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  • 201. Codemo, Mario
    et al.
    Muschiol, Sandra
    Iovino, Federico
    Nannapaneni, Priyanka
    Plant, Laura
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Henriques-Normark, Birgitta
    Immunomodulatory Effects of Pneumococcal Extracellular Vesicles on Cellular and Humoral Host Defenses2018Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, nr 2, artikel-id e00559-18Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gram-positive bacteria, including the major respiratory pathogen Streptococcus pneumoniae, were recently shown to produce extracellular vesicles (EVs) that likely originate from the plasma membrane and are released into the extracellular environment. EVs may function as cargo for many bacterial proteins, however, their involvement in cellular processes and their interactions with the innate immune system are poorly understood. Here, EVs from pneumococci were characterized and their immunomodulatory effects investigated. Pneumococcal EVs were protruding from the bacterial surface and released into the medium as 25 to 250 nm lipid stained vesicles containing a large number of cytosolic, membrane, and surface-associated proteins. The cytosolic pore-forming toxin pneumolysin was significantly enriched in EVs compared to a total bacterial lysate but was not required for EV formation. Pneumococcal EVs were internalized into A549 lung epithelial cells and human monocyte-derived dendritic cells and induced proinflammatory cytokine responses irrespective of pneumolysin content. EVs from encapsulated pneumococci were recognized by serum proteins, resulting in C3b deposition and formation of C5b-9 membrane attack complexes as well as factor H recruitment, depending on the presence of the choline binding protein PspC. Addition of EVs to human serum decreased opsonophagocytic killing of encapsulated pneumococci. Our data suggest that EVs may act in an immunomodulatory manner by allowing delivery of vesicle-associated proteins and other macromolecules into host cells. In addition, EVs expose targets for complement factors in serum, promoting pneumococcal evasion of humoral host defense.

    Importance: Streptococcus pneumoniae is a major contributor to morbidity and mortality worldwide, being the major cause of milder respiratory tract infections such as otitis and sinusitis and of severe infections such as community-acquired pneumonia, with or without septicemia, and meningitis. More knowledge is needed on how pneumococci interact with the host, deliver virulence factors, and activate immune defenses. Here we show that pneumococci form extracellular vesicles that emanate from the plasma membrane and contain virulence properties, including enrichment of pneumolysin. We found that pneumococcal vesicles can be internalized into epithelial and dendritic cells and bind complement proteins, thereby promoting pneumococcal evasion of complement-mediated opsonophagocytosis. They also induce pneumolysin-independent proinflammatory responses. We suggest that these vesicles can function as a mechanism for delivery of pneumococcal proteins and other immunomodulatory components into host cells and help pneumococci to avoid complement deposition and phagocytosis-mediated killing, thereby possibly contributing to the symptoms found in pneumococcal infections.

  • 202. Collin, Betty
    et al.
    Rehnstam-Holm, Ann-Sofi
    Lindmark, Barbro
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Pal, Amit
    Wai, Sun N.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hernroth, Bodil
    the origin of vibrio cholerae influences uptake and persistence in the blue mussel mytilus edulis2012Ingår i: Journal of Shellfish Research, ISSN 0730-8000, E-ISSN 1943-6319, Vol. 31, nr 1, s. 87-92Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Vibrio cholerae may cause diarrheal diseases and wound infections, both of which have the potential to be fatal. Transmission to humans is often linked to consumption of contaminated shellfish/drinking water or dermal exposure to water (e.g. when swimming). In this study, we investigated whether different isolates of Vibrio cholerae differ in terms of accumulation, persistence, and viability when encountering blue mussels (Mytilus edulis). Mussel uptake and elimination of three different V. cholerae strains were compared: one fatal clinical non-OI/O139 isolate, one highly potent El Tor biotype, and one marine strain isolated from blue mussels. The results showed that the uptake of the marine strain was significantly higher than the clinical strain, but the elimination process of the marine strain was also more efficient. The El Tor strain was not at all ingested by the mussels. In addition, the survival of bacteria when incubated together with M. edulis hemocytes was tested in vitro. The viability of clinical strains was unaffected by the presence of hemocytes, and the marine strains were even more resistant and able to multiply. We conclude that the highly virulent El Tor biotype was not taken up by the mussels and could thereby escape the mussels' elimination process. The potentially fatal non-OI/O139 V. cholerae strain may accumulate in low numbers, but could be very persistent in mussels.

  • 203.
    Colucci, Francesco
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). INSERM U429, Necker Hospital, Paris, France.
    Bergman, Marie-Louise
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lejon, Kristina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Diabetes induction in C57BL/6 mice reconstituted with lymphocytes of nonobese diabetic C57BL/6 mouse embryo aggregation chimeras1998Ingår i: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 48, nr 6, s. 571-576Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To determine whether the genetic background of the insulin-producing beta cells of the pancreas contributes to autoimmune diabetes susceptibility, we have used a model of the disease based on transferring spleen cells from nonobese diabetic (NOD) <--> C57BL/6 (B6) embryo aggregation (EA) chimeras into B6 and NOD irradiated mice. Insulitis and diabetes could be induced into both B6 and NOD hosts, albeit with low incidence. Cyclophosphamide (CY) treatment, known to accelerate diabetes in prediabetic NOD mice, was found to increase diabetes incidence up to 50-60% in both B6 and NOD mice reconstituted with chimeric splenocytes, while diabetes did not occur in CY-treated B6 mice reconstituted with B6 splenocytes. We conclude that the genetic make-up of the target organ does not affect the final stage of the pathogenesis of insulin-dependent diabetes mellitus.

  • 204.
    Colucci, Francesco
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Bergman, Marie-Louise
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Penha-Gonçalves, Carlos
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Cilio, Corrado M.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked to the Idd5 diabetes susceptibility region1997Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 94, nr 16, s. 8670-8674Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Defects in lymphocyte apoptosis may lead to autoimmune disorders and contribute to the pathogenesis of type 1 diabetes. Lymphocytes of nonobese diabetic (NOD) mice, an animal model of autoimmune diabetes, have been found resistant to various apoptosis signals, including the alkylating drug cyclophosphamide. Using an F2 intercross between the apoptosis-resistant NOD mouse and the apoptosis-susceptible C57BL/6 mouse, we define a major locus controlling the apoptosis-resistance phenotype and demonstrate its linkage (logarithm of odds score = 3.9) to a group of medial markers on chromosome 1. The newly defined gene cannot be dissociated from Ctla4 and Cd28 and in fact marks a 20-centimorgan region encompassing Idd5, a previously postulated diabetes susceptibility locus. Interestingly, we find that the CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and the CD28 costimulatory molecules are defectively expressed in NOD mice, suggesting that one or both of these molecules may be involved in the control of apoptosis resistance and, in turn, in diabetes susceptibility.

  • 205.
    Colucci, Francesco
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cilio, Corrado M.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Pediatrics, University of Rome ‘La Sapienza’ Rome, Italy.
    Lejon, Kristina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Penha Gonçalves, Carlos
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Bergman, Marie-Louise
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Programmed cell death in the pathogenesis of murine IDDM: resistance to apoptosis induced in lymphocytes by cyclophosphamide1996Ingår i: Journal of Autoimmunity, ISSN 0896-8411, E-ISSN 1095-9157, Vol. 9, nr 2, s. 271-276Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The non-obese diabetic (NOD) mouse displays several immune related defects, each of which could potentially contribute to the immunopathogenesis of diabetes that spontaneously develops in these mice. The reported resistance of NOD-lymphocytes to several apoptosis-inducing signals constitutes one such factor. Apoptosis plays a key role in the homeostasis of the immune system, as a means of selecting lymphocyte repertoires both in primary lymphoid organs and in the periphery; distortions in the apoptotic machinery may therefore be hypothesized to be implicated in the pathogenesis of autoimmune disorders. We now report that cyclophosphamide constitutes an apoptosis signal to peripheral lymphocytes and we provide evidence that NOD B cells as well as both CD4 and CD8 T cells display resistance to cyclophosphamide-induced apoptosis. These observations support the notion that apoptosis resistance in NOD mice exists at various levels, and suggest that the CY-sensitive lymphoid population, believed to play an important role in inhibiting the disease in diabetes resistant NOD mice (particularly males), may be controlled by mechanisms that are mediated by apoptosis.

