A small RNA controls expression of the chitinase ChiA in Listeria monocytogenes. Listeria monocytogenes

In recent years, more than 60 small RNAs (sRNAs) have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo0850, which encodes a small protein of unknown function. LhrA controls the translation and degradation of the lmo0850 mRNA by an antisense mechanism, and it depends on the RNA chaperone Hfq for efficient binding to its target. In the present study, we sought to gain more insight into the functional role of LhrA in L. monocytogenes. To this end, we determined the effects of LhrA on global-wide gene expression. We observed that nearly 300 genes in L. monocytogenes are either positively or negatively affected by LhrA. Among these genes, we identified lmo0302 and chiA as direct targets of LhrA, thus establishing LhrA as a multiple target regulator. Lmo0302 encodes a hypothetical protein with no known function, whereas chiA encodes one of two chitinases present in L. monocytogenes. We show here that LhrA acts as a post-transcriptional regulator of lmo0302 and chiA by interfering with ribosome recruitment, and we provide evidence that both LhrA and Hfq act to down-regulate the expression of lmo0302 and chiA. Furthermore, in vitro binding experiments show that Hfq stimulates the base pairing of LhrA to chiA mRNA. Finally, we demonstrate that LhrA has a negative effect on the chitinolytic activity of L. monocytogenes. In marked contrast to this, we found that Hfq has a stimulating effect on the chitinolytic activity, suggesting that Hfq plays multiple roles in the complex regulatory pathways controlling the chitinases of L. monocytogenes. Overall design: cDNA from 4 independent replicates of wild type EGD was directly compared to cDNA from 4 independent replicates of EGD delta LhrA

近年来，研究人员已在革兰氏阳性人类致病菌单核细胞增生李斯特菌（Listeria monocytogenes）中鉴定出60余种小RNA（sRNA），但绝大多数小RNA的推测功能与作用机制仍不明晰。此前已有研究表明，小RNA LhrA是单基因lmo0850的转录后调控因子，该基因编码一种功能未知的小型蛋白质。LhrA通过反义机制调控lmo0850 mRNA的翻译与降解过程，且其与靶标结合需依赖RNA分子伴侣Hfq以实现高效结合。本研究旨在深入解析LhrA在单核细胞增生李斯特菌中的功能角色。为此，我们分析了LhrA对全基因组基因表达的影响。研究发现，单核细胞增生李斯特菌中近300个基因的表达受LhrA的正向或负向调控。在这些基因中，我们鉴定出lmo0302与chiA为LhrA的直接靶标，由此证实LhrA属于多靶标调控因子。Lmo0302编码一种功能未知的假设蛋白，而chiA则编码单核细胞增生李斯特菌中的两种几丁质酶之一。本研究证实，LhrA通过干扰核糖体招募实现对lmo0302与chiA的转录后调控，且实验证据表明LhrA与Hfq均可下调lmo0302和chiA的表达水平。此外，体外结合实验证实，Hfq可促进LhrA与chiA mRNA的碱基配对。最终，我们证实LhrA对单核细胞增生李斯特菌的几丁质酶活性具有抑制作用。与此形成鲜明对比的是，Hfq可提升该菌的几丁质酶活性，这表明Hfq在调控单核细胞增生李斯特菌几丁质酶的复杂信号通路中发挥多重作用。实验整体设计：将野生型EGD的4次独立生物学重复的cDNA与EGD ΔLhrA突变株的4次独立生物学重复的cDNA进行直接比对。