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Non-coding genome in bacterial virulence: characterization of regulatory rnas in listeria monocytogenes
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RESUMO: Listeria monocytogenes é um patógeno alimentar, gram-positivo, que causa listeriose e, em casos graves, meningite e infeções feto-placentárias. Devido à sua capacidade de atravessar as barreiras intestinal, hematoencefálica e placentária, L. monocytogenes serve como um excelente modelo para compreender as interações entre um hospedeiro e um patógeno. Os pequenos RNAs não codificantes (sRNAs) são uma classe de reguladores da expressão génica bacteriana importantes em muitos processos fisiológicos, incluindo a virulência. Mais de 300 sRNAs foram identificados em L. monocytogenes, mas apenas um pequeno número foi caracterizado (53-60), para o seu papel na patogenicidade da bactéria. Neste estudo, uma abordagem de genómica comparativa foi utilizada para identificar sRNAs com elevado potencial de serem novos reguladores de virulência em L. monocytogenes, que estão ausentes no genoma da Listeria innocua, não patogénica. Além disso, 33 sRNAs candidatos de reguladores de virulência foram selecionados através de genómica comparativa entre L. monocytogenes e L. innocua. Critérios rigorosos, incluindo a previsão de alvos de mRNA in silico e a comparação com sRNomas centrais de outras espécies de Listeria patogénicas e não patogénicas, bem como uma estirpe avirulenta de L. monocytogenes, permitiram reduzir a lista de candidatos a 6 sRNAs com maior probabilidade de atuarem como reguladores de virulência: Rli24, Rli77, Rli107, Rli118, Rli133 e Rli142. A construção de mutantes de deleção usando o vetor pMAD e recombinação homóloga na L. monocytogenes EGD-e foi bem-sucedida para Δrli24, Δrli107, Δrli133 e Δrli142. O potencial de virulência desses sRNAs foi testado in vitro usando células epiteliais intestinais humanas da linha celular Caco-2. Enquanto as estirpes Δrli24, Δrli107 e Δrli142 não demonstraram impacto na virulência de L. monocytogenes nas condições testadas, o mutante Δrli133 apresentou uma redução de quase 30% nas bactérias intracelulares pós-infeção. Este trabalho demonstrou que Rli133 é um novo sRNA envolvido na regulação da patogenicidade de L. monocytogenes, destacando o seu potencial papel positivo na ativação de vias de virulência.
ABSTRACT: Listeria monocytogenesis a gram-positive foodborne pathogen causing listeriosis and in severe cases, meningitis and maternofetal infections. Because of its capability of crossing the intestinal, blood-brain, and placental barriers, L. monocytogenes serves as an excellent model for understanding the interactions between a host and a pathogen. Small non-coding RNAs (sRNAs) are a class of bacterial gene expression regulators important in many physiological processes, including virulence. More than 300 sRNAs have been identified in L. monocytogenes but only a small number have been characterized (53-60), highlighting the great potential of sRNAs to act as novel regulators of bacterial pathogenicity. In this study, a comparative genomics approach was used to identify sRNAs with high potential to be new regulators of virulence in L. monocytogenes, which were absent from the genome of the closely related non-pathogenic Listeria innocua. Strict criteria involving in silico mRNA target prediction and comparison to core sRNomes of other pathogenic, non-pathogenic Listeria species and one avirulent L. monocytogenes strain allowed to narrow the list of sRNAs candidates. The top six sRNAs selected for their higher probability to act as virulence regulators of L. monocytogenes were Rli24, Rli77, Rli107, Rli118, Rli133, and Rli142. Deletion mutant construction in L. monocytogenes EGD-e strain were successful for rli24, rli107, rli133, and rli142. The virulence potential of these sRNAs was tested in vitro using the human intestinal epithelial Caco-2 cells. While rli24, rli107 and rli142 strains were not found to affect the infectivity of L. monocytogenes under the conditions tested, the rli133 mutant showed a nearly 30% decrease in intracellular bacteria post-infection. This work demonstrated that Rli133 is a newly sRNA involved in the regulation of L. monocytogenes pathogenicity, highlighting its potential positive role in activating virulence pathways.
ABSTRACT: Listeria monocytogenesis a gram-positive foodborne pathogen causing listeriosis and in severe cases, meningitis and maternofetal infections. Because of its capability of crossing the intestinal, blood-brain, and placental barriers, L. monocytogenes serves as an excellent model for understanding the interactions between a host and a pathogen. Small non-coding RNAs (sRNAs) are a class of bacterial gene expression regulators important in many physiological processes, including virulence. More than 300 sRNAs have been identified in L. monocytogenes but only a small number have been characterized (53-60), highlighting the great potential of sRNAs to act as novel regulators of bacterial pathogenicity. In this study, a comparative genomics approach was used to identify sRNAs with high potential to be new regulators of virulence in L. monocytogenes, which were absent from the genome of the closely related non-pathogenic Listeria innocua. Strict criteria involving in silico mRNA target prediction and comparison to core sRNomes of other pathogenic, non-pathogenic Listeria species and one avirulent L. monocytogenes strain allowed to narrow the list of sRNAs candidates. The top six sRNAs selected for their higher probability to act as virulence regulators of L. monocytogenes were Rli24, Rli77, Rli107, Rli118, Rli133, and Rli142. Deletion mutant construction in L. monocytogenes EGD-e strain were successful for rli24, rli107, rli133, and rli142. The virulence potential of these sRNAs was tested in vitro using the human intestinal epithelial Caco-2 cells. While rli24, rli107 and rli142 strains were not found to affect the infectivity of L. monocytogenes under the conditions tested, the rli133 mutant showed a nearly 30% decrease in intracellular bacteria post-infection. This work demonstrated that Rli133 is a newly sRNA involved in the regulation of L. monocytogenes pathogenicity, highlighting its potential positive role in activating virulence pathways.
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Listeria monocytogenes virulence Regulatory RNAs sRNome of Listeria