RESUMO
CRISPR interference confers adaptive, sequence-based immunity against viruses and plasmids and is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from spacer-repeat units. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here, our studies of crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp. reveal a unique crRNA maturation pathway in which crRNAs are transcribed from promoters that are embedded within each repeat, yielding crRNA 5' ends formed by transcription and not by processing. Although crRNA 3' end formation involves RNase III and trans-encoded tracrRNA, as in other type II CRISPR systems, this processing is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/Cas system characterized to date. Endogenous CRISPR spacers limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in the human pathogen N. meningitidis.
Assuntos
Sequências Repetidas Invertidas/genética , Neisseria meningitidis/genética , RNA Bacteriano/genética , Transformação Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Genes Bacterianos/genética , Interações Hospedeiro-Patógeno , Humanos , Infecções Meningocócicas/microbiologia , Modelos Genéticos , Neisseria meningitidis/patogenicidade , Neisseria meningitidis/fisiologia , Regiões Promotoras Genéticas/genética , Processamento Pós-Transcricional do RNA , RNA Bacteriano/metabolismo , Ribonuclease III/metabolismo , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Virulência/genéticaRESUMO
BACKGROUND: Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. RESULTS: Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region. CONCLUSIONS: Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.
Assuntos
Regulação Bacteriana da Expressão Gênica , Variação Genética , Neisseria meningitidis/genética , Transcriptoma , Virulência/genética , Técnicas de Tipagem Bacteriana , Biomarcadores , Análise por Conglomerados , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Genes Reguladores , Genoma Bacteriano , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Meningite Meningocócica/sangue , Meningite Meningocócica/metabolismo , Meningite Meningocócica/microbiologia , Anotação de Sequência Molecular , Neisseria meningitidis/classificação , Neisseria meningitidis/patogenicidade , Fenótipo , Regiões Promotoras GenéticasRESUMO
A longstanding question in infection biology addresses the genetic basis for invasive behavior in commensal pathogens. A prime example for such a pathogen is Neisseria meningitidis. On the one hand it is a harmless commensal bacterium exquisitely adapted to humans, and on the other hand it sometimes behaves like a ferocious pathogen causing potentially lethal disease such as sepsis and acute bacterial meningitis. Despite the lack of a classical repertoire of virulence genes in N. meningitidis separating commensal from invasive strains, molecular epidemiology suggests that carriage and invasive strains belong to genetically distinct populations. In recent years, it has become increasingly clear that metabolic adaptation enables meningococci to exploit host resources, supporting the concept of nutritional virulence as a crucial determinant of invasive capability. Here, we discuss the contribution of core metabolic pathways in the context of colonization and invasion with special emphasis on results from genome-wide surveys. The metabolism of lactate, the oxidative stress response, and, in particular, glutathione metabolism as well as the denitrification pathway provide examples of how meningococcal metabolism is intimately linked to pathogenesis. We further discuss evidence from genome-wide approaches regarding potential metabolic differences between strains from hyperinvasive and carriage lineages and present new data assessing in vitro growth differences of strains from these two populations. We hypothesize that strains from carriage and hyperinvasive lineages differ in the expression of regulatory genes involved particularly in stress responses and amino acid metabolism under infection conditions.