  • 206.
    Colucci, Francesco
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lejon, Kristina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cilio, Corrado M.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Paediatrics, University of Rome ‘La Sapienza’ Rome, Italy.
    Bergqvist, Ingela
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Matsunaga, Takeshi
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Induction of diabetes in NOD‹–›C57BL/6 embryo aggregation chimeras by cyclophosphamide through preferential depletion of C57BL/6 lymphocytes1996Ingår i: Journal of Autoimmunity, ISSN 0896-8411, E-ISSN 1095-9157, Vol. 9, nr 4, s. 493-499Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The majority of embryo aggregation (EA) mouse chimeras between non-obese diabetic (NOD) mice and C57BL/6 (B6) mice show clear signs of insulitis frequently accompanied by beta-cell destruction. Less than 5% of these chimeras, however, spontaneously progress to autoimmune diabetes, an incidence far lower than observed in NOD mice. The resistance in chimeras can be accounted for by the target organ chimerism and/or the immune system chimerism. To investigate the mechanism(s) controlling diabetes resistance in these mice, we studied a total of 92 NOD<-->B6 EA chimeras that showed overt lymphoid chimerism and treated 34 chimeras with cyclophosphamide (CY), a compound known to precipitate an acute form of insulin-dependent diabetes mellitus (IDDM) in pre-diabetic NOD mice, by interfering with regulatory mechanisms. We found that CY-treated EA chimeras displayed an increase in the NOD:B6 lymphocyte ratio and 32% of them developed diabetes that could be adoptively transferred to irradiated NOD or NOD-rag-2-/- mice. These findings suggest that lymphocyte chimerism rather than beta-cell chimerism accounts for diabetes resistance in NOD<-->B6 EA chimeras and that the susceptibility to CY-induced diabetes may be related to the proportion of NOD versus B6 lymphoid cells.

  • 207.
    Comstedt, Pär
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Biology of Borrelia garinii Spirochetes2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Lyme borreliosis is a tick-transmitted infectious disease. The causative agents are spiral-shaped bacteria and the most common sign of infection is a skin rash at the site of the tick bite. If not treated with antibiotics, the bacteria can disseminate and cause a variety of different manifestations including arthritis, carditis or neurological problems. The disease is a zoonosis and the bacteria are maintained in nature by different vertebrate reservoir host animals. In Europe, three different Borrelia genospecies cause Lyme borreliosis: B. burgdorferi, B. afzelii and B. garinii. The latter depends in part on birds as its reservoir host. B. garinii bacteria have been found in a marine enzootic infection cycle worldwide and also among terrestrial birds. This thesis suggests that passerine birds and seabirds constitute an important reservoir for B. garinii bacteria also with clinical importance. We have found bacteria very similar to Lyme borreliosis causing isolates in ticks infesting migrating passerine birds. The birds not only transport infected ticks, but are competent reservoir hosts, as measured by their ability to infect naïve ticks. Their role as a reservoir host is dependent on their foraging behavior, where ground-dwelling birds are of greater importance than other species. When comparing B. garinii isolates from Europe, the Arctic and North Pacific, and including isolates from seabirds, passerine birds, Ixodes ricinus ticks and Lyme borreliosis patients, we found that phylogenetic grouping was not necessarily dependent on geographical or biological origin. B. garinii from seabirds were very heterogeneous and found in all different groups. Therefore, the marine and the terrestrial infection cycles are likely to overlap. This was supported by the fact that B. garinii isolated from seabirds can establish a long-term infection in mice. Bacteria from the genospecies B. garinii are overrepresented among neuroborreliosis patients. Interestingly, many clinical B. garinii isolates are sensitive to human serum and have shown weak binding to the complement inhibitor protein factor H. By transforming a serum-sensitive B. garinii isolate with a shuttle vector containing the gene for the factor H binding protein OspE from complement-resistant B. burgdorferi, serum resistance could be increased. In addition, neurovirulent B. garinii strains recently isolated from neuroborreliosis patients were shown to express a factor H binding protein, not found in bacteria that had been kept in culture for a long time. This protein may contribute to the virulence of neuroborreliosis-causing B. garinii strains. When testing B. garinii isolates from Lyme borreliosis patients and seabirds for resistance to human serum, all members of the latter group were sensitive to even low levels of serum. This suggests that seabird isolates are not capable of infecting humans. In agreement with this, B. garinii isolated from seabirds do not appear to bind human factor H.

  • 208.
    Comstedt, Pär
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Asokliene, Loreta
    Eliasson, Ingvar
    Olsen, Björn
    Wallensten, Anders
    Bunikis, Jonas
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Complex population structure of Lyme borreliosis group spirochete Borrelia garinii in subarctic Eurasia.2009Ingår i: PloS one, ISSN 1932-6203, Vol. 4, nr 6, s. e5841-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Borrelia garinii, a causative agent of Lyme borreliosis in Europe and Asia, is naturally maintained in marine and terrestrial enzootic cycles, which primarily involve birds, including seabirds and migratory passerines. These bird groups associate with, correspondingly, Ixodes uriae and Ixodes ricinus ticks, of which the latter species may bite and transmit the infection to humans. Studies of the overlap between these two natural cycles of B. garinii have been limited, in part due to the absence of representative collections of this spirochete's samples, as well as of the lack of reliable measure of the genetic heterogeneity of its strains. As a prerequisite for understanding the epidemiological correlates of the complex maintenance of B. garinii, the present study sought to assess the diversity and phylogenetic relationships of this species' strains from its natural hosts and patients with Lyme borreliosis from subarctic Eurasia. We used sequence typing of the partial rrs-rrl intergenic spacer (IGS) of archived and prospective samples of B. garinii from I. uriae ticks collected predominantly on Commander Islands in North Pacific, as well as on the islands in northern Sweden and arctic Norway. We also typed B. garinii samples from patients with Lyme borreliosis and I. ricinus ticks infesting migratory birds in southern Sweden, or found questing in selected sites on the islands in the Baltic Sea and Lithuania. Fifty-two (68%) of 77 B. garinii samples representing wide geographical range and associated with I. ricinus and infection of humans contributed 12 (60%) of total 20 identified IGS variants. In contrast, the remaining 25 (32%) samples recovered from I. uriae ticks from a few islands accounted for as many as 10 (50%) IGS types, suggesting greater local diversity of B. garinii maintained by seabirds and their ticks. Two IGS variants of the spirochete in common for both tick species were found in I. ricinus larvae from migratory birds, an indication that B. garinii strains are exchanged between different ecological niches. Notably, B. garinii variants associated with I. uriae ticks were found in each of the six clusters, representing two phylogenetic lineages of this species identified among the studied samples. Our findings suggest that B. garinii in subarctic Eurasia comprises two partially overlapping populations with different levels of genetic heterogeneity, presumably, due to distinctive selective pressures on the spirochete in its marine and terrestrial enzootic cycles.

