| 1. |
- Francis, Matthew, 1969-, et al.
(författare)
-
A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD
- 2000
-
Ingår i: Molecular Microbiology. - : Blackwell Publishing. - 0950-382X .- 1365-2958. ; 38:1, s. 85-102
-
Tidskriftsartikel (refereegranskat)abstract
- The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersiniaouter protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic α-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic α-helix abolished the YopD–LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.
|
|
| 2. |
- O'Toole, Ronan, et al.
(författare)
-
Visualisation of zebrafish infection by GFP-labelled Vibrio anguillarum.
- 2004
-
Ingår i: Microb Pathog. - : Elsevier BV. - 0882-4010 .- 1096-1208. ; 37:1, s. 41-6
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum is an invasive pathogen of fish causing a septicaemia called vibriosis. In this work, transparent zebrafish were immersed in water containing green fluorescent protein labelled V. anguillarum. The infection was visualised at the whole fish and single bacterium levels using microscopy. The gastrointestinal tract was the first site where the pathogen was detected. This enteric localisation occurred independently of the flagellum or motility. On the other hand, chemotactic motility was essential for association of the pathogen with the fish surface. In conclusion, the zebrafish infection model provides evidence that the intestine and skin represent sites of infection by V. anguillarum and suggests a host site where chemotaxis may function in virulence.
|
|
| 3. |
- Dukuzumuremyi, Jean-Marie, et al.
(författare)
-
The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor
- 2000
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 275:45, s. 35281-35290
-
Tidskriftsartikel (refereegranskat)abstract
- The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.
|
|
| 4. |
- Aili, Margareta, et al.
(författare)
-
GAP activity of Yersinia YopE
- 2002
-
Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 358, s. 359-70
-
Tidskriftsartikel (refereegranskat)
|
|
| 5. |
- Aili, Margareta, et al.
(författare)
-
In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity
- 2003
-
Ingår i: Microbial Pathogenesis. - : Elsevier. - 0882-4010 .- 1096-1208. ; 34:6, s. 297-308
-
Tidskriftsartikel (refereegranskat)abstract
- The YopE cytotoxin of Yersinia is an essential virulence determinant that is translocated into the eukaryotic target cell via a plasmid-encoded type III secretion system. YopE possess a GTPase activating protein activity that in vitro has been shown to down regulate RhoA, Rac1, and Cdc42. Translocated YopE induces de-polymerisation of the actin microfilament structure in the eukaryotic cell which results in a rounding up of infected cells described as a cytotoxic effect. Here, we have investigated the importance of different regions of YopE for induction of cytotoxicity and in vitro GAP activity. Sequential removal of the N- and C-terminus of YopE identified the region between amino acids 90 and 215 to be necessary for induction of cytotoxicity. Internal deletions containing the essential arginine at position 144 resulted in a total loss of cytotoxic response. In-frame deletions flanking the arginine finger defined a region important for the cytotoxic effect to amino acids 166–183. Four triple-alanine substitution mutants in this region, YopE166-8A, 169-71A, 175-7A and 178-80A were still able to induce cytotoxicity on HeLa cells although they did not show any in vitro GAP activity towards RhoA, Rac1 or Cdc42. A substitution mutant in position 206-8A showed the same phenotype, ability to induce cytotoxic response but no in vitro GAP activity. We speculate that YopE may have additional unidentified targets within the eukaryotic cell.
|
|
| 6. |
- Deleuil, Fabienne, et al.
