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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, 359-70 p.Article 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, 183-192 p.Article 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, 1020-1033 p.Article 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.
    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, 1639-1644 p.Article 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.

  • 5. Andersson, K
    et al.
    Carballeira Suarez, N
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Magnusson, K E
    Persson, C
    Stendahl, O
    Wolf-Watz, H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fällman, M
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signalling associated with phagocytosis.1996In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 20, no 5, 1057-69 p.Article in journal (Refereed)
    Abstract [en]

    The PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild-type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild-type yopH restored the anti-phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopHC403A was without effect. The PTPase inhibitor orthovanadate impaired the anti-phagocytic effect of the wild-type strain, further demonstrating the importance of bacteria-derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild-type Y. pseudotuberculosis caused a YopH-dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface-located protein invasin. Co-infection with the phagocytosable strain and the wild-type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH-mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis-linked signalling in the phagocyte.

  • 6.
    Antti, Henrik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Näsström, Elin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kouremenos, Konstantinos
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sundén-Cullberg, Jonas
    Guo, Yongzhi
    Moritz, Thomas
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Metabolic profiling for detection of staphylococcus aureus infection and antibiotic resistance2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2, e56971Article in journal (Refereed)
    Abstract [en]

    Due to slow diagnostics, physicians must optimize antibiotic therapies based on clinical evaluation of patients without specific information on causative bacteria. We have investigated metabolomic analysis of blood for the detection of acute bacterial infection and early differentiation between ineffective and effective antibiotic treatment. A vital and timely therapeutic difficulty was thereby addressed: the ability to rapidly detect treatment failures because of antibiotic-resistant bacteria. Methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) were used and for infecting mice, while natural MSSA infection was studied in humans. Samples of bacterial growth media, the blood of infected mice and of humans were analyzed with combined Gas Chromatography/Mass Spectrometry. Multivariate data analysis was used to reveal the metabolic profiles of infection and the responses to different antibiotic treatments. experiments resulted in the detection of 256 putative metabolites and mice infection experiments resulted in the detection of 474 putative metabolites. Importantly, ineffective and effective antibiotic treatments were differentiated already two hours after treatment start in both experimental systems. That is, the ineffective treatment of MRSA using cloxacillin and untreated controls produced one metabolic profile while all effective treatment combinations using cloxacillin or vancomycin for MSSA or MRSA produced another profile. For further evaluation of the concept, blood samples of humans admitted to intensive care with severe sepsis were analyzed. One hundred thirty-three putative metabolites differentiated severe MSSA sepsis (n = 6) from severe sepsis (n = 10) and identified treatment responses over time. Combined analysis of human, , and mice samples identified 25 metabolites indicative of effective treatment of sepsis. Taken together, this study provides a proof of concept of the utility of analyzing metabolite patterns in blood for early differentiation between ineffective and effective antibiotic treatment in acute infections.

  • 7.
    Bailey, Leslie
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gylfe, Asa
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sundin, Charlotta
    Muschiol, Sandra
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nordström, Peter
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine. Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Geriatric Medicine.
    Henriques-Normark, Birgitta
    Lugert, Raimond
    Waldenström, Anders
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Molecular Biology.
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle2007In: FEBS Lett, ISSN 0014-5793, Vol. 581, no 4, 587-595 p.Article in journal (Refereed)
    Abstract [en]

    Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re-differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti-chlamydial drugs.

  • 8.
    Björnfot, Ann-Catrin
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lavander, Moa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). 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 Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Autoproteolysis of YscU of Yersinia pseudotuberculosis is important for regulation of expression and secretion of Yop proteins2009In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, no 13, 4259-4267 p.Article in journal (Refereed)
    Abstract [en]

    YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU(CC). Autoproteolysis occurs at the conserved N downward arrowPTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca(2+) conditions) and calcium-depleted medium (-Ca(2+) conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca(2+)-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca(2+) conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under -Ca(2+) conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.

  • 9.
    Björnfot, Ann-Catrin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Login, Frédéric H.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Edgren, Tomas
    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).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Involvement of the heat shock proteins DnaK/DnaJ in Yersinia T3SManuscript (preprint) (Other academic)
    Abstract [en]

    Yersinia  pseudotuberculosis  uses  a  type  III  secretion  system  (T3SS)  to  secrete  and  deliver  effectors  called Yops into target cells. These processes are highly regulated and the pathogen senses cell contact and respond accordingly by inducing Yop-effector expression.  A key component of the T3SS is the YscF needle present on the  surface of  the  pathogen. It has  been  suggested  that the  bacterium  can  switch  from  needle  export  to  Yop expression  and  secretion  and  that this  substrate  switch  is  important  for proper  regulation  during infection. YscU  is  an  essential  protein  regulating  the  substrate  switch  and  autoproteolysis  of  YscU  is  essential  for accurate  T3SS  regulation.  To  study  regulation  of  Yop  translocation  in  more  detail,  we  generated  mutants defective for expression of the heat shock proteins (HSPs) DnaJ and DnaK, since earlier studies had indicated a role of these proteins in regulation of effector translocation in Salmonella. The dnaJ mutant and the double dnaK/J  mutant  showed  significant  defects  in  Yop  translocation,  but  surprisingly  both  mutants  were  able  to secrete Yops in vitro much like the wild type.  However, both mutants showed a changed export pattern of the YscF  needle  with  a  pronounced  increased  export  of  the  YscF  needle  protein  after  incubation  in  calcium containing media. This phenotype was linked to defects in YscU autoproteolysis and in this respect the  hsp-mutants  were  identical  to  earlier  identified  autoprocessing  defective  mutants  in  YscU  (Single  amino  acid exchange mutants N263A and P264A). The hsp-mutants and the processing mutants accumulated full-length YscU,  which  surprisingly  was  associated  with  the  outer  membrane,  while  the  processed  form  of  YscU  was found  in  the  inner  membrane  fraction.  The  dnaJ  and  dnaK/J  mutants  were  strongly  affected  in  YscU autoproteolysis, which indicates a possible direct role for DnaJ in this process. Indeed a specific interaction between  YscU  and  DnaJ  could  be  found  suggesting  a  direct  role  of  the  HSPs  in  regulation  of  the  substrate switch in the T3SS.

