Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN, tyeA, sycN, yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies

Day, James B.; Ferracci, Franco; Plano, Gregory V.

doi:10.1046/j.1365-2958.2003.03343.x

Cited by 77 publications

(124 citation statements)

References 84 publications

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“…Y. pestis KIM5-3173 is YopJ -(DJ) due to a bacteriophage Mu dI1734 insertion in yopJ of pCD1 [62]. The secretion-and injection-competent poly-yop deletion mutant Y. pestis KIM5-3001.48 (sycE yopE::km yopJ yopT yopM yopH ypkA lcrQ) and the injection-defective poly-yop deletion mutant KIM5-3001.49 (sycE yopE::km yopJ yopT yopM yopH ypkA lcrQ yopB) have been previously described [26]. Plasmids pYopJ-GSK encodes fulllength YopJ carrying a phosphorylatable C-terminal 13 residue GSK-tag that can be detected by phospho-specific anti-GSK antibodies (Cell Signaling, Beverly, MA).…”

Section: Methodsmentioning

confidence: 99%

“…Because the same intracellular signaling pathways appear to be activated in K562 and primary precursors during DC differentiation, and because K562 are a homogenous population, this cell line is a readily accessible model to study YopJ's effects on DC signal transduction. We first examined the effect of YopJ on DC differentiation by infecting K562 with injection competent (Y. pestis KIM5-3001.48; YopB + YopJ + ) and injection defective (Y. pestis KIM5-3001.49; YopB -YopJ + ) poly-yop-deleted strains expressing a functional plasmid-expressed GSK epitope-tagged YopJ protein [26,27]. Both strains carry deletions in pCD1 genes encoding the six effector Yops; therefore, YopJ-GSK is the only effector Yop injected.…”

Section: Effect Of Injection Competent Vs Incompetent Strains Of Y mentioning

confidence: 99%

“…YopB is an essential component of the T3SS injection machinery [28,29], and thus the YopB -strains cannot inject YopJ-GSK into eukaryotic cells. We utilized the GSK-tag reporter system to identify injected YopJ [26,27]. The GSK tag is only phosphorylated within eukaryotic cells [30,31], and can be detected using phospho GSK-specific antibodies.…”

Section: Effect Of Injection Competent Vs Incompetent Strains Of Y mentioning

confidence: 99%

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Modulation of dendritic cell differentiation and function by YopJ of Yersinia pestis

et al. 2007

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Yersinia pestis evades immune responses in part by injecting into host immune cells several effector proteins called Yersinia outer proteins (Yops) that impair cellular function. This has been best characterized in the innate effector cells, but much less so for cells involved in adaptive immune responses. Dendritic cells (DC) sit at the crossroads between innate and adaptive immunity, and can function to initiate or inhibit adaptive immune responses. Although Y. pestis can target and inactivate DC, the mechanism responsible for this remains unclear. We have found that injection of Y. pestis YopJ into DC progenitors disrupts key signal transduction pathways and interferes with DC differentiation and subsequent function. YopJ injection prevents upregulation of the NF-jB transcription factor Rel B and inhibits MAPK/ERK activationboth having key roles in DC differentiation. Furthermore, YopJ injection prevents costimulatory ligand up-regulation, LPS-induced cytokine expression, and yields differentiated DC with diminished capability to induce T cell proliferation and IFN-c induction. By modulating DC function through YopJ-mediated disruption of signaling pathways during progenitor to DC differentiation, Yersinia may interfere with the adaptive responses necessary to clear the infection as well as establish a tolerant immune environment that leads to chronic infection/carrier state in the surviving host.

