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  • 1. Aili, Margareta
    et al.
    Hallberg, Bengt
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rosqvist, Roland
    GAP activity of Yersinia YopE2002In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 358, p. 359-70Article in journal (Refereed)
  • 2.
    Aili, Margareta
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Isaksson, Elin L
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Carlsson, Sara E
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Regulation of Yersinia Yop-effector delivery by translocated YopE2008In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 298, no 3-4, p. 183-192Article in journal (Refereed)
    Abstract [en]

    The bacterial pathogen Yersinia pseudotuberculosis uses a type III secretion (T3S) system to translocate Yop effectors into eukaryotic cells. Effectors are thought to gain access to the cytosol via pores formed in the host cell plasma membrane. Translocated YopE can modulate this pore formation through its GTPase-activating protein (GAP) activity. In this study, we analysed the role of translocated YopE and all the other known Yop effectors in the regulation of effector translocation. Elevated levels of Yop effector translocation into HeLa cells occurred by YopE-defective strains, but not those defective for other Yop effectors. Only Yersinia devoid of YopK exhibits a similar hyper-translocation phenotype. Since both yopK and yopE mutants also failed to down-regulate Yop synthesis in the presence of eukaryotic cells, these data imply that translocated YopE specifically regulates subsequent effector translocation by Yersinia through at least one mechanism that involves YopK. We suggest that the GAP activity of YopE might be working as an intra-cellular probe measuring the amount of protein translocated by Yersinia during infection. This may be a general feature of T3S-associated GAP proteins, since two homologues from Pseudomonas aeruginosa, exoenzyme S (ExoS) and exoenzyme T (ExoT), can complement the hyper-translocation phenotypes of the yopE GAP mutant.

  • 3.
    Aili, Margareta
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Isaksson, Elin L
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis2006In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 8, no 6, p. 1020-1033Article in journal (Refereed)
    Abstract [en]

    YopE of Yersinia pseudotuberculosis inactivates three members of the small RhoGTPase family (RhoA, Rac1 and Cdc42) in vitro and mutation of a critical arginine abolishes both in vitro GTPase-activating protein (GAP) activity and cytotoxicity towards HeLa cells, and renders the pathogen avirulent in a mouse model. To understand the functional role of YopE, in vivo studies of the GAP activity in infected eukaryotic cells were conducted. Wild-type YopE inactivated Rac1 as early as 5 min after infection whereas RhoA was down regulated about 30 min after infection. No effect of YopE was found on the activation state of Cdc42 in Yersinia-infected cells. Single-amino-acid substitution mutants of YopE revealed two different phenotypes: (i) mutants with significantly lowered in vivo GAP activity towards RhoA and Rac1 displaying full virulence in mice, and (ii) avirulent mutants with wild-type in vivo GAP activity towards RhoA and Rac1. Our results show that Cdc42 is not an in vivo target for YopE and that YopE interacts preferentially with Rac1, and to a lesser extent with RhoA, during in vivo conditions. Surprisingly, we present results suggesting that these interactions are not a prerequisite to establish infection in mice. Finally, we show that avirulent yopE mutants translocate YopE in about sixfold higher amount compared with wild type. This raises the question whether YopE's primary function is to sense the level of translocation rather than being directly involved in downregulation of the host defence.

  • 4.
    Aili, Margareta
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Telepnev, Max
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity2003In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 34, no 6, p. 297-308Article in journal (Refereed)
    Abstract [en]

    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.

  • 5.
    Akopyan, Karen
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Edgren, Tomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Wang-Edgren, Helen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Translocation of surface-localized effectors in type III secretion2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 4, p. 1639-1644Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells. Upon host cell contact, purified YopH coated on Y. pseudotuberculosis was specifically and rapidly translocated across the target-cell membrane, which led to a physiological response in the infected cell. In addition, translocation of externally added YopH required a functional T3SS and a specific translocation domain in the effector protein. Efficient, T3SS-dependent translocation of purified YopH added in vitro was also observed when using coated Salmonella typhimurium strains, which implies that T3SS-mediated translocation of extracellular effector proteins is conserved among T3SS-dependent pathogens. Our results demonstrate that polarized T3SS-dependent translocation of proteins can be achieved through an intermediate extracellular step that can be reconstituted in vitro. These results indicate that translocation can occur by a different mechanism from the assumed single-step conduit model.

  • 6. Dukuzumuremyi, Jean-Marie
    et al.
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Åkerström, Bo
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Schesser, Kurt
    The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 45, p. 35281-35290Article in journal (Refereed)
    Abstract [en]

    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.