  • 10. Boehme, Katja
    et al.
    Steinmann, Rebekka
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Kortmann, Jens
    Seekircher, Stephanie
    Heroven, Ann Kathrin
    Berger, Evelin
    Pisano, Fabio
    Thiermann, Tanja
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Narberhaus, Franz
    Dersch, Petra
    Concerted Actions of a Thermo-labile Regulator and a Unique Intergenic RNA Thermosensor Control Yersinia Virulence2012In: PLOS PATHOGENS, ISSN 1553-7366, Vol. 8, no 2, e1002518- p.Article in journal (Refereed)
    Abstract [en]

    Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein-and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique cis-acting RNA element located within the intergenic region of the yscW-lcrF transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25 degrees C. The second hairpin sequesters the lcrF ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37 degrees C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different Y. pseudotuberculosis patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyer's patches, liver and spleen and have fully lost their lethality. Intriguingly, Yersinia strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency.

  • 11.
    Costa, Tiago
    et al.
    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).
    Amer, Ayad
    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).
    Farag, Salah
    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).
    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, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Edgren, Tomas
    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).
    Francis, Matthew
    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).
    Type III secretion translocon assemblies that attenuate Yersinia virulence2013In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, no 7, 1088-1110 p.Article in journal (Refereed)
    Abstract [en]

    Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α-helix motif at the C-terminus. Mutants YopD(I262P) and YopD(K267P) poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopD(A263P) and YopD(A270P) had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.

  • 12.
    Deleuil, Fabienne
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Mogemark, Lena
    Umeå University, Faculty of Medicine, Molecular Biology.
    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).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Interaction between the Yersinia protein tyrosine phosphatase YopH and eukaryotic Cas/Fyb is an important virulence mechanism2003In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 5, no 1, 53-64 p.Article in journal (Refereed)
    Abstract [en]

    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.

  • 13. 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, 35281-35290 p.Article 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.

  • 14.
    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, e1002669- p.Article in journal (Refereed)
  • 15.
    Engström, Patrik
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Krishnan, K. Syam
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ngyuen, Bidong D.
    Chorell, Erik
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Normark, Johan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Silver, Jim
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bastidas, Robert J.
    Welch, Matthew D.
    Hultgren, Scott J.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Valdivia, Raphael H.
    Almqvist, Fredrik
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    A 2-Pyridone-Amide Inhibitor Targets the Glucose Metabolism Pathway of Chlamydia trachomatis2015In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 6, no 1, e02304-14Article in journal (Refereed)
    Abstract [en]

    In a screen for compounds that inhibit infectivity of the obligate intracellular pathogen Chlamydia trachomatis, we identified the 2-pyridone amide KSK120. A fluorescent KSK120 analogue was synthesized and observed to be associated with the C. trachomatis surface, suggesting that its target is bacterial. We isolated KSK120-resistant strains and determined that several resistance mutations are in genes that affect the uptake and use of glucose-6-phosphate (G-6P). Consistent with an effect on G-6P metabolism, treatment with KSK120 blocked glycogen accumulation. Interestingly, KSK120 did not affect Escherichia coli or the host cell. Thus, 2-pyridone amides may represent a class of drugs that can specifically inhibit C. trachomatis infection. IMPORTANCE Chlamydia trachomatis is a bacterial pathogen of humans that causes a common sexually transmitted disease as well as eye infections. It grows only inside cells of its host organism, within a parasitophorous vacuole termed the inclusion. Little is known, however, about what bacterial components and processes are important for C. trachomatis cellular infectivity. Here, by using a visual screen for compounds that affect bacterial distribution within the chlamydial inclusion, we identified the inhibitor KSK120. As hypothesized, the altered bacterial distribution induced by KSK120 correlated with a block in C. trachomatis infectivity. Our data suggest that the compound targets the glucose-6-phosphate (G-6P) metabolism pathway of C. trachomatis, supporting previous indications that G-6P metabolism is critical for C. trachomatis infectivity. Thus, KSK120 may be a useful tool to study chlamydial glucose metabolism and has the potential to be used in the treatment of C. trachomatis infections.

  • 16.
    Engström, Patrik
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nguyen, Bidong D.
    Normark, Johan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Nilsson, Ingela
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bastidas, Robert J.
    Gylfe, Åsa
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Elofsson, Mikael
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Fields, Kenneth A.
    Valdivia, Raphael H.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bergström, Sven
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Mutations in hemG Mediate Resistance to Salicylidene Acylhydrazides, Demonstrating a Novel Link between Protoporphyrinogen Oxidase (HemG) and Chlamydia trachomatis Infectivity2013In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 195, no 18, 4221-4230 p.Article in journal (Refereed)
    Abstract [en]

    Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear. We investigated here whether SAHs exhibit a function on C. trachomatis that goes beyond iron chelation. We found that the iron-saturated SAH INP0341 (IS-INP0341) specifically affects C. trachomatis infectivity with reduced generation of infectious elementary body (EB) progeny. Selection and isolation of spontaneous SAH-resistant mutant strains revealed that mutations in hemG suppressed the reduced infectivity caused by IS-INP0341 treatment. Structural modeling of C. trachomatis HemG predicts that the acquired mutations are located in the active site of the enzyme, suggesting that IS-INP0341 inhibits this domain of HemG and that protoporphyrinogen oxidase (HemG) and heme metabolism are important for C. trachomatis infectivity.

