Investigation of the invasion mechanism mediated by the outer membrane protein PagN of Salmonella Typhimurium

Abstract

Background Salmonella can invade host cells via a type three secretion system called T3SS-1 and its outer membrane proteins, PagN and Rck. However, the mechanism of PagN-dependent invasion pathway used by Salmonella enterica, subspecies enterica serovar Typhimurium remains unclear. Results Here, we report that PagN is well conserved and widely distributed among the different species and subspecies of Salmonella. We showed that PagN of S. Typhimurium was sufficient and necessary to enable non-invasive E. coli over-expressing PagN and PagN-coated beads to bind to and invade different non-phagocytic cells. According to the literature, PagN is likely to interact with heparan sulfate proteoglycan (HSPG) as PagN-mediated invasion could be inhibited by heparin treatment in a dose-dependent manner. This report shows that this interaction is not sufficient to allow the internalization mechanism. Investigation of the role of β1 integrin as co-receptor showed that mouse embryo fibroblasts genetically deficient in β1 integrin were less permissive to PagN-mediated internalization. Moreover, PagN-mediated internalization was fully inhibited in glycosylation-deficient pgsA-745 cells treated with anti-β1 integrin antibody, supporting the hypothesis that β1 integrin and HSPG cooperate to induce the PagN-mediated internalization mechanism. In addition, use of specific inhibitors and expression of dominant-negative derivatives demonstrated that tyrosine phosphorylation and class I phosphatidylinositol 3-kinase were crucial to trigger PagN-dependent internalization, as for the Rck internalization mechanism. Finally, scanning electron microscopy with infected cells showed microvillus-like extensions characteristic of Zipper-like structure, engulfing PagN-coated beads and E. coli expressing PagN, as observed during Rck-mediated internalization. Conclusions Our results supply new comprehensions into T3SS-1-independent invasion mechanisms of S. Typhimurium and highly indicate that PagN induces a phosphatidylinositol 3-kinase signaling pathway, leading to a Zipper-like entry mechanism as the Salmonella outer membrane protein Rck.

Cross-Talk Between the Intestinal Epithelium and Salmonella Typhimurium

“…The second invasion mechanism used by STm is a “zipper mechanism,” which requires a host membrane receptor. The interaction of the outer membrane proteins Rck and PagN with EGFR and proteoglycans/β1 integrin respectively, activates signaling pathways, involving GTPases and actin polymerization ( Lambert and Smith, 2008 ; Hume et al, 2017 ; Mambu et al, 2017 ; Barilleau et al, 2021 ). This mechanism is accompanied by a weaker remodeling of the membrane which is characteristic of a “zipper mechanism” ( Figure 2 ; Rosselin et al, 2010 ; Barilleau et al, 2021 ).…”

“…The interaction of the outer membrane proteins Rck and PagN with EGFR and proteoglycans/β1 integrin respectively, activates signaling pathways, involving GTPases and actin polymerization ( Lambert and Smith, 2008 ; Hume et al, 2017 ; Mambu et al, 2017 ; Barilleau et al, 2021 ). This mechanism is accompanied by a weaker remodeling of the membrane which is characteristic of a “zipper mechanism” ( Figure 2 ; Rosselin et al, 2010 ; Barilleau et al, 2021 ). Furthermore, studies using a STm mutant strain, which does not express Rck, PagN and a functional T3SS-1, reveal the existence of hitherto unidentified invasion factors causing weak or massive membrane rearrangements at distinct entry sites ( Rosselin et al, 2011 ; Roche et al, 2018 ).…”

Understanding the Mechanism of Antimicrobial Resistance and Pathogenesis of Salmonella enterica Serovar Typhi

“…T3SS-1 enables the injection of bacterial effector proteins directly into the host cells, promoting actin polymerization and ruffling membrane rearrangements, thereby leading to bacterial internalization. This mechanism is known as the trigger mechanism and is widely dependent on host cells [ 106 ]. Postinfection, the incubation phase of the pathogen may not always be demonstrated by a symptomatic phase.…”

Establishment of indirect ELISA method for Salmonella antibody detection from ducks based on PagN protein

“…PagN is an outer membrane protein that contributes to adhesion to and invasion of epithelial cells by Salmonella . The PagN as conserved gene is localized on the specific centisome 7 genomic island and widely distributed among the different Salmonella serovars [ 20 , 21 ]. The PagN is a PhoP-regulated gene that is up-regulated during growth within macrophages and in vivo in murine models of infection [ 22 ].…”

“…The PagN is a PhoP-regulated gene that is up-regulated during growth within macrophages and in vivo in murine models of infection [ 22 ]. Some studies have shown that the PagN protein utilizes heparinated proteoglycans to adhere and invade mammalian cells [ 21 , 23 ]. The PagN protein contains a transmembrane region and four extracellular loops.…”