  • 7.
    Edgren, Tomas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Type III Secretion in Yersinia: Injectisome or Not?2012In: PLoS pathogens, ISSN 1553-7374, Vol. 8, no 5, p. e1002669-Article in journal (Refereed)
  • 8.
    Forsberg, Karin
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Jonsson, P Andreas
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Andersen, Peter M
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Neurology.
    Bergemalm, Daniel
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Graffmo, Karin S
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Hultdin, Magnus
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Jacobsson, Johan
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Neurology.
    Rosquist, Roland
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Marklund, Stefan L
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Brännström, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients2010In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 7, p. e11552-Article in journal (Refereed)
    Abstract [en]

    Mutations in CuZn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) and are found in 6% of ALS patients. Non-native and aggregation-prone forms of mutant SOD1s are thought to trigger the disease. Two sets of novel antibodies, raised in rabbits and chicken, against peptides spaced along the human SOD1 sequence, were by enzyme-linked immunosorbent assay and an immunocapture method shown to be specific for denatured SOD1. These were used to examine SOD1 in spinal cords of ALS patients lacking mutations in the enzyme. Small granular SOD1-immunoreactive inclusions were found in spinal motoneurons of all 37 sporadic and familial ALS patients studied, but only sparsely in 3 of 28 neurodegenerative and 2 of 19 non-neurological control patients. The granular inclusions were by confocal microscopy found to partly colocalize with markers for lysosomes but not with inclusions containing TAR DNA binding protein-43, ubiquitin or markers for endoplasmic reticulum, autophagosomes or mitochondria. Granular inclusions were also found in carriers of SOD1 mutations and in spinobulbar muscular atrophy (SBMA) patients and they were the major type of inclusion detected in ALS patients homozygous for the wild type-like D90A mutation. The findings suggest that SOD1 may be involved in ALS pathogenesis in patients lacking mutations in the enzyme.

  • 9.
    Frithz-Lindsten, Elisabet
    et al.
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Holmström, Anna
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Jacobsson, Lars
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Soltani, Mehnam
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Olsson, Jan
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Functional conservation of the effector protein translocators PopB/YopB and PopD/YopD of Pseudomonas aeruginosa and Yersinia pseudotuberculosis.1998In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 29, no 5, p. 1155-1165Article in journal (Refereed)
    Abstract [en]

    Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa, on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact-dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia, YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa. Expression of the pcrGVHpopBD operon in defined translocation-deficient mutants (yopB/yopD) of Yersinia resulted in complete complementation of the cell contact-dependent, YopE-induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact-dependent toxin-targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.

  • 10.
    Gu, Weigang
    et al.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Neurology. Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Brännström, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wester, Per
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Cell division in the cerebral cortex of adult rats after photothrombotic ring stroke.2009In: Stem Cell Research, ISSN 1876-7753, Vol. 2, no 1, p. 68-77Article in journal (Refereed)
    Abstract [en]

    Neurogenesis has been shown to occur in the cerebral cortex in adult rats after ischemic stroke. The origin of the newborn neurons is largely unknown. This study aimed to explore cell division in the poststroke penumbral cortex. Adult male Wistar rats were subjected to photothrombotic ring stroke. After repeated delivery of the DNA duplication marker BrdU, the animals were sacrificed at various times poststroke. BrdU was detected by immunohistochemistry/immunofluorescence labeling, as was the M-phase marker Phos H3 and the spindle components alpha-tubulin/gamma-tubulin. DNA damage was examined by TUNEL staining. Cell type was ascertained by double immunolabeling with the neuronal markers Map-2ab/beta-tubulin III and NeuN/Hu or the astrocyte marker GFAP. From 16h poststroke, BrdU-immunolabeled cells appeared in the penumbral cortex. From 24h, Phos H3 was colocalized with BrdU in the nuclei. Mitotic spindles immunolabeled by alpha-tubulin/gamma-tubulin appeared inside the cortical cells containing BrdU-immunopositive nuclei. Unexpectedly, the markers of neuronal differentiation, Map-2ab/beta-tubulin III/NeuN/Hu, were expressed in the Phos H3-immunolabeled cells, and NeuN was detected in some cells containing spindles. This study suggests that in response to a sublethal ischemic insult, endogenous cells with neuronal immunolabeling may duplicate their nuclear DNA and commit cell mitosis to generate daughter neurons in the penumbral cortex in adult rats.

