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1.
BMC Microbiol ; 1: 22, 2001.
Article in English | MEDLINE | ID: mdl-11696238

ABSTRACT

BACKGROUND: Several bacterial pathogens express antihost factors that likely decrease both their maximal growth rate (due to metabolic costs) as well as their mortality rate (by neutralizing host defenses). The pathogenic yersiniae make a huge metabolic investment expressing virulence proteins (referred to as Yops) that are directly injected into eukaryotic cells and that modulate host defense responses such as phagocytosis and stress-activated signaling pathways. Although host-cell contact enhanced Yop expression as well as the cellular activities of several Yops have recently been described, a clear link between these phenomena and bacterial survival and/or proliferation remains to be established RESULTS: We show that the proliferation of Y. pseudotuberculosis is compromised when the bacterium is growing in association with eukaryotic cells compared to free-living bacteria. One factor likely limiting Yersinia proliferation is the metabolically taxing expression of yopE which we show using flow cytometry increases in individual bacteria following their contact with cultured macrophage-like cells. An additional factor limiting Y. pseudotuberculosis proliferation are host cell defense systems which can be significantly ameliorated by disrupting the host cell cytoskeletal system by either exogenously added toxins or by the bacterial-mediated injection of YopE or YopH. CONCLUSIONS: Our results demonstrate that despite their metabolic costs the Yop virulence proteins play an important role in enabling Y. pseudotuberculosis to survive and proliferate when confronted with the antimicrobial activities of the eukaryotic cell.


Subject(s)
Bacterial Outer Membrane Proteins/physiology , Eukaryotic Cells/microbiology , Protein Tyrosine Phosphatases/physiology , Yersinia/physiology , Animals , Cell Division , Cell Survival , Cytoskeleton/physiology , Eukaryotic Cells/physiology
2.
J Biol Chem ; 275(45): 35281-90, 2000 Nov 10.
Article in English | MEDLINE | ID: mdl-10950948

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.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/physiology , Virulence , Yersinia/enzymology , Yersinia/pathogenicity , rhoA GTP-Binding Protein/metabolism , Actins/metabolism , Amino Acid Sequence , Cell Membrane/chemistry , Cell Membrane/metabolism , Enzyme Activation , Glutathione Transferase/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Guanosine Diphosphate/metabolism , HeLa Cells , Humans , Image Processing, Computer-Assisted , Kinetics , Microscopy, Confocal , Molecular Sequence Data , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Stress Fibers/metabolism , Time Factors , Transfection , Two-Hybrid System Techniques , Yersinia/metabolism , cdc42 GTP-Binding Protein/metabolism , rac GTP-Binding Proteins/metabolism
3.
Microb Pathog ; 28(2): 59-70, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10644492

ABSTRACT

The YopJ protein of Yersinia pseudotuberculosis inhibits several eukaryotic signalling pathways that are normally activated in cells following their contact with bacteria. Salmonella encodes a protein, AvrA, that is secreted by the typeIII inv/spa secretion system which is clearly homologous to YopJ (56% identical, 87% similarity). Since AvrA and YopJs similarity also encompassed a region of YopJ that had previously been shown to be critical for its biological activity, we were interested whether AvrA and YopJ provoked similar responses in eukaryotic cells. Two different approaches were used to determine whether AvrA possesses YopJ-like activity in modulating cytokine expression or killing macrophages. An avrA strain of Salmonella dublin was constructed and its activity was compared to an isogenic wildtype counterpart in cellular response assays. In a complementary approach, AvrA was expressed in and delivered into eukaryotic cells by a yopJ strain of Yersinia pseudotuberculosis. We show here that AvrA affects neither cytokine expression or plays a role in macrophage killing when expressed by either Salmonella or Yersinia. Additionally, AvrA does not possess SopB/D-like activity in promoting fluid secretion into infected calf ileal loops. These data indicate that Salmonella and Yersinia trigger and/or modulate eukaryotic cell responses by different typeIII-secreted proteins and suggests that despite their close evolutionary relatedness, AvrA and YopJ perform different functions for Salmonella and Yersinia, respectively.


Subject(s)
Bacterial Proteins/metabolism , Salmonella enterica/metabolism , Yersinia pseudotuberculosis/metabolism , Apoptosis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cell Line , Cytokines/metabolism , HeLa Cells , Humans , Ileum/metabolism , Ileum/microbiology , Macrophages/microbiology , Molecular Sequence Data , Plasmids/genetics , Salmonella enterica/genetics , Sequence Homology, Amino Acid , Yersinia pseudotuberculosis/genetics
4.
Cell Microbiol ; 2(3): 231-8, 2000 Jun.
Article in English | MEDLINE | ID: mdl-11207579