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Section: Methodsmentioning

confidence: 99%

Section: Effect Of Injection Competent Vs Incompetent Strains Of Y mentioning

confidence: 99%

Section: Effect Of Injection Competent Vs Incompetent Strains Of Y mentioning

confidence: 99%

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Modulation of dendritic cell differentiation and function by YopJ of Yersinia pestis

et al. 2007

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“…KIM5 derivatives carrying a YopE-Elk-encoding plasmid (pYopE 129 -Elk) (19) were cultivated overnight in HIB at 28°C, diluted to an OD 620 of 0.15 in HIB, and incubated for 3 to 4 more hours at 28°C. HEp-2 or THP-1 cells were cultured in six-well tissue culture plates until they reached 100% or 60% confluence, respectively.…”

Section: Vol 77 2009mentioning

confidence: 99%

TheYersinia pestisAil Protein Mediates Binding and Yop Delivery to Host Cells Required for Plague Virulence

2009

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Although adhesion to host cells is a critical step in the delivery of cytotoxic Yop proteins by Yersinia pestis, the mechanism has not been defined. To identify adhesins critical for Yop delivery, we initiated two transposon mutagenesis screens using the mariner transposon. To avoid redundant cell binding activities, we initiated the screen with a strain deleted for two known adhesins, pH 6 antigen and the autotransporter, YapC, as well as the Caf1 capsule, which is known to obscure some adhesins. The mutants that emerged contained insertions within the ail (attachment and invasion locus) gene of Y. pestis. A reconstructed mutant with a single deletion in the ail locus (y1324) was severely defective for delivery of Yops to HEp-2 human epithelial cells and significantly defective for delivery of Yops to THP-1 human monocytes. Specifically, the Yop delivery defect was apparent when cell rounding and translocation of an ELK-tagged YopE derivative into host cells were monitored. Although the ail mutant showed only a modest decrease in cell binding capacity in vitro, the KIM5 ⌬ail mutant exhibited a >3,000-fold-increased 50% lethal dose in mice. Mice infected with the ⌬ail mutant also had 1,000-fold fewer bacteria in their spleens, livers, and lungs 3 days after infection than did those infected with the parental strain, KIM5. Thus, the Ail protein is critical for both Y. pestis type III secretion in vitro and infection in mice.

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“…Three pathogenic Yersinia spp., Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, employ type III secretion machines to transport Yop proteins into the cytoplasm of immune cells, thereby evading innate immune responses in their mammalian hosts (Rosqvist et al, 1988;Sory and Cornelis, 1994;Skrzypek et al, 1998;Day et al, 2003;Marketon et al, 2005). The 70 kb virulence plasmid of yersiniae encodes type III secretion substrates (Yops, Yersinia outer proteins), secretion machinery components (ysc, Yop secretion), regulatory genes and chaperones for secretion substrates (Cornelis and Wolf-Watz, 1997;Cornelis et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Ubiquitin‐Yop hybrids as probes for post‐translational transport by the Yersinia type III secretion pathway

Quenee

Schneewind²

2007

Molecular Microbiology

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SummaryYersinia enterocolitica uses type III secretion to transport Yop proteins into the cytoplasm of host cells. Previous work generated hypotheses for both co-and post-translational transport mechanisms in the Yersinia type III pathway. Here, we used ubiquitin (Ub) and UBP1, the Ub-specific protease, to examine whether Yops can be secreted when synthesized prior to recognition by the type III machinery. Fusion of Ub to the N-terminus of Yops blocked substrate recognition and secretion of hybrids generated with YopE, YopQ or YopR. UBP1 removed Ub from the N-terminus of these hybrids and allowed YopE, YopQ or YopR cleavage products to enter the secretion pathway. Following the release of Ub, Yersinia type III machines also transported the YopE cleavage product into the cytosol of tissue culture cells. Minimal secretion signals were also examined with the Ub/UBP1 system and some, but not all, of these signals promoted type III secretion even after polypeptides had been freed from Ub. These results suggest that recognition and secretion of Yop substrates by the type III machinery can occur by a post-translational mechanism.

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Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN, tyeA, sycN, yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies

Cited by 77 publications

References 84 publications

Modulation of dendritic cell differentiation and function by YopJ of Yersinia pestis

Modulation of dendritic cell differentiation and function by YopJ of Yersinia pestis

TheYersinia pestisAil Protein Mediates Binding and Yop Delivery to Host Cells Required for Plague Virulence

Ubiquitin‐Yop hybrids as probes for post‐translational transport by the Yersinia type III secretion pathway

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