  • 11.
    Henriksson, M L
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Telepnev, Maxim
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras2000In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 347, no 1, p. 217-222Article in journal (Refereed)
  • 12.
    Holmström, Anna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Petterson, Jonas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Håkansson, Sebastian
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tafazoli, Farideh
    Department of Medical Microbiology, Linköping University, S-581 85 Linköping, Sweden.
    Fällman, Maria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Magnusson, Karl-Eric
    Department of Medical Microbiology, Linköping University, S-581 85 Linköping, Sweden.
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Department of Microbiology, National Defence Research Establishment, S-901 82 Umeå, Sweden..
    YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane.1997In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 24, no 1, p. 73-91Article in journal (Refereed)
    Abstract [en]

    Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia, the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK-mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK-mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.

  • 13.
    Holmström, Anna
    et al.
    Umeå University, Faculty of Medicine, Microbiology.
    Rosqvist, Roland
    Umeå University, Faculty of Medicine, Microbiology.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Microbiology.
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Microbiology.
    Virulence plasmid-encoded YopK is essential for Yersinia pseudotuberculosis to cause systemic infection in mice.1995In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 63, no 6, p. 2269-2276Article in journal (Refereed)
    Abstract [en]

    The virulence plasmid common to pathogenic Yersinia species encodes a number of secreted proteins denoted Yops (Yersinia outer proteins). Here, we identify and characterize a novel plasmid-encoded virulence determinant of Yersinia pseudotuberculosis, YopK. The yopK gene was found to be conserved among the three pathogenic Yersinia species and to be homologous to the previously described yopQ and yopK genes of Y. enterocolitica and Y. pestis, respectively. Similar to the other Yops, YopK expression and secretion were shown to be regulated by temperature and by the extracellular Ca2+ concentration; thus, yopK is part of the yop regulon. In addition, YopK secretion was mediated by the specific Yop secretion system. In Y. pseudotuberculosis, YopK was shown neither to have a role in this bacterium's ability to resist phagocytosis by macrophages nor to cause cytotoxicity in HeLa cells. YopK was, however, shown to be required for the bacterium to cause a systemic infection in both intraperitoneally and orally infected mice. Characterization of the infection kinetics showed that, similarly to the wild-type strain, the yopK mutant strain colonized and persisted in the Peyer's patches of orally infected mice. A yopE mutant which is impaired in cytotoxicity and in antiphagocytosis was, however, found to be rapidly cleared from these lymphoid organs. Neither the yopK nor the yopE mutant strain could overcome the primary host defense and reach the spleen. This finding implies that YopK acts at a different level during the infections process than the antiphagocytic YopE cytotoxin does.

  • 14.
    Isaksson, Elin L
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aili, Margareta
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Carlsson, Sara E
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    The membrane localization domain is required for intracellular localization and autoregulation of YopE in Yersinia pseudotuberculosis.2009In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 77, no 11, p. 4740-4749Article in journal (Refereed)
    Abstract [en]

    Recent work has shown that a domain of YopE of Yersinia pseudotuberculosis ranging from amino acids 54 to 75 (R. Krall, Y. Zhang, and J. T. Barbieri, J. Biol. Chem. 279:2747-2753, 2004) is required for proper localization of YopE after ectopic expression in eukaryotic cells. This domain, called the membrane localization domain (MLD), has not been extensively studied in Yersinia. Therefore, an in cis MLD deletion mutant of YopE was created in Y. pseudotuberculosis. The mutant was found to secrete and translocate YopE at wild-type levels. However, the mutant was defective in the autoregulation of YopE expression after the infection of HeLa cells. Although the mutant translocated YopE at wild-type levels, it showed a delayed HeLa cell cytotoxicity. This delay was not caused by a change in GTPase activating protein (GAP) activity, since the mutant showed wild-type YopE GAP activity toward Rac1 and RhoA. The MLD mutant displayed a changed intracellular localization pattern of YopE in HeLa cells after infection, and the YopEDeltaMLD protein was found to be dispersed within the whole cell, including the nucleus. In contrast, wild-type YopE was found to localize to the perinuclear region of the cell and was not found in the nucleus. In addition, the yopEDeltaMLD mutant was avirulent. Our results suggest that YopE must target proteins other than RhoA and Rac1 and that the MLD is required for the proper targeting and hence virulence of YopE during infection. Our results raise the question whether YopE is a regulatory protein or a "true" virulence effector protein.

  • 15.
    O'Toole, Ronan
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Von Hofsten, Jonas
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Olsson, Per-Erik
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Visualisation of zebrafish infection by GFP-labelled Vibrio anguillarum.2004In: Microb Pathog, ISSN 0882-4010, Vol. 37, no 1, p. 41-6Article in journal (Refereed)
    Abstract [en]

    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.

