• A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum.

      Fabiani, Florian D; Renault, Thibaud T; Peters, Britta; Dietsche, Tobias; Gálvez, Eric J C; Guse, Alina; Freier, Karen; Charpentier, Emmanuelle; Strowig, Till; Franz-Wachtel, Mirita; et al. (2017-08)
      Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP-FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.
    • Identification of endoribonuclease specific cleavage positions reveals novel targets of RNase III in Streptococcus pyogenes.

      Le Rhun, Anaïs; Lécrivain, Anne-Laure; Reimegård, Johan; Proux-Wéra, Estelle; Broglia, Laura; Della Beffa, Cristina; Charpentier, Emmanuelle; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-03-17)
      A better understanding of transcriptional and post-transcriptional regulation of gene expression in bacteria relies on studying their transcriptome. RNA sequencing methods are used not only to assess RNA abundance but also the exact boundaries of primary and processed transcripts. Here, we developed a method, called identification of specific cleavage position (ISCP), which enables the identification of direct endoribonuclease targets in vivo by comparing the 5΄ and 3΄ ends of processed transcripts between wild type and RNase deficient strains. To demonstrate the ISCP method, we used as a model the double-stranded specific RNase III in the human pathogen Streptococcus pyogenes. We mapped 92 specific cleavage positions (SCPs) among which, 48 were previously described and 44 are new, with the characteristic 2 nucleotides 3΄ overhang of RNase III. Most SCPs were located in untranslated regions of RNAs. We screened for RNase III targets using transcriptomic differential expression analysis (DEA) and compared those with the RNase III targets identified using the ISCP method. Our study shows that in S. pyogenes, under standard growth conditions, RNase III has a limited impact both on antisense transcripts and on global gene expression with the expression of most of the affected genes being downregulated in an RNase III deletion mutant.
    • Host-derived extracellular RNA promotes adhesion of Streptococcus pneumoniae to endothelial and epithelial cells.

      Zakrzewicz, Dariusz; Bergmann, Simone; Didiasova, Miroslava; Giaimo, Benedetto Daniele; Borggrefe, Tilman; Mieth, Maren; Hocke, Andreas C; Lochnit, Guenter; Schaefer, Liliana; Hammerschmidt, Sven; et al. (2016-11-28)
      Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. The infection process involves bacterial cell surface receptors, which interact with host extracellular matrix components to facilitate colonization and dissemination of bacteria. Here, we investigated the role of host-derived extracellular RNA (eRNA) in the process of pneumococcal alveolar epithelial cell infection. Our study demonstrates that eRNA dose-dependently increased S. pneumoniae invasion of alveolar epithelial cells. Extracellular enolase (Eno), a plasminogen (Plg) receptor, was identified as a novel eRNA-binding protein on S. pneumoniae surface, and six Eno eRNA-binding sites including a C-terminal 15 amino acid motif containing lysine residue 434 were characterized. Although the substitution of lysine 434 for glycine (K434G) markedly diminished the binding of eRNA to Eno, the adherence to and internalization into alveolar epithelial cells of S. pneumoniae strain carrying the C-terminal lysine deletion and the mutation of internal Plg-binding motif were only marginally impaired. Accordingly, using a mass spectrometric approach, we identified seven novel eRNA-binding proteins in pneumococcal cell wall. Given the high number of eRNA-interacting proteins on pneumococci, treatment with RNase1 completely inhibited eRNA-mediated pneumococcal alveolar epithelial cell infection. Our data support further efforts to employ RNAse1 as an antimicrobial agent to combat pneumococcal infectious diseases.
    • Adaptation in CRISPR-Cas Systems.

      Sternberg, Samuel H; Richter, Hagen; Charpentier, Emmanuelle; Qimron, Udi; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-03-17)
      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.
    • RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.

      Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-02)
      Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.
    • A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus.

      Resch, Ulrike; Tsatsaronis, James Anthony; Le Rhun, Anaïs; Stübiger, Gerald; Rohde, M; Kasvandik, Sergo; Holzmeister, Susanne; Tinnefeld, Philip; Wai, Sun Nyunt; Charpentier, Emmanuelle; et al. (2016)
      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.
    • Interferon-γ-inducible Rab20 regulates endosomal morphology and EGFR degradation in macrophages.