  • 17.
    Eriksson, Jonas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer-Eriksson, A Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer, Uwe H
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    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).
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Small Molecule Screening for Inhibitors of the YopH Phosphatase of Yersinia pseudotuberculosis2012In: Advances in Yersinia Research, New York: Springer, 2012, Vol. 954, 357-363 p.Chapter in book (Refereed)
    Abstract [en]

    Bacterial virulence systems are attractive targets for development of new antibacterial agents. Yersinia spp. utilize the type III secretion (T3S) system to secrete and translocate Yersinia outer proteins (Yop effectors) into the cytosol of the target cell and thereby overcome host defenses to successfully establish an infection. Thus, the Yop effectors constitute attractive targets for drug development. In the present study we apply small molecule screening to identify inhibitors of one of the secreted proteins YopH, a tyrosine phosphatase required for virulence. Characterization of seven inhibitors indicated that both competitive and noncompetitive inhibitors were identified with IC50 values of 6–20 μM.

  • 18. Fernebro, Jenny
    et al.
    Blomberg, Christel
    Morfeldt, Eva
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, 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).
    Normark, Staffan
    Normark, Birgitta Henriques
    The influence of in vitro fitness defects on pneumococcal ability to colonize and to cause invasive disease.2008In: BMC microbiology, ISSN 1471-2180, Vol. 8, 65- p.Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Streptococcus pneumoniae is a genetically diverse major human pathogen, yet a common colonizer of the nasopharynx. Here we analyzed the influence of defects affecting in vitro growth rate, on the ability of S. pneumoniae to colonize and to cause invasive disease in vivo. RESULTS: Of eleven different clinical isolates one serotype 14 carrier isolate showed a significantly longer generation time as compared to other isolates, and was severely attenuated in mice. To directly investigate the impact of growth rate on virulence, a panel of mutants in five non-essential housekeeping genes was constructed in the virulent TIGR4 background by insertion-deletion mutagenesis. Three of these mutants (ychF, hemK and yebC) were, to different degrees, growth defective, and showed a reduced invasiveness in an intranasal murine challenge model that correlated to their in vitro growth rate, but remained capable of colonizing the upper airways. The growth defect, as well as virulence defect of the hemK insertion-deletion mutant, was mediated by polarity effects on the downstream yrdC gene, encoding a probable chaperone in ribosome assembly. CONCLUSION: We conclude that large fitness defects are needed to completely prevent pneumococci from causing invasive disease after intranasal challenge. However, even severe growth defects still allow pneumococci to persistently colonize the upper airways.

  • 19.
    Francis, Matthew S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lloyd, Scott A
    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).
    The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis2001In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 42, no 4, 1075-1093 p.Article in journal (Refereed)
    Abstract [en]

    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.

  • 20.
    Francis, Matthew S
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Schesser, Kurt
    Forsberg, Åke
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Type III secretion systems in animal- and plant-interacting bacteria2004In: Cellular Microbiology, ASM Press, Washington, D.C. , 2004, 362-392 p.Chapter in book (Other (popular science, discussion, etc.))
  • 21.
    Francis, Matthew S
    et al.
    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).
    YopD of Yersinia pseudotuberculosis is translocated into the cytosol of HeLa epithelial cells: evidence of a structural domain necessary for translocation.1998In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 29, no 3, 799-813 p.Article in journal (Refereed)
    Abstract [en]

    Yersinia pseudotuberculosis YopB and YopD proteins are essential for translocation of Yop effector proteins into the target cell cytosol. YopB is suggested to mediate pore formation in the target cell plasma membrane, allowing translocation of Yop effector proteins, although the function of YopD is unclear. To investigate the role in translocation for YopD, a mutant strain in Y. pseudotuberculosis was constructed containing an in frame deletion of essentially the entire yopD gene. As shown recently for the Y. pestis YopD protein, we found that the in vitro low calcium response controlling virulence gene expression was negatively regulated by YopD. This yopD null mutant (YPIII/pIB621) was also non-cytotoxic towards HeLa cell monolayers, supporting the role for YopD in the translocation process. Although other constituents of the Yersinia translocase apparatus (YopB, YopK and YopN) are not translocated into the host cell cytosol, fractionation of infected HeLa cells allowed us to identify the cytosolic localization of YopD by the wild-type strain (YPIII/pIB102), but not by strains defective in either YopD or YopB. YopD was also identified by immunofluorescence in the cytoplasm of HeLa cell monolayers infected with a multiple yop mutant strain (YPIII/pIB29MEKA). These results demonstrate a dual function for YopD in negative regulation of Yop production and Yop effector translocation, including the YopD protein itself. To investigate whether an amphipathic domain near the C-terminus of YopD is involved in the translocation process, a mutant strain (YPIII/pIB155deltaD278-292) was constructed that is devoid of this region. Phenotypically, this small in frame deltayopD278-292 deletion mutant was indistinguishable from the yopD null mutant. The truncated YopD protein and Yop effectors were not translocated into the cytosol of HeLa cell monolayers infected with this mutant. The comparable regulatory and translocation phenotypes displayed by the small in frame deltayopD278-292 deletion and deltayopD null mutants suggest that regulation of Yop synthesis and Yop translocation are intimately coupled. We present an intriguing scenario to the Yersinia infection process that highlights the need for polarized translocation of YopD to specifically establish translocation of Yop effectors. These observations are contrary to previous suggestions that members of the translocase apparatus were not translocated into the host cell cytosol.

