• 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.
    • 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.
    • Tumor necrosis factor alpha modulates the dynamics of the plasminogen-mediated early interaction between Bifidobacterium animalis subsp. lactis and human enterocytes.

      Centanni, Manuela; Bergmann, Simone; Turroni, Silvia; Hammerschmidt, Sven; Chhatwal, Gursharan Singh; Brigidi, Patrizia; Candela, Marco; Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy. (2012-04)
      The capacity to intervene with the host plasminogen system has recently been considered an important component in the interaction process between Bifidobacterium animalis subsp. lactis and the human host. However, its significance in the bifidobacterial microecology within the human gastrointestinal tract is still an open question. Here we demonstrate that human plasminogen favors the B. animalis subsp. lactis BI07 adhesion to HT29 cells. Prompting the HT29 cell capacity to activate plasminogen, tumor necrosis factor alpha (TNF-α) modulated the plasminogen-mediated bacterium-enterocyte interaction, reducing the bacterial adhesion to the enterocytes and enhancing migration to the luminal compartment.
    • 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.
    • Variability in the distribution of genes encoding virulence factors and putative extracellular proteins of Streptococcus pyogenes in India, a region with high streptococcal disease burden, and implication for development of a regional multisubunit vaccine.

      Sagar, Vivek; Bergmann, René; Nerlich, Andreas; McMillan, David J; Nitsche Schmitz, D Patric; Chhatwal, Gursharan S; Department of Medical Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2012-11)
      Streptococcus pyogenes causes a wide variety of human diseases and is a significant cause of morbidity and mortality. Attempts to develop a vaccine were hampered by the genetic diversity of S. pyogenes across different regions of the world. This study sought to identify streptococcal antigens suitable for a region-specific vaccine in India. We used a two-step approach, first performing epidemiological analysis to identify the conserved antigens among Indian isolates. The second step consisted of validating the identified antigens by serological analysis. The 201 streptococcal clinical isolates from India used in this study represented 69 different emm types, with emm12 being the most prevalent. Virulence profiling of the North and South Indian S. pyogenes isolates with a custom-designed streptococcal virulence microarray identified seven conserved putative vaccine candidates. Collagen-like surface protein (SCI), putative secreted 5'-nucleotidase (PSNT), and C5a peptidase were found in 100% of the isolates, while R28, a putative surface antigen (PSA), and a hypothetical protein (HYP) were found in 90% of the isolates. A fibronectin binding protein, SfbI, was present in only 78% of the isolates. In order to validate the identified potential vaccine candidates, 185 serum samples obtained from patients with different clinical manifestations were tested for antibodies. Irrespective of clinical manifestations, serum samples showed high antibody titers to all proteins except for SCI and R28. Thus, the data indicate that PSNT, C5a peptidase, PSA, HYP, and SfbI are promising candidates for a region-specific streptococcal vaccine for the different parts of India.
    • Virulence gene pool detected in bovine group C Streptococcus dysgalactiae subsp. dysgalactiae isolates by use of a group A S. pyogenes virulence microarray.

      Rato, Márcia G; Nerlich, Andreas; Bergmann, René; Bexiga, Ricardo; Nunes, Sandro F; Vilela, Cristina L; Santos-Sanches, Ilda; Chhatwal, Gursharan S; Centro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal. (2011-07)
      A custom-designed microarray containing 220 virulence genes of Streptococcus pyogenes (group A Streptococcus [GAS]) was used to test group C Streptococcus dysgalactiae subsp. dysgalactiae (GCS) field strains causing bovine mastitis and group C or group G Streptococcus dysgalactiae subsp. equisimilis (GCS/GGS) isolates from human infections, with the latter being used for comparative purposes, for the presence of virulence genes. All bovine and all human isolates carried a fraction of the 220 genes (23% and 39%, respectively). The virulence genes encoding streptolysin S, glyceraldehyde-3-phosphate dehydrogenase, the plasminogen-binding M-like protein PAM, and the collagen-like protein SclB were detected in the majority of both bovine and human isolates (94 to 100%). Virulence factors, usually carried by human beta-hemolytic streptococcal pathogens, such as streptokinase, laminin-binding protein, and the C5a peptidase precursor, were detected in all human isolates but not in bovine isolates. Additionally, GAS bacteriophage-associated virulence genes encoding superantigens, DNase, and/or streptodornase were detected in bovine isolates (72%) but not in the human isolates. Determinants located in non-bacteriophage-related mobile elements, such as the gene encoding R28, were detected in all bovine and human isolates. Several virulence genes, including genes of bacteriophage origin, were shown to be expressed by reverse transcriptase PCR (RT-PCR). Phylogenetic analysis of superantigen gene sequences revealed a high level (>98%) of identity among genes of bovine GCS, of the horse pathogen Streptococcus equi subsp. equi, and of the human pathogen GAS. Our findings indicate that alpha-hemolytic bovine GCS, an important mastitis pathogen and considered to be a nonhuman pathogen, carries important virulence factors responsible for virulence and pathogenesis in humans.