Abstract

The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca2+-binding sites per BIg domain formed by conserved aspartate or glutamate residues. In a mutational analysis Ca2+-binding sites were disrupted by aspartate to serine exchange at various positions in the BIg domains of SiiE. Amounts of secreted SiiE diminish with a decreasing number of intact Ca2+-binding sites. BIg domains of SiiE contain distinct Ca2+-binding sites, with type I sites being similar to other T1SS-secreted proteins and type II sites newly identified in SiiE. We functionally and structurally dissected the roles of type I and type II Ca2+-binding sites in SiiE, as well as the importance of Ca2+-binding sites in various positions of SiiE. Type I Ca2+-binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion.

Highlights

  • Salmonella enterica is a food-borne Gram-negative pathogen which causes self-limiting gastroenteritis

  • The interaction of Salmonella enterica with polarized epithelial cells depends on the function of SiiE, a 595 kDa adhesin containing 53 repeats of a bacterial immunoglobulin (BIg) domain

  • We found that BIg domains contain amino acid residues forming binding sites for Ca2+ ions

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Summary

Introduction

Salmonella enterica is a food-borne Gram-negative pathogen which causes self-limiting gastroenteritis. A Salmonella pathogenicity island (SPI) 1-encoded type 3 secretion system (T3SS) is necessary for invasion [2]. This protein secretion system is capable to secrete a distinct cocktail of effector proteins, which manipulate the host cell. SiiE mediates the first intimate contact to the host cell through binding to glycostructures containing N-acetyl-glucosamine and/or α2,3-linked sialic acid [5]. This contact positions the SPI1-T3SS to efficiently translocate effector proteins which lead to actin remodeling and macropinocytosis of the bacteria. This channel may use the proton motive force (PMF) at the cytoplasmic membrane to regulate the retention of SiiE, either through sensing the physiological state of the cell or by inducing conformational changes to binding partners [7]

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