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  • 1.
    Aili, Margareta
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Role of YopE and LcrH in effector translocation, HeLa cell cytotoxicity and virulence2005Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In order to establish an extra-cellular infection the gram-negative bacteria Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to translocate a set of anti-host effectors into eukaryotic cells. The toxins disrupt signalling pathways important for phagocytosis, cytokine production and cell survival. Secretion and translocation via this T3SS is strictly regulated on several levels. In this context, the function of YopE and LcrH during Yersinia infections has been analysed.

    YopE is an essential translocated effector that disrupts the actin cytoskeleton of infected eukaryotic cells, by inactivating small GTPases through its GTPase activating protein (GAP) activity. However, cytotoxicity can be uncoupled from in vitro GAP activity towards the RhoA, Rac1 and Cdc42 GTPases. Furthermore, in vivo studies of the YopE GAP activity revealed that only RhoA and Rac1 are targeted, but this is not a pre-requisite for Yersinia virulence. Hence, YopE must target one or more additional GTPases to cause disease in mice.

    YopE was the only Yersinia effector that blocks LDH release from infected cells. Moreover, translocated YopE could regulate the level of subsequent effector translocation by a mechanism that involved the YopE GAP function and another T3S component, YopK. Loss of translocation control elevated total T3S gene expression in the presence of eukaryotic cells. This indicated the existence of a regulatory loop for feedback control of T3S gene expression in the bacteria that originates from the interior of the eukaryotic cell after effector translocation is completed. This might represent the true virulence function of YopE.

    Exoenzyme S (ExoS) of Pseudomonas aeruginosa has a YopE-like GAP domain with similar activity towards RhoA, Rac1 and Cdc42. However, ExoS is unable to complement hyper-translocation resulting from loss of YopE. This indicates a unique function for YopE in translocation control in Yersinia that might be dependent on correct intracellular localisation. It follows that the Membrane Localisation Domain in YopE was important for translocation control, but dispensable for cytotoxicity and blockage of LDH release.

    YopD and its cognate chaperone LcrH are negative regulatory elements of the T3S regulon and together with YopB, are involved in the effector translocation process. Randomly generated point mutants in LcrH specifically effected stability and secretion of both the YopB and YopD substrates in vitro and prevented their apparent insertion as translocon pores in the membranes of infected cells. Yet, these mutants still produced stable substrates in the presence of eukaryotic cells and most could mediate at least partial effector translocation. Thus, only minimal amounts of the YopB and YopD translocator proteins are needed for translocation and the LcrH chaperone may regulate this process from inside the bacteria.

  • 2. Aili, Margareta
    et al.
    Hallberg, Bengt
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Rosqvist, Roland
    GAP activity of Yersinia YopE2002Inngår i: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 358, s. 359-70Artikkel i tidsskrift (Fagfellevurdert)
  • 3.
    Aili, Margareta
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Isaksson, Elin L
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Carlsson, Sara E
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Regulation of Yersinia Yop-effector delivery by translocated YopE2008Inngår i: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 298, nr 3-4, s. 183-192Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 4.
    Aili, Margareta
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Isaksson, Elin L
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hallberg, Bengt
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis2006Inngår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 8, nr 6, s. 1020-1033Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 5.
    Aili, Margareta
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Telepnev, Max
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hallberg, Bengt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity2003Inngår i: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 34, nr 6, s. 297-308Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The YopE cytotoxin of Yersinia is an essential virulence determinant that is translocated into the eukaryotic target cell via a plasmid-encoded type III secretion system. YopE possess a GTPase activating protein activity that in vitro has been shown to down regulate RhoA, Rac1, and Cdc42. Translocated YopE induces de-polymerisation of the actin microfilament structure in the eukaryotic cell which results in a rounding up of infected cells described as a cytotoxic effect. Here, we have investigated the importance of different regions of YopE for induction of cytotoxicity and in vitro GAP activity. Sequential removal of the N- and C-terminus of YopE identified the region between amino acids 90 and 215 to be necessary for induction of cytotoxicity. Internal deletions containing the essential arginine at position 144 resulted in a total loss of cytotoxic response. In-frame deletions flanking the arginine finger defined a region important for the cytotoxic effect to amino acids 166–183. Four triple-alanine substitution mutants in this region, YopE166-8A, 169-71A, 175-7A and 178-80A were still able to induce cytotoxicity on HeLa cells although they did not show any in vitro GAP activity towards RhoA, Rac1 or Cdc42. A substitution mutant in position 206-8A showed the same phenotype, ability to induce cytotoxic response but no in vitro GAP activity. We speculate that YopE may have additional unidentified targets within the eukaryotic cell.

  • 6.
    Akopyan, Karen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Edgren, Tomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Wang-Edgren, Helen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Translocation of surface-localized effectors in type III secretion2011Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, nr 4, s. 1639-1644Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 7.
    Alenius, Mattias
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Bohm, Staffan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Differential function of RNCAM isoforms in precise target selection of olfactory sensory neurons2003Inngår i: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 130, nr 5, s. 917-927Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Olfactory sensory neurons (OSNs) are individually specified to express one odorant receptor (OR) gene among similar to1000 different and project with precision to topographically defined convergence sites, the glomeruli, in the olfactory bulb. Although ORs partially determine the location of convergence sites, the mechanism ensuring that axons with different OR identities do not co-converge is unknown. RNCAM (OCAM, NCAM2) is assumed to regulate a broad zonal segregation of projections by virtue of being a homophilic cell adhesion molecule that is selectively expressed on axons terminating in a defined olfactory bulb region. We have identified NADPH diaphorase activity as being an independent marker for RNCAM-negative axons. Analyses of transgenic mice that ectopically express RNCAM in NADPH diaphorasepositive OSNs show that the postulated function of RNCAM in mediating zone-specific segregation of axons is unlikely. Instead, analyses of one OR-specific OSN subpopulation (P2) reveal that elevated RNCAM levels result in an increased number of P2 axons that incorrectly co-converge with axons of other OR identities. Both Gpianchored and transmembrane-bound RNCAM isoforms are localized on axons in the nerve layer, while the transmembrane-bound RNCAM is the predominant isoform on axon terminals within glomeruli. Overexpressing transmembrane-bound RNCAM results in co-convergence events close to the correct target glomeruli. By contrast, overexpression of Gpi-anchored RNCAM results in axons that can bypass the correct target before co-converging on glomeruli located at a distance. The phenotype specific for Gpi-anchored RNCAM is suppressed in mice overexpressing both isoforms, which suggests that two distinct RNCAM isoform-dependent activities influence segregation of OR-defined axon subclasses.

  • 8.
    Alenius, Mattias
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå University.
    Bohm, Staffan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Identification of a novel neural cell adhesion molecule-related gene with a potential role in selective axonal projection1997Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, nr 42, s. 26083-26086Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We describe here the cloning of mouse complementary DNAs encoding a novel protein, Rb-8 neural cell adhesion molecule (RNCAM), with a predicted extracellular region of five immunoglobulin Ca-type domains followed by two fibronectin type III domains, Alternative splicing is likely to generate two RNCAM isoforms, which are differently attached to the cell membrane, These structural features and overall sequence identity identify this protein as a novel member of a cell adhesion molecule subgroup together with vertebrate neural cell adhesion molecule, Aplysia cell adhesion molecule, and Drosophila fasciclin II, In insects, fasciclin II is present on a restricted subset of embryonic central nervous system axons where it controls selective axon fasciculation. Intriguingly, RNCAM likewise is expressed in subsets of olfactory and vomeronasal neurons with topographically defined axonal projections, The spatial expression RNCAM corresponds precisely to that of certain odorant receptor expression zones of the olfactory epithelium. These expression patterns thus render RNCAM the first described cell adhesion molecule with a potential regulatory role in formation of selective axonal projections important for olfactory sensory information coding.

