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  • 1. Charpentier, E
    et al.
    Courvalin, P
    Antibiotic resistance in Listeria spp.1999In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 43, no 9, p. 2103-2108Article in journal (Refereed)
  • 2. Charpentier, E
    et al.
    Courvalin, P
    Emergence of the trimethoprim resistance gene dfrD in Listeria monocytogenes BM4293.1997In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 41, no 5, p. 1134-1136Article in journal (Refereed)
    Abstract [en]

    The sequence of the trimethoprim resistance gene of the 3.7-kb plasmid (pIP823) that confers high-level resistance (MIC, 1,024 microg/ml) to Listeria monocytogenes BM4293 was determined. The gene was identical to dfrD recently detected in Staphylococcus haemolyticus MUR313. The corresponding protein, S2DHFR, represents the second class of high-level trimethoprim-resistant dihydrofolate reductase identified in gram-positive bacteria. We propose that trimethoprim resistance in L. monocytogenes BM4293 could originate in staphylococci.

  • 3. Charpentier, E
    et al.
    Gerbaud, G
    Courvalin, P
    Characterization of a new class of tetracycline-resistance gene tet(S) in Listeria monocytogenes BM4210.1993In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 131, no 1, p. 27-34Article in journal (Refereed)
    Abstract [en]

    The nucleotide sequence of the tetracycline (Tc)-minocycline (Mc)-resistance determinant of plasmid pIP811 from Listeria monocytogenes BM4210 has been determined. The gene, designated tet(S), was identified by analysis of the start and stop codons as a coding sequence of 1923 bp, corresponding to a protein with a calculated M(r) of 72,912. The apparent 68-kDa size estimated by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of the protein characterized in a cell-free coupled transcription-translation system was in good agreement with the calculated value. The tet(S) gene product exhibits 79 and 72% amino acid identity with Tet(M) from Streptococcus pneumoniae and Tet(O) from Campylobacter coli, respectively. The distribution of tet(S) in strains of Gram+ and Gram- genera resistant to Tc (TcR) and Mc (McR) was studied by hybridization under high stringency using a 590-bp intragenic probe. Homology with tet(S) was detected in two clinical isolates of L. monocytogenes isolated in different geographical areas.

  • 4. Charpentier, E
    et al.
    Gerbaud, G
    Courvalin, P
    Presence of the Listeria tetracycline resistance gene tet(S) in Enterococcus faecalis.1994In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 38, no 10, p. 2330-2335Article in journal (Refereed)
    Abstract [en]

    Two hundred thirty-eight tetracycline- and minocycline-resistant clinical isolates of Enterococcus and Streptococcus spp. were investigated by dot blot hybridization for the presence of nucleotide sequences related to tet(S) (first detected in Listeria monocytogenes BM4210), tet(K), tet(L), tet(M), tet(O), tet(P), and tet(Q) genes. The tet(S) determinant was found in 22 strains of Enterococcus faecalis, associated with tet(M) in 9 of these isolates and further associated with tet(L) in 3 of these strains. tet(M) was detected in all strains of Streptococcus spp. and in all but 10 isolates of Enterococcus spp.; tet(L) was found in 93 enterococci and tet(O) was found in single isolates of E. faecalis and Streptococcus milleri. No hybridization with the tet(K), tet(P), and tet(Q) probes was observed. Transfer of tet(S) by conjugation to E. faecalis or to E. faecalis and L. monocytogenes was obtained from 8 of the 10 E. faecalis strains harboring only this tet gene. Hybridization experiments with DNAs of four donors and of the corresponding transconjugants suggested that tet(S) was located in the chromosome. These results indicate that the genetic support of tet(S) in E. faecalis is different from that in L. monocytogenes, where it is carried by self-transferable plasmids, and confirm the notion of exchange of genetic information between Enterococcus and Listeria spp. in nature.