  • 22.
    Francis, Matthew S
    et al.
    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).
    Forsberg, Ake
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Regulation of type III secretion systems2002In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 5, no 2, 166-172 p.Article in journal (Refereed)
    Abstract [en]

    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.

  • 23.
    Frost, Stefan
    et al.
    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).
    Ho, Oanh
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Login, Frédéric H
    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).
    Weise, Christoph F
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    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).
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Autoproteolysis and Intramolecular Dissociation of Yersinia YscU Precedes Secretion of Its C-Terminal Polypeptide YscU CC2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, e49349Article in journal (Refereed)
    Abstract [en]

    Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscUCC. Here we show that depletion of calcium induces intramolecular dissociation of YscUCC from YscU followed by secretion of the YscUCC polypeptide. Thus, YscUCC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscUCC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscUCC dissociation for Yop secretion. We propose that YscUCC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.

  • 24.
    Fällman, M
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Persson, C
    Umeå University, Faculty of Medicine, Molecular Biology.
    Schesser, K
    Umeå University, Faculty of Medicine, Molecular Biology.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Molecular Biology.
    Bidirectional signaling between Yersinia and its target cell.1998In: Folia microbiologica (Prague), ISSN 0015-5632, E-ISSN 1874-9356, Vol. 43, no 3, 263-273 p.Article in journal (Refereed)
    Abstract [en]

    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.

  • 25.
    Fällman, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Persson, Cathrine
    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).
    Yersinia proteins that target host cell signaling pathways1997In: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 99, no 6, 1153-1157 p.Article, review/survey (Refereed)
  • 26. Galán, Jorge E
    et al.
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Protein delivery into eukaryotic cells by type III secretion machines.2006In: Nature, ISSN 1476-4687, Vol. 444, no 7119, 567-73 p.Article in journal (Refereed)
    Abstract [en]

    Bacteria that have sustained long-standing close associations with eukaryotic hosts have evolved specific adaptations to survive and replicate in this environment. Perhaps one of the most remarkable of those adaptations is the type III secretion system (T3SS)--a bacterial organelle that has specifically evolved to deliver bacterial proteins into eukaryotic cells. Although originally identified in a handful of pathogenic bacteria, T3SSs are encoded by a large number of bacterial species that are symbiotic or pathogenic for humans, other animals including insects or nematodes, and plants. The study of these systems is leading to unique insights into not only organelle assembly and protein secretion but also mechanisms of symbiosis and pathogenesis.

  • 27.
    Garbom, Sara
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Forsberg, Ake
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Kihlberg, Britt-Marie
    Identification of novel virulence-associated genes via genome analysis of hypothetical genes.2004In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 72, no 3, 1333-1340 p.Article in journal (Refereed)
    Abstract [en]

    The sequencing of bacterial genomes has opened new perspectives for identification of targets for treatment of infectious diseases. We have identified a set of novel virulence-associated genes (vag genes) by comparing the genome sequences of six human pathogens that are known to cause persistent or chronic infections in humans: Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi, Streptococcus pneumoniae, and Treponema pallidum. This comparison was limited to genes annotated as hypothetical in the T. pallidum genome project. Seventeen genes with unknown functions were found to be conserved among these pathogens. Insertional inactivation of 14 of these genes generated nine mutants that were attenuated for virulence in a mouse infection model. Out of these nine genes, five were found to be specifically associated with virulence in mice as demonstrated by infection with Yersinia pseudotuberculosis in-frame deletion mutants. In addition, these five vag genes were essential only in vivo, since all the mutants were able to grow in vitro. These genes are broadly conserved among bacteria. Therefore, we propose that the corresponding vag gene products may constitute novel targets for antimicrobial therapy and that some vag mutants could serve as carrier strains for live vaccines.

  • 28.
    Garbom, Sara
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Olofsson, Martina
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björnfot, Ann-Catrin
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Srivastava, Manoj Kumar
    Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, S-90183 Umeå, Sweden.
    Robinson, Victoria L
    Biomedical Sciences, Dstl Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
    Oyston, Petra C F
    Biomedical Sciences, Dstl Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
    Titball, Richard W
    Biomedical Sciences, Dstl Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Phenotypic characterization of a virulence-associated protein, VagH, of Yersinia pseudotuberculosis reveals a tight link between VagH and the type III secretion system.2007In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 153, no Pt 5, 1464-73 p.Article in journal (Refereed)
    Abstract [en]

    Recently, a number of attenuated mutants of Yersinia pseudotuberculosis have been identified using a bioinformatics approach. One of the target genes identified in that study was vagH, which the authors now characterized further. VagH shows homology to HemK of Escherichia coli, possessing methyltransferase activity similar to that of HemK, and targeting release factors 1 and 2. Microarray studies comparing the wild-type and the vagH mutant revealed that the mRNA levels of only a few genes were altered in the mutant. By proteome analysis, expression of the virulence determinant YopD was found to be increased, indicating a possible connection between VagH and the virulence plasmid-encoded type III secretion system (T3SS). Further analysis showed that Yop expression and secretion were repressed in a vagH mutant. This phenotype could be suppressed by trans-complementation with the wild-type vagH gene or by deletion of the negative regulator yopD. Also, in a similar manner to a T3SS-negative mutant, the avirulent vagH mutant was rapidly cleared from Peyer's patches and could not reach the spleen after oral infection of mice. In a manner analogous to that of T3SS mutants, the vagH mutant could not block phagocytosis by macrophages. However, a vagH mutant showed no defects in the T3SS-independent ability to proliferate intracellularly and replicated to levels similar to those of the wild-type in macrophages. In conclusion, the vagH mutant exhibits a virulence phenotype similar to that of a T3SS-negative mutant, indicating a tight link between VagH and type III secretion in Y. pseudotuberculosis.

