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  • 51. Makarova, Kira S
    et al.
    Haft, Daniel H
    Barrangou, Rodolphe
    Brouns, Stan J J
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Horvath, Philippe
    Moineau, Sylvain
    Mojica, Francisco J M
    Wolf, Yuri I
    Yakunin, Alexander F
    van der Oost, John
    Koonin, Eugene V
    Evolution and classification of the CRISPR-Cas systems2011In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 9, no 6, p. 467-477Article in journal (Refereed)
    Abstract [en]

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR-Cas systems and Cas proteins. Three major types of CRISPR-Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR-Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a 'polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR-cas loci.

  • 52. Makarova, Kira S
    et al.
    Wolf, Yuri I
    Alkhnbashi, Omer S
    Costa, Fabrizio
    Shah, Shiraz A
    Saunders, Sita J
    Barrangou, Rodolphe
    Brouns, Stan J J
    Charpentier, Emmanuelle
    Department of Regulation in Infection, Max Planck Institute for Infection Biology.
    Haft, Daniel H
    Horvath, Philippe
    Moineau, Sylvain
    Mojica, Francisco J M
    Terns, Rebecca M
    Terns, Michael P
    White, Malcolm F
    Yakunin, Alexander F
    Garrett, Roger A
    van der Oost, John
    Backofen, Rolf
    Koonin, Eugene V
    An updated evolutionary classification of CRISPR-Cas systems2015In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 13, no 11, p. 722-736Article in journal (Refereed)
    Abstract [en]

    The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

  • 53. Mangold, Monika
    et al.
    Siller, Maria
    Roppenser, Bernhard
    Vlaminckx, Bart J M
    Penfound, Tom A
    Klein, Reinhard
    Novak, Rodger
    Novick, Richard P
    Charpentier, Emmanuelle
    Synthesis of group A streptococcal virulence factors is controlled by a regulatory RNA molecule.2004In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 53, no 5, p. 1515-1527Article in journal (Refereed)
    Abstract [en]

    The capacity of pathogens to cause disease depends strictly on the regulated expression of their virulence factors. In this study, we demonstrate that the untranslated mRNA of the recently described streptococcal pleiotropic effect locus (pel), which incidentally contains sagA, the structural gene for streptolysin S, is an effector of virulence factor expression in group A beta-haemolytic streptococci (GAS). Our data suggest that the regulation by pel RNA occurs at both transcriptional (e.g. emm, sic, nga) and post-transcriptional (e.g. SpeB) levels. We could exclude the possibility that the pel phenotype was linked to a polar effect on downstream genes (sagB-I). Remarkably, the RNA effector is regulated in a growth phase-dependent fashion and we provide evidence that pel RNA expression is induced by conditioned media.

  • 54. Novak, R
    et al.
    Braun, J S
    Charpentier, E
    Tuomanen, E
    Penicillin tolerance genes of Streptococcus pneumoniae: the ABC-type manganese permease complex Psa.1998In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 29, no 5, p. 1285-1296Article in journal (Refereed)
    Abstract [en]

    Downregulation of the major autolysin in Streptococcus pneumoniae leads to penicillin tolerance, a feature that is characterized by the ability to survive but not grow in the presence of antibiotic. Screening a library of mutants in pneumococcal surface proteins for the ability to survive 10x minimum inhibitory concentration (MIC) of penicillin revealed over 10 candidate tolerance genes. One such mutant contained an insertion in the known gene psaA, which is part of the psa locus. This locus encodes an ABC-type Mn permease complex. Sequence analysis of adjacent DNA extended the known genetic organization of the locus to include two new open reading frames (ORFs), psaB, which encodes an ATP-binding protein, and psaC, which encodes a hydrophobic transmembrane protein. Mutagenesis of psaB, psaC, psaA and downstream psaD resulted in penicillin tolerance. Defective adhesion and reduced transformation efficiency, as reported previously for a psaA- mutant, were phenotypes shared by psaB-, psaC- and psaD- knockout mutants. Western blot analysis demonstrated that the set of mutants expressed RecA, but none of them showed translation of the autolysin gene, which is located downstream of recA. The addition of manganese (Mn) failed to correct the abnormal physiology. These results suggest that this ABC-type Mn permease complex has a pleiotropic effect on pneumococcal physiology including adherence and autolysis. These are the first genes suggested as being involved in triggering autolysin. The results raise the possibility that loss of function of PsaA, by vaccine-induced antibody for instance, may promote penicillin tolerance.