  • 5. Charpentier, E
    et al.
    Gerbaud, G
    Jacquet, C
    Rocourt, J
    Courvalin, P
    Incidence of antibiotic resistance in Listeria species.1995In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 172, no 1, p. 277-281Article in journal (Refereed)
    Abstract [en]

    To define the prevalence of antibiotic resistance in Listeria species pathogenic for humans and animals, 1100 isolates (60 from cases of listeriosis and 1040 from food and environment) collected worldwide were screened. Of the 61 tetracycline- and minocycline-resistant strains (37 Listeria monocytogenes), 57 harbored tet(M); 4 non-L. monocytogenes isolates contained tet(S). One Listeria innocua isolate was also resistant to streptomycin and contained the tet(M) and aad6 genes. An L. monocytogenes isolate was trimethoprim-resistant, a characteristic not reported previously in Listeria species, because of the presence of a yet-uncharacterized gene. Three clinical isolates of L. monocytogenes were resistant to low levels of streptomycin. Since the tet(M), tet(S), and aad6 genes are common in enterococci and streptococci, these data suggest transfer from the latter to Listeria species. Uniform susceptibility to tetracycline, minocycline, trimethoprim, and streptomycin cannot be assumed any longer for Listeria species.

  • 6. Charpentier, E
    et al.
    Lavker, R M
    Acquista, E
    Cowin, P
    Plakoglobin suppresses epithelial proliferation and hair growth in vivo.2000In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 149, no 2, p. 503-520Article in journal (Refereed)
    Abstract [en]

    Plakoglobin regulates cell adhesion by providing a modulatable connection between both classical and desmosomal cadherins and their respective cytoskeletal linker proteins. Both plakoglobin and the related protein beta-catenin are posttranscriptionally upregulated in response to Wnt-1 in cultured cells. Upregulation of beta-catenin has been implicated in potentiating hyperproliferation and tumor formation. To investigate the role of plakoglobin in these functions we expressed a full-length (PG) and an NH(2)-terminally truncated form of plakoglobin (DeltaN80PG) in mouse epidermis and hair follicles, tissues which undergo continuous and easily observed postnatal renewal and remodeling. Expression of these constructs results in stunted hair growth, a phenotype that has also been observed in transgenic mice expressing Wnt3 and Dvl2 (Millar et al. 1999). Hair follicles from PG and DeltaN80PG mice show premature termination of the growth phase (anagen) of the hair cycle, an event that is regulated in part by FGF5 (Hebert et al. 1994). The proliferative rate of the epidermal cells was reduced and apoptotic changes, which are associated with entry into the regressive phase of the hair follicle cycle (catagen), occurred earlier than usual.

  • 7. Charpentier, E
    et al.
    Novak, R
    Tuomanen, E
    Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC.2000In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 37, no 4, p. 717-26Article in journal (Refereed)
    Abstract [en]

    The ClpC ATPase is a subfamily of HSP100/Clp molecular chaperones-regulators of proteolysis. By screening a library of loss of function mutants for the ability to survive treatment with penicillin, we identified the gene clpC. The corresponding protein was identified as a ClpC ATPase, sharing strong peptide sequence identity with ClpC of Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis. Northern blot experiments showed that expression of clpC was induced in response to high temperature (40-42 degrees C) versus 37 degrees C, suggesting that ClpC is a heat shock protein. Insertional duplication mutagenesis of clpC resulted in increased tolerance to high temperature; a result in contrast to other bacterial Clp proteases. The clpC-deficient mutant formed long chains and failed to undergo lysis after treatment with penicillin or vancomycin. The effect of the clpC mutation extended to deficiency of adherence to the human type II alveolar cells. Finally, the clpC disruption resulted in decreased genetic transformation. Western blot analysis demonstrated that the mutant failed to express pneumolysin and the choline-binding proteins LytA, CbpA, CbpE, CbpF, CbpJ. These results suggest that the heat shock protein ClpC plays an essential complex pleiotropic role in pneumococcal physiology, including cell growth under heat stress, cell division, autolysis, adherence and transformation.

