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  • 101.
    Chylinski, Krzysztof
    et al.
    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).
    Le Rhun, Anaïs
    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).
    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).
    The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems2013Inngår i: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 10, nr 5, s. 726-737Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    CRISPR-Cas is a rapidly evolving RNA-mediated adaptive immune system that protects bacteria and archaea against mobile genetic elements. The system relies on the activity of short mature CRISPR RNAs (crRNAs) that guide Cas protein(s) to silence invading nucleic acids. A set of CRISPR-Cas, type II, requires a trans-activating small RNA, tracrRNA, for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following co-processing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. Here, we screened available genomes for type II CRISPR-Cas loci by searching for Cas9 orthologs. We analyzed 75 representative loci, and for 56 of them we predicted novel tracrRNA orthologs. Our analysis demonstrates a high diversity in cas operon architecture and position of the tracrRNA gene within CRISPR-Cas loci. We observed a correlation between locus heterogeneity and Cas9 sequence diversity, resulting in the identification of various type II CRISPR-Cas subgroups. We validated the expression and co-processing of predicted tracrRNAs and pre-crRNAs by RNA sequencing in five bacterial species. This study reveals tracrRNA family as an atypical, small RNA family with no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. The tracrRNA family is however characterized by the conserved feature to base-pair to cognate pre-crRNA repeats, an essential function for crRNA maturation and DNA silencing by dual-RNA:Cas9. The large panel of tracrRNA and Cas9 ortholog sequences should constitute a useful database to improve the design of RNA-programmable Cas9 as genome editing tool.

  • 102.
    Chylinski, Krzysztof
    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). Max F. Perutz Laboratories, University of Vienna, Austria .
    Makarova, Kira S.
    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 Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany ; Hannover Medical School, Hannover, Germany .
    Koonin, Eugene V.
    Classification and evolution of type II CRISPR-Cas systems2014Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, nr 10, s. 6091-6105Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The CRISPR-Cas systems of archaeal and bacterial adaptive immunity are classified into three types that differ by the repertoires of CRISPR-associated (cas) genes, the organization of cas operons and the structure of repeats in the CRISPR arrays. The simplest among the CRISPR-Cas systems is type II in which the endonuclease activities required for the interference with foreign deoxyribonucleic acid (DNA) are concentrated in a single multidomain protein, Cas9, and are guided by a co-processed dual-tracrRNA: crRNA molecule. This compact enzymatic machinery and readily programmable site-specific DNA targeting make type II systems top candidates for a new generation of powerful tools for genomic engineering. Here we report an updated census of CRISPR-Cas systems in bacterial and archaeal genomes. Type II systems are the rarest, missing in archaea, and represented in similar to 5% of bacterial genomes, with an over-representation among pathogens and commensals. Phylogenomic analysis suggests that at least three cas genes, cas1, cas2 and cas4, and the CRISPR repeats of the type II-B system were acquired via recombination with a type I CRISPR-Cas locus. Distant homologs of Cas9 were identified among proteins encoded by diverse transposons, suggesting that type II CRISPR-Cas evolved via recombination of mobile nuclease genes with type I loci.

  • 103. 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 Defenses2018Inngår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, nr 2, artikkel-id e00559-18Artikkel i tidsskrift (Fagfellevurdert)
    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.

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  • 104.
    Corkery, Dale
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Castro-Gonzalez, Sergio
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Knyazeva, Anastasia
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Herzog, Laura K.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes2023Inngår i: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 24, nr 9, artikkel-id e56841Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Lysosomal membrane damage represents a threat to cell viability. As such, cells have evolved sophisticated mechanisms to maintain lysosomal integrity. Small membrane lesions are detected and repaired by the endosomal sorting complex required for transport (ESCRT) machinery while more extensively damaged lysosomes are cleared by a galectin-dependent selective macroautophagic pathway (lysophagy). In this study, we identify a novel role for the autophagosome-lysosome tethering factor, TECPR1, in lysosomal membrane repair. Lysosomal damage promotes TECPR1 recruitment to damaged membranes via its N-terminal dysferlin domain. This recruitment occurs upstream of galectin and precedes the induction of lysophagy. At the damaged membrane, TECPR1 forms an alternative E3-like conjugation complex with the ATG12-ATG5 conjugate to regulate ATG16L1-independent unconventional LC3 lipidation. Abolishment of LC3 lipidation via ATG16L1/TECPR1 double knockout impairs lysosomal recovery following damage.

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  • 105.
    Corkery, Dale
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Nadeem, Aftab
    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).
    Aung, Kyaw Min
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Hassan, Ahmed
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Liu, Tao
    Cervantes-Rivera, Ramón
    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).
    Lystad, Alf Håkon
    Wang, Hui
    Persson, Karina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Puhar, Andrea
    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).
    Simonsen, Anne
    Uhlin, Bernt Eric
    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).
    Wai, Sun Nyunt
    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).
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy2021Inngår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 134, nr 5, artikkel-id jcs252015Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Autophagy plays an essential role in the defense against manymicrobial pathogens as a regulator of both innate and adaptive immunity. Some pathogens have evolved sophisticated mechanisms that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, motility-associatedkilling factor A (MakA). pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 (herein referring to MAP1LC3B) lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex, required for LC3 lipidation at the membranous aggregate, resulted in an inhibition of both canonical autophagy and autophagy-related processes, including the unconventional secretion of interleukin-1β (IL-1β). These findings identify a novel mechanismof host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.

  • 106.
    Corkery, Dale P.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    ATG12–ATG5-TECPR1: an alternative E3-like complex utilized during the cellular response to lysosomal membrane damage2023Inngår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    ATG16L1 is an essential component of the Atg8-family protein conjugation machinery, providing membrane targeting for the ATG12–ATG5 conjugate. Recently, we identified an alternative E3-like complex that functions independently of ATG16L1. This complex utilizes the autophagosome-lysosome tethering factor TECPR1 for membrane targeting. TECPR1 is recruited to damaged lysosomal membranes via a direct interaction with sphingomyelin. At the damaged membrane, TECPR1 assembles into an E3-like complex with ATG12–ATG5 to regulate unconventional LC3 lipidation and promote efficient lysosomal repair.

    Fulltekst (pdf)
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  • 107.
    Corkery, Dale P.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Eating while intoxicated: characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy2023Inngår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 19, nr 6, s. 1885-1886Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Extracellular pathogens utilize secreted virulence factors to regulate host cell function. Recently we characterized the molecular mechanism behind host macroautophagy/autophagy regulation by the Vibrio cholerae toxin MakA. Cholesterol binding at the plasma membrane induces MakA endocytosis and pH-dependent pore assembly. Membrane perforation of late endosomal membranes induces cellular membrane repair pathways and V-ATPase-dependent unconventional LC3 lipidation on damaged membranes.