  • 9.
    Alexeyev, Oleg A
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Lundskog, Bertil
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Ganceviciene, Ruta
    Palmer, Ruth H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    McDowell, Andrew
    Patrick, Sheila
    Zouboulis, Christos
    Golovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
    Pattern of tissue invasion by Propionibacterium acnes in acne vulgaris2012Inngår i: Journal of dermatological science (Amsterdam), ISSN 0923-1811, E-ISSN 1873-569X, Vol. 67, nr 1, s. 63-66Artikkel i tidsskrift (Fagfellevurdert)
  • 10.
    Amer, Ayad
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Controlling substrate export by the Ysc-Yop type III secretion system in Yersinia2013Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Several pathogenic Gram-negative bacteria invest in sophisticated type III secretion systems (T3SS) to incapacitate their eukaryotic hosts. T3SSs can secrete protein cargo outside the bacterial cell and also target many of them into the eukaryotic cell interior. Internalized proteins promote bacterial colonization, survival and transmission, and can often cause severe disease. An example is the Ysc-Yop T3SS apparatus assembled by pathogenic Yersinia spp. A correctly assembled Ysc-Yop T3SS spans the Yersinia envelope and also protrudes from the bacterial surface. Upon host cell contact, this system is competent to secrete hydrophobic translocators that form a translocon pore in the host cell membrane to complete the delivery channel bridging both bacterial and host cells. Newly synthesized effector Yops may pass through this channel to gain entry into the host cell cytosol.As type III secretion (T3S) substrates function sequentially during infection, it is hypothesized that substrate export is temporally controlled to ensure that those required first are prioritized for secretion. On this basis three functional groups are classified as early (i.e. structural components), middle (i.e. translocators) and late (i.e. effectors). Factors considered to orchestrate the T3S of substrates are many, including the intrinsic substrate secretion signal sequences, customized chaperones, and recognition/sorting platforms at the base of the assembled T3SS. Investigating the interplay between these elements is critical for a better understanding of the molecular mechanisms governing export control during Yersinia T3S.To examine the composition of the N-terminal T3S signals of the YscX early substrate and the YopD middle substrate, these segments were altered by mutagenesis and the modified substrates analyzed for their T3S. Translational fusions between these signals and a signalless β-Lactamase were used to determine their optimal length required for efficient T3S. This revealed that YscX and YopD export is most efficiently supported by their first 15 N-terminal residues. At least for YopD, this is a peptide signal and not base upon information in the mRNA sequence. Moreover, features within and upstream of this segment contribute to their translational control. In parallel, bacteria were engineered to produce substrate chimeras where the N-terminal segments were exchanged between substrates of different classes in an effort to examine the temporal dynamics of T3S. In several cases, Yersinia producing chimeric substrates were defective in T3S activity, which could be a consequence of disturbing a pre-existing hierarchal secretion mechanism.YopN and TyeA regulatory molecules can be naturally produced as a 42 kDa YopN-TyeA hybrid, via a +1 frame shift event somewhere at the 5’-end of yopN. To study this event, Yersinia were engineered to artificially produce this hybrid, and these maintained in vitro T3S control of both middle and late substrates. However, modestly diminished directed targeting of effectors into eukaryotic cells correlated to virulence attenuation in vivo. Upon further investigation, a YopN C-terminal segment encompassing residues 278 to 287 was probably responsible, as this region is critical for YopN to control T3S, via enabling a specific interaction with TyeA.Investigated herein were molecular mechanisms to orchestrate substrate export by the T3SS of Yersinia. While N-terminal secretion signals may contribute to specific substrate order, the YopN and TyeA regulatory molecules do not appear to distinguish between the different substrate classes.

  • 11.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Costa, Tiago
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Farag, Salah
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Avican, Ummehan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis2013Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 10, artikkel-id e77767Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.

  • 12.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Costa, Tiago
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Gurung,, Jyoti
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Avican, Ummehan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Functional consequences of site-directed mutagenesis in theC-terminus of YopN, a Yersinia pseudotuberculosis regulator ofYop secretionManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Pathogenic Yersinia spp. utilizes the Ysc-Yop type III secretion system to targetYop effector proteins into the cytosol of host immune cells. Internalizedeffectors alter specific signaling pathways to neutralize immune cell-dependentphagocytosis, killing and pro-inflammatory responsiveness. This enablesextracellular bacterial multiplication and survival in immune tissue. Central tothe temporal control of Yop type III secretion is the regulator YopN. Incomplex with TyeA, YopN acts to plug the inner face of the type III secretionchannel, denying entry to other Yop substrates until after YopN has beensecreted. A +1 frameshift event in the 3-prime end of yopN results in thesynthesis of a singular secreted YopN-TyeA polypeptide chimera that retainssome regulatory function. As the C-terminal coding sequence of YopN in thishybrid product differs greatly from native sequence, we used site-directedmutagenesis to determine the functional significance of this segment. YopNtruncated at residue 287 or containing a shuffled sequence covering 288 to 293retains full function both in vitro and in vivo. Thus, the extreme C-terminus isapparently superfluous to YopN function. In contrast, a YopN varianttruncated after residue 278 was completely unstable, and these bacteria hadlost all control of T3S activity, and failed to defend against immune cell killing.Interestingly, inclusion of a shuffled sequence from residues 279 to 287recovered some T3S control over function. Hence, the YopN segmentencompassing 279 to 287 is essential for full function, although the exact aminoacid sequence is less important.

  • 13.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Gurung, Jyoti
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Costa, Tiago
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ruuth, Kristina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Zavialov, Anton
    Joint Biotechnology Laboratory, Department of Chemistry, University of Turku, Turku, Finland.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis2016Inngår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, artikkel-id 66Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopNW279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.

  • 14.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Gurung, Jyoti
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Yersinia pseudotuberculosis type III secretion is reliant upon anauthentic N‐terminal YscX secretor domainManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Certain Gram‐negative bacteria use type III secretion systems to deliver effectorproteins into eukaryotic cells, serving either parasitic or mutualistic roles inside the hostcell. About 25 structural proteins are needed to assemble and deliver effector proteins.Collections of these proteins are quite well characterized, although the function ofsome continues to remain obscure. This is true for the Yersinia Ysc‐Yop systemcomponents YscX, a secreted substrate and YscY, its cognate non‐secreted chaperone.Despite recent evidence suggesting that they might coordinate Yop substrate secretion,YscX and YscY remain poorly characterized. To further investigate the function of theseproteins in the enteropathogen Y. pseudotuberculosis, we explored correlationsbetween the YscX N‐terminal segment, YscX secretion, as well as the secretion of otherYops. Analysis of a series of chimeric substrates in which the extreme YscX N‐terminushad been exchanged with equivalent functional secretion signals of other Ysc‐Yopsubstrates revealed that this segment contains non‐redundant information needed forYscX function, which includes permitting surface polymerization of the YscF needle andYops secretion. Further, in cis deletion of the YscX N‐terminus and ectopic expression ofepitope tagged YscX variants again correlated stable YscX production but not secretionto the type III secretion of Yops. Despite this, the first 5 codons were determined toconstitute a minimal signal capable of promoting secretion of the signalless ‐lactamasereporter. Hence, YscX does contain a fully equipped N‐terminal secretor domain topromote secretion of self. Nevertheless, the primary role of this N‐terminal segmentmust be to assemble an operational secretion system, and this occurs independently ofYscX secretion.