  • 209.
    Comstedt, Pär
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Bergström, Sven
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Olsen, Björn
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Infektionssjukdomar.
    Garpmo, Ulf
    Marjavaara, Lisette
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Mejlon, Hans
    Barbour, Alan G
    Bunikis, Jonas
    Migratory passerine birds as reservoirs of Lyme borreliosis in Europe.2006Ingår i: Emerging Infectious Diseases, ISSN 1080-6040, Vol. 12, nr 7, s. 1087-95Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To define the role of birds as reservoirs and disseminators of Borrelia spirochetes, we characterized tick infestation and reservoir competence of migratory passerine birds in Sweden. A total of 1,120 immature Ixodes ricinus ticks were removed from 13,260 birds and assayed by quantitative polymerase chain reaction (PCR) for Borrelia, followed by DNA sequencing for species and genotype identification. Distributions of ticks on birds were aggregated, presumably because of varying encounters with ticks along migratory routes. Lyme borreliosis spirochetes were detected in 160 (1.4%) ticks. Borrelia garinii was the most common species in PCR-positive samples and included genotypes associated with human infections. Infestation prevalence with infected ticks was 5 times greater among ground-foraging birds than other bird species, but the 2 groups were equally competent in transmitting Borrelia. Migratory passerine birds host epidemiologically important vector ticks and Borrelia species and vary in effectiveness as reservoirs on the basis of their feeding behavior.

  • 210.
    Comstedt, Pär
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Larsson, Christer
    Meri, Taru
    Meri, Seppo
    Bergström, Sven
    Borrelia garinii isolated from seabirds can infect rodents but is sensitive to normal human serumManuskript (Övrigt vetenskapligt)
  • 211. Coppi, Alida
    et al.
    Natarajan, Ramya
    Pradel, Gabriele
    Bennett, Brandy L.
    James, Eric R.
    Roggero, Mario A.
    Corradin, Giampietro
    Persson, Cathrine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Tewari, Rita
    Sinnis, Photini
    The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host2011Ingår i: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 208, nr 2, s. 341-356Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Plasmodium sporozoites make a remarkable journey from the mosquito midgut to the mammalian liver. The sporozoite's major surface protein, circumsporozoite protein (CSP), is a multifunctional protein required for sporozoite development and likely mediates several steps of this journey. In this study, we show that CSP has two conformational states, an adhesive conformation in which the C-terminal cell-adhesive domain is exposed and a nonadhesive conformation in which the N terminus masks this domain. We demonstrate that the cell-adhesive domain functions in sporozoite development and hepatocyte invasion. Between these two events, the sporozoite must travel from the mosquito midgut to the mammalian liver, and N-terminal masking of the cell-adhesive domain maintains the sporozoite in a migratory state. In the mammalian host, proteolytic cleavage of CSP regulates the switch to an adhesive conformation, and the highly conserved region I plays a critical role in this process. If the CSP domain architecture is altered such that the cell-adhesive domain is constitutively exposed, the majority of sporozoites do not reach their target organs, and in the mammalian host, they initiate a blood stage infection directly from the inoculation site. These data provide structure-function information relevant to malaria vaccine development.

  • 212.
    Costa, Tiago
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Amer, Ayad
    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). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    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, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Fahlgren, Anna
    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).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Type III secretion translocon assemblies that attenuate Yersinia virulence2013Ingår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, nr 7, s. 1088-1110Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 213.
    Costa, Tiago R. D.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Amer, Ayad A. A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Coiled-coils in the YopD translocator family: A predicted structure unique to the YopD N-terminus contributes to full virulence of Yersinia pseudotuberculosis2012Ingår i: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 12, nr 8, s. 1729-1742Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pathogenic Yersinia all harbor a virulence plasmid-encoded Ysc–Yop T3SS. In this system, translocator function is performed by the hydrophobic proteins YopB and YopD. With the goal to better understand how YopD orchestrates yop-regulatory control, translocon pore formation and Yop effector translocation, we performed an in silico prediction of coiled-coil motifs in YopD and YopD-like sequences from other bacteria. Of interest was a predicted N-terminal coiled-coil that occurred solely in Yersinia YopD sequences. To investigate if this unique feature was biologically relevant, two in cis point mutations were generated with a view to disrupting this putative structure. Both mutants maintained full T3SS function in vitro in terms of environmental control of Yops synthesis and secretion, effector toxin translocation and evasion of phagocytosis and killing by cultured immune cells. However, these same mutants were attenuated for virulence in a murine oral-infection model. The cause of this tardy disease progression is unclear. However, these data indicate that any structural flaw in this element unique to the N-terminus will subtly compromise an aspect of YopD biology. Sub-optimal T3SSs are then formed that are unable to fortify Yersinia against attack by the host innate and adaptive immune response.

  • 214. Crawford, Aaron C.
    et al.
    Lehtovirta-Morley, Laura E.
    Alamir, Omran
    Niemiec, Maria Joanna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Research Group Microbial Immunology, Leibniz Institute for Natural Product Research and Infection Biology–Hans Knoell Institute, Jena, Germany; Center for Sepsis Control and Care (CSCC), University Hospital, Jena, Germany.
    Alawfi, Bader
    Alsarraf, Mohammad
    Skrahina, Volha
    Costa, Anna C. B. P.
    Anderson, Andrew
    Yellagunda, Sujan
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ballou, Elizabeth R.
    Hube, Bernhard
    Urban, Constantin F.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Wilson, Duncan
    Biphasic zinc compartmentalisation in a human fungal pathogen2018Ingår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 14, nr 5, artikel-id e1007013Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2 Delta growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrtl/Zrt2-cellular import, followed by Zrcl-dependent intracellular compartmentalisation.

  • 215. Crowe-McAuliffe, Caillan
    et al.
    Graf, Michael
    Huter, Paul
    Takada, Hiraku
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Abdelshahid, Maha
    Novácek, Jirí
    Murina, Victoriia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Atkinson, Gemma C.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Institute of Technology, University of Tartu, 50411 Tartu, Estonia.
    Wilson, Daniel N.
    Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR2018Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, nr 36, s. 8978-8983Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ(2) mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).

  • 216.
    Croxatto, Antony
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Weber, Barbara
    Chen, Chang
    Milton, Debra L
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Post-transcriptional regulation of the Vibrio anguillarum quorum-sensing regulator vanT by RpoSManuskript (Övrigt vetenskapligt)
  • 217. Cuenca, Miguelangel
    et al.
    Pfister, Simona P.
    Buschor, Stefanie
    Bayramova, Firuza
    Hernandez, Sara B.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kuru, Erkin
    Van Nieuwenhze, Michael S.
    Brun, Yves V.
    Coelho, Fernanda M.
    Hapfelmeier, Siegfried
    D-Alanine-Controlled Transient Intestinal Mono-Colonization with Non-Laboratory-Adapted Commensal E. coli Strain HS2016Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, nr 3, artikel-id e0151872Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an already microbially colonized individual again-germ-free. We previously developed the in vivo growth-incompetent E. coli K-12 strain HA107 that is auxotrophic for the peptidoglycan components D-alanine (D-Ala) and meso-diaminopimelic acid (Dap) and can be used to transiently associate germ-free animals with live bacteria, without permanent loss of germ-free status. Here we describe the translation of this experimental model from the laboratory-adapted E. coli K-12 prototype to the better gut-adapted commensal strain E. coli HS. In this genetic background it was necessary to complete the D-Ala auxotrophy phenotype by additional knockout of the hypothetical third alanine racemase metC. Cells of the resulting fully auxotrophic strain assembled a peptidoglycan cell wall of normal composition, as long as provided with D-Ala and Dap in the medium, but could not proliferate a single time after D-Ala/Dap removal. Yet, unsupplemented bacteria remained active and were able to complete their cell cycle with fully sustained motility until immediately before autolytic death. Also in vivo, the transiently colonizing bacteria retained their ability to stimulate a live-bacteria-specific intestinal Immunoglobulin (Ig) A response. Full D-Ala auxotrophy enabled rapid recovery to again-germ-free status. E. coli HS has emerged from human studies and genomic analyses as a paradigm of benign intestinal commensal E. coli strains. Its reversibly colonizing derivative may provide a versatile research tool for mucosal bacterial conditioning or compound delivery without permanent colonization.