  • 29.
    Garbom, Sara
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Olofsson, Martina
    Björnfot, Ann-Catrin
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Srivastava, Manoj Kumar
    Robinson, Victoria
    Oyston, Petra
    Titbull, Richard
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Phenotypic characterization of a virulence associated protein, VagH, of Yersinia pseudotuberculosis reveals a tight link between VagH and the T3SSManuscript (Other academic)
  • 30.
    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, 73-91 p.Article 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.

  • 31.
    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, 2269-2276 p.Article 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.

  • 32. Hudson, Debra L
    et al.
    Layton, Abigail N
    Field, Terry R
    Bowen, Alison J
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Elofsson, Mikael
    Chemistry.
    Stevens, Mark P
    Galyov, Edouard E
    Inhibition of Type III Secretion in Salmonella enterica Serovar Typhimurium by Small-Molecule Inhibitors2007In: Antimicrobial Agents and Chemotherapy, Vol. 51, no 7, 2631-5 p.Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SS) are conserved in many pathogenic gram-negative bacteria. Small molecules that specifically target T3SS in Yersinia and Chlamydia spp. have recently been identified. Here we show that two such compounds inhibit Salmonella T3SS-1, preventing secretion of T3SS-1 effectors, invasion of cultured epithelial cells, and enteritis in vivo.

  • 33.
    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, 4740-4749 p.Article 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.

  • 34. Kauppi, Anna
    et al.
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Hägglund, U
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Elofsson, Mikael
    Chemistry.
    Salicylanilides are potent inhibitors of type III secretion in Yersinia2003In: Advances in Experimental Medicine and Biology, Vol. 529, 97-100 p.Article in journal (Refereed)
  • 35.
    Kauppi, Anna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Uvell, Hanna
    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).
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Targeting bacterial Virulence:  Inhibitors of type III secretion in Yersinia2003In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 10, no 3, 241-249 p.Article in journal (Refereed)
    Abstract [en]

    Agents that target bacterial virulence without detrimental effect on bacterial growth are useful chemical probes for studies of virulence and potential candidates for drug development. Several gram-negative pathogens employ type III secretion to evade the innate immune response of the host. Screening of a chemical library with a luciferase reporter gene assay in viable Yersinia pseudotuberculosis furnished several compounds that inhibit the reporter gene signal expressed from the yopE promoter and effector protein secretion at concentrations with no or modest effect on bacterial growth. The selectivity patterns observed for inhibition of various reporter gene strains indicate that the compounds target the type III secretion machinery at different levels. Identification of this set of inhibitors illustrates the approach of utilizing cell-based assays to identify compounds that affect complex bacterial virulence systems.

  • 36. Keyser, P
    et al.
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Rosell, S
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, 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).
    Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria2008In: Journal of Internal Medicine, Vol. 264, no 1, 17-29 p.Article in journal (Refereed)
    Abstract [en]

    In recent years mounting problems related to antibiotic-resistant bacteria have resulted in the prediction that we are entering the preantibiotic era. A way of preventing such a development would be to introduce novel antibacterial medicines with modes of action distinct from conventional antibiotics. Recent studies of bacterial virulence factors and toxins have resulted in increased understanding of the way in which pathogenic bacteria manipulate host cellular processes. This knowledge may now be used to develop novel antibacterial medicines that disarm pathogenic bacteria. The type III secretion system (T3SS) is known to be a potent virulence mechanism shared by a broad spectrum of pathogenic Gram-negative bacteria that interact with human, animal and plant hosts by injecting effector proteins into the cytosol of host cells. Diseases, such as bubonic plague, shigellosis, salmonellosis, typhoid fever, pulmonary infections, sexually transmitted chlamydia and diarrhoea largely depend on the bacterial proteins injected by the T3SS machinery. Recently a number of T3SS inhibitors have been identified using screening-based approaches. One class of inhibitors, the salicylidene acylhydrazides, has been subjected to chemical optimization and evaluation in several in vitro and ex vivo assays in multiple bacterial species including Yersinia spp., Chlamydia spp., Salmonella spp. and Pseudotuberculosis aeruginosa. Reports published up to date indicate that T3SS inhibitors have the potential to be developed into novel antibacterial therapeutics.