  • 15.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Åhlund, Monika
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Bröms, Jeanette
    Department of Medical Countermeasures, Swedish Defense Research Agency, Division of NBC12 Defense, Umeå, Sweden.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Impact of the N-terminal secretor domain on YopD translocator function in Yersinia pseudotuberculosis type III secretion2011Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 23, s. 6683-6700Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5′ mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from Yersinia pseudotuberculosis. Mutations in the 5′ end of yopD that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for yopD translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the in vitro secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of yopD translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.

  • 16.
    Andersson, Agneta
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå marina forskningscentrum (UMF).
    Hajdu, Susanna
    Inst. f. Systemekologi, Stockholms universitet.
    Haecky, Pia
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå marina forskningscentrum (UMF).
    Kuparinen, Jorma
    Wikner, Johan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå marina forskningscentrum (UMF).
    Succession and growth limitation of phytoplankton in the Gulf of Bothnia (Baltic Sea)1996Inngår i: Marine Biology, ISSN 0025-3162, E-ISSN 1432-1793, Vol. 126, nr 4, s. 791-801Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A one year field study of four stations in the Gulf of Bothnia during 1991 showed that the biomass was ca. two times, and primary productivity ca, four times, lower in the north (Bothnian Bay) than in the south (Bothnian Sea) during the summer. Nutrient addition experiments indicated phosphorus limitation of phytoplankton in the Bothnian Bay and the coastal areas in the northern Bothnian Sea, but nitrogen limitation in the open Bothnian Sea. A positive correlation between the phosphate concentration and the production/biomass ratio of phytoplankton was demonstrated, which partly explained the differences in the specific growth rate of the phytoplankton during the summer. Differences in photosynthetic active radiation between the stations also showed a covariation with the primary productivity. The relative importance of nutrient or light limitation for photosynthetic carbon fixation could not, however, be conclusively determined from this study. Marked differences in phytoplankton species composition from north to south were also observed. The number of dominating species was higher in the Bothnian Sea than in the Bothnian Bay. The distribution of some species could be explained as due to nutrient availability (e.g. Nodularia spumigena, Aphanizomenon sp.), while salinity probably limits the distribution of some limnic as well as marine species. The potentially toxic phytoplankton N. spumigena, Dinophysis acuminata and Chrysochromulina spp. were common in the Bothnian Sea but not in the Bothnian Bay. The pico- and nanoplankton biomass during late summer was higher than previously reported due to a revised carbon/volume ratio.

  • 17.
    Andersson Escher, Stefan
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Ekenstedt, J
    Elberg, K
    Saura, Anssi
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The Drosophilidae (Diptera) of Estonia2006Inngår i: Entomologica Fennica, Vol. 17, s. 13-20Artikkel i tidsskrift (Fagfellevurdert)
  • 18.
    Andersson Escher, Stefan
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Rasmuson-Lestander, Å
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The Drosophila glucose transporter gene: cDNA sequence, phylogenetic comparisons, analysis of functional sites and secondary structures.1999Inngår i: Hereditas, ISSN 0018-0661, Vol. 130, nr 2, s. 95-103Artikkel i tidsskrift (Fagfellevurdert)
  • 19. Andersson, K
    et al.
    Carballeira Suarez, N
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Magnusson, K E
    Persson, C
    Stendahl, O
    Wolf-Watz, H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Fällman, M
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signalling associated with phagocytosis.1996Inngår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 20, nr 5, s. 1057-69Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 20. Andres Valderrama, J
    et al.
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Carmona, Manuel
    Diaz, Eduardo
    AccR is a master regulator involved in carbon catabolite repression of the anaerobic catabolism of aromatic compounds in Azoarcus sp CIB2014Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, nr 4, s. 1892-1904Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Here we characterized the first known transcriptional regulator that accounts for carbon catabolite repression (CCR) control of the anaerobic catabolism of aromatic compounds in bacteria. The AccR response regulator of Azoarcus sp. CIB controls succinate-responsive CCR of the central pathways for the anaerobic catabolism of aromatics by this strain. Phosphorylation of AccR to AccR-P triggers a monomer-to-dimer transition as well as the ability to bind to the target promoter and causes repression both in vivo and in vitro. Substitution of the Asp(60) phosphorylation target residue of the N-terminal receiver motif of AccR to a phosphomimic Glu residue generates a constitutively active derivative that behaves as a superrepressor of the target genes. AccR-P binds in vitro to a conserved inverted repeat (ATGCA-N-6-TGCAT) present at two different locations within the P-N promoter of the bzd genes for anaerobic benzoate degradation. Because the DNA binding-proficient C-terminal domain of AccR is monomeric, we propose an activation mechanism in which phosphorylation of Asp(60) of AccR alleviates interdomain repression mediated by the N-terminal domain. The presence of AccR-like proteins encoded in the genomes of other -proteobacteria of the Azoarcus/Thauera group further suggests that AccR constitutes a master regulator that controls anaerobic CCR in these bacteria.

  • 21. Atkins, John F
    et al.
    Björk, Glenn R
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    A gripping tale of ribosomal frameshifting: extragenic suppressors of frameshift mutations spotlight P-site realignment2009Inngår i: Microbiology and molecular biology reviews, ISSN 1092-2172, E-ISSN 1098-5557, Vol. 73, nr 1, s. 178-210Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Mutants of translation components which compensate for both -1 and +1 frameshift mutations showed the first evidence for framing malleability. Those compensatory mutants isolated in bacteria and yeast with altered tRNA or protein factors are reviewed here and are considered to primarily cause altered P-site realignment and not altered translocation. Though the first sequenced tRNA mutant which suppressed a +1 frameshift mutation had an extra base in its anticodon loop and led to a textbook "yardstick" model in which the number of anticodon bases determines codon size, this model has long been discounted, although not by all. Accordingly, the reviewed data suggest that reading frame maintenance and translocation are two distinct features of the ribosome. None of the -1 tRNA suppressors have anticodon loops with fewer than the standard seven nucleotides. Many of the tRNA mutants potentially affect tRNA bending and/or stability and can be used for functional assays, and one has the conserved C74 of the 3' CCA substituted. The effect of tRNA modification deficiencies on framing has been particularly informative. The properties of some mutants suggest the use of alternative tRNA anticodon loop stack conformations by individual tRNAs in one translation cycle. The mutant proteins range from defective release factors with delayed decoding of A-site stop codons facilitating P-site frameshifting to altered EF-Tu/EF1alpha to mutant ribosomal large- and small-subunit proteins L9 and S9. Their study is revealing how mRNA slippage is restrained except where it is programmed to occur and be utilized.