  • 218. Davidson, Iain F.
    et al.
    Goetz, Daniela
    Zaczek, Maciej P.
    Molodtsov, Maxim I.
    in't Veld, Pim J. Huis
    Weissmann, Florian
    Litos, Gabriele
    Cisneros, David A.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Research Institute of Molecular Pathology (IMP), Vienna, Austria.
    Ocampo-Hafalla, Maria
    Ladurner, Rene
    Uhlmann, Frank
    Vaziri, Alipasha
    Peters, Jan-Michael
    Rapid movement and transcriptional re-localization of human cohesin on DNA2016Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 35, nr 24, s. 2671-2685Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA. It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.

  • 219.
    de Pedro, Miguel A.
    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).
    Cava, Felipe
    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).
    Structural constraints and dynamics of bacterial cell wall architecture2015Ingår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, artikel-id 449Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The peptidoglycan wall (PG) is a unique structure which confers physical strength and defined shape to bacteria. It consists of a net-like macromolecule of peptide interlinked glycan chains overlying the cell membrane. The structure and layout of the PG dictates that the wall has to be continuously modified as bacteria go through division, morphological differentiation, and adaptive responses. The PG is poorly known in structural terms. However, to understand morphogenesis a precise knowledge of glycan strand arrangement and of local effects of the different kinds of subunits is essential. The scarcity of data led to a conception of the PG as a regular, highly ordered structure which strongly influenced growth models. Here, we review the structure of the PG to define a more realistic conceptual framework. We discuss the consequences of the plasticity of murein architecture in morphogenesis and try to define a set of minimal structural constraints that must be fulfilled by any model to be compatible with present day information.

  • 220. Debruyne, L
    et al.
    Broman, T
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olsen, B
    Univ Uppsala Hosp, Dept Med Sci, Infect Dis Sect.
    On, SLW
    Vandamme, P
    Campylobacter volucris sp nov., isolated from black-headed gulls (Larus ridibundus)2010Ingår i: International Journal of Systematic and Evolutionary Microbiology, ISSN 1466-5026, E-ISSN 1466-5034, Vol. 60, nr 8, s. 1870-1875Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    During a study of the prevalence of Campylobacter jejuni in black-headed gulls (Larus ridibundus) in Sweden, three isolates, strains LMG 24379, LMG 24380(T) and LMG 24381, were initially identified as Campylobacter lari. Further characterization by both AFLP and whole-cell protein SDS-PAGE analyses revealed that they formed a distinct group in the genus Cam pylobacter. This unique position was confirmed by phenotypic characterization, 16S rRNA and hsp60 gene sequence analysis and DNA-DNA hybridizations. The combined data confirm that these isolates represent a novel species within the genus Campylobacter, for which the name Campylobacter volucris sp. nov. is proposed. The type strain is LMG 24380(T) (=CCUG 57498(T)).

  • 221. Debruyne, Lies
    et al.
    Broman, Tina
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olsen, Björn
    On, Stephen L W
    Vandamme, Peter
    Campylobacter subantarcticus sp nov., isolated from birds in the sub-Antarctic region2010Ingår i: International Journal of Systematic and Evolutionary Microbiology, ISSN 1466-5026, E-ISSN 1466-5034, Vol. 60, s. 815-819Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Six Gram-stain-negative, spiral-shaped, microaerobic isolates were obtained during a sampling from wild birds in the sub-Antarctic region. Based on initial observations, these isolates were classified as Campylobacter lari-like. The isolates were further characterized by whole-cell protein and amplified fragment length polymorphism (AFLP) analysis, which revealed that they were distinct from C. lari and all other known species of the genus Campylobacter. Here, we present comprehensive phylogenetic, genomic and phenotypic evidence that these isolates represent a novel species within the genus Campylobacter, for which the name Campylobacter subantarcticus sp. nov. is proposed. The type strain is R-3023T (=LMG 24377T =CCUG 38513T).

  • 222.
    Deleuil, Fabienne
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Mogemark, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). 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).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Interaction between the Yersinia protein tyrosine phosphatase YopH and eukaryotic Cas/Fyb is an important virulence mechanism2003Ingår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 5, nr 1, s. 53-64Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The tyrosine phosphatase YopH is an essential virulence factor produced by pathogenic Yersinia species. YopH is translocated into host cells via a type III secretion system and its dephosphorylating activity causes disruption of focal complex structures and blockage of the phagocytic process. Among the host cell targets of YopH are the focal adhesion proteins Crk-associated substrate (p130Cas) and focal adhesion kinase (FAK) in epithelial cells, and p130Cas and Fyn-binding protein (Fyb) in macrophages. Previous studies have shown that the N-terminal domain of YopH acts as a substrate-binding domain. In this study, the mechanism and biological importance of the targeting of YopH to focal complexes relative to its interaction with p130Cas/Fyb was elucidated. Mutants of YopH that were defective in p130Cas/Fyb binding but otherwise indistinguishable from wild type were constructed. Mutants unable to bind p130Cas did not localize to focal complex structures in infected cells, indicating that the association with p130Cas is critical for appropriate subcellular localization of YopH. These yopH mutants were also clearly attenuated in virulence, showing that binding to p130Cas and/or Fyb is biologically relevant in Yersinia infections.

  • 223.
    Deltcheva, Elitza
    et al.
    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).
    Chylinski, Krzysztof
    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).
    Sharma, Cynthia M
    Gonzales, Karine
    Chao, Yanjie
    Pirzada, Zaid A
    Eckert, Maria R
    Vogel, Jörg
    Charpentier, Emmanuelle
    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).
    CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III2011Ingår i: Nature, Vol. 471, nr 7340, s. 602-607Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.

  • 224. Demuth, Andreas
    et al.
    Aharonowitz, Yair
    Bachmann, Till T
    Blum-Oehler, Gabriele
    Buchrieser, Carmen
    Covacci, Antonello
    Dobrindt, Ulrich
    Emödy, Levente
    van der Ende, Arie
    Ewbank, Jonathan
    Fernández, Luis Angel
    Frosch, Matthias
    Portillo, Francisco García-Del
    Gilmore, Michael S
    Glaser, Philippe
    Goebel, Werner
    Hasnain, Seyed E
    Heesemann, Jürgen
    Islam, Khalid
    Korhonen, Timo
    Maiden, Martin
    Meyer, Thomas F
    Montecucco, Cesare
    Oswald, Eric
    Parkhill, Julian
    Pucciarelli, M Graciela
    Ron, Eliora
    Svanborg, Catharina
    Uhlin, Bernt Eric
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Wai, Sun Nyunt
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Wehland, Jürgen
    Hacker, Jörg
    Pathogenomics: an updated European Research Agenda.2008Ingår i: Infect Genet Evol, ISSN 1567-1348, Vol. 8, nr 3, s. 386-93Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The emerging genomic technologies and bioinformatics provide novel opportunities for studying life-threatening human pathogens and to develop new applications for the improvement of human and animal health and the prevention, treatment, and diagnosis of infections. Based on the ecology and population biology of pathogens and related organisms and their connection to epidemiology, more accurate typing technologies and approaches will lead to better means of disease control. The analysis of the genome plasticity and gene pools of pathogenic bacteria including antigenic diversity and antigenic variation results in more effective vaccines and vaccine implementation programs. The study of newly identified and uncultivated microorganisms enables the identification of new threats. The scrutiny of the metabolism of the pathogen in the host allows the identification of new targets for anti-infectives and therapeutic approaches. The development of modulators of host responses and mediators of host damage will be facilitated by the research on interactions of microbes and hosts, including mechanisms of host damage, acute and chronic relationships as well as commensalisms. The study of multiple pathogenic and non-pathogenic microbes interacting in the host will improve the management of multiple infections and will allow probiotic and prebiotic interventions. Needless to iterate, the application of the results of improved prevention and treatment of infections into clinical tests will have a positive impact on the management of human and animal disease. The Pathogenomics Research Agenda draws on discussions with experts of the Network of Excellence "EuroPathoGenomics" at the management board meeting of the project held during 18-21 April 2007, in the Villa Vigoni, Menaggio, Italy. Based on a proposed European Research Agenda in the field of pathogenomics by the ERA-NET PathoGenoMics the meeting's participants updated the established list of topics as the research agenda for the future.