      Pei, Gang; Schnettger, Laura; Bronietzki, Marc; Repnik, Urska; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel; Helmholtz Centre for infection research (HZI), 38124 Braunschweig, Germany. (2015-09-01)
      Little is known about the molecular players that regulate changes in the endocytic pathway during immune activation. Here we investigate the role of Rab20 in the endocytic pathway during activation of macrophages. Rab20 is associated with endocytic structures, but the function of this Rab GTPase in the endocytic pathway remains poorly characterized. We find that in macrophages, Rab20 expression and endosomal association significantly increase after interferon-γ (IFN-γ) treatment. Moreover, IFN-γ and Rab20 expression induce a dramatic enlargement of endosomes. These enlarged endosomes are the result of homotypic fusion promoted by Rab20 expression. The expression of Rab20 or the dominant-negative mutant Rab20T19N does not affect transferrin or dextran 70 kDa uptake. However, knockdown of Rab20 accelerates epidermal growth factor (EGF) trafficking to LAMP-2-positive compartments and EGF receptor degradation. Thus this work defines a function for Rab20 in the endocytic pathway during immune activation of macrophages.
    • Toward Whole-Transcriptome Editing with CRISPR-Cas9.

      Heckl, Dirk; Charpentier, Emmanuelle; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-05-21)
      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.
    • Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity.

      Charpentier, Emmanuelle; Richter, Hagen; van der Oost, John; White, Malcolm F; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015-05)
      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.
    • CRISPR-Cas9: how research on a bacterial RNA-guided mechanism opened new perspectives in biotechnology and biomedicine.

      Charpentier, Emmanuelle; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-04)
    • Experimental selection of long-term intracellular mycobacteria.

      Vázquez, Cristina L; Lerner, Thomas R; Kasmapour, Bahram; Pei, Gang; Gronow, Achim; Bianco, Maria V; Blanco, Federico C; Geffers, Robert; Geffers, Robert; Bigi, Fabiana; et al. (2014-09)
      Some intracellular bacteria are known to cause long-term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long-term intracellular lifestyle. Selected Mycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re-infection and also with the specific expression of stress- and survival-related genes. Our findings identify bacterial traits implicated in the establishment of long-term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments.
    • Increased neutrophil extracellular trap-mediated Staphylococcus aureus clearance through inhibition of nuclease activity by clindamycin and immunoglobulin.

      Schilcher, Katrin; Andreoni, Federica; Uchiyama, Satoshi; Ogawa, Taiji; Schuepbach, Reto A; Zinkernagel, Annelies S; Helmholtz Centre for infection reseach, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-08-01)
      The Gram-positive human pathogen Staphylococcus aureus causes a variety of human diseases such as skin infections, pneumonia, and endocarditis. The micrococcal nuclease Nuc1 is one of the major S. aureus virulence factors and allows the bacterium to avoid neutrophil extracellular trap (NET)-mediated killing. We found that addition of the protein synthesis inhibitor clindamycin to S. aureus LAC cultures decreased nuc1 transcription and subsequently blunted nuclease activity in a molecular beacon-based fluorescence assay. We also observed reduced NET degradation through Nuc1 inhibition translating into increased NET-mediated clearance. Similarly, pooled human immunoglobulin specifically inhibited nuclease activity in a concentration-dependent manner. Inhibition of nuclease activity by clindamycin and immunoglobulin enhanced S. aureus clearance and should be considered in the treatment of S. aureus infections.
    • Identification of an immune-regulated phagosomal Rab cascade in macrophages.