  • 22.
    Avican, Ummehan
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Twin-arginine translocation in Yersinia: the substrates and their role in virulence2016Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Pathogenic Yersinia cause a manifold of diseases in humans ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to pneumonic and bubonic plague (Y. pestis), while all three have a common virulence strategy that relies on a well-studied type III secretion system and its effector proteins to colonize the host and evade immune responses. However, the role of other protein secretion and/or translocation systems in virulence of Yersinia species is not well known. In this thesis, we sought to investigate the contribution of twin-arginine translocation (Tat) pathway and its secreted substrates to the physiology and virulence of Y. pseudotuberculosis. Tat pathway uniquely exports folded proteins including virulence factors across the cytoplasmic membranes of bacteria. The proteins exported by Tat pathway contain a highly conserved twin-arginine motif in the N-terminal signal peptide. We found that the loss of Tat pathway causes a drastic change of the transcriptome of Y. pseudotuberculosis in stationary phase at environmental temperature with differential regulation of genes involved in virulence, carbon metabolism and stress responses. Phenotypic analysis revealed novel phenotypes of the Tat-deficient strain with defects in iron acquisition, acid resistance, copper oxidation and envelope integrity, which we were partly able to associate with the related Tat substrates. Moreover, increased glucose consumption and accumulation of intracellular fumarate were observed in response to inactivation of Tat pathway implicating a generic effect in cellular physiology. We evaluated the direct role of 22 in silico predicted Tat substrate mutants in the mouse infection model and found only one strain, ΔsufI, exhibited a similar degree of attenuation as Tat-deficient strain. Comparative in vivo characterization studies demonstrated a minor defect for ΔsufI in colonization of intestinal tissues compared to the Tat-deficient strain during early infection, whereas both SufI and TatC were required for dissemination from mesenteric lymph nodes and further systemic spread during late infection. This verifies that SufI has a major role in attenuation seen for the Tat deficient strain both during late infection and initial colonization. It is possible that other Tat substrates such as those involved in iron acquisition and copper resistance also has a role in establishing infection. Further phenotypic analysis indicated that SufI function is required for cell division and stress-survival. Transcriptomic analysis revealed that the highest number of differentially regulated genes in response to loss of Tat and SufI were involved in metabolism and transport. Taken together, this thesis presents a thorough analysis of the involvement of Tat pathway in the overall physiology and virulence strategies of Y. pseudotuberculosis. Finally, we propose that strong effects in virulence render TatC and SufI as potential targets for development of novel antimicrobial compounds

  • 23.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Avican, Kemal
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Transcriptomic and phenotypic analysis of sufI and tatC mutants of Yersinia pseudotuberculosisManuskript (preprint) (Annet vitenskapelig)
  • 24.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Beckstette, Michael
    Heroven, Ann Kathrin
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Dersch, Petra
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis2016Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, nr 20, s. 2876-2886Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The Twin-arginine translocation (Tat) system mediates secretion of folded proteins that in bacteria, plants and archaea are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and ∆tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26oC and 37oC. The most significant changes in the transcriptome of the ∆tatC mutant were seen at 26oC during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper conditions as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.

  • 25.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Doruk, Tugrul
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Östberg, Yngve
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    The Tat substrate SufI is critical for the ability of Yersinia pseudotuberculosis to cause systemic infection2017Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 85, nr 4, artikkel-id e00867-16Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The twin arginine translocation (Tat) system targets folded proteins across the inner membrane and is crucial for virulence in many important humanpathogenic bacteria. Tat has been shown to be required for the virulence of Yersinia pseudotuberculosis, and we recently showed that the system is critical for different virulence-related stress responses as well as for iron uptake. In this study, we wanted to address the role of the Tat substrates in in vivo virulence. Therefore, 22 genes encoding potential Tat substrates were mutated, and each mutant was evaluated in a competitive oral infection of mice. Interestingly, a.sufI mutant was essentially as attenuated for virulence as the Tat-deficient strain. We also verified that SufI was Tat dependent for membrane/periplasmic localization in Y. pseudotuberculosis. In vivo bioluminescent imaging of orally infected mice revealed that both the.sufI and Delta tatC mutants were able to colonize the cecum and Peyer's patches (PPs) and could spread to the mesenteric lymph nodes (MLNs). Importantly, at this point, neither the Delta tatC mutant nor the Delta sufI mutant was able to spread systemically, and they were gradually cleared. Immunostaining of MLNs revealed that both the Delta tatC and Delta sufI mutants were unable to spread from the initial infection foci and appeared to be contained by neutrophils, while wild-type bacteria readily spread to establish multiple foci from day 3 postinfection. Our results show that SufI alone is required for the establishment of systemic infection and is the major cause of the attenuation of the Delta tatC mutant.

  • 26.
    Bailey, Leslie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Engström, Patrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Nordström, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för samhällsmedicin och rehabilitering, Rehabiliteringsmedicin. Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap, Idrottsmedicin.
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Waldenström, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Medicin.
    Nordström, Peter
    Umeå universitet, Medicinska fakulteten, Institutionen för samhällsmedicin och rehabilitering, Geriatrik.
    Chlamydia pneumoniae infection results in generalized bone loss in mice2008Inngår i: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 10, nr 10-11, s. 1175-1181Artikkel i tidsskrift (Fagfellevurdert)
  • 27.
    Bailey, Leslie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gylfe, Åsa
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sundin, Charlotta
    Muschiol, Sandra
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Nordström, Peter
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Medicin.
    Henriques-Normark, Birgitta
    Lugert, Raimond
    Waldenström, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Medicin.
    Wolf-Watz, Hans
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle2007Inngår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, nr 4, s. 587-595Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 28. Baker-Austin, Craig
    et al.
    Dopson, Mark
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Life in acid: pH homeostasis in acidophiles.2007Inngår i: Trends Microbiol, ISSN 0966-842X, Vol. 15, nr 4, s. 165-71Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Microorganisms that have a pH optimum for growth of less than pH 3 are termed "acidophiles". To grow at low pH, acidophiles must maintain a pH gradient of several pH units across the cellular membrane while producing ATP by the influx of protons through the F(0)F(1) ATPase. Recent advances in the biochemical analysis of acidophiles coupled to sequencing of several genomes have shed new insights into acidophile pH homeostatic mechanisms. Acidophiles seem to share distinctive structural and functional characteristics including a reversed membrane potential, highly impermeable cell membranes and a predominance of secondary transporters. Also, once protons enter the cytoplasm, methods are required to alleviate effects of a lowered internal pH. This review highlights recent insights regarding how acidophiles are able to survive and grow in these extreme conditions.

  • 29. Baker-Austin, Craig
    et al.
    Dopson, Mark
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wexler, Margaret
    Sawers, R Gary
    Bond, Philip L
    Molecular insight into extreme copper resistance in the extremophilic archaeon 'Ferroplasma acidarmanus' Fer1.2005Inngår i: Microbiology, ISSN 1350-0872, Vol. 151, nr Pt 8, s. 2637-46Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    'Ferroplasma acidarmanus' strain Fer1 is an extremely acidophilic archaeon involved in the genesis of acid mine drainage, and was isolated from copper-contaminated mine solutions at Iron Mountain, CA, USA. Here, the initial proteomic and molecular investigation of Cu(2+) resistance in this archaeon is presented. Analysis of Cu(2+) toxicity via batch growth experiments and inhibition of oxygen uptake in the presence of ferrous iron demonstrated that Fer1 can grow and respire in the presence of 20 g Cu(2+) l(-1). The Fer1 copper resistance (cop) loci [originally detected by Ettema, T. J. G., Huynen, M. A., de Vos, W. M. & van der Oost, J. Trends Biochem Sci 28, 170-173 (2003)] include genes encoding a putative transcriptional regulator (copY), a putative metal-binding chaperone (copZ) and a putative copper-transporting P-type ATPase (copB). Transcription analyses demonstrated that copZ and copB are co-transcribed, and transcript levels were increased significantly in response to exposure to high levels of Cu(2+), suggesting that the transport system is operating for copper efflux. Proteomic analysis of Fer1 cells exposed to Cu(2+) revealed the induction of stress proteins associated with protein folding and DNA repair (including RadA, thermosome and DnaK homologues), suggesting that 'Ferroplasma acidarmanus' Fer1 uses multiple mechanisms for resistance to high levels of copper.