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  • 108.
    Corkery, Dale
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ursu, Andrei
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Lucas, Belén
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Grigalunas, Michael
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Kriegler, Simon
    Physical Chemistry I – Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany, Germany.
    Oliva, Rosario
    Physical Chemistry I – Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany, Germany; Department of Chemical Sciences, University of Naples Federico II, Via Cintia 4, Naples, Italy.
    Dec, Robert
    Physical Chemistry I – Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany, Germany.
    Koska, Sandra
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Pahl, Axel
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Sievers, Sonja
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Ziegler, Slava
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Winter, Roland
    Physical Chemistry I – Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany, Germany.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Waldmann, Herbert
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany; Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, Dortmund, Germany.
    Inducin triggers LC3-lipidation and ESCRT-mediated lysosomal membrane repair2023Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 24, nr 24, artikkel-id e202300579Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death.

    Fulltekst (pdf)
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  • 109.
    Corkery, Dale
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Dowaidar, Moataz
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)2021Inngår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 17, nr 1, s. 1-382Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

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  • 110.
    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 virulence2013Inngår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, nr 7, s. 1088-1110Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 111.
    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). Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College, London, UK.
    Francis, Monika K.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Region Västerbotten.
    Farag, Salah
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Edgren, Tomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Measurement of Yersinia translocon pore formation in erythrocytes2019Inngår i: Pathogenic Yersinia: methods and protocols / [ed] Viveka Vadyvaloo and Matthew B. Lawrenz, New York, NY, U.S.A.: Humana Press, 2019, s. 211-229Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    Many Gram-negative pathogens produce a type III secretion system capable of intoxicating eukaryotic cells with immune-modulating effector proteins. Fundamental to this injection process is the prior secretion of two translocator proteins destined for injectisome translocon pore assembly within the host cell plasma membrane. It is through this pore that effectors are believed to travel to gain access to the host cell interior. Yersinia species especially pathogenic to humans and animals assemble this translocon pore utilizing two hydrophobic translocator proteins-YopB and YopD. Although a full molecular understanding of the biogenesis, function and regulation of this translocon pore and subsequent effector delivery into host cells remains elusive, some of what we know about these processes can be attributed to studies of bacterial infections of erythrocytes. Herein we describe the methodology of erythrocyte infections by Yersinia, and how analysis of the resultant contact-dependent hemolysis can serve as a relative measurement of YopB- and YopD-dependent translocon pore formation.

  • 112.
    Costa, Tiago R. D.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    YopD translocator function in Yersinia pseudotuberculosis type III secretion2012Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Type III secretion systems (T3SS) are a common feature of Gram-negative bacteria, allowing them to inject anti-host effectors into the interior of infected eukaryotic cells. By this mechanism, these virulence factors help the bacteria to modulate eukaryotic cell function in its favor and subvert host innate immunity. This promotes a less hostile environment in which infecting bacteria can colonize and cause disease.

    In pathogenic Yersinia, a crucial protein in this process is YopD. YopD is a T3S substrate that, together with YopB, forms a translocon pore in the host cell membrane through which the Yop effectors may gain access to the target-cell cytosol. The assembly of the translocator pore in plasma membranes is considered a fundamental feature of all T3SSs. How the pore is formed, what determines the correct size and ultimately the stoichiometry between YopD YopB, is still unknown. Portions of YopD are also observed inside HeLa cells. Moreover, YopD functions together with its T3S chaperone, LcrH, to control Yops synthesis in the bacterial cytoplasm. The multifunctional YopD may influence all these processes by compartmentalizing activities into discrete modular domains along the protein length. Therefore, understanding how particular domains and/or residues within these regions coordinate multiple functions of the protein will provide a platform to improve our knowledge of the molecular mechanisms behind translocation through T3SSs.

    Comprehensive site-directed mutagenesis of the YopD C-terminal amphipathic α-helix domain, pinpointed hydrophobic residues as important for YopD function. Some YopD variants were defective in self-assembly and in the ability to interact with the needle tip protein, LcrV, which were required to facilitate bacterial T3S activity. A similar mutagenesis approach was used to understand the role of the two predicted coiled-coils located at the N-terminal and C-terminal region of YopD. The predicted N-terminal element that occurs solely in the Yersinia YopD translocator family is essential for optimal T3SS and full disease progression. The predicted YopD C-terminal coiled-coil shapes a functional translocon inserted into host cell membranes. This translocon was seen to be a dynamic structure facilitating at least two roles during effectors delivery into cells; one to guarantee translocon pore insertion into target cell membranes and the other to promote targeted activity of internalized effector toxins.

    In Yersinia expression of yop genes and secretion of the corresponding polypeptides is tightly regulated at a transcriptional and post-transcriptional level. If T3S chaperones of the translocator class are known to influence transcriptional output of T3SS genes in other bacteria, we show that in Yersinia the class II T3S chaperone LcrH has no such effect on the LcrF transcriptional activator activity. We also demonstrate that there are possibly additional yop-regulatory roles for the LcrH chaperone besides forming a stable complex with YopD to impose post-transcriptional silencing on Yops synthesis. This mechanism that relies upon an active T3SS, might act independently of both YopD and the regulatory element LcrQ.

    In conclusion, this work has sought to delineate the encrypted functions of the YopD translocator that contribute to Yersinia T3SS-dependent pathogenesis. Contributions of the YopD cognate chaperone LcrH in yop regulatory control are also presented.  

    Fulltekst (pdf)
    YopD translocator function in Yersinia pseudotuberculosis type III secretion
  • 113.
    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).
    Farag, Salah I.
    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, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Fahlgren, Anna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). 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 S.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Active type III translocon assemblies that attenuate Yersinia virulenceManuskript (preprint) (Annet vitenskapelig)
    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 a-helix motif at the C-terminus. Mutants YopDI262P and YopDK267P poorly localised 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 YopDA263P and YopDA270P 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. It is therefore probable that an active translocon makes a range of contributions during bacteria-host cell contact that extends beyond effector delivery per se.

  • 114.
    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 pseudotuberculosis2012Inngår i: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 12, nr 8, s. 1729-1742Artikkel i tidsskrift (Fagfellevurdert)
    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.