  • 55. Novak, R
    et al.
    Cauwels, A
    Charpentier, E
    Tuomanen, E
    Identification of a Streptococcus pneumoniae gene locus encoding proteins of an ABC phosphate transporter and a two-component regulatory system.1999In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 181, no 4, p. 1126-1133Article in journal (Refereed)
    Abstract [en]

    The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.

  • 56. Novak, R
    et al.
    Charpentier, E
    Braun, J S
    Park, E
    Murti, S
    Tuomanen, E
    Masure, R
    Extracellular targeting of choline-binding proteins in Streptococcus pneumoniae by a zinc metalloprotease.2000In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 36, no 2, p. 366-376Article in journal (Refereed)
    Abstract [en]

    A genetic-based search for surface proteins of Streptococcus pneumoniae involved in adhesion identified a putative zinc metalloprotease (ZmpB). ZmpB shared high amino acid sequence similarities with IgA1 proteases of Gram-positive bacteria, but ZmpB had neither IgA1 nor IgA2 protease activity. Analysis of a family of surface-expressed proteins, the choline-binding proteins (Cbp's), in a zmpB-deficient mutant demonstrated a global loss of surface expression of CbpA, CbpE, CbpF and CbpJ. CbpA was detected within the cytoplasm. The zmpB-deficient mutant also failed to lyse with penicillin, a sign of lack of function of the Cbp LytA. Immunodetection studies revealed that the autolysin (LytA), normally located on the cell wall, was trapped in the cytoplasm colocalized with DNA and the transformation protein CinA. Trafficking of CinA and RecA to the cell membrane during genetic competence was also not observed in the zmpB-deficient mutant. These results suggest a protease dependent regulatory mechanism governing the translocation of CinA and the Cbp's LytA and CbpA of S. pneumoniae.

  • 57. Novak, R
    et al.
    Charpentier, E
    Braun, J S
    Tuomanen, E
    Signal transduction by a death signal peptide: uncovering the mechanism of bacterial killing by penicillin.2000In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 5, no 1, p. 49-57Article in journal (Refereed)
    Abstract [en]

    The binding of bactericidal antibiotics like penicillins, cephalosporins, and glycopeptides to their bacterial targets stops bacterial growth but does not directly cause cell death. A second process arising from the bacteria itself is necessary to trigger endogenous suicidal enzymes that dissolve the cell wall during autolysis. The signal and the trigger pathway for this event are completely unknown. Using S. pneumoniae as a model, we demonstrate that signal transduction via the two-component system VncR/S triggers multiple death pathways. We show that the signal sensed by VncR/S is a secreted peptide, Pep27, that initiates the cell death program. These data depict a novel model for the control of bacterial cell death.

  • 58. Novak, R
    et al.
    Henriques, B
    Charpentier, E
    Normark, S
    Tuomanen, E
    Emergence of vancomycin tolerance in Streptococcus pneumoniae.1999In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 399, no 6736, p. 590-593Article in journal (Refereed)
    Abstract [en]