  • 30. Baker-Austin, Craig
    et al.
    Dopson, Mark
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wexler, Margaret
    Sawers, R Gary
    Stemmler, Ann
    Rosen, Barry P
    Bond, Philip L
    Extreme arsenic resistance by the acidophilic archaeon 'Ferroplasma acidarmanus' Fer1.2007Inngår i: Extremophiles, ISSN 1431-0651, Vol. 11, nr 3, s. 425-34Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    'Ferroplasma acidarmanus' Fer1 is an arsenic-hypertolerant acidophilic archaeon isolated from the Iron Mountain mine, California; a site characterized by heavy metals contamination. The presence of up to 10 g arsenate per litre [As(V); 133 mM] did not significantly reduce growth yields, whereas between 5 and 10 g arsenite per litre [As(III); 67-133 mM] significantly reduced the yield. Previous bioinformatic analysis indicates that 'F. acidarmanus' Fer1 has only two predicted genes involved in arsenic resistance and lacks a recognizable gene for an arsenate reductase. Biochemical analysis suggests that 'F. acidarmanus' Fer1 does not reduce arsenate indicating that 'F. acidarmanus' Fer1 has an alternative resistance mechanism to arsenate other than reduction to arsenite and efflux. Primer extension analysis of the putative ars transcriptional regulator (arsR) and efflux pump (arsB) demonstrated that these genes are co-transcribed, and expressed in response to arsenite, but not arsenate. Two-dimensional polyacrylamide gel electrophoresis analysis of 'F. acidarmanus' Fer1 cells exposed to arsenite revealed enhanced expression of proteins associated with protein refolding, including the thermosome Group II HSP60 family chaperonin and HSP70 DnaK type heat shock proteins. This report represents the first molecular and proteomic study of arsenic resistance in an acidophilic archaeon.

  • 31. Baker-Austin, Craig
    et al.
    Potrykus, Joanna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wexler, Margaret
    Bond, Philip L
    Dopson, Mark
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). School of Biological Sciences, University of East Anglia, Norwich, UK .
    Biofilm development in the extremely acidophilic archaeon 'Ferroplasma acidarmanus' Fer12010Inngår i: Extremophiles, ISSN 1431-0651, E-ISSN 1433-4909, Vol. 14, nr 6, s. 485-491Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    'Ferroplasma acidarmanus' Fer1 is an ironoxidizing extreme acidophile isolated from the Iron Mountain mine, California, USA This archaeon is predominantly found in biofilm-associated structures in the environment, and produces two distinct biofilm morphologies Bioinformatic analysis of the acidarmanus' Fer1 genome Identified genes annotated as involved in attachment and biofilm formation No putative quorum sensing signaling genes were identified and no N-acyl homoserine lactone-like compounds were found in acidarmanus' Fer1 biofilm supernatant Scanning confocal microscopy analysis of biofilm development on the surface of pyrite demonstrated the temporal and spatial development of biofilm growth Furthermore, two-dimensional polyacrylamide gel electrophoresis was used to examine differential protein expression patterns between biofilm and planktonic populations Ten up-regulated proteins were identified that included six enzymes associated with anaerobic growth, suggesting that the dominating phenotype in the mature biofilm was associated with anaerobic modes of growth This report increases our knowledge of the genetic and proteomic basis of biofilm formation in an extreme acidophilic archaeon.

  • 32.
    Bamyaci, Sarp
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ekestubbe, Sofie
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Nordfelth, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ertmann, Saskia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Edgren, Tomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    YopN is required for efficient translocation and virulence in Yersinia pseudotuberculosisManuskript (preprint) (Annet vitenskapelig)
  • 33.
    Bamyaci, Sarp
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ekestubbe, Sofie
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Nordfelth, Roland
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Erttmann, Saskia F.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Edgren, Tomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis2018Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 86, nr 8, artikkel-id e00957-17Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion systems (T3SSs) are used by various Gram-negative pathogens to subvert the host defense by a host cell contact-dependent mechanism to secrete and translocate virulence effectors. While the effectors differ between pathogens and determine the pathogenic life style, the overall mechanism of secretion and translocation is conserved. T3SSs are regulated at multiple levels, and some secreted substrates have also been shown to function in regulation. In Yersinia, one of the substrates, YopN, has long been known to function in the host cell contact-dependent regulation of the T3SS. Prior to contact, through its interaction with TyeA, YopN blocks secretion. Upon cell contact, TyeA dissociates from YopN, which is secreted by the T3SS, resulting in the induction of the system. YopN has also been shown to be translocated into target cells by a T3SS-dependent mechanism. However, no intracellular function has yet been assigned to YopN. The regulatory role of YopN involves the N-terminal and C-terminal parts, while less is known about the role of the central region of YopN. Here, we constructed different in-frame deletion mutants within the central region. The deletion of amino acids 76 to 181 resulted in an unaltered regulation of Yop expression and secretion but triggered reduced YopE and YopH translocation within the first 30 min after infection. As a consequence, this deletion mutant lost its ability to block phagocytosis by macrophages. In conclusion, we were able to differentiate the function of YopN in translocation and virulence from its function in regulation.

  • 34.
    Bamyaci, Sarp
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Nordfelth, Roland
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Identification of specific sequence motif of YopN of Yersinia pseudotuberculosis required for systemic infection2019Inngår i: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 10, nr 1, s. 10-25Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion systems (T3SSs) are tightly regulated key virulence mechanisms shared by many Gram-negative pathogens. YopN, one of the substrates, is also crucial in regulation of expression, secretion and activation of the T3SS of pathogenic Yersinia species. Interestingly, YopN itself is also targeted into host cells but so far no activity or direct role for YopN inside host cells has been described. Recently, we were able show that the central region of YopN is required for efficient translocation of YopH and YopE into host cells. This was also shown to impact the ability of Yersinia to block phagocytosis. One difficulty in studying YopN is to generate mutants that are not impaired in regulation of the T3SS. In this study we extended our previous work and were able to generate specific mutants within the central region of YopN. These mutants were predicted to be crucial for formation of a putative coiled-coil domain (CCD). Similar to the previously described deletion mutant of the central region, these mutants were all impaired in translocation of YopE and YopH. Interestingly, these YopN variants were not translocated into host cells. Importantly, when these mutants were introduced in cis on the virulence plasmid, they retained full regulatory function of T3SS expression and secretion. This allowed us to evaluate one of the mutants, yopNGAGA, in the systemic mouse infection model. Using in vivo imaging technology we could verify that the mutant was also attenuated in vivo and highly impaired to establish systemic infection.