    Fulltekst (pdf)
    fulltext
  • 115.
    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).
    Carlsson, Katrin E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Edqvist, Petra J.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew S.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Influence of the LcrH chaperone on type III secretion system regulation in Yersinia pseudotuberculosisManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Human pathogenic Yersiniae share a common virulence plasmid that encodes for the Ysc-Yop type III secretion system (T3SS). Control of yop expression involves several pathways in which their cross-talk is not completely understood. LcrF, an AraC-like transcriptional activator, is required for temperature-dependent yop-transcription. In contrast, a repressive effect of the T3S chaperone LcrH and the cognate translocator substrate YopD occurs through binding to yop mRNA and inhibiting translation; a process that is also thought to involve LcrQ. Several homologous members of the LcrH family of translocator-class of T3S chaperones can act as a cofactor to amplify the activity of transcriptional activators analogous to LcrF. However, we show here in Y. pseudotuberculosis that LcrH does not induce LcrF-dependent transcription of target genes. Moreover, a full length DlcrH null mutant in which YopB and YopD are rapidly degraded is totally de-repressed for Yop synthesis even though the anti-activator LcrQ is forced to accumulate in the cytoplasm through rendering the Ysc-Yop T3SS non-functional or ectopically producing LcrQ in trans. Typically, this mutant cannot grow at 37°C. Thus, in all respects, the DlcrH null mutant mirrors the regulatory defects established for Yersinia lacking the translocator and anti-activator YopD. On the other hand, Y. pseudotuberculosis producing the LcrHE30G point mutant that is defective for YscY chaperone binding exhibits a mild regulatory defect that permits some growth at 37°C, but is blind to the cytoplasmic accumulation of LcrQ. Critically however, this mutant still responds to repression caused by YopD accumulation, which is stably produced and efficiently secreted by this strain. Thus, our work with LcrHE30G indicates an additional regulatory function of this versatile T3S chaperone that is independent of the LcrF transcription factor and the YopD anti-activator.

     

  • 116.
    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).
    Edqvist, Petra J
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette E
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Åhlund, Monika K
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew S
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis2010Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, nr 33, s. 25269-25284Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.

  • 117. 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 pathogen2018Inngår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 14, nr 5, artikkel-id e1007013Artikkel i tidsskrift (Fagfellevurdert)
    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.

    Fulltekst (pdf)
    fulltext
  • 118.
    Crowe-McAuliffe, Caillan
    et al.
    Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, Hamburg, Germany.
    Murina, Victoriia
    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).
    Turnbull, Kathryn Jane
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark.
    Huch, Susanne
    SciLifeLab, Department of Microbiology, Tumor and Cell Biology. Karolinska Institutet, Solna, Sweden.
    Kasari, Marje
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). University of Tartu, Institute of Technology, Tartu, Estonia.
    Takada, Hiraku
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo, Motoyama, Kita-ku, Kyoto, Japan.
    Nersisyan, Lilit
    SciLifeLab, Department of Microbiology, Tumor and Cell Biology. Karolinska Institutet, Solna, Sweden.
    Sundsfjord, Arnfinn
    Department of Microbiology and Infection Control, Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, University Hospital of North Norway, Tromsø, Norway; Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø, Norway.
    Hegstad, Kristin
    Department of Microbiology and Infection Control, Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, University Hospital of North Norway, Tromsø, Norway; Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø, Norway.
    Atkinson, Gemma C.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Pelechano, Vicent
    SciLifeLab, Department of Microbiology, Tumor and Cell Biology. Karolinska Institutet, Solna, Sweden.
    Wilson, Daniel N.
    Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, Hamburg, Germany.
    Hauryliuk, Vasili
    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). University of Tartu, Institute of Technology, Tartu, Estonia; Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Structural basis for PoxtA-mediated resistance to phenicol and oxazolidinone antibiotics2022Inngår i: Nature Communications, E-ISSN 2041-1723, Vol. 13, nr 1, artikkel-id 1860Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF). They confer resistance to oxazolidinone and phenicol antibiotics, such as linezolid and chloramphenicol, which stall translating ribosomes when certain amino acids are present at a defined position in the nascent polypeptide chain. These proteins are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria, and are thought to confer resistance by binding to the ribosome and dislodging the bound antibiotic. However, the mechanistic basis of this resistance remains unclear. Here we refine the PoxtA spectrum of action, demonstrate alleviation of linezolid-induced context-dependent translational stalling, and present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome. PoxtA perturbs the CCA-end of the P-site tRNA, causing it to shift by ∼4 Å out of the ribosome, corresponding to a register shift of approximately one amino acid for an attached nascent polypeptide chain. We postulate that the perturbation of the P-site tRNA by PoxtA thereby alters the conformation of the attached nascent chain to disrupt the drug binding site.

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  • 119.
    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 (Annet vitenskapelig)
  • 120.
    Curtis, Michael W.
    et al.
    Department of Microbiology and Immunology, Medical College of Wisconsin, WI, Milwaukee, United States.
    Fierros, Christa H.
    Department of Microbiology and Immunology, Medical College of Wisconsin, WI, Milwaukee, United States.
    Hahn, Beth L.
    Department of Medicine, Division of Infectious Diseases, Medical College of Wisconsin, WI, Milwaukee, United States.
    Surdel, Matthew C.
    Department of Medicine, Division of Infectious Diseases, Medical College of Wisconsin, WI, Milwaukee, United States.
    Kessler, Julie
    Department of Medicine, Division of Infectious Diseases, Medical College of Wisconsin, WI, Milwaukee, United States.
    Anderson, Phillip N.
    Department of Medicine, Division of Infectious Diseases, Medical College of Wisconsin, WI, Milwaukee, United States.
    Vandewalle-Capo, Marine
    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).
    Bonde, Mari
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Zhu, Jieqing
    Blood Research Institute, WI, Milwaukee, United States; Department of Biochemistry, Medical College of Wisconsin, WI, Milwaukee, United States.
    Bergström, Sven
    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).
    Coburn, Jenifer
    Department of Microbiology and Immunology, Medical College of Wisconsin, WI, Milwaukee, United States; Department of Medicine, Division of Infectious Diseases, Medical College of Wisconsin, WI, Milwaukee, United States.
    Identification of amino acid domains of Borrelia burgdorferi P66 that are surface exposed and important for localization, oligomerization, and porin function of the protein2022Inngår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 12, artikkel-id 991689Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    P66, a bifunctional integral outer membrane protein, is necessary for Borrelia burgdorferi to establish initial infection and to disseminate in mice. The integrin binding function of P66 facilitates extravasation and dissemination, but the role of its porin function during murine infection has not been investigated. A limitation to studying P66 porin function during mammalian infection has been the lack of structural information for P66. In this study, we experimentally characterized specific domains of P66 with regard to structure and function. First, we aligned the amino acid sequences of P66 from Lyme disease-causing Borrelia and relapsing fever-causing Borrelia to identify conserved and unique domains between these disease-causing clades. Then, we examined whether specific domains of P66 are exposed on the surface of the bacteria by introducing c-Myc epitope tags into each domain of interest. The c-Myc epitope tag inserted C-terminally to E33 (highly conserved domain), to T187 (integrin binding region domain and a non-conserved domain), and to E334 (non-conserved domain) were all detected on the surface of Borrelia burgdorferi. The c-Myc epitope tag inserted C-terminally to E33 and D303 in conserved domains disrupted P66 oligomerization and porin function. In a murine model of infection, the E33 and D303 mutants exhibited decreased infectivity and dissemination. Taken together, these results suggest the importance of these conserved domains, and potentially P66 porin function, in vivo.