    Streptococcus pneumoniae, the pneumococcus, is the most common cause of sepsis and meningitis. Multiple-antibiotic-resistant strains are widespread, and vancomycin is the antibiotic of last resort. Emergence of vancomycin resistance in this community-acquired bacterium would be catastrophic. Antibiotic tolerance, the ability of bacteria to survive but not grow in the presence of antibiotics, is a precursor phenotype to resistance. Here we show that loss of function of the VncS histidine kinase of a two-component sensor-regulator system in S. pneumoniae produced tolerance to vancomycin and other classes of antibiotic. Bacterial two-component systems monitor environmental parameters through a sensor histidine-kinase/phosphatase, which phosphorylates/dephosphorylates a response regulator that in turn mediates changes in gene expression. These results indicate that signal transduction is critical for the bactericidal activity of antibiotics. Experimental meningitis caused by the vncS mutant failed to respond to vancomycin. Clinical isolates tolerant to vancomycin were identified and DNA sequencing revealed nucleotide alterations in vncS. We conclude that broad antibiotic tolerance of S. pneumoniae has emerged in the community by a molecular mechanism that eliminates sensitivity to the current cornerstone of therapy, vancomycin.

  • 59. Novak, R
    et al.
    Tuomanen, E
    Charpentier, E
    The mystery of psaA and penicillin tolerance in Streptococcus pneumoniae.2000In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 36, no 6, p. 1504-1505Article in journal (Refereed)
  • 60. Plagens, Andre
    et al.
    Richter, Hagen
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Randau, Lennart
    DNA and RNA interference mechanisms by CRISPR-Cas surveillance complexes2015In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 39, no 3, p. 442-463Article, review/survey (Refereed)
    Abstract [en]

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) adaptive immune systems use small guide RNAs, the CRISPR RNAs (crRNAs), to mark foreign genetic material, e.g. viral nucleic acids, for degradation. Archaea and bacteria encode a large variety of Cas proteins that bind crRNA molecules and build active ribonucleoprotein surveillance complexes. The evolution of CRISPR-Cas systems has resulted in a diversification of cas genes and a classification of the systems into three types and additional subtypes characterized by distinct surveillance and interfering complexes. Recent crystallographic and biochemical advances have revealed detailed insights into the assembly and DNA/RNA targeting mechanisms of the various complexes. Here, we review our knowledge on the molecular mechanism involved in the DNA and RNA interference stages of type I (Cascade: CRISPR-associated complex for antiviral defense), type II (Cas9) and type III (Csm, Cmr) CRISPR-Cas systems. We further highlight recently reported structural and mechanistic themes shared among these systems.

  • 61. Reimer, Jana
    et al.
    Knoess, Sabine
    Labuhn, Maurice
    Charpentier, Emmanuelle M.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Max Planck Institute for Infection Biology, Berlin, Germany.
    Goehring, Gudrun
    Schlegelberger, Brigitte
    Klusmann, Jan-Henning
    Heckl, Dirk
    CRISPR-Cas9-induced t(11;19)/MLL-ENL translocations initiate leukemia in human hematopoietic progenitor cells in vivo2017In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 102, no 9, p. 1558-1566Article in journal (Refereed)
    Abstract [en]

    Chromosomal translocations that generate oncogenic fusion proteins are causative for most pediatric leukemias and frequently affect the MLL/ KMT2A gene. In vivo modeling of bona fide chromosomal translocations in human hematopoietic stem and progenitor cells is challenging but essential to determine their actual leukemogenic potential. We therefore developed an advanced lentiviral CRISPR-Cas9 vector that efficiently transduced human CD34(+) hematopoietic stem and progenitor cells and induced the t(11; 19)/MLL-ENL translocation. Leveraging this system, we could demonstrate that hematopoietic stem and progenitor cells harboring the translocation showed only a transient clonal growth advantage in vitro. In contrast, t(11; 19)/MLL-ENL-harboring CD34(+) hematopoietic stem and progenitor cells not only showed longterm engraftment in primary immunodeficient recipients, but t(11; 19)/ MLL-ENL also served as a first hit to initiate a monocytic leukemia-like disease. Interestingly, secondary recipients developed acute lymphoblastic leukemia with incomplete penetrance. These findings indicate that environmental cues not only contribute to the disease phenotype, but also to t(11; 19)/ MLL-ENL-mediated oncogenic transformation itself. Thus, by investigating the true chromosomal t(11; 19) rearrangement in its natural genomic context, our study emphasizes the importance of environmental cues for the pathogenesis of pediatric leukemias, opening an avenue for novel treatment options.