  • 35.
    Bartilson, M
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Nordlund, I
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Shingler, V
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Location and organization of the dimethylphenol catabolic genes of Pseudomonas CF600.1990Inngår i: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 220, nr 2Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The gene organization of the phenol catabolic pathway of Pseudomonas CF600 has been investigated. This strain can grow on phenol and some methylated phenols by virtue of an inducible phenol hydroxylase and metacleavage pathway enzymes. The genes coding for these enzymes are located on pVI150, an IncP-2 degradative mega plasmid of this strain. Twenty-three kilobases of contiguous DNA were isolated from lambda libraries constructed from strains harbouring wild type and Tn5 insertion mutants of pVI150. A 19.9 kb region of this DNA has been identified which encodes all the catabolic genes of the pathway. Using transposon mutagenesis, polypeptide analysis and expression of subfragments of DNA, the genes encoding the first four enzymatic steps of the pathway have been individually mapped and found to lie adjacent to each other. The order of these genes is the same as that for isofunctional genes of TOL plasmid pWWO and plasmid NAH7.

  • 36.
    Bartilson, M
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Shingler, V
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Nucleotide sequence and expression of the catechol 2,3-dioxygenase-encoding gene of phenol-catabolizing Pseudomonas CF600.1989Inngår i: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 85, nr 1Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pseudomonas CF600 degrades phenol and some of its methylated derivatives via a plasmid-encoded catabolic pathway. The catechol 2,3-dioxygenase (C23O) enzyme of this pathway catalyses the conversion of catechol to 2-hydroxymuconic semialdehyde. We have determined the nucleotide (nt) sequence of the dmpB structural gene for this enzyme, and expressed and identified its polypeptide product in Escherichia coli. The xylE gene of TOL plasmid pWWO and the nahH gene of plasmid NAH7 encode analogous C23O enzymes. Comparison of these three genes shows homology of 78-81% on the nt level and 83-87% homology on the amino acid level.

  • 37.
    Bartra, Sara Schesser
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Styer, Katie L
    O'Bryant, Deanna M
    Nilles, Matthew L
    Hinnebusch, B Joseph
    Aballay, Alejandro
    Plano, Gregory V
    Resistance of Yersinia pestis to complement-dependent killing is mediated by the Ail outer membrane protein2008Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 76, nr 2, s. 612-622Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Yersinia pestis, the causative agent of plague, must survive in blood in order to cause disease and to be transmitted from host to host by fleas. Members of the Ail/Lom family of outer membrane proteins provide protection from complement-dependent killing for a number of pathogenic bacteria. The Y. pestis KIM genome is predicted to encode four Ail/Lom family proteins. Y. pestis mutants specifically deficient in expression of each of these proteins were constructed using lambda Red-mediated recombination. The Ail outer membrane protein was essential for Y. pestis to resist complement-mediated killing at 26 and 37 degrees C. Ail was expressed at high levels at both 26 and 37 degrees C, but not at 6 degrees C. Expression of Ail in Escherichia coli provided protection from the bactericidal activity of complement. High-level expression of the three other Y. pestis Ail/Lom family proteins (the y1682, y2034, and y2446 proteins) provided no protection against complement-mediated bacterial killing. A Y. pestis ail deletion mutant was rapidly killed by sera obtained from all mammals tested except mouse serum. The role of Ail in infection of mice, Caenorhabditis elegans, and fleas was investigated.

  • 38. Beljantseva, Jelena
    et al.
    Kudrin, Pavel
    Andresen, Liis
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Shingler, Vicky
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Atkinson, Gemma C.
    Tenson, Tanel
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Institute of Technology, University of Tartu, 50411 Tartu, Estonia.
    Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA2017Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 14, s. 3726-3731Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ: RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5 alpha. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes.

  • 39. Beljantseva, Jelena
    et al.
    Kudrin, Pavel
    Jimmy, Steffi
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ehn, Marcel
    Pohl, Radek
    Varik, Vallo
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). 1University of Tartu, Institute of Technology, Tartu, Estonia.
    Tozawa, Yuzuru
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Tenson, Tanel
    Rejman, Dominik
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). 1University of Tartu, Institute of Technology, Tartu, Estonia.
    Molecular mutagenesis of ppGpp: turning a RelA activator into an inhibitor2017Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikkel-id 41839Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The alarmone nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p) ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p) ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work.

  • 40. Bento-Abreu, Andre
    et al.
    Jager, Gunilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Swinnen, Bart
    Rué, Laura
    Hendrickx, Stijn
    Jones, Ashley
    Staats, Kim A.
    Taes, Ines
    Eykens, Caroline
    Nonneman, Annelies
    Nuyts, Rik
    Timmers, Mieke
    Silva, Lara
    Chariot, Alain
    Nguyen, Laurent
    Ravits, John
    Lemmens, Robin
    Cabooter, Deirdre
    Van Den Bosch, Ludo
    Van Damme, Philip
    Al-Chalabi, Ammar
    Bystrom, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Robberecht, Wim
    Elongator subunit 3 (ELP3) modifies ALS through tRNA modification.2018Inngår i: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 27, nr 7, s. 1276-1289Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder of which the progression is influenced by several disease-modifying factors. Here, we investigated ELP3, a subunit of the elongator complex that modifies tRNA wobble uridines, as one of such ALS disease modifiers. ELP3 attenuated the axonopathy of a mutant SOD1, as well as of a mutant C9orf72 ALS zebrafish model. Furthermore, the expression of ELP3 in the SOD1G93A mouse extended the survival and attenuated the denervation in this model. Depletion of ELP3 in vitro reduced the modified tRNA wobble uridine mcm5s2U and increased abundance of insoluble mutant SOD1, which was reverted by exogenous ELP3 expression. Interestingly, the expression of ELP3 in the motor cortex of ALS patients was reduced and correlated with mcm5s2U levels. Our results demonstrate that ELP3 is a modifier of ALS and suggest a link between tRNA modification and neurodegeneration.

  • 41.
    Berg, A. H.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Thomas, P.
    Olsson, P-E.
    Characterization of the 17,20β-dihydroxy-4-pregnen-3-one membrane receptor in Arctic char (Salvelinus alpinus) ovaries and its upregulation during gonadotropin induction of oocyte maturationManuskript (Annet vitenskapelig)
  • 42.
    Berg, A. H.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Westerlund, L.
    Olsson, P-E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Regulation of Arctic char (Salvelinus alpinus) egg shell proteins and vitellogenin during reproduction and in response to 17β-estradiol and cortisol2004Inngår i: General and Comparative Endocrinology, ISSN 0016-6480, E-ISSN 1095-6840, Vol. 135, nr 3, s. 276-285Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Estrogens induce both vitellogenin (Vtg) and egg shell (zona pellucida; ZP) protein synthesis in salmonids. However, while Vtg is strictly under estrogenic control, recent reports suggest that additional mechanisms are involved in ZP protein synthesis. During sexual maturation both estrogen and glucocorticoid levels increase in the circulation of female fish. As glucocorticoids have been shown to interfere with Vtg induction in fish we investigated whether cortisol (F) had similar effects on ZP regulation. In the present study we determined both the natural variation in Vtg and ZP during an annual reproductive cycle in female Arctic char (Salvelinus alpinus), and the effect of co-treatment of juvenile Arctic char with 17β-estradiol (E2) and F. During sexual maturation the expression of Vtg and ZP correlated to plasma levels of E2 and F. Determination of Vtg and ZP protein levels following co-treatment with E2 and F showed that F antagonized E2 induction of Vtg. However, F was observed to potentiate the expression of ZP protein in the same fish. These results indicate that in Arctic char Vtg and ZP proteins are not regulated by the same mechanisms and suggest that ZP protein expression does not necessarily imply exposure to estrogenic compounds alone, and may thus not be ideally suited as a biomarker of exposure to estrogenic compounds.

