Real-time imaging of type III secretion: Salmonella SipA injection into host cells.
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ABSTRACT:
SUBMITTER: Schlumberger MC
PROVIDER: S-EPMC1194920 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Gram-negative pathogenic bacteria such as Salmonella utilize the type III secretion system to inject bacterial effector proteins into a host cell. Upon entry, these effectors bind mammalian cell proteins, hijack cellular signaling pathways, and redirect cellular function, thus enabling bacterial infection. In this study we use the FlAsH/tetracysteine labeling system to fluorescently tag specific effectors in Salmonella to observe real-time secretion of these proteins into a mammalian host cell. The tetracysteine tag is genomically incorporated, thus preserving endogenous control of bacterial effectors. We demonstrate that two effectors, SopE2 and SptP, exhibit different secretion kinetics, as well as different rates of degradation within the host cell. These proteins respectively activate and suppress GTPase Cdc42, suggesting that there is a temporal hierarchy for effector delivery and persistence within the cell that is directly related to effector function.
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Project description:The type-III secretion system (T3SS) enables gram-negative bacteria to inject effector proteins into eukaryotic host cells. Upon entry, T3SS effectors work cooperatively to reprogram host cells, enabling bacterial survival. Progress in understanding when and where effectors localize in host cells has been hindered by a dearth of tools to study these proteins in the native cellular environment. We report a method to label and track T3SS effectors during infection using a split-GFP system. We demonstrate this technique by labeling three effectors from Salmonella enterica (PipB2, SteA and SteC) and characterizing their localization in host cells. PipB2 displayed highly dynamic behavior on tubules emanating from the Salmonella-containing vacuole labeled with both endo- and exocytic markers. SteA was preferentially enriched on tubules localizing with Golgi markers. This segregation suggests that effector targeting and localization may have a functional role during infection.
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Project description:Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems (T3SSs), encoded in pathogenicity islands 1 (SPI1) and 2 (SPI2), respectively. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with certain host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both T3SSs. Nothing is known about the function of this protein inside the host cells. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in epithelial cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also represses genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. Consisted with this analysis a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, reduction of cytotoxicity, cell-cell adhesion and migration capability, and increase in endocytosis.
2014-05-28 | GSE51043 | GEO
Project description:Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems (T3SSs), encoded in pathogenicity islands 1 (SPI1) and 2 (SPI2), respectively. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with certain host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both T3SSs. Nothing is known about the function of this protein inside the host cells. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in epithelial cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also represses genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. Consisted with this analysis a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, reduction of cytotoxicity, cell-cell adhesion and migration capability, and increase in endocytosis. Three experiments. Each experiment replicated three times. Control: HeLa cell transfected with plasmid pBABE without steA, Experiment 1:HeLa line 2 transfected with plasmid pBABE with steA. Experiment 2: HeLa line 4 transfected with plasmid pBABE with steA
2014-05-28 | E-GEOD-51043 | biostudies-arrayexpress
Project description:Many important gram-negative bacterial pathogens use highly sophisticated type III protein secretion systems (T3SSs) to establish complex host-pathogen interactions. Bacterial-host cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and host cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella-host cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the host cell membrane, and the establishment of an intimate association between the bacteria and the target cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation.
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Project description:Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex. The flagellar type-III secretion apparatus utilizes both the energy of the proton motive force and ATP hydrolysis to energize substrate unfolding and translocation. We report formation of functional flagella in the absence of type-III ATPase activity by mutations that increased the proton motive force and flagellar substrate levels. We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion. Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process.
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Project description:Protein secretion, a key intercellular event for transducing cellular signals, is thought to be strictly regulated. However, secretion dynamics at the single-cell level have not yet been clarified because intercellular heterogeneity results in an averaging response from the bulk cell population. To address this issue, we developed a novel assay platform for real-time imaging of protein secretion at single-cell resolution by a sandwich immunoassay monitored by total internal reflection microscopy in sub-nanolitre-sized microwell arrays. Real-time secretion imaging on the platform at 1-min time intervals allowed successful detection of the heterogeneous onset time of nonclassical IL-1? secretion from monocytes after external stimulation. The platform also helped in elucidating the chronological relationship between loss of membrane integrity and IL-1? secretion. The study results indicate that this unique monitoring platform will serve as a new and powerful tool for analysing protein secretion dynamics with simultaneous monitoring of intracellular events by live-cell imaging.
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Project description:Salmonella enterica pathogenesis is dependent on its ability to enter and replicate inside host cells. Replication occurs inside the Salmonella-containing vacuole (SCV), a vacuolar compartment that is modified by bacterial effectors secreted through the two type III secretion systems (T3SS-1 and T3SS-2). Type III effectors interact with the host cell endocytic pathway to aid replication. We investigated whether Salmonella effector proteins may also interact with the host's exocytic pathway. A secreted alkaline phosphatase (SEAP) assay indicated three Salmonella effectors inhibited the secretory pathway, although only Salmonella outer protein B (SopB) was confirmed to block exocytosis using a vesicular stomatitis virus glycoprotein-green fluorescent protein (VSVG-GFP) transport assay. The 4-phosphatase activity of SopB was crucial to its effect on exocytosis. The interaction with the secretory pathway could potentially be important for providing replicating Salmonella with nutrients, contributing membrane material necessary for SCV biogenesis, altering antibacterial peptide/protein secretion or manipulating cell surface proteins important in the host response to infection.
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Project description:The type III secretion systems (TTSS) encoded in Salmonella pathogenicity island-1 and -2 (SPI-1 and -2) are virulence factors required for specific phases of Salmonella infection in animal hosts. However, the host cell types targeted by the TTSS have not been determined. To investigate this, we have constructed translational fusions between the beta-lactamase reporter and a broad array of TTSS effectors secreted via SPI-1, SPI-2, or both. Secretion of the fusion protein to a host cell was determined by cleavage of a specific fluorescent substrate. In cultured cells, secretion of all six effectors could be observed. However, two to four days following i.p. infection of mice, only effectors secreted by SPI-2 were detected in spleen cells. The cells targeted were identified via staining with nine different cell surface markers followed by FACS analysis as well as by conventional cytological methods. The targeted cells include B and T lymphocytes, neutrophils, monocytes, and dendritic cells, but not mature macrophages. To further investigate replication in these various cell types, Salmonella derivatives were constructed that express a red fluorescent protein. Bacteria could be seen in each of the cell types above; however, most viable bacteria were present in neutrophils. We find that Salmonella is capable of targeting most phagocytic and non-phagocytic cells in the spleen but has a surprisingly high preference for neutrophils. These findings suggest that Salmonella specifically target splenic neutrophils presumably to attenuate their microbicidal functions, thereby promoting intracellular survival and replication in the mouse.
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