  • 62.
    Resch, Ulrike
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Vascular Biology and Thrombosis Research, Medical University Vienna, Vienna, Austria.
    Tsatsaronis, James Anthony
    Le Rhun, Anais
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Stuebiger, Gerald
    Rohde, Manfred
    Kasvandik, Sergo
    Holzmeister, Susanne
    Tinnefeld, Philip
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Hannover Medical School, Hannover, Germany.
    A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 6, article id e00207-16Article in journal (Refereed)
    Abstract [en]

    Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to "anchorless surface proteins." Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response. IMPORTANCE Group A streptococcus (GAS) is a Gram-positive bacterial pathogen responsible for more than 500,000 deaths annually. Establishment of GAS infection is dependent on a suite of proteins exported via the general secretory pathway. Here, we show that GAS naturally produces extracellular vesicles with a unique lipid composition that are laden with proteins and RNAs. Interestingly, both virulence-associated proteins and RNA species were found to be differentially abundant in vesicles relative to the bacteria. Furthermore, we show that genetic disruption of the virulence-associated two-component regulator CovRS leads to an increase in vesicle production. This study comprehensively describes the protein, RNA, and lipid composition of GAS-secreted MVs and alludes to a regulatory system impacting this process.

  • 63. Richter, Florian
    et al.
    Fonfara, Ines
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Inst Infect Biol, D-10117 Berlin, Germany.
    Bouazza, Boris
    Schumacher, Charlotte Helene
    Bratovic, Majda
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Inst Infect Biol, D-10117 Berlin, Germany.
    Moeglich, Andreas
    Engineering of temperature- and light-switchable Cas9 variants2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 20, p. 10003-10014Article in journal (Refereed)
    Abstract [en]

    Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via amechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of RsLOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Es-cherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29 degrees C but negligible activity at 37. C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, RsLOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity.

  • 64. Richter, Florian
    et al.
    Fonfara, Ines
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Institute for Infection Biology, 10117 Berlin, Germany.
    Gelfert, Renate
    Nack, Jennifer
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Institute for Infection Biology, 10117 Berlin, Germany.
    Möglich, Andreas
    Switchable Cas92017In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 48, p. 119-126Article, review/survey (Refereed)
    Abstract [en]

    Ever since its discovery, Cas9 from Streptococcus pyogenes has revolutionized biology by enabling analysis and engineering of genomes with unprecedented precision and ease. To fine-tune on-target effects and to mitigate adverse effects caused by untimely and off-target action of Cas9, strategies have been developed to control its activity at the post-translational stage via external trigger signals. Control is either achieved by modifying the Cas9 protein itself or its programmable RNA molecules. To date, switchable Cas9 variants responding to small ligands, light or temperature have been engineered. With these variants in hand, the regulation and modification of genomes can be accomplished in graded and ever more precise manner.

  • 65.
    Romby, Pascale
    et al.
    Architecture et Re´activite´ de l’ARN, Universite´ de Strasbourg, Strasbourg, France.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    An overview of RNAs with regulatory functions in gram-positive bacteria.2010In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 67, no 2, p. 217-237Article in journal (Refereed)
    Abstract [en]

    During the last decade, RNA molecules with regulatory functions on gene expression have benefited from a renewed interest. In bacteria, recent high throughput computational and experimental approaches have led to the discovery that 10-20% of all genes code for RNAs with critical regulatory roles in metabolic, physiological and pathogenic processes. The trans-acting RNAs comprise the noncoding RNAs, RNAs with a short open reading frame and antisense RNAs. Many of these RNAs act through binding to their target mRNAs while others modulate protein activity or target DNA. The cis-acting RNAs include regulatory regions of mRNAs that can respond to various signals. These RNAs often provide the missing link between sensing changing conditions in the environment and fine-tuning the subsequent biological responses. Information on their various functions and modes of action has been well documented for gram-negative bacteria. Here, we summarize the current knowledge of regulatory RNAs in gram-positive bacteria.

