| 1. |
- Bröms, Jeanette E, et al.
(författare)
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Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion
- 2005
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 187:22, s. 7738-7752
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Tidskriftsartikel (refereegranskat)abstract
- Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of Yersinia pseudotuberculosis. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native Pseudomonas aeruginosa or when produced in Yersinia. In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the Yersinia type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of P. aeruginosa. YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of Yersinia and Pcr3 of P. aeruginosa. Still, Pcr4 was unable to complement a DeltayscY null mutant defective for type III secretion and yop-regulatory control in Yersinia, despite the ability of YscY to function in P. aeruginosa. Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to Yersinia type III secretion.
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| 2. |
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| 3. |
- Bröms, Jeanette E, et al.
(författare)
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Tetratricopeptide repeats are essential for PcrH chaperone function in Pseudomonas aeruginosa type III secretion.
- 2006
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Ingår i: FEMS Microbiol Lett. - 0378-1097. ; 256:1, s. 57-66
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Tidskriftsartikel (refereegranskat)abstract
- The type III secretion system (T3SS) is a specialized apparatus evolved by Gram-negative bacteria to deliver effector proteins into host cells, thus facilitating the establishment of an infection. Effector translocation across the target cell plasma membrane is believed to occur via pores formed by at least two secreted translocator proteins, the functions of which are dependent upon customized class II T3SS chaperones. Recently, three internal tetratricopeptide repeats (TPRs) were identified in this class of chaperones. Here, defined mutagenesis of the class II chaperone PcrH of Pseudomonas aeruginosa revealed these TPRs to be essential for chaperone activity towards the translocator proteins PopB and PopD and subsequently for the translocation of exoenzymes into host cells.
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| 4. |
- Carlsson, Katrin E, et al.
(författare)
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Extracytoplasmic-stress-responsive pathways modulate type III secretion in Yersinia pseudotuberculosis.
- 2007
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Ingår i: Infect Immun. - 0019-9567. ; 75:8, s. 3913-24
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Tidskriftsartikel (refereegranskat)abstract
- Three signal transduction pathways, the two-component systems CpxRA and BaeSR and the alternative sigma factor sigma(E), respond to extracytoplasmic stress that facilitates bacterial adaptation to changing environments. At least the CpxRA and sigma(E) pathways control the production of protein-folding and degradation factors that counter the effects of protein misfolding in the periplasm. This function also influences the biogenesis of multicomponent extracellular appendages that span the bacterial envelope, such as various forms of pili. Herein, we investigated whether any of these regulatory pathways in the enteropathogen Yersinia pseudotuberculosis affect the functionality of the Ysc-Yop type III secretion system. This is a multicomponent molecular syringe spanning the bacterial envelope used to inject effector proteins directly into eukaryotic cells. Disruption of individual components revealed that the Cpx and sigma(E) pathways are important for Y. pseudotuberculosis type III secretion of Yops (Yersinia outer proteins). In particular, a loss of CpxA, a sensor kinase, reduced levels of structural Ysc (Yersinia secretion) components in bacterial membranes, suggesting that these mutant bacteria are less able to assemble a functional secretion apparatus. Moreover, these bacteria were no longer capable of localizing Yops into the eukaryotic cell interior. In addition, a cpxA lcrQ double mutant engineered to overproduce and secrete Yops was still impaired in intoxicating cells. Thus, the Cpx pathway might mediate multiple influences on bacterium-target cell contact that modulate Yersinia type III secretion-dependent host cell cytotoxicity.
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| 5. |
- Carlsson, Katrin E, et al.
(författare)
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Influence of the Cpx extracytoplasmic-stress-responsive pathway on Yersinia sp.-eukaryotic cell contact.
- 2007
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Ingår i: Infect Immun. - 0019-9567. ; 75:9, s. 4386-99
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Tidskriftsartikel (refereegranskat)abstract
- The extracytoplasmic-stress-responsive CpxRA two-component signal transduction pathway allows bacteria to adapt to growth in extreme environments. It controls the production of periplasmic protein folding and degradation factors, which aids in the biogenesis of multicomponent virulence determinants that span the bacterial envelope. This is true of the Yersinia pseudotuberculosis Ysc-Yop type III secretion system. However, despite using a second-site suppressor mutation to restore Yop effector secretion by yersiniae defective in the CpxA sensor kinase, these bacteria poorly translocated Yops into target eukaryotic cells. Investigation of this phenotype herein revealed that the expression of genes which encode several surface-located adhesins is also influenced by the Cpx pathway. In particular, the expression and surface localization of invasin, an adhesin that engages beta1-integrins on the eukaryotic cell surface, are severely restricted by the removal of CpxA. This reduces bacterial association with eukaryotic cells, which could be suppressed by the ectopic production of CpxA, invasin, or RovA, a positive activator of inv expression. In turn, these infected eukaryotic cells then became susceptible to intoxication by translocated Yop effectors. In contrast, bacteria harboring an in-frame deletion of cpxR, which encodes the cognate response regulator, displayed an enhanced ability to interact with cell monolayers, as well as elevated inv and rovA transcription. This phenotype could be drastically suppressed by providing a wild-type copy of cpxR in trans. We propose a mechanism of inv regulation influenced by the direct negative effects of phosphorylated CpxR on inv and rovA transcription. In this fashion, sensing of extracytoplasmic stress by CpxAR contributes to productive Yersinia sp.-eukaryotic cell interactions.
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| 6. |
- Costa, Tiago R. D., et al.
(författare)
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Influence of the LcrH chaperone on type III secretion system regulation in Yersinia pseudotuberculosis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Human pathogenic Yersiniae share a common virulence plasmid that encodes for the Ysc-Yop type III secretion system (T3SS). Control of yop expression involves several pathways in which their cross-talk is not completely understood. LcrF, an AraC-like transcriptional activator, is required for temperature-dependent yop-transcription. In contrast, a repressive effect of the T3S chaperone LcrH and the cognate translocator substrate YopD occurs through binding to yop mRNA and inhibiting translation; a process that is also thought to involve LcrQ. Several homologous members of the LcrH family of translocator-class of T3S chaperones can act as a cofactor to amplify the activity of transcriptional activators analogous to LcrF. However, we show here in Y. pseudotuberculosis that LcrH does not induce LcrF-dependent transcription of target genes. Moreover, a full length DlcrH null mutant in which YopB and YopD are rapidly degraded is totally de-repressed for Yop synthesis even though the anti-activator LcrQ is forced to accumulate in the cytoplasm through rendering the Ysc-Yop T3SS non-functional or ectopically producing LcrQ in trans. Typically, this mutant cannot grow at 37°C. Thus, in all respects, the DlcrH null mutant mirrors the regulatory defects established for Yersinia lacking the translocator and anti-activator YopD. On the other hand, Y. pseudotuberculosis producing the LcrHE30G point mutant that is defective for YscY chaperone binding exhibits a mild regulatory defect that permits some growth at 37°C, but is blind to the cytoplasmic accumulation of LcrQ. Critically however, this mutant still responds to repression caused by YopD accumulation, which is stably produced and efficiently secreted by this strain. Thus, our work with LcrHE30G indicates an additional regulatory function of this versatile T3S chaperone that is independent of the LcrF transcription factor and the YopD anti-activator.
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| 7. |
- Costa, Tiago R D, et al.
(författare)
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YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis
- 2010
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 285:33, s. 25269-25284
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Tidskriftsartikel (refereegranskat)abstract
- YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.
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