ABSTRACT

Bacterially encoded proteins are known to affect eukaryotic signalling pathways and thus cell growth and differentiation. The enteric pathogen Yersinia pseudotuberculosis (YP) can translocate Yersinia outer proteins (Yops) into eukaryotic cells. Recently, MKK proteins have been identified as tentative targets of YopJ-mediated inhibition of ligand receptor-dependent signal transduction in mammalian cells. These results prompted us to assess whether multiple signal transduction pathways and their downstream target genes would also be subject to regulation by YopJ. Here, we show that YopJ effectively blocks the lipopolysaccharide (LPS) receptor, the interleukin (IL)-1beta receptor and the UVC-induced activation of the transcription receptor cAMP response element-binding protein (CREB). In addition, by abrogating the phosphorylation of CREB and thus activating protein (AP)-1-dependent transcription, YopJ can block LPS-induced clonal expansion that is associated with an adaptive immune response. Thus, YopJ interferes with multiple pathways converging on the transcription factor CREB. Our data are discussed in the context of YopJ acting as an antagonist to circumvent innate and adaptive immune responses at multiple levels.


Subject(s)
Bacterial Proteins/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Signal Transduction , Yersinia pseudotuberculosis/metabolism , Yersinia pseudotuberculosis/pathogenicity , Bacterial Proteins/genetics , Blotting, Western , Cyclic AMP Response Element-Binding Protein/genetics , HeLa Cells , Humans , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Reverse Transcriptase Polymerase Chain Reaction , Spleen/cytology , Transcription Factor AP-1/metabolism , Tumor Cells, Cultured , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis Infections/microbiology , p38 Mitogen-Activated Protein Kinases
5.
Immunol Lett ; 68(2-3): 247-9, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10424427

ABSTRACT

Inflammatory bowel disease (IBD) comprises different diseases in the gastrointestinal tract in human, of which Crohn's disease (CD) and ulcerative colitis (UC) are the most prominent. A key factor in the etiology of IBD is the chronic inflammatory process, and a large body of evidence suggests that the transcription factor nuclear factor-kappa B (NF-kappaB) is the key regulator of responses determining the clinical inflammatory condition. Recent findings using antisense oligonucleotides provide direct evidence that the p65 subunit of NF-kappaB plays a central role in chronic intestinal inflammation. It has previously been shown that the Gram negative bacteria Yersinia pseudotubercolosis targets the eukaryotic signal transduction pathway(s) that lead to NF-kappaB activation (and thus avoid an anti-bacterial inflammatory response). In this paper, growth-based selected Salmonella typhimurium clones have been used to generate a clearer picture of the molecular mechanisms involved in host-parasite interactions. From the results presented here, S. typhimurium and Y. pseudotubercolosis may use the same mechanism to block NF-kappaB activation, following host cell infection. A new adaptational feature could also be shown, where a growth-based selected bacteria avoided the normally induced translocation of NF-kappaB in host cells.


Subject(s)
I-kappa B Proteins , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Salmonella typhimurium/pathogenicity , Biological Transport , Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Inflammatory Bowel Diseases/etiology , NF-KappaB Inhibitor alpha , Signal Transduction , Transcription Factor RelA
6.
Folia Microbiol (Praha) ; 43(3): 263-73, 1998.
Article in English | MEDLINE | ID: mdl-9717253

ABSTRACT

Preventing the early host immune defense allows pathogenic Yersinia to proliferate in lymphatic tissue. This ability depends on signaling that occurs between the bacteria and the host cells. Following intimate contact with the target cell a signal is generated within the bacterium that results in increased expression of virulence-associated proteins that are subsequently delivered into the infected cell. These proteins, designated Yops, interfere with the host-cell signaling pathways that are normally activated to eliminate infectious agents.


Subject(s)
Lymphatic System/cytology , Lymphatic System/microbiology , Signal Transduction/physiology , Yersinia Infections/microbiology , Yersinia/physiology , Humans
7.
Mol Microbiol ; 28(6): 1067-79, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9680199

ABSTRACT

Upon exposure to bacteria, eukaryotic cells activate signalling pathways that result in the increased expression of several defence-related genes. Here, we report that the yopJ locus of the enteropathogen Yersinia pseudotuberculosis encodes a protein that inhibits the activation of NF-kappaB transcription factors by a mechanism(s), which prevents the phosphorylation and subsequent degradation of the inhibitor protein IkappaB. Consequently, eukaryotic cells infected with YopJ-expressing Yersinia become impaired in NF-kappaB-dependent cytokine expression. In addition, the blockage of inducible cytokine production coincides with yopJ-dependent induction of apoptosis. Interestingly, the YopJ protein contains a region that resembles a src homology domain 2 (SH2), and we show that a wild-type version of this motif is required for YopJ activity in suppressing cytokine expression and inducing apoptosis. As SH2 domains are found in several eukaryotic signalling proteins, we propose that YopJ, which we show is delivered into the cytoplasm of infected cells, interacts directly with signalling proteins involved in inductive cytokine expression. The repressive activity of YopJ on the expression of inflammatory mediators may account for the lack of an inflammatory host response observed in experimental yersiniosis. YopJ-like activity may also be a common feature of commensal bacteria that, like Yersinia, do not provoke a host inflammatory response.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Cytokines/metabolism , NF-kappa B/metabolism , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis/physiology , src Homology Domains/genetics , Amino Acid Sequence , Apoptosis/physiology , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/physiology , Cells, Cultured , Flow Cytometry , Gene Expression Regulation, Bacterial , Genes, Reporter , HeLa Cells , Humans , Macrophages , Molecular Sequence Data , Plasmids , Transcriptional Activation , Transfection , Tumor Necrosis Factor-alpha/analysis , Virulence , Yersinia pseudotuberculosis/pathogenicity
8.
J Bacteriol ; 178(24): 7227-33, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8955406