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  • 121.
    Dahlberg, Tobias
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Baker, Joseph
    Department of Chemistry, The College of New Jersey, Ewing, New Jersey.
    Bullitt, Esther
    Department of Physiology & Biophysics, Boston University School of Medicine, Boston, Massachusetts.
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Unveiling molecular interactions that stabilize bacterial adhesion pili2022Inngår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 121, nr 11, s. 2096-2106Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Adhesion pili assembled by the chaperone-usher pathway are superelastic helical filaments on the surface of bacteria, optimized for attachment to target cells. Here, we investigate the biophysical function and structural interactions that stabilize P pili from uropathogenic bacteria. Using optical tweezers, we measure P pilus subunit-subunit interaction dynamics and show that pilus compliance is contour-length dependent. Atomic details of subunit-subunit interactions of pili under tension are shown using steered molecular dynamics (sMD) simulations. sMD results also indicate that the N-terminal “staple” region of P pili, which provides interactions with pilins that are four and five subunits away, significantly stabilizes the helical filament structure. These data are consistent with previous structural data, and suggest that more layer-to-layer interactions could compensate for the lack of a staple in type 1 pili. This study informs our understanding of essential structural and dynamic features of adhesion pili, supporting the hypothesis that the function of pili is critically dependent on their structure and biophysical properties.

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  • 122.
    Dahlgren, Markus K
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Zetterström, Caroline E
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gylfe, Åsa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Linusson, Anna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Statistical molecular design of a focused salicylidene acylhydrazide library and multivariate QSAR of inhibition of type III secretion in the Gram-negative bacterium Yersinia2010Inngår i: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 18, nr 7, s. 2686-2703Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A combined application of statistical molecular design (SMD), quantitative structure-activity relationship (QSAR) modeling and prediction of new active compounds was effectively used to develop salicylidene acylhydrazides as inhibitors of type III secretion (T3S) in the Gram-negative pathogen Yersinia pseudotuberculosis. SMD and subsequent synthesis furnished 50 salicylidene acylhydrazides in high purity. Based on data from biological evaluation in T3S linked assays 18 compounds were classified as active and 25 compounds showed a dose-dependent inhibition. The 25 compounds were used to compute two multivariate QSAR models and two multivariate discriminant analysis models were computed from both active and inactive compounds. Three of the models were used to predict 4416 virtual compounds in consensus and eight new compounds were selected as an external test set. Synthesis and biological evaluation of the test set in Y. pseudotuberculosis and the intracellular pathogen Chlamydia trachomatis validated the models. Y. pseudotuberculosis and C. trachomatis cell-based infection models showed that compounds identified in this study are selective and non-toxic inhibitors of T3S dependent virulence.

  • 123.
    Dahmane, Selma
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Shankar, Kasturika
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Carlson, Lars-Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    A 3D view of how enteroviruses hijack autophagy2023Inngår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 19, nr 7, s. 2156-2158Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Viruses are masters at using cellular pathways to aid their replication. Cryo-electron tomography of poliovirus-infected cells revealed how it utilizes macroautophagy to its advantage. Assembly of these non-enveloped virions takes place directly on membranes and requires PIK3C3/VPS34 activity to be completed, whereas the canonical autophagy inducer ULK1 restricts virus assembly. The tomograms further revealed that enterovirus-induced autophagy is selective for RNA-loaded virions, which may help ensure maximum infectivity of the virus-laden vesicles released through secretory autophagy.

  • 124.
    Dai, Yunfei
    et al.
    Department of Biology, Northeastern University, MA, Boston, United States.
    Pinedo, Victor
    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).
    Tang, Amy Y.
    Department of Biology, Northeastern University, MA, Boston, United States.
    Cava, Felipe
    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).
    Geisinger, Edward
    Department of Biology, Northeastern University, MA, Boston, United States.
    A New Class of Cell Wall-Recycling L,D-Carboxypeptidase Determines β-Lactam Susceptibility and Morphogenesis in Acinetobacter baumannii2021Inngår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 12, nr 6, artikkel-id e0278621Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for maintaining a characteristic short rod shape and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell wall-modifying L,D-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic L,D-carboxypeptidase (LDC) that provides a critical step in cell wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, morphology, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including L,D-cross-link formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on envelope physiology. We thus reveal a novel class of cell wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria.

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  • 125.
    Daitch, Allison K.
    et al.
    Department of Biological Chemistry, Johns Hopkins University School of Medicine, MD, Baltimore, United States.
    Orsburn, Benjamin C.
    Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, MD, Baltimore, United States.
    Chen, Zan
    Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, MD, Baltimore, United States.
    Alvarez, Laura
    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). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Eberhard, Colten D.
    Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, MD, Baltimore, United States.
    Sundararajan, Kousik
    Department of Biological Chemistry, Johns Hopkins University School of Medicine, MD, Baltimore, United States.
    Zeinert, Rilee
    Department of Biochemistry and Molecular Biology, University of Massachusetts-Amherst, MA, Amherst, United States.
    Kreitler, Dale F.
    National Synchrotron Light Source II, Brookhaven National Laboratory, P.O. Box 5000, NY, Upton, United States.
    Jakoncic, Jean
    National Synchrotron Light Source II, Brookhaven National Laboratory, P.O. Box 5000, NY, Upton, United States.
    Chien, Peter
    Department of Biochemistry and Molecular Biology, University of Massachusetts-Amherst, MA, Amherst, United States.
    Cava, Felipe
    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).
    Gabelli, Sandra B.
    Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N Wolfe Street, MD, Baltimore, United States; Department of Oncology, Johns Hopkins University School of Medicine, 725 N Wolfe Street, MD, Baltimore, United States; Department of Medicine, Johns Hopkins University School of Medicine, MD, Baltimore, United States.
    Goley, Erin D.
    Department of Biological Chemistry, Johns Hopkins University School of Medicine, MD, Baltimore, United States.
    EstG is a novel esterase required for cell envelope integrity in Caulobacter2023Inngår i: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 33, nr 2, s. 228-240.e7Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the ⍺-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

