Pathogenicity Islands play a major role in the pathogenesis of infections by Salmonella enterica. The molecular function of Salmonella Pathogenicity Island 4 (SPI4) is largely unknown, but recent work indicated a role of SPI4 for Salmonella pathogenesis in certain animal models. We analysed the virulence functions of SPI4 and observed that SPI4 is contributing to intestinal inflammation in a mouse model. On a cellular level, SPI4 mediates adhesion to epithelial cells. We demonstrate the function of SPI4-encoded proteins as a type I secretion system (T1SS) and identify SiiE as the substrate protein of the T1SS. SiiE is secreted into the culture medium but mediates contact-dependent adhesion to epithelial cell surfaces. SiiE is a very large non-fimbrial adhesin of 600 kDa and consists of 53 repeats of Ig domains. Our study describes the first T1SS-secreted protein that functions as a non-fimbrial adhesin in binding to eukaryotic cells. The SPI4-encoded T1SS and SiiE might functionally resemble the type I fimbrial adhesins.

The host's immune system plays a key role in modulating growth of pathogens and the intestinal microbiota in the gut. In particular, inflammatory bowel disorders and pathogen infections induce shifts of the resident commensal microbiota which can result in overgrowth of Enterobacteriaceae (“inflammation-inflicted blooms”). Here, we investigated competition of the human pathogenic Salmonella enterica serovar Typhimurium strain SL1344 (S. Tm) and commensal E. coli in inflammation-inflicted blooms. S. Tm produces colicin Ib (ColIb), which is a narrow-spectrum protein toxin active against related Enterobacteriaceae. Production of ColIb conferred a competitive advantage to S. Tm over sensitive E. coli strains in the inflamed gut. In contrast, an avirulent S. Tm mutant strain defective in triggering gut inflammation did not benefit from ColIb. Expression of ColIb (cib) is regulated by iron limitation and the SOS response. CirA, the cognate outer membrane receptor of ColIb on colicin-sensitive E. coli, is induced upon iron limitation. We demonstrate that growth in inflammation-induced blooms favours expression of both S. Tm ColIb and the receptor CirA, thereby fuelling ColIb dependent competition of S. Tm and commensal E. coli in the gut. In conclusion, this study uncovers a so-far unappreciated role of inflammation-inflicted blooms as an environment favouring ColIb-dependent competition of pathogenic and commensal representatives of the Enterobacteriaceae family. Colicins are bacterial protein toxins which show potent activity against sensitive strains in vitro. Ecological models suggest that colicins play a major role in modulating dynamics of bacterial populations in the gut. However, previous studies could not readily confirm these predictions by respective in vivo experiments. In animal models, colicin-producing strains only show a minor or even absent fitness benefit over sensitive competitors. Here, we propose that the gut environment plays a crucial role in generating conditions for bacterial competition by colicin Ib (ColIb). Gut inflammation favours overgrowth of Enterobacteriaceae (“inflammation-inflicted Enterobacterial blooms”). We show that a pathogenic Salmonella Typhimurium (S. Tm) strain benefits from ColIb production in competition against commensal E. coli upon growth in inflammation-inflicted blooms. In the absence of gut inflammation, ColIb production did not confer a competitive advantage to S. Tm. In the inflamed gut, the genes for ColIb production in S. Tm and its corresponding ColIb-surface receptor CirA in E. coli were markedly induced, as compared to the non-inflamed gut. Therefore, environmental conditions in inflammation-inflicted blooms favour colicin-dependent competition of Enterobacteriaceae by triggering ColIb production and susceptibility at the same time. Our findings reveal a role of colicins as important bacterial fitness factors in inflammation-induced blooms.

Invasion is an important microbial virulence strategy to overcome the barrier formed by polarized epithelial cells. Salmonella enterica is a food-borne pathogen that deploys a type III secretion system for the manipulation of the actin cytoskeleton and to trigger internalization into epithelial cells. Here we show that this function is not sufficient to enter polarized cells and report that penetration of epithelia from the luminal side requires both the type III secretion system and novel virulence functions conferred by Salmonella pathogenicity island 4. Salmonella pathogenicity island 4 encodes a type I secretion system for the giant non-fimbrial adhesin SiiE that mediates intimate contact of Salmonella to microvilli on the apical membrane. Mutant strains lacking SiiE fail to invade polarized cells, to destroy epithelial barrier functions and to efface the apical brush border. Deletion analyses of repetitive domains in SiiE indicate that the large size of the adhesin is of functional importance. Our observations demonstrate that efficient penetration of epithelial barriers requires the cooperative activity of two Salmonella pathogenicity islands encoding different secretion systems. These findings underline the role of the epithelial brush border and reveal a new mechanism used by bacterial pathogens to overcome this barrier.

Direct manipulation of bacterial chromosomes by recombination-based techniques has become increasingly important for both cognitive and applied research. Here we demonstrate, for the first time, the combination of the Red recombinase system with I-SceI endonuclease-based selection of successful recombinants after electroporation with short synthetic olignucleotides. We show the generation of scarless gene knockouts as well as site-directed mutagenesis using the Salmonella virulence-associated two component signaling system PhoPQ. The presented approach is very versatile for generating in-frame deletions, point mutations or insertions within bacterial chromosomes.

Salmonella pathogenicity island 4 (SPI4) encodes a type I secretion system and the cognate substrate protein, SiiE. We have recently demonstrated that SiiE is a giant nonfimbrial adhesin involved in the adhesion of Salmonella enterica serovar Typhimurium to polarized epithelial cells. We also observed that under in vitro culture conditions, the synthesis and secretion of SiiE coincided with the activation of Salmonella invasion genes. These observations prompted us to investigate the regulation of SPI4 genes in detail. A novel approach for the generation of reporter gene fusions was employed to generate single-copy chromosomal fusions to various genes within SPI4, and the expression of these fusions was investigated. We analyzed the regulation of SPI4 genes and the roles of various regulatory systems for SPI4 expression. Our data show that the expression of SPI4 genes is coregulated with SPI1 invasion genes by the global regulator SirA. Expression of a SPI4 gene was also reduced in the absence of HilA, the central local regulator of SPI1 gene expression. Both SirA and HilA functions were required for the secretion of SiiE and the SPI4-mediated adhesion. Our data demonstrate that SPI4-mediated adhesion, as well as SPI1-mediated invasion, are tightly coregulated by the same regulatory circuits and induced under similar environmental conditions.