  • 37. Lavander, Moa
    et al.
    Sundberg, Lena
    Umeå University, Faculty of Medicine, Molecular Biology.
    Edqvist, Petra J
    Umeå University, Faculty of Medicine, Molecular Biology.
    Lloyd, Scott A
    Umeå University, Faculty of Medicine, Molecular Biology.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Ake
    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).
    Characterisation of the type III secretion protein YscU in Yersinia pseudotuberculosis: YscU cleavage--dispensable for TTSS but essential for survival2003In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 529, 109-112 p.Article in journal (Other academic)
  • 38. Lloyd, Scott A
    et al.
    Forsberg, Åke
    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).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Targeting exported substrates to the Yersinia TTSS: different functions for different signals?2001In: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 9, no 8, 367-371 p.Article in journal (Refereed)
    Abstract [en]

    Many Gram-negative pathogens utilize a type III secretion system (TTSS) to inject toxins into the cytosol of eukaryotic cells. Previous studies have indicated that exported substrates are targeted to the Yersinia TTSS via the coding regions of their 5' mRNA sequences, as well as by their cognate chaperones. However, recent results from our laboratory have challenged the role of mRNA targeting signals, as we have shown that the amino termini of exported substrates are crucial for type III secretion. Here, we discuss the nature of these amino-terminal secretion signals and propose a model for the secretion of exported substrates by amino-terminal and chaperone-mediated signals. In addition, we discuss the roles of chaperones as regulators of virulence gene expression and present models suggesting that such regulation can occur independently of the delivery of their substrates to the secretion apparatus.

  • 39.
    Lloyd, Scott A
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sjöström, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, Sara
    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).
    Molecular characterization of type III secretion signals via analysis of synthetic N-terminal amino acid sequences2002In: Molecular Microbiology, Vol. 43, no 1, 51-9 p.Article in journal (Refereed)
    Abstract [en]

    Yersinia species utilize a type III secretion system to inject toxins, called Yops (Yersinia outer proteins), into eukaryotic cells. The N-termini of the Yops serve as type III secretion signals, but they do not share a consensus sequence. To simplify the analysis of type III secretion signals, we replaced amino acids 2–8 of the secreted protein YopE with all permutations (27 or 128) of synthetic serine/isoleucine sequences. The results demonstrate that amphipathic N-terminal sequences, containing four or five serine residues, have a much greater probability than hydrophobic or hydrophilic sequences to target YopE for secretion. Multiple linear regression analysis of the synthetic sequences was used to obtain a model for N-terminal secretion signals. The model accurately classifies the N-terminal sequences of native type III substrates as efficient secretion signals.

  • 40.
    Login, Frederic H.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 43, 26282-26291 p.Article in journal (Refereed)
    Abstract [en]

    All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscU(CC), the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca2+ -depleted medium at 37 degrees C. Here, we investigated the secretion of this early T3S-substrate and showed that YscU(CC) encompasses a specific C-terminal T3S signal within the 15 last residues (U-15). U-15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the "classical" N-terminal secretion signal, U-15 interacted with the ATPase YscN. Although U-15 is critical for YscU(CC) secretion, deletion of the C-terminal secretion signal of YscU(CC) did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion.

  • 41.
    Marcinowska, Renata
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    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).
    Mortiz, Thomas
    Surowiec, Izabella
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Optimization of a sample preparation method for the metabolomic analysis of clinically relevant bacteria2011In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 87, no 1, 24-31 p.Article in journal (Refereed)
    Abstract [en]

    Metabolomics, or metabolite profiling, is an approach that is increasingly used to study the metabolism of diverse organisms, elucidate biological processes and/or find characteristic biomarkers of physiological states. Here, we describe the optimization of a method for global metabolomic analysis of bacterial cultures, with the following steps. Cells are grown to log-phase, starting from an overnight culture and bacterial concentrations are monitored by measuring the optical density of the cultures at 600nm. At an appropriate density they are harvested by centrifugation, washed three times with NaCl solution and metabolites are extracted using methanol and a bead-mill. Dried extracts are methoxymated and derivatized with methyltrimethylsilyltrifluoroacetamide (MSTFA) then analyzed using gas chromatography coupled to time-of-flight mass spectrometry (GC-MS/TOF). Finally, patterns in the acquired data are examined by multivariate data modeling. This method enabled us to obtain reproducible metabolite profiles of Yersinia pseudotuberculosis, with about 25% compound identification, based on comparison with entries in available GC-MS libraries. To assess the potential utility of the method for comparative analysis of other bacterial species we analyzed cultures of Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and methicillin-sensitive Staphylococcus aureus (MSSA). Multivariate analysis of the acquired data showed that it was possible to differentiate the species according to their metabolic profiles. Our results show that the presented procedure can be used for metabolomic analysis of a wide range of bacterial species of clinical interest.

  • 42. Meinzer, Ulrich
    et al.
    Barreau, Frederick
    Esmiol-Welterlin, Sophie
    Jung, Camille
    Villard, Claude
    Leger, Thibaut
    Ben-Mkaddem, Sanah
    Berrebi, Dominique
    Dussaillant, Monique
    Alnabhani, Ziad
    Roy, Maryline
    Bonacorsi, Stephane
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Perroy, Julie
    Ollendorff, Vincent
    Hugot, Jean-Pierre
    Yersinia pseudotuberculosis Effector YopJ Subverts the Nod2/RICK/TAK1 Pathway and Activates Caspase-1 to Induce Intestinal Barrier Dysfunction2012In: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 11, no 4, 337-351 p.Article in journal (Refereed)
    Abstract [en]

    Yersinia pseudotuberculosis is an enteropathogenic bacteria that disrupts the intestinal barrier and invades its host through gut-associated lymphoid tissue and Peyer's patches (PP). We show that the Y. pseudotuberculosis effector YopJ induces intestinal barrier dysfunction by subverting signaling of the innate immune receptor Nod2, a phenotype that can be reversed by pretreating with the Nod2 ligand muramyl-dipeptide. YopJ, but not the catalytically inactive mutant YopJ(C172A), acetylates critical sites in the activation loops of the RICK and TAK1 kinases, which are central mediators of Nod2 signaling, and decreases the affinity of Nod2 for RICK. Concomitantly, Nod2 interacts with and activates caspase-1, resulting in increased levels of IL-1 beta. Finally, IL-1 beta within PP plays an essential role in inducing intestinal barrier dysfunction. Thus, YopJ alters intestinal permeability and promotes the dissemination of Yersinia as well as commensal bacteria by exploiting the mucosal inflammatory response.