  • 126.
    Dang, Hung The
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Chorell, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, USA.
    Uvell, Hanna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pinkner, Jerome S.
    Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, USA.
    Hultgren, Scott J.
    Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, USA.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Syntheses and biological evaluation of 2-amino-3-acyl-tetrahydrobenzothiophene derivatives: antibacterial agents with antivirulence activity2014Inngår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 12, nr 12, s. 1942-1956Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Developing new compounds targeting virulence factors (e.g., inhibition of pilus assembly by pilicides) is a promising approach to combating bacterial infection. A high-throughput screening campaign of a library of 17 500 small molecules identified 2-amino-3-acyl-tetrahydrobenzothiophene derivatives (hits 2 and 3) as novel inhibitors of pili-dependent biofilm formation in a uropathogenic Escherichia coli strain UTI89. Based on compounds 2 and 3 as the starting point, we designed and synthesized a series of structurally related analogs and investigated their activity against biofilm formation of E. coli UTI89. Systematic structural modification of the initial hits provided valuable information on their SARs for further optimization. In addition, small structural changes to the parent molecules resulted in low micromolar inhibitors (20-23) of E. coli biofilm development without an effect on bacterial growth. The hit compound 3 and its analog 20 were confirmed to prevent pili formation in a hemagglutination (HA) titer assay and electron microscopy (EM) measurements. These findings suggest that 2-amino-3-acyl-tetrahydrobenzothiophenes may serve as a new class of compounds for further elaboration as antibacterial agents with antivirulence activity.

  • 127.
    de Oliveira, Ana H.
    et al.
    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).
    Tiensuu, Teresa
    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).
    Guerreiro, Duarte
    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). Bacterial Stress Response Group, Microbiology, School of Biological and Chemical Sciences, University of Galway, Galway, Ireland.
    Tükenmez, Hasan
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Dessaux, Charlotte
    Laboratory of Intracellular Bacterial Pathogens, National Center of Biotechnology (CNB)-CSIC, Madrid, Spain.
    García-Del Portillo, Francisco
    Laboratory of Intracellular Bacterial Pathogens, National Center of Biotechnology (CNB)-CSIC, Madrid, Spain.
    O'Byrne, Conor
    Bacterial Stress Response Group, Microbiology, School of Biological and Chemical Sciences, University of Galway, Galway, Ireland.
    Johansson, Jörgen
    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).
    The virulence and infectivity of Listeria monocytogenes are not substantially altered by elevated SigB activity2023Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 91, nr 6Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Listeria monocytogenes is a bacterial pathogen capable of causing severe infections but also thriving outside the host. To respond to different stress conditions, L. monocytogenes mainly utilizes the general stress response regulon, which largely is controlled by the alternative sigma factor Sigma B (SigB). In addition, SigB is important for virulence gene expression and infectivity. Upon encountering stress, a large multicomponent protein complex known as the stressosome becomes activated, ultimately leading to SigB activation. RsbX is a protein needed to reset a "stressed"stressosome and prevent unnecessary SigB activation in nonstressed conditions. Consequently, absence of RsbX leads to constitutive activation of SigB even without prevailing stress stimulus. To further examine the involvement of SigB in the virulence of this pathogen, we investigated whether a strain with constitutively active SigB would be affected in virulence factor expression and/or infectivity in cultured cells and in a chicken embryo infection model. Our results suggest that increased SigB activity does not substantially alter virulence gene expression compared with the wild-type (WT) strain at transcript and protein levels. Bacteria lacking RsbX were taken up by phagocytic and nonphagocytic cells at a similar frequency to WT bacteria, both in stressed and nonstressed conditions. Finally, the absence of RsbX only marginally affected the ability of bacteria to infect chicken embryos. Our results suggest only a minor role of RsbX in controlling virulence factor expression and infectivity under these conditions.

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  • 128.
    de Oliveira, Ana Henriques
    et al.
    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). Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden;Department of Molecular Biology, Umeå University, Umeå, Sweden;Umeå Centre of Microbial Research, Umeå University, Umeå, Sweden.
    Tiensuu, Teresa
    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).
    Guerreiro, Duarte N.
    Bacterial Stress Response Group, Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Tükenmez, Hasan
    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). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dessaux, Charlotte
    Laboratory of Intracellular Bacterial Pathogens, National Center of Biotechnology, (CNB)-CSIC, Spain.
    García-del Portillo, Francisco
    Laboratory of Intracellular Bacterial Pathogens, National Center of Biotechnology, (CNB)-CSIC, Spain.
    O’Byrne, Conor
    Bacterial Stress Response Group, Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Johansson, Jörgen
    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).
    Listeria monocytogenes requires the RsbX protein to prevent SigB-activation under non-stressed conditions2022Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 204, nr 1, artikkel-id e00486-21Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The survival of microbial cells under changing environmental conditions requires an efficient reprogramming of transcription, often mediated by alternative sigma factors. The Gram-positive human pathogen Listeria monocytogenes senses and responds to environmental stress mainly through the alternative sigma factor σB (SigB), which controls expression of the general stress response regulon. SigB activation is achieved through a complex series of phosphorylation/dephosphorylation events culminating in the release of SigB from its anti-sigma factor RsbW. At the top of the signal transduction pathway lies a large multi-protein complex known as the stressosome that is believed to act as a sensory hub for stresses. Following signal detection, stressosome proteins become phosphorylated. Resetting of the stressosome is hypothesized to be exerted by a putative phosphatase, RsbX, which presumably removes phosphate groups from stressosome proteins post-stress.We addressed the role of the RsbX protein in modulating the activity of the stressosome and consequently regulating SigB activity in L. monocytogenes. We show that RsbX is required to reduce SigB activation/levels under non-stress conditions and that it is required for appropriate SigB mediated stress-adaptation. A strain lacking RsbX displayed impaired motility and biofilm formation, but also an increased survival at low pH. Our results could suggest that absence of RsbX alter the multi-protein composition of the stressosome without dramatically affecting its phosphorylation status. Overall the data show that RsbX plays a critical role in modulating the signal transduction pathway by blocking SigB activation under non-stressed conditions.

    Fulltekst (pdf)
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  • 129.
    de Oliveira, Ana Henriques
    et al.
    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). Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Center of Microbial Research, Umeå, Sweden.
    Tiensuu, Teresa
    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).
    Tükenmez, Hasan
    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). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dessaux, Charlotte
    Laboratory of Intracellular Bacterial Pathogens, National Center of Biotechnology, (CNB)-CSIC, Spain.
    Portillo, Francisco García-del
    Laboratory of Intracellular Bacterial Pathogens, National Center of Biotechnology, (CNB)-CSIC, Spain.
    Johansson, Jörgen
    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).
    A minor role of RsbX in Listeria monocytogenes’ virulenceManuskript (preprint) (Annet vitenskapelig)
  • 130.
    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 architecture2015Inngår i: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 6, artikkel-id 449Artikkel, forskningsoversikt (Fagfellevurdert)
    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.