  • 8. Charpentier, E
    et al.
    Tuomanen, E
    Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae.2000In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 2, no 15, p. 1855-64Article in journal (Refereed)
    Abstract [en]

    Streptococcus pneumoniae is a major pathogen causing potentially life-threatening community-acquired diseases in both the developed and developing world. Since 1967, there has been a dramatic increase in the incidence of penicillin-resistant and multiply antibiotic-resistant pneumococci worldwide. Prevention of access of the antibiotic to the target, inactivation of the antibiotic and alteration of the target are mechanisms that S. pneumoniae has developed to resist antibiotics. Recent studies on antibiotic-tolerant pneumococcal mutants permitted development of a novel model for the control of bacterial cell death.

  • 9.
    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). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; Hannover Medical School, Hannover, Germany.
    CRISPR-Cas9: how research on a bacterial RNA-guided mechanism opened new perspectives in biotechnology and biomedicine2015In: EMBO Molecular Medicine, ISSN 1757-4676, E-ISSN 1757-4684, Vol. 7, no 4, p. 363-365Article in journal (Refereed)
  • 10.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Programmable RNA-guided Cas9 endonuclease: a novel tool for genome engineering2014In: Transgenic research, ISSN 0962-8819, E-ISSN 1573-9368, Vol. 23, no 1, p. 188-189Article in journal (Other academic)
  • 11. Charpentier, Emmanuelle
    et al.
    Anton, Ana I
    Barry, Peter
    Alfonso, Berenice
    Fang, Yuan
    Novick, Richard P
    Novel cassette-based shuttle vector system for Gram-positive bacteria.2004In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 10, p. 6076-6085Article in journal (Refereed)
    Abstract [en]

    Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible P(cad)-cadC and constitutive P(blaZ) promoters were designed and analyzed in transcriptional fusions to the staphylococcal beta-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.

  • 12. Charpentier, Emmanuelle
    et al.
    Courvalin, P
    Antibiotic resistance in Listeria spp1995In: Med. Maladies. Infect., Vol. 25, p. 225-232Article in journal (Refereed)
  • 13.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Doudna, Jennifer A.
    Biotechnology: rewriting a genome2013In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 495, no 7439, p. 50-51Article in journal (Other academic)
  • 14. Charpentier, Emmanuelle
    et al.
    Gerbaud, G
    Courvalin, P
    Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among gram-positive and gram-negative bacteria.1999In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 181, no 11, p. 3368-3374Article in journal (Refereed)
    Abstract [en]

    We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.

  • 15.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Marraffini, Luciano A.
    Editorial overview: Novel technologies in microbiology: Recent advances in techniques in microbiology2014In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 19, p. VIII-XArticle in journal (Refereed)
  • 16.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Marraffini, Luciano A.
    Harnessing CRISPR-Cas9 immunity for genetic engineering2014In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 19, p. 114-119Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas encodes an adaptive immune system that defends prokaryotes against infectious viruses and plasmids. Immunity is mediated by Cas nucleases, which use small RNA guides (the crRNAs) to specify a cleavage site within the genome of invading nucleic acids. In type II CRISPR-Cas systems, the DNA-cleaving activity is performed by a single enzyme Cas9 guided by an RNA duplex. Using synthetic single RNA guides, Cas9 can be reprogrammed to create specific double-stranded DNA breaks in the genomes of a variety of organisms, ranging from human cells to bacteria, and thus constitutes a powerful tool for genetic engineering. Here we describe recent advancements in our understanding of type II CRISPR-Cas immunity and how these studies led to revolutionary genome editing applications.

  • 17. Charpentier, Emmanuelle
    et al.
    Novak, R
    Bacterial death and antibiotics of the ß-lactam family2000In: Med Sciences, ISSN 0767-0974, Vol. 16, no 10, p. 1125-1127Article in journal (Refereed)
  • 18.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Richter, Hagen
    van der Oost, John
    White, Malcolm F.
    Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity2015In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 39, no 3, p. 428-441Article, review/survey (Refereed)
    Abstract [en]

    CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types.

