhesin SiiE to quantify T1SS-dependent secretion. Previous results from the analysis of different SiiE deletion mutants with ELISA underscored the importance of the C-terminal part, including the complete Ig53 domain for secretion of the adhesin (28). We addressed this question in a different way, defining the SiiE C-terminal region including Ig53 to be essential and sufficient for mediating T1S of a heterologous N-terminal domain (Gluc).…”

“…The T1SS substrate SiiE, a giant nonfimbrial adhesion, is secreted via a C-terminal signal sequence and subsequently mediates intimate contact to the apical host cell membrane, allowing SPI-1-dependent effector injection and bacterial invasion (6). SiiE is of repetitive structure, comprising 53 domains of the immunoglobulin (Ig) fold as well as beta-sheets and coiled-coils within its N terminus (28).…”

Gallbladder Epithelium as a Niche for Chronic Salmonella Carriage

“…These DGEC have been intensively used for physiological studies, which supported their use as a model for human gallbladder epithelial cells (24)(25)(26)(27)(28)(29). The percentage of S. Typhimurium invasion obtained in this study is relatively lower than results of other studies with polarized epithelial (30)(31)(32).…”