  • 225. Desmarais, Samantha M.
    et al.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    de Pedro, Miguel A.
    Huang, Kerwyn Casey
    Isolation and preparation of bacterial cell walls for compositional analysis by Ultra Performance Liquid Chromatography2014Ingår i: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, nr 83, s. UNSP e51183-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan's central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

  • 226.
    Desmarais, Samantha M
    et al.
    Department of Bioengineering, Stanford University, Stanford, CA, USA.
    De Pedro, Miguel A
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Huang, Kerwyn Casey
    Department of Bioengineering, Stanford University, Stanford, CA, USA ; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
    Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly2013Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 89, nr 1, s. 1-13Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell wall synthesis and cell growth. High-performance liquid chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques.

  • 227. Desmarais, Samantha M
    et al.
    Tropini, Carolina
    Miguel, Amanda
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Monds, Russell D
    de Pedro, Miguel A
    Huang, Kerwyn Casey
    High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography2015Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 52, s. 31090-31100Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial cell wall is a network of glycan strands crosslinked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.

  • 228. Dimova, Lidiya G.
    et al.
    Zlatkov, Nikola
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Verkade, Henkjan J.
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Tietge, Uwe J. F.
    High- cholesterol diet does not alter gut microbiota composition in mice2017Ingår i: Nutrition & Metabolism, ISSN 1743-7075, E-ISSN 1743-7075, Vol. 14, artikel-id 15Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Introduction: Western diet containing both saturated fat and cholesterol impairs cardio- metabolic health partly by modulating diversity and function of the microbiota. While diet containing only high fat has comparable effects, it is unclear how diets only enriched in cholesterol impact the microbiota. Therefore, we aimed to characterize the response of host and microbiota to a high cholesterol ( HC) diet in mice susceptible to cardio- metabolic disease. Methods: LDLR knockout mice received either 1.25% HC or no cholesterol containing control diet ( NC) for 12 weeks before characterizing host cholesterol metabolism and intestinal microbiota composition ( next generation sequencing). Results: HC diet substantially increased plasma ( 1.6- fold) and liver cholesterol levels ( 21- fold), biliary cholesterol secretion ( 4.5- fold) and fecal neutral sterol excretion ( 68- fold, each p < 0.001) but not fecal bile acid excretion. Interestingly, despite the profound changes in intestinal cholesterol homeostasis no differences in microbial composition between control and HC- fed mice were detected. In both groups the main phyla were Bacteroidetes ( 55%), Firmicutes ( 27%) and Verrucomicrobia ( 14%). Conclusion: Our results demonstrate that in mice HC diet alone does not alter the microbiota composition despite inducing substantial adaptive changes in whole body cholesterol homeostasis. The impact of Western diet on intestinal microbiota thus appears to be mediated exclusively by its high fat content.

  • 229.
    Dongre, Mitesh
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Singh, Bhupender
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Aung, Kyaw Min
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Larsson, Per
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Miftakhova, Regina R.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Persson, Karina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Askarian, Fatemeh
    Johannessen, Mona
    von Hofsten, Jonas
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Persson, Jenny L.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Erhardt, Marc
    Tuck, Simon
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Flagella-mediated secretion of a novel Vibrio cholerae cytotoxin affecting both vertebrate and invertebrate hosts2018Ingår i: Communications Biology, ISSN 2399-3642, Vol. 1, artikel-id 59Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Using Caenorhabditis elegans as an infection host model for Vibrio cholerae predator interactions, we discovered a bacterial cytotoxin, MakA, whose function as a virulence factor relies on secretion via the flagellum channel in a proton motive force-dependent manner. The MakA protein is expressed from the polycistronic makDCBA (motility-associated killing factor) operon. Bacteria expressing makDCBA induced dramatic changes in intestinal morphology leading to a defecation defect, starvation and death in C. elegans. The Mak proteins also promoted V. cholerae colonization of the zebrafish gut causing lethal infection. A structural model of purified MakA at 1.9 Å resolution indicated similarities to members of a superfamily of bacterial toxins with unknown biological roles. Our findings reveal an unrecognized role for V. cholerae flagella in cytotoxin export that may contribute both to environmental spread of the bacteria by promoting survival and proliferation in encounters with predators, and to pathophysiological effects during infections.

  • 230.
    Dongre, Mitesh
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Bacterial nanotubes for intimate sharing2011Ingår i: Frontiers in microbiology, ISSN 1664-302X, Vol. 2, s. 108-Artikel i tidskrift (Refereegranskat)
  • 231.
    Dongre, Mitesh
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    The Protease of Vibrio cholerae (PrtV)2013Ingår i: Handbook of proteolytic enzymes: volume 1 / [ed] Neil D. Rawlings, Guy Salvesen, Elsevier, 2013, 3, Vol. 1, s. 1219-1225Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 850 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 273 is The Protease of Vibrio cholerae (PrtV). Keywords Auto-proteolysis, Caenorhabditis elegans, cytokine induction, fibrinogen, fibronectin, HapR. Vibrio cholerae cytolysin (VCC), M6-peptidase family, plasminogen, polycystic kidney disease (PKD) domains, Quorum Sensing (QS), zinc-dependent metalloproteases.

  • 232.
    Dorafshan Esfahani, Eshagh
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå University.
    Methyltransferase Ash1, histone methylation and their impact on Polycomb repression2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Antagonistic interactions between Polycomb Group (PcG) and Trithorax Group (TrxG) proteins orchestrate the expression of key developmental genes. Distinct maternally loaded repressors establish the silenced state of these genes in cells where they should not be expressed and later PcG proteins sense whether a target gene is inactive and maintain the repression throughout multiple cell divisions. PcG proteins are targeted to genes by DNA elements called Polycomb Response Elements (PREs). The proteins form two major classes of complexes, namely Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2). Mechanistic details of Polycomb repression are not fully understood, however, tri-methylation of Lysine 27 of histone H3 (H3K27me3) is essential for this process. Using Drosophila cell lines deficient for either PRC1 or PRC2, I investigated the role of H3K27 methylation and the interdependence of PRC1 complexes for their recruitment to PREs. My results indicate that recruitment of PcG complexes to PREs proceed via multiple pathways and that H3K27 methylation is not needed for their targeting. However, the methylation is required to stabilize interactions of PRE-anchored PcG complexes with surrounding chromatin.