  • 19.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Schroeder, Renée
    RNA techniques for bacteria2007In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 10, no 3, p. 254-256Article in journal (Other academic)
  • 20.
    Chylinski, Krzysztof
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Le Rhun, Anaïs
    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).
    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).
    The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems2013In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 10, no 5, p. 726-737Article in journal (Refereed)
    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.

  • 21.
    Chylinski, Krzysztof
    et al.
    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). Max F. Perutz Laboratories, University of Vienna, Austria .
    Makarova, Kira S.
    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). 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 systems2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 10, p. 6091-6105Article in journal (Refereed)
    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.

  • 22.
    Deltcheva, Elitza
    et al.
    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).
    Chylinski, Krzysztof
    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).
    Sharma, Cynthia M
    Gonzales, Karine
    Chao, Yanjie
    Pirzada, Zaid A
    Eckert, Maria R
    Vogel, Jörg
    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).
    CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III2011In: Nature, Vol. 471, no 7340, p. 602-607Article in journal (Refereed)
    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.

  • 23.
    Doudna, Jennifer A.
    et al.
    Univ Calif Berkeley, Berkeley, CA 94720 USA.
    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). 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-Cas92014In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 346, no 6213, p. 1077-+Article, review/survey (Refereed)
    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.

  • 24. Fineran, Peter C.
    et al.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information2012In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 434, no 2, p. 202-209Article, review/survey (Refereed)
    Abstract [en]

    Multiple organisms face the threat of viral infections. To combat phage invasion, bacteria and archaea have evolved an adaptive mechanism of protection against exogenic mobile genetic elements, called CRISPR-Cas. In this defense strategy, phage infection is memorized via acquisition of a short invader sequence, called a spacer, into the CRISPR locus of the host genome. Upon repeated infection, the 'vaccinated' host expresses the spacer as a precursor RNA, which is processed into a mature CRISPR RNA (crRNA) that guides an endonuclease to the matching invader for its ultimate destruction. Recent efforts have uncovered molecular details underlying the crRNA biogenesis and interference steps. However, until recently the step of adaptation had remained largely uninvestigated. In this minireview, we focus on recent publications that have begun to reveal molecular insights into the adaptive step of CRISPR-Cas immunity, which is required for the development of the heritable memory of the host against viruses. 

  • 25.
    Fonfara, Ines
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany.
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany.
    Chylinski, Krzysztof
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Deptartment of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Austria.
    Makarova, Kira S.
    Lécrivain, Anne-Laure
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bzdrenga, Janek
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Koonin, Eugene V.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany ; Hannover Medical School, Hannover, Germany .
    Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, p. 2577-2590Article in journal (Refereed)
    Abstract [en]

    The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA: crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA: Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

  • 26. Fuhrmann, Jakob
    et al.
    Mierzwa, Beata
    Trentini, Debora B.
    Spiess, Silvia
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lehner, Anita
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Clausen, Tim
    Structural Basis for Recognizing Phosphoarginine and Evolving Residue-Specific Protein Phosphatases in Gram-Positive Bacteria2013In: Cell Reports, ISSN 2211-1247, Vol. 3, no 6, p. 1832-1839Article in journal (Refereed)
    Abstract [en]

    Many cellular pathways are regulated by the competing activity of protein kinases and phosphatases. The recent identification of arginine phosphorylation as a protein modification in bacteria prompted us to analyze the molecular basis of targeting phosphoarginine. In this work, we characterize an annotated tyrosine phosphatase, YwlE, that counteracts the protein arginine kinase McsB. Strikingly, structural studies of YwlE reaction intermediates provide a direct view on a captured arginine residue. Together with biochemical data, the crystal structures depict the evolution of a highly specific phospho-arginine phosphatase, with the use of a size-and-polarity filter for distinguishing phosphorylated arginine from other phosphorylated side chains. To confirm the proposed mechanism, we performed bioinformatic searches for phosphatases, employing a similar selectivity filter, and identified a protein in Drosophila melanogaster exhibiting robust arginine phosphatase activity. In sum, our findings uncover the molecular framework for specific targeting of phospho-arginine and suggest that protein arginine (de) phosphorylation may be relevant in eukaryotes.