  • 43. Meinzer, Ulrich
    et al.
    Esmiol-Welterlin, Sophie
    Barreau, Frederick
    Berrebi, Dominique
    Dussaillant, Monique
    Bonacorsi, Stephane
    Chareyre, Fabrice
    Niwa-Kawakita, Michiko
    Alberti, Corinne
    Sterkers, Ghislaine
    Villard, Claude
    Lesuffleur, Thecla
    Peuchmaur, Michel
    Karin, Michael
    Eckmann, Lars
    Giovannini, Marco
    Ollendorff, Vincent
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, 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).
    Hugot, Jean-Pierre
    Nod2 mediates susceptibility to Yersinia pseudotuberculosis in mice.2008In: PLoS ONE, ISSN 1932-6203, Vol. 3, no 7, e2769- p.Article in journal (Refereed)
    Abstract [en]

    Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs) in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs. We investigated the role of Nod2 during Y. pseudotuberculosis infection. Death was delayed in Nod2 deleted and Crohn's disease associated Nod2 mutated mice orogastrically inoculated with Y. pseudotuberculosis. In PPs, the local immune response was characterized by a higher KC level and a more intense infiltration by neutrophils and macrophages. The apoptotic and bacterial cell counts were decreased. Finally, Nod2 deleted mice had a lower systemic bacterial dissemination and less damage of the haematopoeitic organs. This resistance phenotype was lost in case of intraperitoneal infection. We concluded that Nod2 contributes to the susceptibility to Y. pseudotuberculosis in mice.

  • 44. Muschiol, Sandra
    et al.
    Bailey, Leslie
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gylfe, Asa
    Sundin, Charlotta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hultenby, Kjell
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Normark, Staffan
    Henriques-Normark, Birgitta
    A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis2006In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 39, 14566-14571 p.Article in journal (Refereed)
    Abstract [en]

    The intracellular pathogen Chlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs). INP0400, at high concentration, given at the time of infection, partially blocked entry of elementary bodies into host cells. Early treatment inhibited the localization of the mammalian protein 14-3-3beta to the inclusions, indicative of absence of the early induced TTS effector IncG from the inclusion membrane. Treatment with INP0400 during chlamydial mid-cycle prevented secretion of the TTS effector IncA and homotypic vesicular fusions mediated by this protein. INP0400 given during the late phase resulted in the detachment of RBs from the inclusion membrane concomitant with an inhibition of RB to elementary body conversion causing a marked decrease in infectivity.

  • 45. Muschiol, Sandra
    et al.
    Bailey, Leslie
    Gylfe, Åsa
    Sundin, Charlotta
    Hultenby, Kjell
    Bergström, Sven
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Hans
    Normark, Staffan
    Henriques-Normark, Birgitta
    A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis2006In: National Academy of Sciences, USA: A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis, 2006, 14566-71 p.Conference paper (Other academic)
    Abstract [en]

    The intracellular pathogen Chlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs). INP0400, at high concentration, given at the time of infection, partially blocked entry of elementary bodies into host cells. Early treatment inhibited the localization of the mammalian protein 14-3-3beta to the inclusions, indicative of absence of the early induced TTS effector IncG from the inclusion membrane. Treatment with INP0400 during chlamydial mid-cycle prevented secretion of the TTS effector IncA and homotypic vesicular fusions mediated by this protein. INP0400 given during the late phase resulted in the detachment of RBs from the inclusion membrane concomitant with an inhibition of RB to elementary body conversion causing a marked decrease in infectivity.

  • 46. Najdenski, H
    et al.
    Golkocheva-Markova, E
    Kussovski, V
    Vesselinova, A
    Garbom, Sara
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Attenuation and preserved immunogenic potential of Yersinia pseudotuberculosis mutant strains evidenced in oral pig model2009In: Zoonoses and Public Health, ISSN 1863-1959, E-ISSN 1863-2378, Vol. 56, no 4, 157-168 p.Article in journal (Refereed)
    Abstract [en]

    Experimental oral infection of pigs with a parental Yersinia pseudotuberculosis strain pIB102, serotype O:3 and two mutant isogenic strains - pIB155,DeltayopK and pIB44,DeltaypkA has been carried out. Clinical findings, microbiological and immunological parameters were examined in dynamics from day 7 to day 60 post-infection (p.i.). All types of infections ran asymptomatically, without hyperthermia, loss of appetite, etc. Experiments on the blood parameters demonstrated a transient leucocytosis with lymphocytosis and monocytosis better expressed after yopK infection. Even though pig is usually known as a reservoir of yersiniae, bacterial colonization was found in mesenterial lymph nodes and tonsils on day 7, respectively 14 p.i. with parental strain, and only in tonsils on day 14 p.i. with both mutant strains. The augmented sensitivity of mutants to the bactericidal effect of leukocytes and blood sera is the characteristic feature of attenuation in their pathogenicity, compared to the parental strain. Comparative in vitro experiments on the immune response and immunostimulating capacity of Y. pseudotuberculosis mutant strains verify their preserved immunogenic potential, predominantly in case of yopK. Hyperplasia and strong activation of the lymph tissue of Peyer's patches, mesenterial lymph nodes, tonsils and spleen of pigs challenged with both mutant strains were proved as immunomorphological rearrangements. The results obtained give the reason to claim that the genetically constructed yopK null mutant strain is significantly attenuated but is still immunogenic and has the potential for a live vaccine carrier strain.