  • 16.
    Persson, Cathrine
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Holmström, Anna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Håkansson, Sebastian
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cell-surface-bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into the target cell1995In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 18, no 1, p. 135-150Article in journal (Refereed)
    Abstract [en]

    YopH is translocated by cell-surface-bound bacteria through the plasma membrane to the cytosol of the HeLa cell. The transfer mechanism is contact dependent and polarizes the translocation to only occur at the contact zone between the bacterium and the target cell. More than 99% of the PTPase activity is associated with the HeLa cells. In contrast to the wild-type strain, the yopBD mutant cannot deliver YopH to the cytosol. Instead YopH is deposited in localized areas in the proximity of cell-associated bacteria. A yopN mutant secretes 40% of the total amount of YopH to the culture medium, suggesting a critical role of YopN in regulation of the polarized translocation. Evidence for a region in YopH important for its translocation through the plasma membrane of the target cell but not for secretion from the pathogen is provided.

  • 17. Shrestha, Niraj
    et al.
    Boucher, Justin
    Bahnan, Wael
    Clark, Emily S.
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fields, Kenneth A.
    Khan, Wasif N.
    Schesser, Kurt
    The Host-Encoded Heme Regulated Inhibitor (HRI) Facilitates Virulence-Associated Activities of Bacterial Pathogens2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 7, p. e68754-Article in journal (Refereed)
    Abstract [en]

    Here we show that cells lacking the heme-regulated inhibitor (HRI) are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S) system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria. For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2 alpha kinase) are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.

  • 18.
    Von Pawel-Rammingen, Ulrich
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Telepnev, Maxim V
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Schmidt, Gudula
    Aktories, Klaus
    Wolf-Watz, Hans
    Rosqvist, Roland
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway: a mechanism for disruption of actin microfilament structure.2000In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 36, no 3, p. 737-748Article in journal (Refereed)
    Abstract [en]

    The YopE cytotoxin of Yersinia pseudotuberculosis is an essential virulence determinant that is injected into the eukaryotic target cell via a plasmid-encoded type III secretion system. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. Here, we show that YopE exhibits a GTPase-activating protein (GAP) activity and that the presence of YopE stimulates downregulation of Rho, Rac and Cdc42 activity. YopE has an arginine finger motif showing homology with those found in other GAP proteins. Exchange of arginine 144 with alanine, located in this arginine finger motif, results in an inactive form of YopE that can no longer stimulate GTP hydrolysis by the GTPase. Furthermore, a yopE(R144A) mutant is unable to induce cytotoxicity on cultured HeLa cells in contrast to the corresponding wild-type strain. Expression of wild-type YopE in cells of Saccharomyces cerevisiae inhibits growth, while in contrast, expression of the inactive form of YopE, YopE(R144A), does not affect the yeast cells. Co-expression of proteins belonging to the Rho1 pathway of yeast, Rho1, Rom2p, Bck1 and Ste20, suppressed the growth phenotype of YopE in yeast cells. These results provide evidence that YopE exhibits a GAP activity to inactivate RhoGTPases, leading to depolymerization of the actin stress fibres in eukaryotic cells and growth inhibition in yeast.

  • 19. Wiley, David J
    et al.
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Schesser, Kurt
    Induction of the Yersinia type 3 secretion system as an all-or-none phenomenon2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 373, no 1, p. 27-37Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia spp. possess a protein secretion system, designated as type 3, that plays a clear role in promoting their survival vis-à-vis the macrophage. Inductive expression of the Yersinia type 3 secretion system (T3SS), triggered either by host cell contact, or, in the absence of host cells, by a reduction in extracellular calcium ion levels, is accompanied by a withdrawal from the bacterial division cycle. Here, we analyzed Ca2+-dependent induction of the T3SS at the single-cell level to understand how Yersinia coordinates pro-survival and growth-related activities. We utilized a novel high-throughput quantitative microscopy approach as well as flow cytometry to determine how Ca2+ levels, T3SS expression, and cellular division are interrelated. Our analysis showed that there is a high degree of homogeneity in terms of T3SS expression levels among a population of Y. pseudotuberculosis cells following the removal of Ca2+, and that T3SS expression appears to be independent of the cellular division cycle. Unexpectedly, our analysis showed that Ca2+ levels are inversely related to the initiation of inductive T3SS expression, and not to the intensity of activation once initiated, thus providing a basis for the seemingly graded response of T3SS activation observed in bulk-level analyses. The properties of the system described here display both similarities to and differences from that of the lac operon first described 50 years ago by Novick and Weiner.

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