(författare)
-
Interaction between the Yersinia protein tyrosine phosphatase YopH and eukaryotic Cas/Fyb is an important virulence mechanism
- 2003
-
Ingår i: Cellular Microbiology. - : Blackwell Publishing. - 1462-5814 .- 1462-5822. ; 5:1, s. 53-64
-
Tidskriftsartikel (refereegranskat)abstract
- The tyrosine phosphatase YopH is an essential virulence factor produced by pathogenic Yersinia species. YopH is translocated into host cells via a type III secretion system and its dephosphorylating activity causes disruption of focal complex structures and blockage of the phagocytic process. Among the host cell targets of YopH are the focal adhesion proteins Crk-associated substrate (p130Cas) and focal adhesion kinase (FAK) in epithelial cells, and p130Cas and Fyn-binding protein (Fyb) in macrophages. Previous studies have shown that the N-terminal domain of YopH acts as a substrate-binding domain. In this study, the mechanism and biological importance of the targeting of YopH to focal complexes relative to its interaction with p130Cas/Fyb was elucidated. Mutants of YopH that were defective in p130Cas/Fyb binding but otherwise indistinguishable from wild type were constructed. Mutants unable to bind p130Cas did not localize to focal complex structures in infected cells, indicating that the association with p130Cas is critical for appropriate subcellular localization of YopH. These yopH mutants were also clearly attenuated in virulence, showing that binding to p130Cas and/or Fyb is biologically relevant in Yersinia infections.
|
|
| 7. |
- Edqvist, Petra, et al.
(författare)
-
YscP and YscU Regulate Substrate Specificity of the Yersinia Type III Secretion System
- 2003
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 185:7, s. 2259-2266
-
Tidskriftsartikel (refereegranskat)abstract
- Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37 degrees C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.
|
|
| 8. |
- Francis, Matthew S, et al.
(författare)
-
Regulation of type III secretion systems
- 2002
-
Ingår i: Current Opinion in Microbiology. - 1369-5274 .- 1879-0364. ; 5:2, s. 166-172
-
Tidskriftsartikel (refereegranskat)abstract
- Type III secretion systems are utilised by numerous Gram-negative bacteria to efficiently interact with a host. Appropriate expression of type III genes is achieved through the integration of several regulatory pathways that ultimately co-ordinate the activity of a central transcriptional activator usually belonging to the AraC family. The complex regulatory cascades allow this virulence strategy to be utilised by different bacteria even if they occupy diverse niches that define a unique set of environmental cues. Simulating the appropriate combination of signals in vitro to allow a meaningful interpretation of the type III assembly and secretion regulatory cascade remains a common goal for researchers. Pieces of the puzzle slowly emerge to provide insightful views into the complex regulatory networks that allow bacteria to assemble and utilise type III secretion to efficiently colonise a host.
|
|
| 9. |
- Francis, Matthew S, et al.
(författare)
-
The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis
- 2001
-
Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 42:4, s. 1075-1093
-
Tidskriftsartikel (refereegranskat)abstract
- The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens secrete and subsequently translocate antihost effector proteins into target eukaryotic cells by a common type III secretion system (TTSS). In this process, YopD (Yersinia outer protein D) is essential to establish regulatory control of Yop synthesis and the ensuing translocation process. YopD function depends upon the non-secreted TTSS chaperone LcrH (low-calcium response H), which is required for presecretory stabilization of YopD. However, as a new role for TTSS chaperones in virulence gene regulation has been proposed recently, we undertook a detailed analysis of LcrH. A lcrH null mutant constitutively produced Yops, even when this strain was engineered to produce wild-type levels of YopD. Furthermore, the YopD-LcrH interaction was necessary to regain the negative regulation of virulence associated genes yops). This finding was used to investigate the biological significance of several LcrH mutants with varied YopD binding potential. Mutated LcrH alleles were introduced in trans into a lcrH null mutant to assess their impact on yop regulation and the subsequent translocation of YopE, a Rho-GTPase activating protein, across the plasma membrane of eukaryotic cells. Two mutants, LcrHK20E, E30G, I31V, M99V, D136G and LcrHE30G lost all regulatory control, even though YopD binding and secretion and the subsequent translocation of YopE was indistinguishable from wild type. Moreover, these regulatory deficient mutants showed a reduced ability to bind YscY in the two-hybrid assay. Collectively, these findings confirm that LcrH plays an active role in yop regulation that might be mediated via an interaction with the Ysc secretion apparatus. This chaperone-substrate interaction presents an innovative means to establish a regulatory hierarchy in Yersinia infections. It also raises the question as to whether or not LcrH is a true chaperone involved in stabilization and secretion of YopD or a regulatory protein responsible for co-ordinating synthesis of Yersinia virulence determinants. We suggest that LcrH can exhibit both of these activities.
|
|
| 10. |
|
|
