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SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of Salmonella-containing vacuoles in vivo.

ABSTRACT: Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.

SUBMITTER: Fulde M

PROVIDER: S-EPMC8475570 | biostudies-literature | 2021 Jan-Dec

REPOSITORIES: biostudies-literature

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SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of Salmonella-containing vacuoles in vivo.

Fulde Marcus M van Vorst Kira K Zhang Kaiyi K Westermann Alexander J AJ Busche Tobias T Huei Yong Chiun YC Welitschanski Katharina K Froh Isabell I Pägelow Dennis D Plendl Johanna J Pfarrer Christiane C Kalinowski Jörn J Vogel Jörg J Valentin-Weigand Peter P Hensel Michael M Tedin Karsten K Repnik Urska U Hornef Mathias W MW

Gut microbes 20210101 1

Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary ...[more]

PMID: 34542008

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Project description:Following infection of mammalian cells, Salmonella enterica serovar Typhimurium (S Typhimurium) replicates within membrane-bound compartments known as Salmonella-containing vacuoles (SCVs). The Salmonella pathogenicity island 2 type III secretion system (SPI-2 T3SS) translocates approximately 30 different effectors across the vacuolar membrane. SseF and SseG are two such effectors that are required for SCVs to localize close to the Golgi network in infected epithelial cells. In a yeast two-hybrid assay, SseG and an N-terminal variant of SseF interacted directly with mammalian ACBD3, a multifunctional cytosolic Golgi network-associated protein. Knockdown of ACBD3 by small interfering RNA (siRNA) reduced epithelial cell Golgi network association of wild-type bacteria, phenocopying the effect of null mutations of sseG or sseF Binding of SseF to ACBD3 in infected cells required the presence of SseG. A single-amino-acid mutant of SseG and a double-amino-acid mutant of SseF were obtained that did not interact with ACBD3 in Saccharomyces cerevisiae When either of these was produced together with the corresponding wild-type effector by Salmonella in infected cells, they enabled SCV-Golgi network association and interacted with ACBD3. However, these properties were lost and bacteria displayed an intracellular replication defect when cells were infected with Salmonella carrying both mutant genes. Knockdown of ACBD3 resulted in a replication defect of wild-type bacteria but did not further attenuate the growth defect of a ΔsseFG mutant strain. We propose a model in which interaction between SseF and SseG enables both proteins to bind ACBD3, thereby anchoring SCVs at the Golgi network and facilitating bacterial replication. Upon invasion of epithelial cells, the majority of vacuoles containing Salmonella enterica migrate to the perinuclear region-located Golgi network and remain in this region of the cell during the first few rounds of bacterial replication, forming a clustered microcolony of vacuoles. This process requires the action of SseF and SseG, two effector proteins that are translocated by the Salmonella SPI-2 type III secretion system. However, little is known about how they function. Here, we show that both proteins interact with the mammalian Golgi network-associated protein ACBD3. To our knowledge, the SseF-SseG-ACBD3 interaction is the first example of a tethering complex between a pathogen-containing vacuole and a host cell organelle.

Project description:BackgroundThe monolayer of intestinal epithelial cells (IECs) plays a crucial role in controlling intestinal homeostasis, also by its interaction with the immune system, via paracrine cytokine production, thus driving innate responses by tissue-resident immune cells. Here, using a co-culture model, we investigated the interactions between differentiated Caco-2 cells in monolayer and macrophages, by mimicking the cross-talk between enterocytes and immune cells during gastrointestinal (GI) tract inflammation.MethodsCaco-2 mature monolayers grown on Transwell membranes were challenged with apical or basolateral LPS. After stimulations, the enterocyte-like monolayers were transferred in co-culture with THP-1 derived macrophages. The functional impact of treatments was evaluated in terms of monolayer's permeability, expression of mRNAs related to inflammation and immune responses and analysis of immune soluble factors present in the co-culture media.ResultsLPS effectively affected monolayer's permeability and induced a pro-inflammatory transcriptional program in Caco-2 monolayers. Remarkably, THP-1 derived macrophages differentially responded based on the diverse directional source of LPS, previously administered to the Caco-2 monolayers. Basolateral sensing of LPS, by Caco-2 monolayers, induced specific increase of several pro-inflammatory factors such as NF-kB1, IL-6 and IL-8, at transcript level, in macrophages, while apical sensing triggering targeted increase of IL-1β expression. Significantly, the analysis of immune factors secreted in the co-culture media suggested that paracrine interactions between enterocyte-like monolayers and macrophages are differently driven based on the basolateral vs. apical inflammation, previously triggered by LPS against the epithelial monolayer, and thus involving different immune gene networks.ConclusionsTaken together, our results suggest a framework of interactions between IECs and macrophages, depending upon the "polarized" inflammatory dysregulation.

Project description:The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukaryotic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA' reporter of Bordetella pertussis are often used. CyaA' is a calmodulin-dependent adenylate cyclase that is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA' can be evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids pCyaA'-Kan and pCyaA'-Cam, which contain the ORF encoding CyaA' adjacent to a cassette that confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target (FRT) sites. A PCR product from pCyaA'-Kan or pCyaA'-Cam containing these genetic elements can be introduced into the bacterial chromosome to generate gene fusions by homologous recombination using the Red recombination system from bacteriophage λ. Subsequently, the resistance cassette can be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept, the plasmids pCyaA'-Kan and pCyaA'-Cam were used to generate unmarked chromosomal fusions of 10 T3SS effectors to CyaA' in S. Typhimurium. Each fusion protein was detected by Western blot using an anti-CyaA' monoclonal antibody when the corresponding mutant strain was grown under conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of fusion protein SipA-CyaA' during in vitro growth was verified by Western blot analysis of culture supernatants. Finally, efficient translocation of SipA-CyaA' into HeLa cells was evidenced by increased intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA'-Kan and pCyaA'-Cam can be used to generate unmarked chromosomal cyaA' translational fusion to study regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells.

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SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of Salmonella-containing vacuoles in vivo.

Publications

SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of <i>Salmonella</i>-containing vacuoles <i>in vivo</i>.

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