  • 27. Fuhrmann, Jakob
    et al.
    Schmidt, Andreas
    Spiess, Silvia
    Lehner, Anita
    Turgay, Kürsad
    Mechtler, Karl
    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).
    Clausen, Tim
    McsB is a protein arginine kinase that phosphorylates and inhibits the heat-shock regulator CtsR2009In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 324, no 5932, p. 1323-1327Article in journal (Refereed)
    Abstract [en]

    All living organisms face a variety of environmental stresses that cause the misfolding and aggregation of proteins. To eliminate damaged proteins, cells developed highly efficient stress response and protein quality control systems. We performed a biochemical and structural analysis of the bacterial CtsR/McsB stress response. The crystal structure of the CtsR repressor, in complex with DNA, pinpointed key residues important for high-affinity binding to the promoter regions of heat-shock genes. Moreover, biochemical characterization of McsB revealed that McsB specifically phosphorylates arginine residues in the DNA binding domain of CtsR, thereby impairing its function as a repressor of stress response genes. Identification of the CtsR/McsB arginine phospho-switch expands the repertoire of possible protein modifications involved in prokaryotic and eukaryotic transcriptional regulation.

  • 28. Garnier, Fabien
    et al.
    Janapatla, Rajendra Prasad
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Masson, Geoffrey
    Grélaud, Carole
    Stach, Jean François
    Denis, François
    Ploy, Marie-Cécile
    Insertion sequence 1515 in the ply gene of a type 1 clinical isolate of Streptococcus pneumoniae abolishes pneurnolysin expression2007In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 45, no 7, p. 2296-2297Article in journal (Refereed)
    Abstract [en]

    Abstract: A serotype 1 Streptococcus pneumoniae strain isolated by blood culture from a woman with pneumonia was found to harbor insertion sequence (IS) 1515 in the pneumolysin gene, abolishing pneumolysin expression. To our knowledge, this is the first report of an IS in the pneumolysin gene of S. pneumoniae.

  • 29. Gratz, Nina
    et al.
    Siller, Maria
    Schaljo, Barbara
    Pirzada, Zaid A
    Gattermeier, Irene
    Vojtek, Ivo
    Kirschning, Carsten J
    Wagner, Hermann
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kovarik, Pavel
    Akira, Shizuo
    Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR92008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 29, p. 19879-19887Article in journal (Refereed)
    Abstract [en]

    Bacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-kappaB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins.

  • 30. Heckl, Dirk
    et al.
    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). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Germany ; Department of Regulation in Infection Biology, Hannover Medical School, Hannover, Germany.
    Toward whole-transcriptome editing with CRISPR-Cas92015In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 58, no 4, p. 560-562Article in journal (Other academic)
    Abstract [en]

    Targeted regulation of gene expression holds huge promise for biomedical research. In a series of recent publications (Gilbert et al., 2014; Konermann et al., 2015; Zalatan et al., 2015), sophisticated, multiplex-compatible transcriptional activator systems based on the CRISPR-Cas9 technology and genome-scale libraries advance the field toward whole-transcriptome control.

  • 31. Jinek, Martin
    et al.
    Chylinski, Krzysztof
    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).
    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).
    Hauer, Michael
    Doudna, Jennifer A
    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).
    A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity2012In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 337, no 6096, p. 816-821Article in journal (Refereed)
    Abstract [en]

    Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

  • 32. Jinek, Martin
    et al.
    Jiang, Fuguo
    Taylor, David W.
    Sternberg, Samuel H.
    Kaya, Emine
    Ma, Enbo
    Anders, Carolin
    Hauer, Michael
    Zhou, Kaihong
    Lin, Steven
    Kaplan, Matias
    Iavarone, Anthony T.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Nogales, Eva
    Doudna, Jennifer A.
    Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation2014In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 343, no 6176, p. 1215-Article in journal (Refereed)
    Abstract [en]

    Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

  • 33.
    Latvala, S.
    et al.
    Natl Inst Hlth & Welf, Dept Infect Dis Surveillance & Control, Virol Unit, Helsinki, Finland.
    Makela, S. M.
    Natl Inst Hlth & Welf, Dept Infect Dis Surveillance & Control, Virol Unit, Helsinki, Finland.
    Miettinen, M.
    Valio Ltd R&D, Helsinki, Finland.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Julkunen, I.
    Natl Inst Hlth & Welf THL, Dept Infect Dis Surveillance & Control, Virol Unit, Helsinki 00271, Finland; Univ Turku, Dept Virol, Turku, Finland.
    Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus pyogenes-infected human macrophages2014In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 178, no 2, p. 320-333Article in journal (Refereed)
    Abstract [en]

    In the present study, we have analysed the ability of Streptococcus pyogenes [Group A streptococcus (GAS)] to activate the NACHT-domain-, leucinerich repeat-and PYD-containing protein 3 (NALP3) inflammasome complex in human monocyte-derived macrophages and the molecules and signalling pathways involved in GAS-induced inflammatory responses. We focused upon analysing the impact of dynamin-dependent endocytosis and the role of major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in the immune responses induced by GAS. These virulence factors are involved in immune evasion by forming pores in host cell membranes, and aid the bacteria to escape from the endosome-lysosome pathway. We analysed cytokine gene expression in human primary macrophages after stimulation with live or inactivated wild-type GAS as well as with live SLO and SLS defective bacteria. Interleukin (IL)-1 beta, IL-10, tumour necrosis factor (TNF)-alpha and chemokine (C-X-C motif) ligand (CXCL)-10 cytokines were produced after bacterial stimulation in a dose-dependent manner and no differences in cytokine levels were seen between live, inactivated or mutant bacteria. These data suggest that streptolysins or other secreted bacterial products are not required for the inflammatory responses induced by GAS. Our data indicate that inhibition of dynamin-dependent endocytosis in macrophages attenuates the induction of IL-1 beta, TNF-alpha, interferon (IFN)-beta and CXCL-10 mRNAs. We also observed that pro-IL-1 beta protein was expressed and efficiently cleaved into mature-IL-1 beta via inflammasome activation after bacterial stimulation. Furthermore, we demonstrate that multiple signalling pathways are involved in GAS-stimulated inflammatory responses in human macrophages.

  • 34.
    Le Rhun, Anaïs
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    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).
    Small RNAs in streptococci2012In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 9, no 4, p. 414-426Article in journal (Refereed)
    Abstract [en]

    The group of streptococci includes species responsible for severe diseases in humans. To adapt to their environment and infect their hosts, streptococci depend on precise regulation of gene expression. The last decade has witnessed increasing findings of small RNAs (sRNAs) having regulatory functions in bacteria. More recently, genome-wide screens revealed that streptococcal genomes also encode multiple sRNAs. Some sRNAs including the class of CRISPR RNAs (crRNAs) play critical roles in streptococcal adaptation and virulence. Analysis of sRNA mechanisms uncovered three sRNAs that target in trans mRNA (FasX), sRNA (tracrRNA) and DNA (crRNA). Overall, the current understanding of sRNA-mediated regulation in streptococci remains very limited. Given the complexity of regulatory networks and the number of recently predicted sRNAs, future research should reveal new functions and mechanisms for the streptococcal sRNAs. Here, we provide a comprehensive summary of the information available on the topic.

  • 35. 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.

  • 36. 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.

  • 37. 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.

  • 38. 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.

  • 39. 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.

  • 40. 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.

  • 41. 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.

  • 42. 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)
  • 43. 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.

  • 44.
    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.

  • 45. 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.

  • 46. 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.

  • 47. 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.

  • 48. 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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