Peñaloza Diego, Acuña Lillian G, Barros M José, Núñez Paula, Montt Fernanda, Gil Fernando, Fuentes Juan A, Calderón Iván L
Laboratorio de RNAs Bacterianos, Departamento de Ciencias Biológicas, Facultad de Ciencias de la Vida, Universidad Andres Bello, 8370186 Santiago, Chile.
Microbiota-Host Interactions and Clostridia Research Group, Departamento de Ciencias Biológicas, Facultad de Ciencias de la Vida, Universidad Andres Bello, 8370186 Santiago, Chile.
Microorganisms. 2021 Mar 18;9(3):635. doi: 10.3390/microorganisms9030635.
Growing evidence indicates that small noncoding RNAs (sRNAs) play important regulatory roles during bacterial infection. In Typhimurium, several sRNAs are strongly up-regulated within macrophages, but little is known about their role during the infection process. Among these sRNAs, the well-characterized paralogs RyhB-1 and RyhB-2 are two regulators of gene expression mainly related with the response to iron availability. To investigate the role of the sRNAs RyhB-1 and RyhB-2 from . Typhimurium in the infection of RAW264.7 macrophages, we analyzed several phenotypic traits from intracellular mutant strains lacking one and both sRNAs. Deletion of RyhB-1 and/or RyhB-2 resulted in increased intracellular survival and faster replication within macrophages. The bacterial metabolic status inside macrophages was also analyzed, revealing that all the mutant strains exhibited higher intracellular levels of ATP and lower NAD/NADH ratios than the wild type. Expression analyses from bacteria infecting macrophages showed that RyhB-1 and RyhB-2 affect the intra-macrophage expression of bacterial genes associated with the pathogenicity island 1 (SPI-1) and the type III secretion system (T3SS). With a two-plasmid system and compensatory mutations, we confirmed that RyhB-1 and RyhB-2 directly interact with the mRNAs of the invasion chaperone SicA and the regulatory protein RtsB. Altogether, these results indicate that the RyhB homologs contribute to the . Typhimurium virulence modulation inside macrophages by reducing the intracellular growth and down-regulating the SPI-1 gene expression.
越来越多的证据表明，小非编码RNA（sRNA）在细菌感染过程中发挥着重要的调节作用。在鼠伤寒沙门氏菌中，几种sRNA在巨噬细胞内强烈上调，但其在感染过程中的作用却知之甚少。在这些sRNA中，特征明确的旁系同源物RyhB-1和RyhB-2是主要与铁可用性反应相关的两个基因表达调节因子。为了研究鼠伤寒沙门氏菌的sRNA RyhB-1和RyhB-2在RAW264.7巨噬细胞感染中的作用，我们分析了缺乏一种和两种sRNA的细胞内突变菌株的几种表型特征。RyhB-1和/或RyhB-2的缺失导致细胞内存活率增加和在巨噬细胞内更快地复制。还分析了巨噬细胞内细菌的代谢状态，发现所有突变菌株的细胞内ATP水平均高于野生型，而NAD/NADH比值则低于野生型。对感染巨噬细胞的细菌进行的表达分析表明，RyhB-1和RyhB-2影响与致病岛1（SPI-1）和III型分泌系统（T3SS）相关的细菌基因在巨噬细胞内的表达。通过双质粒系统和补偿性突变，我们证实RyhB-1和RyhB-2直接与侵袭伴侣蛋白SicA和调节蛋白RtsB的mRNA相互作用。总之，这些结果表明，RyhB同源物通过降低细胞内生长和下调SPI-1基因表达，有助于调节鼠伤寒沙门氏菌在巨噬细胞内的毒力。