ABSTRACT

Pathogenic yersiniae deliver a number of different effector molecules, which are referred to as Yops, into the cytosol of eukaryotic cells via a type III secretion system. To identify the regions of YopE from Yersinia pseudotuberculosis that are necessary for its translocation across the bacterial and eukaryotic cellular membranes, we constructed a series of hybrid genes which consisted of various amounts of yopE fused to the adenylate cyclase-encoding domain of the cyclolysin gene (cyaA) of Bordetella pertussis. By assaying intact cells for adenylate cyclase activity, we show that a YopE-Cya protein containing just the 11 amino-terminal residues of YopE is efficiently exported to the exterior surface of the bacterial cell. Single amino acid replacements of the first seven YopE residues significantly decreased the amount of reporter protein detected on the cell surface, suggesting that the extreme amino-terminal region of YopE is recognized by the secretion machinery. As has recently been shown for the Y. enterocolitica YopE protein (M.-P. Sory, A. Boland, I. Lambermont, and G. R. Cornelis, Proc. Natl. Acad. Sci. USA 92:11998-12002, 1995), we found that export to the cell surface was not sufficient for YopE-Cya proteins to be delivered into the eukaryotic cytoplasm. For traversing the HeLa cell membrane, at least 49 yopE-encoded residues were required. Replacement of leucine 43 of YopE with glycine severely affected the delivery of the reporter protein into HeLa cells. Surprisingly, export from the bacterial cell was also not sufficient for YopE-Cya proteins to be released from the bacterial cell surface into the culture supernatant. At least 75 residues of YopE were required to detect activity of the corresponding reporter protein in the culture supernatant, suggesting that a release domain exists in this region of YopE. We also show that the chaperone-like protein YerA required at least 75 YopE residues to form a stable complex in vitro with YopE-Cya proteins and, furthermore, that YerA is not required to target YopE-Cya proteins to the secretion complex. Taken together, our results suggest that traversing the bacterial and eukaryotic membranes occurs by separate processes that recognize distinct domains of YopE and that these processes are not dependent on YerA activity.


Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Yersinia pseudotuberculosis/metabolism , Adenylyl Cyclases/genetics , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cell Membrane/metabolism , Gene Expression , HeLa Cells , Humans , Mutagenesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Trans-Activators/metabolism , Yersinia pseudotuberculosis/genetics
9.
EMBO J ; 15(21): 5812-23, 1996 Nov 01.
Article in English | MEDLINE | ID: mdl-8918459

ABSTRACT

During infection of cultured epithelial cells, surface-located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non-polar yopB mutant strain displays a wild-type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage-like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia-induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB-dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell-to-cell transfer of Yop effector proteins.


Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Yersinia pseudotuberculosis/metabolism , Animals , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/toxicity , Biological Transport, Active , Cell Membrane/drug effects , Cell Membrane/metabolism , Gene Deletion , Genes, Bacterial , HeLa Cells , Hemolysis/drug effects , Humans , In Vitro Techniques , Mice , Microscopy, Confocal , Phagocytosis , Protein Tyrosine Phosphatases/metabolism , Sheep , Virulence/genetics , Virulence/physiology , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis/pathogenicity
10.
Appl Environ Microbiol ; 57(2): 553-6, 1991 Feb.
Article in English | MEDLINE | ID: mdl-16348420

ABSTRACT

Gaeumannomyces graminis, the causative agent of take-all disease of wheat, barley, and oats, was detected in infected wheat seedlings by using the polymerase chain reaction to amplify Gaeumannomyces-specific DNA fragments. Nested primers and two rounds of amplification were used to amplify two fragments, approximately 287 and 188 bp in size, from G. graminis-infected wheat seedlings. The use of nested primers greatly decreased the number of nonspecific amplification products. Polymerase chain reaction products were not obtained with DNA from seedlings infected with several other phytopathogenic fungi or with DNA from uninfected seedlings. Amplified products were visualized on agarose gels, and their identities were confirmed by DNA hybridization. This method did not require culturing the fungus and has potential for detecting G. graminis in infested wheat, barley, or oat fields.

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