  • 47. Navarro, Lorena
    et al.
    Koller, Antonius
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Taylor, Susan
    Dixon, Jack E
    Identification of a molecular target for the Yersinia protein kinase A.2007In: Mol Cell, ISSN 1097-2765, Vol. 26, no 4, 465-77 p.Article in journal (Refereed)
    Abstract [en]

    Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject bacterial effector proteins directly into the host cytosol. One of these effectors, the Yersinia serine/threonine protein kinase YpkA, is an essential virulence determinant involved in host actin cytoskeletal rearrangements and in inhibition of phagocytosis. Here we report that YpkA inhibits multiple Galphaq signaling pathways. The kinase activity of YpkA is required for Galphaq inhibition. YpkA phosphorylates Ser47, a key residue located in the highly conserved diphosphate binding loop of the GTPase fold of Galphaq. YpkA-mediated phosphorylation of Ser47 impairs guanine nucleotide binding by Galphaq. Y. pseudotuberculosis expressing wild-type YpkA, but not a catalytically inactive YpkA mutant, interferes with Galphaq-mediated signaling pathways. Identification of a YpkA-mediated phosphorylation site in Galphaq sheds light on the contribution of the kinase activity of YpkA to Yersinia pathogenesis.

  • 48. Negrea, Aurel
    et al.
    Bjur, Eva
    Eriksson Ygberg, Sofia
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Chemistry.
    Wolf-Watz, Hans
    Molecular Biology (Faculty of Science and Technology).
    Rhen, Mikael
    Salicylidene Acylhydrazides That Affect Type III Protein Secretion in Salmonella enterica Serovar Typhimurium2007In: Antimicrobial Agents and Chemotherapy, Vol. 51, no 8, 2867-76 p.Article in journal (Refereed)
    Abstract [en]

    A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector ß-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1. Salicylidene acylhydrazides also affected intracellular bacterial replication in murine macrophage-like cells and blocked the transport of an epitope-tagged SPI2 effector protein. Two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We conclude that salicylidene acylhydrazides affect bacterial T3SS activity in S. enterica and hence could be used as lead substances when designing specific inhibitors of bacterial T3SSs in order to pharmaceutically intervene with bacterial virulence.

  • 49.
    Nordfelth, R.
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Kauppi, Anna M.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Norberg, H. A.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Molecular Biology.
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Small-molecule inhibitors specifically targeting type III secretion2005In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 73, no 5, 3104-3114 p.Article in journal (Refereed)
    Abstract [en]

    The type III secretion (TTS) system is used by several animal and plant pathogens to deliver effector proteins into the cytosol of the eukaryotic target cell as a strategy to evade the defense reactions elicited by the infected organism. The fact that these systems are highly homologous implies that novel antibacterial agents that chemically attenuate the pathogens via a specific interaction with the type III secretion mechanism can be identified. A number of small organic molecules having this potential have recently been identified (A. M. Kauppi, R. Nordfelth, H. Uvell, H. Wolf-Watz, and M. Elofsson, Chem. Biol. 10:241-249, 2003). Using different reporter gene constructs, we showed that compounds that belong to a class of acylated hydrazones of different salicylaldehydes target the TTS system of Yersinia pseudotuberculosis. One of these compounds, compound 1, was studied in detail and was found to specifically block Yop effector secretion under in vitro conditions by targeting the TTS system. In this respect the drug mimics the well-known effect of calcium on Yop secretion. In addition, compound I inhibits Yop effector translocation after infection of HeLa cells without affecting the eukaryotic cells or the bacteria. A HeLa cell model that mimics in vivo conditions showed that compound 1 chemically attenuates the pathogen to the advantage of the eukaryotic cell. Thus, our results show proof of concept, i.e., that small compounds targeting the TTS system can be identified, and they point to the possible use of TTS inhibitors as a novel class of antibacterial agents.

  • 50.
    Olsson, Jan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Edqvist, Petra J
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bröms, Jeanette E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Ake
    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).
    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).
    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).
    The YopD translocator of Yersinia pseudotuberculosis is a multifunctional protein comprised of discrete domains.2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 13, 4110-4123 p.Article in journal (Refereed)
    Abstract [en]

    To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III translocon to microinject several anti-host Yop effectors into the cytosol of target eukaryotic cells. YopD has been implicated in several key steps during Yop effector translocation, including maintenance of yop regulatory control and pore formation in the target cell membrane through which effectors traverse. These functions are mediated, in part, by an interaction with the cognate chaperone, LcrH. To gain insight into the complex molecular mechanisms of YopD function, we performed a systematic mutagenesis study to search for discrete functional domains. We highlighted amino acids beyond the first three N-terminal residues that are dispensable for YopD secretion and confirmed that an interaction between YopD and LcrH is essential for maintenance of yop regulatory control. In addition, discrete domains within YopD that are essential for both pore formation and translocation of Yop effectors were identified. Significantly, other domains were found to be important for effector microinjection but not for pore formation. Therefore, YopD is clearly essential for several discrete steps during efficient Yop effector translocation. Recognition of this modular YopD domain structure provides important insights into the function of YopD.

12 1 - 50 of 61
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