  • 43.
    Berg, Håkan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Teleost reproduction: Aspects of Arctic char (Salvelinus alpinus) oocyte growth and maturation2003Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In all vertebrate species, reproduction is a hormonally controlled process, important for growth and maturation of gonads and germ cells. Production of functional germ cells is of outmost importance to secure the survival of a species. Fish comprises 50% of the known vertebrates and are found in aquatic habitats all over the world. Even though fish have evolved a wide variety of morphological and physiological characteristics, due to large differences in the living environment, the growth an maturation of germ cells follows the same pattern in all species. In this thesis the focus has been directed on oocyte growth and development in Arctic char (Salvelinus alpinus), and if stress might inflict disturbances on the reproductive systems.

    All sexually mature female egg laying vertebrates produces yolky eggs surrounded by an eggshell. Production of yolk and egg shell is under estrogenic control and it is known that production of egg components can be induced in male and juvenile fish by estrogenic substances. Many manmade chemicals have been found to interfere with hormonally controlled processes. Therefore production of the egg yolk precursor, vitellogenin (VTG), and the egg shell components, vitelline envelope proteins (VEP), have been used as biomarkers for estrogenic effect. Exposure to endocrine disrupting substances (EDS) does not only give rise to hormonal effects on the organism, but in addition it also gives rise to an increase in stress hormone, cortisol (F), levels.

    It is evident that a wide variety of substances may affect Arctic char oocyte growth and maturation. VTG and VEP production is found to be under dose dependent estrogenic control, but the production was directly affected by F. Under natural condition it has been found that F increases towards ovulation. Even though both VTG and VTG is under estrogenic control, these studies showed that stress lead to a decrease of VTG while the VEP production increased. These effects was only observed on protein levels indicating that a post transcriptional down regulation of VTG production is mediated by F in Arctic char.

    In order for an egg to become fertilizatible, it must undergo a maturation phase. This maturation phase is primarily induced by gonadotropins, which in turn induce the production of species specific maturation inducing substances (MIS). To investigate oocyte development in Arctic char a characterization of its MIS receptor was made. The MIS receptor is localized on the oocyte surface and displays a single class of high affinity and low capacity binding sites. The binding moieties displays association and dissociation kinetics typical of steroid membrane receptors.

    Even though high specificity for Arctic char MIS was observed, it was found that some EDS bind to the Arctic char oocyte membrane receptor. This suggest that certain EDS might affect oocyte maturation and thereby might alter the reproductive success. Furthermore, it was found that F did not bind to the MIS receptor in Arctic char. It is therefore suggested that oocytes are more sensitive to stress during the growth phase than during maturation

  • 44.
    Berg, Håkan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Department of Marine Science, University of Texas Marine Science Institute, University of Texas, Port Aransas, Texas, USA.
    Olsson, Per-Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Natural Science, Unit of Molecular Biology, Orebro University, Orebro, Sweden.
    Modig, Carina
    17ß-estradiol induced vitellogenesis is inhibited by cortisol at the post-transcriptional level in Arctic char (Salvelinus alpinus)2004Inngår i: Reproductive Biology and Endocrinology, ISSN 1477-7827, E-ISSN 1477-7827, Vol. 2, nr 62, s. 1-10Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    This study was performed to investigate stress effects on the synthesis of egg yolk precursor, vitellogenin (Vtg) in Arctic char (Salvelinus alpinus). In particular the effect of cortisol (F) was determined since this stress hormone has been suggested to interfere with vitellogenesis and is upregulated during sexual maturation in teleosts. Arctic char Vtg was purified and polyclonal antibodies were produced in order to develop tools to study regulation of vitellogenesis. The Vtg antibodies were used to develop an enzyme-linked immunosorbent assay. The corresponding Vtg cDNA was cloned from a hepatic cDNA library in order to obtain DNA probes to measure Vtg mRNA expression. Analysis of plasma from juvenile Arctic char, of both sexes, exposed to different steroids showed that production of Vtg was induced in a dose dependent fashion by 17β-estradiol (E2), estrone and estriol. Apart from estrogens a high dose of F also upregulated Vtg. In addition, F, progesterone (P) and tamoxifen were tested to determine these compounds ability to modulate E2 induced Vtg synthesis at both the mRNA and protein level. Tamoxifen was found to inhibit E2 induced Vtg mRNA and protein upregulation. P did not alter the Vtg induction while F reduced the Vtg protein levels without affecting the Vtg mRNA levels. Furthermore the inhibition of Vtg protein was found to be dose dependent. Thus, the inhibitory effect of F on Vtg appears to be mediated at the post-transcriptional level.

  • 45. Berger, Susanne
    et al.
    Schäfer, Gritt
    Kesper, Dörthe A
    Holz, Anne
    Eriksson, Therese
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Palmer, Ruth H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Beck, Lothar
    Klämbt, Christian
    Renkawitz-Pohl, Renate
    Onel, Susanne-Filiz
    WASP and SCAR have distinct roles in activating the Arp2/3 complex during myoblast fusion2008Inngår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 121, nr Pt 8, s. 1303-1313Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Myoblast fusion takes place in two steps in mammals and in Drosophila. First, founder cells (FCs) and fusion-competent myoblasts (FCMs) fuse to form a trinucleated precursor, which then recruits further FCMs. This process depends on the formation of the fusion-restricted myogenic-adhesive structure (FuRMAS), which contains filamentous actin (F-actin) plugs at the sites of cell contact. Fusion relies on the HEM2 (NAP1) homolog Kette, as well as Blow and WASP, a member of the Wiskott-Aldrich-syndrome protein family. Here, we show the identification and characterization of schwächling--a new Arp3-null allele. Ultrastructural analyses demonstrate that Arp3 schwächling mutants can form a fusion pore, but fail to integrate the fusing FCM. Double-mutant experiments revealed that fusion is blocked completely in Arp3 and wasp double mutants, suggesting the involvement of a further F-actin regulator. Indeed, double-mutant analyses with scar/WAVE and with the WASP-interacting partner vrp1 (sltr, wip)/WIP show that the F-actin regulator scar also controls F-actin formation during myoblast fusion. Furthermore, the synergistic phenotype observed in Arp3 wasp and in scar vrp1 double mutants suggests that WASP and SCAR have distinct roles in controlling F-actin formation. From these findings we derived a new model for actin regulation during myoblast fusion.

  • 46.
    Berghard, Anna
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hägglund, Anna-Carin
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Bohm, Staffan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Carlsson, Leif
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Lhx2-dependent specification of olfactory sensory neurons is required for successful integration of olfactory, vomeronasal, and GnRH neurons2012Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, nr 8, s. 3464-3472Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Inactivation of the LIM-homeodomain 2 gene (Lhx2) results in a severe defect in specification of olfactory sensory neurons (OSNs). However, the ramifications of lack of Lhx2-dependent OSN specification for formation of the primary olfactory pathway have not been addressed, since mutant mice die in utero. We have analyzed prenatal and postnatal consequences of conditionally inactivating Lhx2 selectively in OSNs. A cell-autonomous effect is that OSN axons cannot innervate their target, the olfactory bulb. Moreover, the lack of Lhx2 in OSNs causes unpredicted, non-cell-autonomous phenotypes. First, the olfactory bulb shows pronounced hypoplasia in adults, and the data suggest that innervation by correctly specified OSNs is necessary for adult bulb size and organization. Second, absence of an olfactory nerve in the conditional mutant reveals that the vomeronasal nerve is dependent on olfactory nerve formation. Third, the lack of a proper vomeronasal nerve prevents migration of gonadotropin-releasing hormone (GnRH) cells the whole distance to their final positions in the hypothalamus during embryo development. As adults, the conditional mutants do not pass puberty, and these findings support the view of an exclusive nasal origin of GnRH neurons in the mouse. Thus, Lhx2 in OSNs is required for functional development of three separate systems.—Berghard, A., Hägglund, A.-C., Bohm, S., and Carlsson, L. Lhx2-dependent specification of olfactory sensory neurons is required for successful integration of olfactory, vomeronasal, and GnRH neurons.