    Fulltekst (pdf)
    fulltext
  • 131.
    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 III2011Inngår i: Nature, Vol. 471, nr 7340, s. 602-607Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 132.
    Devnath, Popy
    et al.
    College of Veterinary Medicine, Washington State University, WA, Pullman, United States; Department of Microbiology, Noakhali Science and Technology University, Noakhali, Bangladesh.
    Karah, Nabil
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Graham, Jay P.
    School of Public Health, University of California, CA, Berkeley, United States.
    Rose, Elizabeth S.
    Vanderbilt Institute for Global Health, Vanderbilt University Medical Center, TN, Nashville, United States.
    Asaduzzaman, Muhammad
    Department of Community Medicine and Global Health, Institute of Health and Society, Faculty of Medicine, University of Oslo, Oslo, Norway; Planetary Health Alliance, MA, Boston, United States; Planetary Health Working Group, Be-Cause Health, Institute of Tropical Medicine, Nationalestraat 155, Antwerp, Belgium.
    Evidence of antimicrobial resistance in bats and its planetary health impact for surveillance of zoonotic spillover events: a scoping review2023Inngår i: International Journal of Environmental Research and Public Health, ISSN 1661-7827, E-ISSN 1660-4601, Vol. 20, nr 1, artikkel-id 243Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    As a result of the COVID-19 pandemic, as well as other outbreaks, such as SARS and Ebola, bats are recognized as a critical species for mediating zoonotic infectious disease spillover events. While there is a growing concern of increased antimicrobial resistance (AMR) globally during this pandemic, knowledge of AMR circulating between bats and humans is limited. In this paper, we have reviewed the evidence of AMR in bats and discussed the planetary health aspect of AMR to elucidate how this is associated with the emergence, spread, and persistence of AMR at the human–animal interface. The presence of clinically significant resistant bacteria in bats and wildlife has important implications for zoonotic pandemic surveillance, disease transmission, and treatment modalities. We searched MEDLINE through PubMed and Google Scholar to retrieve relevant studies (n = 38) that provided data on resistant bacteria in bats prior to 30 September 2022. There is substantial variability in the results from studies measuring the prevalence of AMR based on geographic location, bat types, and time. We found all major groups of Gram-positive and Gram-negative bacteria in bats, which are resistant to commonly used antibiotics. The most alarming issue is that recent studies have increasingly identified clinically significant multi-drug resistant bacteria such as Methicillin Resistant Staphylococcus aureus (MRSA), ESBL producing, and Colistin resistant Enterobacterales in samples from bats. This evidence of superbugs abundant in both humans and wild mammals, such as bats, could facilitate a greater understanding of which specific pathways of exposure should be targeted. We believe that these data will also facilitate future pandemic preparedness as well as global AMR containment during pandemic events and beyond.

    Fulltekst (pdf)
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  • 133.
    Dingeldein, Artur Peter Günther
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lindberg, Mikael J.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Zhong, Xueyin
    Ruhr University of Bochum, Biomolecular NMR Spectroscopy, Bochum, Germany.
    Stoll, Raphael
    Ruhr University of Bochum, Biomolecular NMR Spectroscopy, Bochum, Germany.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bax to the future: A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies2019Inngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 158, s. 20-26Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli. In this study, we present a novel approach to express and purify full-length Bax with significantly increased yields, when compared to the commonly applied strategy. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we were able to render both Bax termini inactive and prevent disruptive interactions from occurring during gene expression. By deploying an Intein-CBD-tag at the C-terminus we were further able to avoid the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the robustness of the purification procedure. We report yields for pure, full-length Bax protein that are increased by an order of magnitude, when compared to commonly used Bax expression protocols.

  • 134.
    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 sharing2011Inngår i: Frontiers in microbiology, ISSN 1664-302X, Vol. 2, s. 108-Artikkel i tidsskrift (Fagfellevurdert)
    Fulltekst (pdf)
    fulltext
  • 135.
    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)2013Inngår i: Handbook of proteolytic enzymes: volume 1 / [ed] Neil D. Rawlings, Guy Salvesen, Elsevier, 2013, 3, Vol. 1, s. 1219-1225Kapittel i bok, del av antologi (Fagfellevurdert)
    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.

  • 136.
    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-Cas92014Inngår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 346, nr 6213, s. 1077-+Artikkel, forskningsoversikt (Fagfellevurdert)
    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.

  • 137. 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 tolerance2016Inngår 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. 404-409Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 138.
    Edqvist, Petra J
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Bröms, Jeanette E
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Betts, Helen J
    Forsberg, Ake
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Pallen, Mark J
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinsk fakultet, Umeå Centre for Microbial Research (UCMR).
    Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.2006Inngår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 59, nr 1, s. 31-44Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Non-flagellar type III secretion systems (T3SSs) transport proteins across the bacterial cell and into eukaryotic cells. Targeting of proteins into host cells requires a dedicated translocation apparatus. Efficient secretion of the translocator proteins that make up this apparatus depends on molecular chaperones. Chaperones of the translocators (also called class-II chaperones) are characterized by the possession of three tandem tetratricopeptide repeats (TPRs). We wished to dissect the relations between chaperone structure and function and to validate a structural model using site-directed mutagenesis. Drawing on a number of experimental approaches and focusing on LcrH, a class-II chaperone from the Yersinia Ysc-Yop T3SS, we examined the contributions of different residues, residue classes and regions of the protein to chaperone stability, chaperone-substrate binding, substrate stability and secretion and regulation of Yop protein synthesis. We confirmed the expected role of the conserved canonical residues from the TPRs to chaperone stability and function. Eleven mutations specifically abrogated YopB binding or secretion while three mutations led to a specific loss of YopD secretion. These are the first mutations described for any class-II chaperone that allow interactions with one translocator to be dissociated from interactions with the other. Strikingly, all mutations affecting the interaction with YopB mapped to residues with side chains projecting from the inner, concave surface of the modelled TPR structure, defining a YopB interaction site. Conversely, all mutations preventing YopD secretion affect residues that lie on the outer, convex surface of the triple-TPR cluster in our model, suggesting that this region of the molecule represents a distinct interaction site for YopD. Intriguingly, one of the LcrH double mutants, Y40A/F44A, was able to maintain stable substrates inside bacteria, but unable to secrete them, suggesting that these two residues might influence delivery of substrates to the secretion apparatus.