      Pei, Gang; Repnik, Urska; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel; Helmholtz Centre for infection research, Inhoffenstr. 7 , D-38124 Braunschweig, Germany. (2014-05-01)
      Interferon-γ (IFN-γ) has been shown to regulate phagosome trafficking and function in macrophages, but the molecular mechanisms involved are poorly understood. Here, we identify Rab20 as part of the machinery by which IFN-γ controls phagosome maturation. We found that IFN-γ stimulates the association of Rab20 with early phagosomes in macrophages. By using imaging of single phagosomes in live cells, we found that Rab20 induces an early delay in phagosome maturation and extends the time for which Rab5a and phosphatidylinositol 3-phosphate (PI3P) remain associated with phagosomes. Moreover, Rab20 depletion in macrophages abrogates the delay in phagosome maturation induced by IFN-γ. Finally, we demonstrate that Rab20 interacts with the Rab5a guanine nucleotide exchange factor Rabex-5 (also known as RABGEF1) and that Rab20 knockdown impairs the IFN-γ-dependent recruitment of Rabex-5 and Rab5a into phagosomes. Taken together, here, we uncover Rab20 as a key player in the Rab cascade by which IFN-γ induces a delay in phagosome maturation in macrophages.
    • Genome sequence of the moderately thermophilic sulfur-reducing bacterium Thermanaerovibrio velox type strain (Z-9701T) and emended description of the genus Thermanaerovibrio

      Palaniappan, Krishna; Meier-Kolthoff, Jan P.; Teshima, Hazuki; Nolan, Matt; Lapidus, Alla; Tice, Hope; Del Rio, Tijana Glavina; Cheng, Jan-Fang; Han, Cliff; Tapia, Roxanne; et al. (2014-01-06)
    • Low-density lipoprotein receptor-related protein-1 mediates endocytic clearance of tissue inhibitor of metalloproteinases-1 and promotes its cytokine-like activities.

      Thevenard, Jessica; Verzeaux, Laurie; Devy, Jerôme; Etique, Nicolas; Jeanne, Albin; Schneider, Christophe; Hachet, Cathy; Ferracci, Géraldine; David, Marion; Martiny, Laurent; et al. (2014)
      Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the extracellular matrix turnover by inhibiting the proteolytic activity of matrix metalloproteinases (MMPs). TIMP-1 also displays MMP-independent activities that influence the behavior of various cell types including neuronal plasticity, but the underlying molecular mechanisms remain mostly unknown. The trans-membrane receptor low-density lipoprotein receptor-related protein-1 (LRP-1) consists of a large extracellular chain with distinct ligand-binding domains that interact with numerous ligands including TIMP-2 and TIMP-3 and a short transmembrane chain with intracellular motifs that allow endocytosis and confer signaling properties to LRP-1. We addressed TIMP-1 interaction with recombinant ligand-binding domains of LRP-1 expressed by CHO cells for endocytosis study, or linked onto sensor chips for surface plasmon resonance analysis. Primary cortical neurons bound and internalized endogenous TIMP-1 through a mechanism mediated by LRP-1. This resulted in inhibition of neurite outgrowth and increased growth cone volume. Using a mutated inactive TIMP-1 variant we showed that TIMP-1 effect on neurone morphology was independent of its MMP inhibitory activity. We conclude that TIMP-1 is a new ligand of LRP-1 and we highlight a new example of its MMP-independent, cytokine-like functions.
    • The M1 protein of Streptococcus pyogenes triggers an innate uptake mechanism into polarized human endothelial cells.

      Ochel, Anja; Rohde, Manfred; Chhatwal, Gursharan S; Talay, Susanne R; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweíg, Germany. (2014)
      Serotype M1 Streptococcus pyogenes is a major human pathogen associated with severe invasive diseases causing high morbidity and mortality. In a substantial number of cases, invasive disease develops in previously healthy individuals with no obvious port of entry. This has led to the hypothesis that the source of streptococci in these cases is a transient bacteraemia. This study focuses on the analysis of interaction of tissue-invasive serotype M1 S. pyogenes with human endothelial cells (EC) of the vascular system. We identify the M1 surface protein of S. pyogenes as the EC invasin which is recognised by polarized human blood EC, thereby triggering rapid, phagocytosis-like uptake of streptococci into polarized EC layers. Upon internalization, the M1 S. pyogenes serotype is incorporated into phagosomes which traffic via the endosomal/lysosomal pathway. However, some of the streptococci successfully evade this innate killing process and hereby mediate their escape into the cytoplasm of the host cell. The results of this study demonstrate that blood EC possess an efficient uptake mechanism for serotype M1 S. pyogenes. Despite efficient phagocytosis, streptococcal survival within EC constitutes one potential mechanism which favours intracellular persistence and thus facilitates continuous infection and dissemination from the primary side of infection into deep tissue.
    • Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.

