RESUMO
The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in "CRISPRology".
Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Listeria monocytogenes/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA não Traduzido/genética , Proteínas de Bactérias , Proteínas de Ligação a DNA/genética , Genoma Bacteriano , Humanos , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.
Assuntos
Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , RNA Bacteriano/genética , Transcrição Gênica/genética , Animais , Genes Bacterianos/genética , Genoma Bacteriano/genética , Intestinos/microbiologia , Camundongos , Fases de Leitura Aberta/genética , Óperon/genética , RNA Bacteriano/análise , Sequências Reguladoras de Ácido Ribonucleico/genética , Regiões não Traduzidas/genética , Virulência/genéticaRESUMO
Listeria monocytogenes is a human, food-borne pathogen. Genomic comparisons between L. monocytogenes and Listeria innocua, a closely related non-pathogenic species, were pivotal in the identification of protein-coding genes essential for virulence. However, no comprehensive comparison has focused on the non-coding genome. We used strand-specific cDNA sequencing to produce genome-wide transcription start site maps for both organisms, and developed a publicly available integrative browser to visualize and analyze both transcriptomes in different growth conditions and genetic backgrounds. Our data revealed conservation across most transcripts, but significant divergence between the species in a subset of non-coding RNAs. In L. monocytogenes, we identified 113 small RNAs (33 novel) and 70 antisense RNAs (53 novel), significantly increasing the repertoire of ncRNAs in this species. Remarkably, we identified a class of long antisense transcripts (lasRNAs) that overlap one gene while also serving as the 5' UTR of the adjacent divergent gene. Experimental evidence suggests that lasRNAs transcription inhibits expression of one operon while activating the expression of another. Such a lasRNA/operon structure, that we named 'excludon', might represent a novel form of regulation in bacteria.
Assuntos
Listeria/genética , Listeria/patogenicidade , RNA Antissenso/genética , Transcriptoma , Regiões 5' não Traduzidas , Sequência de Bases , Evolução Biológica , Sequência Conservada , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Dados de Sequência Molecular , Óperon , RNA Antissenso/metabolismo , RNA Bacteriano , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Listeria monocytogenes is an intracellular pathogen that can enter and invade host cells. In the course of its infection, RNA-mediated regulatory mechanisms provide a fast and versatile response for both the bacterium and the host. They regulate a variety of processes, such as environment sensing and virulence in pathogenic bacteria, as well as development, cellular differentiation, metabolism and immune responses in eukaryotic cells. The aim of this article is to summarize first the RNA-mediated regulatory mechanisms that play a role in the Listeria lifestyle and in its virulence, and then the host miRNA responses to Listeria infection. Finally, we discuss the potential cross-talk between bacterial RNAs and host RNA regulatory mechanisms as new mechanisms of bacterial virulence.
Assuntos
Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Listeriose/metabolismo , RNA Bacteriano/genética , Interações Hospedeiro-Patógeno , Humanos , Intestinos/microbiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Microbiota , Fatores de VirulênciaRESUMO
In recent years, non-coding RNAs have emerged as key regulators of gene expression. Among these RNAs, the antisense RNAs (asRNAs) are particularly abundant, but in most cases the function and mechanism of action for a particular asRNA remains elusive. Here, we highlight a recently discovered paradigm termed the excludon, which defines a genomic locus encoding an unusually long asRNA that spans divergent genes or operons with related or opposing functions. Because these asRNAs can inhibit the expression of one operon while functioning as an mRNA for the adjacent operon, they act as fine-tuning regulatory switches in bacteria.