  • 139.
    Edwin, Aaron
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Stier, Gunter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer-Eriksson, A Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Domain isolation, expression, purification and proteolytic activity of the metalloprotease PrtV from Vibrio cholerae2014Inngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 96, s. 39-47Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria's ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102kDa) multidomain pre-pro-protein that so far has only been expressed in V. cholerae. Structural studies require high amounts of soluble protein with high purity. Previous attempts for recombinant expression have been hampered by low expression and solubility of protein fragments. Here, we describe results from parallel cloning experiments in Escherichia coli where fusion tagged constructs of PrtV fragments were designed, and protein products tested for expression and solubility. Of more than 100 designed constructs, three produced protein products that expressed well. These include the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25kDa fragment (residues 581-839). The soluble fusion proteins were captured with Ni(2+) affinity chromatography, and subsequently cleaved with tobacco etch virus protease. Purification protocols yielded ∼10-15mg of pure protein from 1L of culture. Proper folding of the shorter domains was confirmed by heteronuclear NMR spectra recorded on (15)N-labeled samples. A modified protocol for the native purification of the secreted 81kDa pro-protein of PrtV is provided. Proteolytic activity measurements suggest that the 37kDa catalytic metalloprotease domain alone is sufficient for activity.

    Fulltekst (pdf)
    fulltext
  • 140.
    Edwin, Aaron
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Persson, Cecilia
    Mayzel, Maxim
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Karlsson, B. Göran
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae2015Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, nr 12, s. 2076-2080Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N- terminal domain (residues 23-103) that contains two short alpha-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.

  • 141.
    Edwin, Aaron
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Rompikuntal, Pramod
    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).
    Björn, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Stier, Gunter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wai, Sun Nyunt
    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).
    Sauer-Eriksson, Elisabeth A.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Calcium binding by the PKD1 domain regulates interdomain flexibility in Vibrio cholerae metalloprotease PrtV2013Inngår i: FEBS Open Bio, E-ISSN 2211-5463, Vol. 3, s. 263-270Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Vibrio cholerae, the causative agent of cholera, releases several virulence factors including secreted proteases when it infects its host. These factors attack host cell proteins and break down tissue barriers and cellular matrix components such as collagen, laminin, fibronectin, keratin, elastin, and they induce necrotic tissue damage. The secreted protease PrtV constitutes one virulence factors of V. cholerae. It is a metalloprotease belonging to the M6 peptidase family. The protein is expressed as an inactive, multidomain, 102 kDa pre-pro-protein that undergoes several N- and C-terminal modifications after which it is secreted as an intermediate variant of 81 kDa. After secretion from the bacteria, additional proteolytic steps occur to produce the 55 kDa active M6 metalloprotease. The domain arrangement of PrtV is likely to play an important role in these maturation steps, which are known to be regulated by calcium. However, the molecular mechanism by which calcium controls proteolysis is unknown. In this study, we report the atomic resolution crystal structure of the PKD1 domain from V. cholera PrtV (residues 755–838) determined at 1.1 Å. The structure reveals a previously uncharacterized Ca2+-binding site located near linker regions between domains. Conformational changes in the Ca2+-free and Ca2+-bound forms suggest that Ca2+-binding at the PKD1 domain controls domain linker flexibility, and plays an important structural role, providing stability to the PrtV protein.

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  • 142. Egge-Jacobsen, Wolfgang
    et al.
    Salomonsson, Emelie Naslund
    Aas, Finn Erik
    Forslund, Anna-Lena
    Winther-Larsen, Hanne C.
    Maier, Josef
    Macellaro, Anna
    Kuoppa, Kerstin
    Oyston, Petra C. F.
    Titball, Richard W.
    Thomas, Rebecca M.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Prior, Joann L.
    Koomey, Michael
    O-Linked glycosylation of the PilA pilin protein of francisella tularensis: identification of the endogenous protein-targeting oligosaccharyltransferase and characterization of the native oligosaccharide2011Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 19, s. 5487-5497Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genus Francisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation in Francisella tularensis and its sufficiency for PilA glycosylation in Escherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica and demonstrated that the protein undergoes multisite, O-linked glycosylation with a pentasaccharide of the structure HexNac-HexHex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains of Francisella subspecies thus express an O-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved in Francisella species, O-linked protein glycosylation may be a feature common to members of this genus.

  • 143. Eggenschwiler, Reto
    et al.
    Moslem, Mohsen
    Fráguas, Mariane Serra
    Galla, Melanie
    Papp, Oliver
    Naujock, Maximilian
    Fonfara, Ines
    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). Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin, Germany.
    Gensch, Ingrid
    Wähner, Annabell
    Beh-Pajooh, Abbas
    Mussolino, Claudio
    Tauscher, Marcel
    Steinemann, Doris
    Wegner, Florian
    Petri, Susanne
    Schambach, Axel
    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).
    Cathomen, Toni
    Cantz, Tobias
    Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase2016Inngår i: Scientific Reports, E-ISSN 2045-2322, Vol. 6, artikkel-id 38198Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puro Delta tk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

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  • 144.
    Ehrenbolger, Kai
    et al.
    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).
    Jespersen, Nathan
    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).
    Sharma, Himanshu
    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).
    Sokolova, Yuliya Y.
    Tokarev, Yuri S.
    Vossbrinck, Charles R.
    Barandun, Jonas
    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).
    Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome2020Inngår i: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 18, nr 10, artikkel-id e3000958Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo-electron microscopy structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein-protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.

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  • 145.
    Ekestubbe, Sofie
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Timing and targeting of Type III secretion translocation of virulence effectors in Yersinia2017Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The Type III secretion system (T3SS) is an important virulence mechanism that allows pathogenic bacteria to translocate virulence effectors directly into the cytoplasm of eukaryotic host cells to manipulate the host cells in favor of the pathogen. Enteropathogenic Yersinia pseudotuberculosis use a T3SS to translocate effectors, Yops, that prevent phagocytosis by immune cells, and is largely dependent on it to establish and sustain an infection in the lymphoid tissues of a mammalian host. Translocation into a host cell requires specific translocator proteins, and is tightly controlled from both the bacterial and host cell cytoplasm. We aimed to investigate two of the regulatory elements, YopN and LcrV, to gain more insight into the translocation mechanism. Two separate regulatory complexes regulate expression and secretion of Yops, however, the processes are linked so that expression is induced when secretion is activated. A complex, including YopD, prevents expression of Yops, while YopN-TyeA and LcrG block secretion. LcrV is required to relieve the secretion block, by sequestering LcrG. We verified that LcrG binds to the C-terminal part of LcrV, which is consistent with what has been shown in Y. pestis. In addition to their regulatory roles, both LcrV and YopD are translocators and are assumed to interact at the bacterial surface, where LcrV promotes insertion of YopB and YopD into the host cell membrane. However, here we show that purified YopD failed to interact with LcrV, instead YopD solely interacted with a complex of LcrV-LcrG. This indicates that LcrV and YopD interact in the bacterial cytosol, which may be important for regulation of Yop expression and secretion. The established role of YopN is to block secretion prior to host cell contact. We found that deleting the central region (amino acids 76-181) had no effect on the regulatory role of YopN in expression and secretion of Yops. Interestingly, we found that, even though the YopN∆76-181 mutant secreted the translocators with similar kinetics as the wild type strain, translocation of the effector YopH, into HeLa cells, was significantly reduced. Consequently, the YopN∆76-181 mutant was unable to block phagocytosis, almost to the same level as the ∆lcrV mutant which is completely unable to translocate YopH. Our results indicate that YopN is involved in the translocation step in addition to its role in regulating secretion. Further, we show that the amino terminal of LcrV, in the context of translocation, is involved in the early intracellular targeting of YopH in order to block phagocytosis efficiently and sustain an in vivo infection. LcrV mutants that failed to efficiently target YopH intracellularly were severely attenuated also for in vivo virulence. All together, we show that LcrV and YopN are involved in more steps in the regulation of translocation, than what was known before. Our studies also highlight that early translocation is essential for Yersinia to block phagocytosis, which in the end is essential for in vivo virulence.