    TrxG proteins prevent erroneous repression of Polycomb target genes where these genes need to be expressed. Ash1 is a TrxG protein which binds Polycomb target genes when they are transcriptionally active. It contains a SET domain which methylates Lysine 36 of histone H3 (H3K36). In vitro, histone H3 methylated at K36 is a poor substrate for H3K27 methylation by PRC2. This prompted a model where Ash1 counteracts Polycomb repression through H3K36 methylation. However, this model was never tested in vivo and does not consider several experimental observations. First, in the ash1 mutant flies the bulk H3K36me2/H3K36me3 levels remain unchanged. Second, in Drosophila, there are two other H3K36-specific histone methyltransferases, NSD and Set2, which should be capable to inhibit PRC2. Third, Ash1 contains multiple evolutionary conserved domains whose roles have not been investigated. Therefore, I asked whether H3K36 methylation is critical for Ash1 to counteract Polycomb repression in vivo and whether NSD and Set2 proteins contribute to this process. I used flies lacking endogenous histone genes and complemented them with transgenic histone genes where Lysine 36 is replaced by Arginine. In these animals, I assayed erroneous repression of HOX genes as a readout for erroneous Polycomb repression. I used the same readout in the NSD or Set2 mutant flies. I also asked if other conserved domains of Ash1 are essential for its function. In addition to SET and domain, Ash1 contains three AT hook motifs as well as BAH and PHD domains. I genetically complemented ash1 loss of function animals with transgenic Ash1 variants, in each, one domain of Ash1 is deleted. I found that Ash1 is the only H3K36-specific histone methyltransferase which counteracts Polycomb repression in Drosophila. My findings suggest that the model, where Ash1 counteracts PcG repression by inhibiting PRC2 via methylation of H3K36, has to be revised. I also showed that, in vivo, Ash1 acts as a multimer and requires SET, BAH and PHD domains to counteract Polycomb repression.

    This work led to two main conclusions. First, trimethylation of H3K27 is not essential for targeting PcG proteins to PREs but acts afterwards to stabilize their interaction with the chromatin of the neighboring genes. Second, while SET domain is essential for Ash1 to oppose Polycomb repression, methylation of H3K36 does not play a central role in the process.

    Publikationen är tillgänglig i fulltext från 2019-12-18 00:01
  • 233.
    Dorafshan, Eshagh
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kahn, Tatyana G.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Glotov, Alexander
    Savitsky, Mikhail
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Schwartz, Yuri B.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Functional dissection of Drosophila Ash1 domainsManuskript (preprint) (Övrigt vetenskapligt)
  • 234.
    Dorafshan, Eshagh
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kahn, Tatyana G.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Glotov, Alexander
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Savitsky, Mikhail
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Walther, Matthias
    Reuter, Gunter
    Schwartz, Yuri B.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ash1 counteracts Polycomb repression independent of histone H3 lysine 36 methylation2019Ingår i: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 20, nr 4, artikel-id e46762Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Polycomb repression is critical for metazoan development. Equally important but less studied is the Trithorax system, which safeguards Polycomb target genes from the repression in cells where they have to remain active. It was proposed that the Trithorax system acts via methylation of histone H3 at lysine 4 and lysine 36 (H3K36), thereby inhibiting histone methyltransferase activity of the Polycomb complexes. Here we test this hypothesis by asking whether the Trithorax group protein Ash1 requires H3K36 methylation to counteract Polycomb repression. We show that Ash1 is the only Drosophila H3K36-specific methyltransferase necessary to prevent excessive Polycomb repression of homeotic genes. Unexpectedly, our experiments reveal no correlation between the extent of H3K36 methylation and the resistance to Polycomb repression. Furthermore, we find that complete substitution of the zygotic histone H3 with a variant in which lysine 36 is replaced by arginine does not cause excessive repression of homeotic genes. Our results suggest that the model, where the Trithorax group proteins methylate histone H3 to inhibit the histone methyltransferase activity of the Polycomb complexes, needs revision.

  • 235.
    Dorafshan, Eshagh
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kahn, Tatyana G.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Glotov, Alexander
    Savitsky, Mikhail
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Walther, Matthias
    Reuter, Gunter
    Schwartz, Yuri B.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Does Ash1 counteract Polycomb repression by methylating H3K36?Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Polycomb repression is critical to maintain cell type specific genome expression programs in a wide range of multicellular animals. Equally important but less studied is the Trithorax group system, which safeguards Polycomb target genes from the repression in cells where they have to remain active. Based on in vitro studies it was proposed that the Trithorax group system acts via methylation of histone H3 at Lysine 4 (H3K4) and Lysine 36 (H3K36) thereby inhibiting histone methyltransferase activity of the Polycomb complexes. This hypothesis is yet to be comprehensively tested in vivo. Here we used the power of the Drosophila model to investigate how the Trithorax group protein Ash1 and the H3K36 methylation counteract Polycomb repression. We show, for the first time, that Ash1 is the only Drosophila H3K36-specific methyltransferase required to prevent excessive Polycomb repression of homeotic genes. Unexpectedly, our experiments revealed no correlation between the extent of H3K36 methylation and the resistance to Polycomb repression. Furthermore, we find that complete substitution of the zygotic histone H3 with a variant in which Lysine 36 is replaced by Arginine does not cause excessive repression of Drosophila homeotic genes. Together with earlier studies, our results suggest that the model, where the Trithorax group proteins methylate histone H3 to inhibit the histone methyltransferase activity of the Polycomb complexes, may need to be reevaluated.

  • 236.
    Dorafshan, Eshagh
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kahn, Tatyana G.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Schwartz, Yuri B.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hierarchical recruitment of Polycomb complexes revisited2017Ingår i: Nucleus, ISSN 1949-1034, E-ISSN 1949-1042, Vol. 8, nr 5, s. 496-505Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Polycomb Group (PcG) proteins epigenetically repress key developmental genes and thereby control alternative cell fates. PcG proteins act as complexes that can modify histones and these histone modifications play a role in transmitting the memory of the repressed state as cells divide. Here we consider mainstream models that link histone modifications to hierarchical recruitment of PcG complexes and compare them to results of a direct test of interdependence between PcG complexes for recruitment to Drosophila genes. The direct test indicates that PcG complexes do not rely on histone modifications to recognize their target genes but use them to stabilize the interactions within large chromatin domains. It also shows that multiple strategies are used to coordinate the targeting of PcG complexes to different genes, which may make the repression of these genes more or less robust.

  • 237. Doublet, Vincent
    et al.
    Poeschl, Yvonne
    Gogol-Doering, Andreas
    Alaux, Cedric
    Annoscia, Desiderato
    Aurori, Christian
    Barribeau, Seth M.
    Bedoya-Reina, Oscar C.
    Brown, Mark J. F.
    Bull, James C.
    Flenniken, Michelle L.
    Galbraith, David A.
    Genersch, Elke
    Gisder, Sebastian
    Grosse, Ivo
    Holt, Holly L.
    Hultmark, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lattorff, H. Michael G.
    Le Conte, Yves
    Manfredini, Fabio
    McMahon, Dino P.
    Moritz, Robin F. A.
    Nazzi, Francesco
    Nino, Elina L.
    Nowick, Katja
    van Rij, Ronald P.
    Paxton, Robert J.
    Grozinger, Christina M.
    Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens2017Ingår i: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 18, artikel-id 207Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Organisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses.

    Results: We identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non-differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses.

    Conclusions: Our meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions.

  • 238.
    Doudna, Jennifer A.
    et al.
    Univ Calif Berkeley, Berkeley, CA 94720 USA.
    Charpentier, Emmanuelle
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Helmholtz Ctr Infect Res, Dept Regulat Infect Biol, D-38124 Braunschweig, Germany; Hannover Med Sch, D-30625 Hannover, Germany.
    The new frontier of genome engineering with CRISPR-Cas92014Ingår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 346, nr 6213, s. 1077-+Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

  • 239. Dukuzumuremyi, Jean-Marie
    et al.
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hallberg, Bengt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Åkerström, Bo
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Schesser, Kurt
    The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor2000Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, nr 45, s. 35281-35290Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.