      Fonfara, Ines; Le Rhun, Anaïs; Chylinski, Krzysztof; Makarova, Kira S; Lécrivain, Anne-Laure; Bzdrenga, Janek; Koonin, Eugene V; Charpentier, Emmanuelle; The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå S-90187, Sweden, Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig D-38124, Germany, Deptartment of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Vienna A-1030, Austria, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA and Hannover Medical School, Hannover D-30625, Germany. (2013-11-22)
      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.
    • Highly stable monodisperse PEGylated iron oxide nanoparticle aqueous suspensions: a nontoxic tracer for homogeneous magnetic bioassays.

      Lak, Aidin; Dieckhoff, Jan; Ludwig, Frank; Scholtyssek, Jan M; Goldmann, Oliver; Lünsdorf, Heinrich; Eberbeck, Dietmar; Kornowski, Andreas; Kraken, Mathias; Litterst, F J; et al. (2013-11-07)
      Uniformly sized and shaped iron oxide nanoparticles with a mean size of 25 nm were synthesized via decomposition of iron-oleate. High resolution transmission electron microscopy and Mössbauer spectroscopy investigations revealed that the particles are spheres primarily composed of Fe3O4 with a small fraction of FeO. From Mössbauer and static magnetization measurements, it was deduced that the particles are superparamagnetic at room temperature. The hydrophobic particles were successfully transferred into water via PEGylation using nitrodopamine as an anchoring group. IR spectroscopy and thermogravimetric analysis showed the success and efficiency of the phase transfer reaction. After PEGylation, the particles retained monodispersity and their magnetic core remained intact as proven by photon cross-correlation spectroscopy, ac susceptibility, and transmission electron microscopy. The particle aqueous suspensions revealed excellent water stability over a month of monitoring and also against temperature up to 40 °C. The particles exhibited a moderate cytotoxic effect on in vitro cultured bone marrow-derived macrophages and no release of inflammatory or anti-inflammatory cytokines. The PEGylated particles were functionalized with Herceptin antibodies via a conjugation chemistry, their response to a rotating magnetic field was studied using a fluxgate-based setup and was compared with the one recorded for hydrophobic and PEGylated particles. The particle phase lag rose after labeling with Herceptin, indicating the successful conjugation of Herceptin antibodies to the particles.
    • Study of the in vivo role of Mce2R, the transcriptional regulator of mce2 operon in Mycobacterium tuberculosis

      Forrellad, Marina A; Bianco, María V; Blanco, Federico C; Nuñez, Javier; Klepp, Laura I; Vazquez, Cristina L; Santangelo, María d l P; Rocha, Rosana V; Soria, Marcelo; Golby, Paul; et al. (2013-09-05)
      Abstract Background Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the agent of human tuberculosis, has developed strategies involving proteins and other compounds called virulence factors to subvert human host defences and damage and invade the human host. Among these virulence-related proteins are the Mce proteins, which are encoded in the mce1, mce2, mce3 and mce4 operons of M. tuberculosis. The expression of the mce2 operon is negatively regulated by the Mce2R transcriptional repressor. Here we evaluated the role of Mce2R during the infection of M. tuberculosis in mice and macrophages and defined the genes whose expression is in vitro regulated by this transcriptional repressor. Results We used a specialized transduction method for generating a mce2R mutant of M. tuberculosis H37Rv. Although we found equivalent replication of the MtΔmce2R mutant and the wild type strains in mouse lungs, overexpression of Mce2R in the complemented strain (MtΔmce2RComp) significantly impaired its replication. During in vitro infection of macrophages, we observed a significantly increased association of the late endosomal marker LAMP-2 to MtΔmce2RComp-containing phagosomes as compared to MtΔmce2R and the wild type strains. Whole transcriptional analysis showed that Mce2R regulates mainly the expression of the mce2 operon, in the in vitro conditions studied. Conclusions The findings of the current study indicate that Mce2R weakly represses the in vivo expression of the mce2 operon in the studied conditions and argue for a role of the proteins encoded in Mce2R regulon in the arrest of phagosome maturation induced by M. tuberculosis.
    • The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems.

      Chylinski, Krzysztof; Le Rhun, Anaïs; Charpentier, Emmanuelle; The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå, Sweden. (2013-05)
      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.