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  • 146.
    Ekestubbe, Sofie
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette E.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Edgren, Tomas
    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, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew S.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence2016Inngår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, artikkel-id 175Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion systems (T3SS) are dedicated to targeting anti-host effector proteins into the cytosol of the host cell to promote bacterial infection. Delivery of the effectors requires three specific translocator proteins, of which the hydrophilic translocator, LcrV, is located at the tip of the T3SS needle and is believed to facilitate insertion of the two hydrophobic translocators into the host cell membrane. Here we used Yersinia as a model to study the role of LcrV in T3SS mediated intracellular effector targeting. Intriguingly, we identified N-terminal IcrV mutants that, similar to the wild-type protein, efficiently promoted expression, secretion and intracellular levels of Yop effectors, yet they were impaired in their ability to inhibit phagocytosis by J774 cells. In line with this, the YopH mediated dephosphorylation of Focal Adhesion Kinase early after infection was compromised when compared to the wild type strain. This suggests that the mutants are unable to promote efficient delivery of effectors to their molecular targets inside the host cell upon host cell contact. The significance of this was borne out by the fact that the mutants were highly attenuated for virulence in the systemic mouse infection model. Our study provides both novel and significant findings that establish a role for LcrV in early targeting of effectors in the host cell.

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  • 147.
    Elbir, Haitham
    et al.
    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).
    Larsson, Pär
    Division of CBRN Security and Defence, FOI Swedish Defence Research Agency, Umeå, Sweden.
    Upreti, Mukunda
    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).
    Normark, Johan
    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).
    Bergström, Sven
    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).
    Genome sequence of the relapsing fever borreliosis species Borrelia hispanica2014Inngår i: Genome Announcements, E-ISSN 2169-8287, Vol. 2, nr 1, artikkel-id e01171-13Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Borrelia hispanica is the etiological pathogen of tick-borne relapsing fever, transmitted to humans by infected Ornithodoros erraticus ticks. Here we present the 1,783,846-bp draft genome sequence, with an average G+C content of 28%. It has 2,140 open reading frames, 3 ribosomal RNAs, and 32 transfer RNAs.

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  • 148.
    El-Schich, Zahra
    et al.
    Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, Malmö, Sweden; Biofilms-Research Center for Biointerfaces, Malmö University, Malmö, Sweden.
    Zhang, Yuecheng
    Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, Malmö, Sweden; Biofilms-Research Center for Biointerfaces, Malmö University, Malmö, Sweden.
    Göransson, Tommy
    Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, Malmö, Sweden.
    Dizeyi, Nishtman
    Department of Translational Medicine, Lund University, Malmö, Sweden.
    Persson, Jenny L.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, Malmö; Biofilms-Research Center for Biointerfaces, Malmö University.
    Johansson, Emil
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Caraballo, Remi
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Shinde, Sudhirkumar
    School of Consciousness, Vishwanath Karad Maharashtra Institute of Technology—World Peace University, Kothrud, Pune, India.
    Sellergren, Börje
    Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, Malmö, Sweden; Biofilms-Research Center for Biointerfaces, Malmö University, Malmö, Sweden.
    Wingren, Anette Gjörloff
    Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, Malmö, Sweden; Biofilms-Research Center for Biointerfaces, Malmö University, Malmö, Sweden.
    Sialic acid as a biomarker studied in breast cancer cell lines in vitro using fluorescent molecularly imprinted polymers2021Inngår i: Applied Sciences, E-ISSN 2076-3417, Vol. 11, nr 7, artikkel-id 3256Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Sialylations are post-translational modifications of proteins and lipids that play important roles in many cellular events, including cell-cell interactions, proliferation, and migration. Tumor cells express high levels of sialic acid (SA), which are often associated with the increased invasive potential in clinical tumors, correlating with poor prognosis. To overcome the lack of natural SA-receptors, such as antibodies and lectins with high enough specificity and sensitivity, we have used molecularly imprinted polymers (MIPs), or “plastic antibodies”, as nanoprobes. Because high expression of epithelial cell adhesion molecule (EpCAM) in primary tumors is often associated with proliferation and a more aggressive phenotype, the expression of EpCAM and CD44 was initially analyzed. The SA-MIPs were used for the detection of SA on the cell surface of breast cancer cells. Lectins that specifically bind to the a-2,3 SA and a-2,6 SA variants were used for analysis of SA expression, with both flow cytometry and confocal microscopy. Here we show a correlation of EpCAM and SA expression when using the SA-MIPs for detection of SA. We also demonstrate the binding pattern of the SA-MIPs on the breast cancer cell lines using confocal microscopy. Pre-incubation of the SA-MIPs with SA-derivatives as inhibitors could reduce the binding of the SA-MIPs to the tumor cells, indicating the specificity of the SA-MIPs. In conclusion, the SA-MIPs may be a new powerful tool in the diagnostic analysis of breast cancer cells.

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  • 149. Elsholz, Alexander K. W.
    et al.
    Birk, Marlene S.
    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). Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Humboldt University, Berlin, Germany.
    Turgay, Kuersad
    Functional Diversity of AAA plus Protease Complexes in Bacillus subtilis2017Inngår i: Frontiers in Molecular Biosciences, E-ISSN 2296-889X, Vol. 4, artikkel-id 44Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics.

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  • 150.
    Emtenäs, Hans
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Soto, Gabe
    Hultgren, Scott J.
    Marshall, Garland R.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Stereoselective Synthesis of Optically Active β-Lactams, Potential Inhibitors of Pilus Assembly in Pathogenic Bacteria2000Inngår i: Organic Letters, Vol. 2, s. 2065-2067Artikkel i tidsskrift (Fagfellevurdert)
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