  • 240.
    Duperthuy, Marylise
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sjöström, Annika E.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sabharwal, Dharmesh
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Damghani, Fatemeh
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Role of the Vibrio cholerae Matrix Protein Bap1 in Cross-Resistance to Antimicrobial Peptides2013Ingår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 9, nr 10, s. e1003620-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Outer membrane vesicles (OMVs) that are released from Gram-negative pathogenic bacteria can serve as vehicles for the translocation of effectors involved in infectious processes. In this study we have investigated the role of OMVs of the Vibrio cholerae O1 El Tor A1552 strain in resistance to antimicrobial peptides (AMPs). To assess this potential role, we grew V. cholerae with sub-lethal concentrations of Polymyxin B (PmB) or the AMP LL-37 and analyzed the OMVs produced and their effects on AMP resistance. Our results show that growing V. cholerae in the presence of AMPs modifies the protein content of the OMVs. In the presence of PmB, bacteria release OMVs that are larger in size and contain a biofilm-associated extracellular matrix protein (Bap1). We demonstrated that Bap1 binds to the OmpT porin on the OMVs through the LDV domain of OmpT. In addition, OMVs from cultures incubated in presence of PmB also provide better protection for V. cholerae against LL-37 compared to OMVs from V. cholerae cultures grown without AMPs or in presence of LL-37. Using a bap1 mutant we showed that cross-resistance between PmB and LL-37 involved the Bap1 protein, whereby Bap1 on OMVs traps LL-37 with no subsequent degradation of the AMP.

  • 241.
    Duperthuy, Marylise
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Biofilm Recruitment of Vibrio cholera by Matrix Proteolysis2015Ingår i: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 23, nr 11, s. 667-668Artikel i tidskrift (Övrigt vetenskapligt)
  • 242.
    Dzhygyr, Ievgen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Functional studies of Escherichia coli stringent response factor RelA2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation ofEscherichia coli to nutritional stress as such as amino acid deficiency. It detects the deficiency of aminoacids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is chargedwith amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to producealarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is aglobal metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolicprocesses, such as DNA replication, gene expression, cell wall synthesis and translation. Thesechanges also include activation of different virulence factors and are proposed to drive formation of abacterial sub-population that is highly resilient to antibiotic treatment, the so-called persisters.

    For a long time the molecular mechanism of RelA’s activation by and interaction with the ribosomedeacylatedtRNA complex was unknown. Only recently several cryo-EM structures of RelA-ribosomecomplex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNAcomplex. Guided by these structures we investigated the role of RelA’s domains in this interaction byconstructing a set of RelA C-terminal truncates and subjecting these to biochemical and microbiologicalexperimentation. These experiments were complemented with mutations in ribosomal RNA atpositions that interact with RelA, namely A-site finger and sarcin-ricin loop.

    We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex,whereas, the set of truncated proteins missing either one, two or three C-terminal domains do notrespond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncatedversions can still be activated by vacant 70S ribosome as well as pppGpp, suggesting that N-terminaldomain of RelA has an allosteric regulation site for (p)ppGpp and is able to interact with the ribosome.The mechanism of this interaction is yet to be elucidated.

    We have shown that A-site finger of the ribosome is required for RelA activation and recruitment tothe ribosome. Using EMSA assays, we have shown that RelA and deacylated tRNA do not form a stablecomplex off the ribosome. His432 located in TGS domain of RelA is crucial for recognition of deacylatedtRNA and a mutation of this histidine to glycine abolishes RelA activation by deacylated tRNA.Since (p)ppGpp plays an important role in bacterial survival and pathogenicity we have also testedseveral strategies for RelA inhibition by antibiotics, which target ribosomes and the interactionbetween RelA and ribosome-deacylated tRNA complex. We have shown that antibiotic thiostreptoninhibits (p)ppGpp synthesis by preventing RelA-tRNA interaction on the ribosome. (p)ppGppproduction is also inhibited by chloramphenicol and tetracycline.

  • 243.
    Dzhygyr, Ievgen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Cut-back analysis reveals the role of RelA's individual domains in its activation by 'starved' ribosomal complexes and pppGpp alarmone nucleotideManuskript (preprint) (Övrigt vetenskapligt)
  • 244. Dörr, Tobias
    et al.
    Alvarez, Laura
    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).
    Delgado, Fernanda
    Davis, Brigid M.
    Cava, Felipe
    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).
    Waldor, Matthew K.
    A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance2016Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, nr 2, s. 404-409Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial cell wall is critical for maintenance of cell shape and survival. Following exposure to antibiotics that target enzymes required for cell wall synthesis, bacteria typically lyse. Although several cell envelope stress response systems have been well described, there is little knowledge of systems that modulate cell wall synthesis in response to cell wall damage, particularly in Gram-negative bacteria. Here we describe WigK/WigR, a histidine kinase/response regulator pair that enables Vibrio cholerae, the cholera pathogen, to survive exposure to antibiotics targeting cell wall synthesis in vitro and during infection. Unlike wild-type V. cholerae, mutants lacking wigR fail to recover following exposure to cell-wall-acting antibiotics, and they exhibit a drastically increased cell diameter in the absence of such antibiotics. Conversely, overexpression of wigR leads to cell slimming. Overexpression of activated WigR also results in increased expression of the full set of cell wall synthesis genes and to elevated cell wall content. WigKR-dependent expression of cell wall synthesis genes is induced by various cell-wall-acting antibiotics as well as by overexpression of an endogenous cell wall hydrolase. Thus, WigKR appears to monitor cell wall integrity and to enhance the capacity for increased cell wall production in response to damage. Taken together, these findings implicate WigKR as a regulator of cell wall synthesis that controls cell wall homeostasis in response to antibiotics and likely during normal growth as well.

  • 245.
    Dörr, Tobias
    et al.
    Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, USA ; Department of Microbiology and Immunobiology, Harvard Medical School and HHMI, Boston, MA, USA .
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). 3 Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain.
    Lam, Hubert
    Discovery Research, Sanofi Pasteur, Cambridge, MA, USA.
    Davis, Brigid M
    Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, USA ; Department of Microbiology and Immunobiology, Harvard Medical School and HHMI, Boston, MA, USA .
    Waldor, Matthew K
    Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, USA ; Department of Microbiology and Immunobiology, Harvard Medical School and HHMI, Boston, MA, USA .
    Substrate specificity of an elongation-specific peptidoglycan endopeptidase and its implications for cell wall architecture and growth of Vibrio cholerae2013Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 89, nr 5, s. 949-962Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial cell wall consists of peptidoglycan (PG), a sturdy mesh of glycan strands cross-linked by short peptides. This rigid structure constrains cell shape and size, yet is sufficiently dynamic to accommodate insertion of newly synthesized PG, which was long hypothesized, and recently demonstrated, to require cleavage of the covalent peptide cross-links that couple previously inserted material. Here, we identify several genes in Vibrio cholerae that collectively are required for growth - particularly elongation - of this pathogen. V. cholerae encodes three putative periplasmic proteins, here denoted ShyA, ShyB, and ShyC, that contain both PG binding and M23 family peptidase domains. While none is essential individually, the absence of both ShyA and ShyC results in synthetic lethality, while the absence of ShyA and ShyB causes a significant growth deficiency. ShyA is a D,d-endopeptidase able to cleave most peptide chain cross-links in V. cholerae's PG. PG from a ∆shyA mutant has decreased average chain length, suggesting that ShyA may promote removal of short PG strands. Unexpectedly, ShyA has little activity against muropeptides containing pentapeptides, which typically characterize newly synthesized material. ShyA's substrate-dependent activity may contribute to selection of cleavage sites in PG, whose implications for the process of side-wall growth are discussed.