  • 66. Schikora, Adam
    et al.
    Carreri, Alessandro
    Charpentier, Emmanuelle
    Department of Microbiology and Immunobiology, Max F. Perutz Laboratories, Vienna, Austria.
    Hirt, Heribert
    The dark side of the salad: Salmonella typhimurium overcomes the innate immune response of Arabidopsis thaliana and shows an endopathogenic lifestyle2008In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 3, no 5, p. e2279-Article in journal (Refereed)
    Abstract [en]

    Salmonella enterica serovar typhimurium contaminated vegetables and fruits are considerable sources of human infections. Bacteria present in raw plant-derived nutrients cause salmonellosis, the world wide most spread food poisoning. This facultative endopathogen enters and replicates in host cells and actively suppresses host immune responses. Although Salmonella survives on plants, the underlying bacterial infection mechanisms are only poorly understood. In this report we investigated the possibility to use Arabidopsis thaliana as a genetically tractable host system to study Salmonella-plant interactions. Using green fluorescent protein (GFP) marked bacteria, we show here that Salmonella can infect various Arabidopsis tissues and proliferate in intracellular cellular compartments. Salmonella infection of Arabidopsis cells can occur via intact shoot or root tissues resulting in wilting, chlorosis and eventually death of the infected organs. Arabidopsis reacts to Salmonella by inducing the activation of mitogen-activated protein kinase (MAPK) cascades and enhanced expression of pathogenesis related (PR) genes. The induction of defense responses fails in plants that are compromised in ethylene or jasmonic acid signaling or in the MKK3-MPK6 MAPK pathway. These findings demonstrate that Arabidopsis represents a true host system for Salmonella, offering unique possibilities to study the interaction of this human pathogen with plants at the molecular level for developing novel drug targets and addressing current safety issues in human nutrition.

  • 67. Schmitz, Franz-Josef
    et al.
    Beyer, Andreas
    Charpentier, Emmanuelle
    Normark, Birgitta Henriques
    Schade, Marc
    Fluit, Ad C
    Hafner, Dieter
    Novak, Rodger
    Toxin-gene profile heterogeneity among endemic invasive European group A streptococcal isolates.2003In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 188, no 10, p. 1578-1586Article in journal (Refereed)
    Abstract [en]

    We determined the toxin-gene profiles of 239 endemic, invasive group A streptococcal (GAS) isolates that circulated, within a 5-year period, in European university hospitals. Profiling was performed by use of multiplex polymerase chain reaction that screened for 9 streptococcal pyrogenic exotoxins (speA, speB, speC, speF, speG, speH, speJ, ssa, and smeZ). Analysis revealed that invasive GAS isolates do not share a common toxin-gene profile. Although all emm types were characterized by several different toxin-gene profiles, a predominance of 1 or 2 toxin-gene profiles could be observed, reflecting that a few invasive clones have spread successfully throughout the world. Remarkably, statistical pair-wise analysis of individual toxin genes revealed that strains that did not share the predominant profile still showed a nonrandom distribution of key toxin genes characteristic of the specific emm type. This could indicate that M proteins function, directly or indirectly, as barriers for horizontal gene exchange.

  • 68. Siller, Maria
    et al.
    Janapatla, Rajendra P
    Pirzada, Zaid A
    Hassler, Christine
    Zinkl, Daniela
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Functional analysis of the group A streptococcal luxS/AI-2 system in metabolism, adaptation to stress and interaction with host cells2008In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 8, p. 188-Article in journal (Refereed)
    Abstract [en]

    Background

    The luxS/AI-2 signaling pathway has been reported to interfere with important physiological and pathogenic functions in a variety of bacteria. In the present study, we investigated the functional role of the streptococcal luxS/AI-2 system in metabolism and diverse aspects of pathogenicity including the adaptation of the organism to stress conditions using two serotypes of Streptococcus pyogenes, M1 and M19.