  • 47.
    Bergman, Marie-Louise
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    A sub-phenotype approach to dissect the genetic control of murine type 1 diabetes2002Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The non-obese diabetic (NOD) mouse is a model for human type 1 diabetes (T1D). The disease in the NOD mouse is polygenic and multifactorial and so far at least 20 insulin dependent diabetes (Idd) susceptibility loci have been identified. However, no etiological mutations have been definitely ascribed to the Idd loci. To identify potential etiological mutations, a sub-phenotype approach was undertaken, consisting of the establishment and genetic mapping of immuno-related sub-phenotypes that may contribute to the pathogenesis of T1D in the NOD mouse model. This thesis presents (1) the results of the identification and genetic mapping of four novel NOD immuno-phenotypes to individual Idd loci, and (2) confirmation of these results by the generation and analysis of congenic strains covering those Idd regions.

    Evidence is provided that gene(s) within the Idd5 region control cyclophosphamide (CY)-induced apoptosis in peripheral lymphocytes and y-irradiation induced apoptosis in NOD thymocytes. Analysis of non-obese resistant (NOR) and NOD-Idd5 congenic mice reveal that CY-induced apoptosis in peripheral lymphocytes and y-irradiation induced apoptosis in thymocytes are controlled by a 20cM and a 6cM region, respectively, both containing the Idd5 region and including the immuno-regulatory Ctla4 gene. Additionally, CTLA4 is shown to be defectively up-regulated in activated NOD peripheral lymphocytes, and CTLA4-deficient mice show similar defects in T cell apoptosis induction. Taken together, these results suggest that a defective up-regulation of CTLA4 mediates apoptosis resistance, contributing to diabetes pathogenesis.

    Moreover, it is shown that gene(s) within the Idd6 region control low proliferation ofNOD immature thymocytes and resistance to dexamethazone-induced apoptosis in immature DP thymocytes. The decrease of diabetes incidence and the restoration of the apoptosis resistance phenotype in reciprocal Idd6 congenic strains further restrict the chromosomal region controlling the Idd6 locus as well as the locus controlling the apoptosis resistance phenotype. In fact, analysis of NOD-Idd6 congenic mice reveal that Dxm-induced apoptosis in thymocytes is controlled by the distal 3cM region of the Idd6 locus. As the thymic selection process is highly dependent on both proliferation and apoptosis, the hypothesis is raised that the Idd6 locus contributes to the pathogenesis of diabetes by altering thymic selection, resulting in an autoimmune prone peripheral T cell repertoire.

  • 48.
    Bergman, Marie-Louise
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Instituto Gulbenkian de Ciencia, Oeiras, Portugal.
    Cilio, Corrado M
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Endocrine Research Unit, Wallenberg Laboratory, Malmö University Hospital MAS, University of Lund, 205 02 Malmö Sweden.
    Penha-Gonçalves, Carlos
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Instituto Gulbenkian de Ciencia, Oeiras, Portugal.
    Lamhamedi-Cherradi, Salah-Eddine
    INSERM U25, Hopital Necker, Paris, France.
    Löfgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Colucci, Francesco
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lejon, Kristina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Garchon, Henri-Jean
    INSERM U25, Hopital Necker, Paris, France.
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Instituto Gulbenkian de Ciencia, Oeiras, Portugal.
    CTLA-4-/- mice display T cell-apoptosis resistance resembling that ascribed to autoimmune-prone non-obese diabetic (NOD) mice2001Inngår i: Journal of Autoimmunity, ISSN 0896-8411, E-ISSN 1095-9157, Vol. 16, nr 2, s. 105-113Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The genes conferring susceptibility to autoimmune (insulin-dependent) diabetes mellitus (IDDM) are, in most cases, not defined. Among the loci so far identified as associated with murine IDDM (Idd1-19), only the nature of Idd1 has been assessed. Here we show that thymocytes and peripheral lymphocytes of the non-obese diabetic (NOD) mouse are relatively resistant to apoptosis induced by gamma-irradiation. By linkage analysis of F2 progeny mice, we map this trait to a locus on chromosome 1 containing the Idd5 diabetes susceptibility region. By the use of congenic mice, we confirm the linkage data and map this locus to a 6 cM region on proximal chromosome 1. Ctla4, being localized in this chromosomal region and mediating crucial functions in T cell biology, is a logical candidate gene in the Idd5 susceptibility region. In line with this, we demonstrate that T cells from Ctla4(-/-)deficient mice show a similar resistance to gamma-irradiation-induced apoptosis as observed in the NOD mice. This reinforces the notion that CTLA-4 contributes to the pathogenesis of autoimmune diabetes.

  • 49.
    Bergman, Marie-Louise
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Penha-Gonçalves, Carlos
    Gulbenkian Institute for Science, Oeiras, Portugal, PT.
    Lejon, Kristina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Low rate of proliferation in immature thymocytes of the non-obese diabetic mouse maps to the Idd6 diabetes susceptibility region2001Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 44, nr 8, s. 1054-1061Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aims/hypothesis: The non-obese diabetic (NOD) mouse spontaneously develops T-cell-dependent autoimmune diabetes. This mouse strain has a number of immune dysfunctions related to T-cell development but so far there are no available data on the proliferation of NOD immature thymocytes. We therefore studied the thymocyte proliferation in the NOD mouse in discrete stages of T-cell development.

    Methods: We depleted thymocytes in vivo and analysed thymocyte proliferation during the thymus recovery from depletion. We used co-segregation analysis and quantitative loci trait analysis to investigate the genetic control of proliferation impairments in NOD thymocytes.

    Results: Immature thymocytes of female NOD mice proliferate with a relatively low rate compared to non-autoimmune C57Bl/6 mice. This aberrant proliferation was most pronounced in CD4 /loCD8+ cells differentiating from the CD4CD8 to the CD4+CD8+ stage. A genetic mapping study using an F2 intercross between the NOD and the C57BL/6 strains showed that a major locus controlling this trait is linked to the insulin-dependent diabetes susceptibility locus Idd6.

    Conclusion/interpretation: Our results suggest that impairment of proliferation of immature thymocytes is one possible mechanism through which the Idd6 locus contributes to the pathogenesis of diabetes.

  • 50. Bernardo, Lisandro
    et al.
    Johansson, Linda U M
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Skärfstad, Eleonore
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    σ54-promoter discrimination and regulation by ppGpp and DksA2009Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, nr 2, s. 828-838Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The sigma(54)-factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of sigma(54)-dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of sigma(54)-dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of sigma(54)-dependent transcription in combination with a series of sigma(54)-promoters with graded affinities for sigma(54)-RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted sigma(54)-transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous sigma(54)-RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive sigma(54)-transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity sigma(54)-promoters that is conferred by the strong DNA binding properties of the P. putida sigma(54)-RNA polymerase holoenzyme.

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