  • 246. Dörr, Tobias
    et al.
    Lam, Hubert
    Alvarez, Laura
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Davis, Brigid M.
    Waldor, Matthew K.
    A novel peptidoglycan binding protein crucial for PBP1A-mediated cell wall biogenesis in Vibrio cholerae2014Ingår i: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, nr 6, s. e1004433-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial cell wall, which is comprised of a mesh of polysaccharide strands crosslinked via peptide bridges (peptidoglycan, PG), is critical for maintenance of cell shape and survival. PG assembly is mediated by a variety of Penicillin Binding Proteins (PBP) whose fundamental activities have been characterized in great detail; however, there is limited knowledge of the factors that modulate their activities in different environments or growth phases. In Vibrio cholerae, the cause of cholera, PG synthesis during the transition into stationary phase is primarily mediated by the bifunctional enzyme PBP1A. Here, we screened an ordered V. cholerae transposon library for mutants that are sensitive to growth inhibition by non-canonical D-amino acids (DAA), which prevent growth and maintenance of cell shape in PBP1A-deficient V. cholerae. In addition to PBP1A and its lipoprotein activator LpoA, we found that CsiV, a small periplasmic protein with no previously described function, is essential for growth in the presence of DAA. Deletion of csiV, like deletion of lpoA or the PBP1A-encoding gene mrcA, causes cells to lose their rod shape in the presence of DAA or the beta-lactam antibiotic cefsulodin, and all three mutations are synthetically lethal with deletion of mrcB, which encodes PBP1B, V. cholerae's second key bifunctional PBP. CsiV interacts with LpoA and PG but apparently not with PBP1A, supporting the hypothesis that CsiV promotes LpoA's role as an activator of PBP1A, and thereby modulates V. cholerae PG biogenesis. Finally, the requirement for CsiV in PBP1A-mediated growth of V. cholerae can be overcome either by augmenting PG synthesis or by reducing PG degradation, thereby highlighting the importance of balancing these two processes for bacterial survival.

  • 247. Dörr, Tobias
    et al.
    Möll, Andrea
    Chao, Michael C.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lam, Hubert
    Davis, Brigid M.
    Waldor, Matthew K.
    Differential Requirement for PBP1a and PBP1b in In Vivo and In Vitro Fitness of Vibrio cholerae2014Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 82, nr 5, s. 2115-2124Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We investigated the roles of the Vibrio cholerae high-molecular-weight bifunctional penicillin binding proteins, PBP1a and PBP1b, in the fitness of this enteric pathogen. Using a screen for synthetic lethality, we found that the V. cholerae PBP1a and PBP1b proteins, like their Escherichia coli homologues, are each essential in the absence of the other and in the absence of the other's putative activator, the outer membrane lipoproteins LpoA and LpoB, respectively. Comparative analyses of V. cholerae mutants suggest that PBP1a/LpoA of V. cholerae play a more prominent role in generating and/or maintaining the pathogen's cell wall than PBP1b/LpoB.V. cholerae lacking PBP1b or LpoB exhibited wild-type growth under all conditions tested. In contrast, V. cholerae lacking PBP1a or LpoA exhibited growth deficiencies in minimal medium, in the presence of deoxycholate and bile, and in competition assays with wild-type cells both in vitro and in the infant mouse small intestine. PBP1a pathway mutants are particularly impaired in stationary phase, which renders them sensitive to a product(s) present in supernatants from stationary-phase wild-type cells. The marked competitive defect of the PBP1a pathway mutants in vivo was largely absent when exponential-phase cells rather than stationary-phase cells were used to inoculate suckling mice. Thus, at least for V. cholerae PBP1a pathway mutants, the growth phase of the inoculum is a key modulator of infectivity.

  • 248.
    Edgren, Tomas
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Type III Secretion in Yersinia: Injectisome or Not?2012Ingår i: PLoS pathogens, ISSN 1553-7374, Vol. 8, nr 5, s. e1002669-Artikel i tidskrift (Refereegranskat)
  • 249.
    Edin, S
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Oruganti, SR
    Grundström, C
    Grundström, T
    Interaction of calmodulin with Bc110 modulates NF-κB activationManuskript (Övrigt vetenskapligt)
  • 250.
    Edin, Sofia
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Calmodulin mediated regulation of NF-kappaB in lymphocytes2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    NF-κB transcription factors are regulators of a wide spectrum of genes involved in immune responses and inflammation as well as cellular proliferation and survival. Transcriptionally competent NF-κB dimers are retained in the cytoplasm of resting cells by binding to inhibitors of NF-κB (IκBs). Stimuli that activate NF-κB converge on the activation of the IκB kinase (IKK), resulting in phosphorylation and subsequent proteasomal degradation of IκB. This releases functional NF-κB dimers that rapidly move to the nucleus where they regulate transcription of NF-κB-dependent target genes. The study of signalling to NF-κB from T and B lymphocyte antigen receptors is a field of intense investigation, and much attention is focused on the complex of the molecular scaffolding proteins Carma1, Bcl10 and MALT1. Together, these are crucial for the organisation of a structure beneath the activated receptor, termed the immunological synapse. IKK is recruited to this structure and becomes activated, subsequently leading to activation of NF-κB.

    Calcium (Ca2+) is a ubiquitous intracellular messenger that is involved in the regulation of numerous aspects of cellular function, including transcription. NF-κB activity is known to be regulated by changes in intracellular Ca2+ levels, such as those created by antigen receptor activation, but the mechanisms are to a large extent undefined. Ca2+ signals in cells are transmitted predominantly by the ubiquitous Ca2+ sensor protein calmodulin (CaM). Signalling that increases the intracellular Ca2+ concentration leads to binding of Ca2+ to CaM, which changes its structure, thereby allowing it to interact with a new range of target proteins.

    The studies of NF-κB signalling in lymphocytes presented here reveal that CaM is involved, both directly and indirectly, in the regulation of NF-κB. CaM was found to interact directly and in a Ca2+-dependent manner with the NF-κB proteins RelA and c-Rel after their signal-induced release from IκB. The interaction of CaM with c-Rel, but not RelA, was found to be inhibitory for its nuclear accumulation and transcriptional activity on Ca2+-regulated IL-2 and GM-CSF promoters; thus, CaM binding was found to differentially regulate c-Rel and RelA in lymphocytes. CaM was also shown to interact directly and in a Ca2+-dependent manner with Bcl10. The interaction was mapped to the Carma1-interacting CARD domain of Bcl10 and was found to have a negative effect on the ability of Bcl10 to bind to Carma1. Binding of CaM to Bcl10 also had a negative effect on activation of NF-κB after T cell receptor stimulation, since a point mutant of Bcl10 with reduced binding to CaM showed increased activation of an NF-κB reporter in Jurkat T cells, which was further enhanced by TCR-activating stimuli.

    In addition, CaM was found to positively regulate NF-κB activation indirectly through CaM-dependent kinase II (CaMKII). Inhibitors of CaM and CaMKII were shown to inhibit IκBα degradation in lymphocytes induced by phorbol ester or T cell receptor stimulation. The actions of CaMKII were mapped to a point upstream of IKK activation and further studies revealed that CaMKII is recruited to the immunological synapse, where it inducibly interacts with and phosphorylates Bcl10 at multiple sites. Phosphorylation of Bcl10 by CaMKII was shown to be important for the ability of Bcl10 to activate NF-κB, since mutation of the phosphorylation sites of Bcl10 inhibited Bcl10-induced transcriptional activity of NF-κB, in part by preventing signalinduced ubiquitination and degradation of Bcl10.

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