    Results

    Exposing wild-type and isogenic luxS-deficient strains to sulfur-limited media suggested a limited role for luxS in streptococcal activated methyl cycle metabolism. Interestingly, loss of luxS led to an increased acid tolerance in both serotypes. Accordingly, luxS expression and AI-2 production were reduced at lower pH, thus linking the luxS/AI-2 system to stress adaptation in S. pyogenes. luxS expression and AI-2 production also decreased when cells were grown in RPMI medium supplemented with 10% serum, considered to be a host environment-mimicking medium. Furthermore, interaction analysis with epithelial cells and macrophages showed a clear advantage of the luxS-deficient mutants to be internalized and survive intracellularly in the host cells compared to the wild-type parents. In addition, our data revealed that luxS influences the expression of two virulence-associated factors, the fasX regulatory RNA and the virulence gene sibA (psp).

    Conclusion

    Here, we suggest that the group A streptococcal luxS/AI-2 system is not only involved in the regulation of virulence factor expression but in addition low level of luxS expression seems to provide an advantage for bacterial survival in conditions that can be encountered during infections.

  • 69.
    Sternberg, Samuel H
    et al.
    Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
    Richter, Hagen
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Qimron, Udi
    Adaptation in CRISPR-Cas Systems2016In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 61, no 6, p. 797-808Article in journal (Refereed)
    Abstract [en]

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in prokaryotes. The system preserves memories of prior infections by integrating short segments of foreign DNA, termed spacers, into the CRISPR array in a process termed adaptation. During the past 3 years, significant progress has been made on the genetic requirements and molecular mechanisms of adaptation. Here we review these recent advances, with a focus on the experimental approaches that have been developed, the insights they generated, and a proposed mechanism for self- versus non-self-discrimination during the process of spacer selection. We further describe the regulation of adaptation and the protein players involved in this fascinating process that allows bacteria and archaea to harbor adaptive immunity.

  • 70. Tsatsaronis, James A.
    et al.
    Franch-Arroyo, Sandra
    Resch, Ulrike
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Extracellular Vesicle RNA: A Universal Mediator of Microbial Communication?2018In: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 26, no 5, p. 401-410Article, review/survey (Refereed)
    Abstract [en]

    Both extracellular RNAs and extracellular vesicles (EVs) have recently garnered attention as novel mediators of intercellular communication in eukaryotes and prokaryotes alike. EVs not only permit export of RNA, but also facilitate delivery and trans-kingdom exchange of these and other biomolecules, for instance between microbes and their hosts. In this Opinion article, we propose that EV-mediated export of RNA represents a universal mechanism for interkingdom and intrakingdom communication that is conserved among bacterial, archaeal, and eukaryotic microbes. We speculate how microbes might use EV RNA to influence target cell gene expression or manipulate host immune responses.

  • 71. Vojtek, Ivo
    et al.
    Pirzada, Zaid A
    Henriques-Normark, Birgitta
    Mastny, Markus
    Janapatla, Rajendra P
    Charpentier, Emmanuelle
    Lysogenic transfer of group A Streptococcus superantigen gene among Streptococci.2008In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 197, no 2, p. 225-34Article in journal (Refereed)
    Abstract [en]

    A group A Streptococcus (GAS) isolate, serotype M12, recovered from a patient with streptococcal toxic shock syndrome was analyzed for superantigen-carrying prophages, revealing phi149, which encodes superantigen SSA. Sequence analysis of the att-L proximal region of phi149 showed that the phage had a mosaic nature. Remarkably, we successfully obtained lysogenic conversion of GAS clinical isolates of various M serotypes (M1, M3, M5, M12, M19, M28, and M94), as well as of group C Streptococcus equisimilis (GCSE) clinical isolates, via transfer of a recombinant phage phi149::Km(r). Phage phi149::Km(r) from selected lysogenized GAS and GCSE strains could be transferred back to M12 GAS strains. Our data indicate that horizontal transfer of lysogenic phages among GAS can occur across the M-type barrier; these data also provide further support for the hypothesis that toxigenic conversion can occur via lysogeny between species. Streptococci might employ this mechanism specifically to allow more efficient adaptation to changing host challenges, potentially leading to fitter and more virulent clones.

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