Abstract:
The causative agent of Legionnaires\' disease, Legionella pneumophila, is an environmental bacterium, that replicates in macrophages, parasitizes amoeba, and forms biofilms. L. pneumophila employs the Legionella quorum sensing (Lqs) system and the transcription factor LvbR to control various bacterial traits, including virulence and biofilm architecture. LvbR negatively regulates the nitric oxide (NO) receptor Hnox1, linking quorum sensing to NO signaling. Here, we assessed the response of L. pneumophila to NO and investigated bacterial receptors underlying this process. Chemical NO donors, such as dipropylenetriamine (DPTA) NONOate and sodium nitroprusside (SNP), delayed and reduced the expression of the promoters for flagellin (PflaA) and the 6S small regulatory RNA (P6SRNA). Marker-less L. pneumophila mutant strains lacking individual (Hnox1, Hnox2, or NosP) or all three NO receptors (triple knockout, TKO) grew like the parental strain in media. However, in the TKO strain, the reduction of PflaA expression by DPTA NONOate was less pronounced, suggesting that the NO receptors are implicated in NO signaling. In the ΔnosP mutant, the lvbR promoter was upregulated, indicating that NosP negatively regulates LvbR. The single and triple NO receptor mutant strains were impaired for growth in phagocytes, and phenotypic heterogeneity of non-growing/growing bacteria in amoebae was regulated by the NO receptors. The single NO receptor and TKO mutant strains showed altered biofilm architecture and lack of response of biofilms to NO. In summary, we provide evidence that L. pneumophila regulates virulence, intracellular phenotypic heterogeneity, and biofilm formation through NO and three functionally non-redundant NO receptors, Hnox1, Hnox2, and NosP.
OBJECTIVE: The highly reactive diatomic gas molecule nitric oxide (NO) is produced by eukaryotes and bacteria to promote short-range and transient signaling within and between neighboring cells. Despite its importance as an inter-kingdom and intra-bacterial signaling molecule, the bacterial response and the underlying components of the signaling pathways are poorly characterized. The environmental bacterium Legionella pneumophila forms biofilms and replicates in protozoan and mammalian phagocytes. L. pneumophila harbors three putative NO receptors, one of which crosstalks with the Legionella quorum sensing (Lqs)-LvbR network to regulate various bacterial traits, including virulence and biofilm architecture. In this study, we used pharmacological, genetic, and cell biological approaches to assess the response of L. pneumophila to NO and to demonstrate that the putative NO receptors are implicated in NO detection, bacterial replication in phagocytes, intracellular phenotypic heterogeneity, and biofilm formation.
摘要:
军团病的病原体,嗜肺军团菌,是一种环境细菌,在巨噬细胞中复制,寄生变形虫,并形成生物膜。嗜肺乳杆菌利用军团菌群体感应(Lqs)系统和转录因子LvbR来控制各种细菌性状,包括毒力和生物膜结构。LvbR负调节一氧化氮(NO)受体Hnox1,将群体感应与NO信号联系起来。这里,我们评估了嗜肺乳杆菌对NO的反应,并研究了这一过程的细菌受体.化学NO供体,如二亚丙基三胺(DPTA)和硝普钠(SNP),延迟并降低了鞭毛蛋白(PflaA)和6S小调节RNA(P6SRNA)启动子的表达。无标记的嗜肺乳杆菌突变株缺乏个体(Hnox1,Hnox2或NosP)或所有三种NO受体(三重敲除,TKO)像媒体中的父母菌株一样成长。然而,在TKO菌株中,DPTAN0NOate对PflaA表达的降低不太明显,这表明NO受体与NO信号有关。在ΔnosP突变体中,lvbR启动子上调,表明NosP负调节LvbR。单个和三个NO受体突变株在吞噬细胞中的生长受损,变形虫中未生长/生长细菌的表型异质性受NO受体调节。单个NO受体和TKO突变菌株显示出改变的生物膜结构和缺乏生物膜对NO的响应。总之,我们提供的证据表明嗜肺乳杆菌调节毒力,细胞内表型异质性,通过NO和三种功能非冗余NO受体形成生物膜,Hnox1、Hnox2和NosP。
目的:高反应性双原子气体分子一氧化氮(NO)由真核生物和细菌产生,以促进相邻细胞内部和之间的短程和瞬时信号传导。尽管它作为王国间和细菌内信号分子的重要性,细菌反应和信号通路的潜在成分特征不充分。环境细菌嗜肺军团菌在原生动物和哺乳动物吞噬细胞中形成生物膜并复制。嗜肺乳杆菌有三种推定的NO受体,其中之一与军团菌群体感应(Lqs)-LvbR网络交叉调节各种细菌性状,包括毒力和生物膜结构。在这项研究中,我们用了药理学,遗传,和细胞生物学方法来评估肺炎支原体对NO的反应,并证明推定的NO受体与NO检测有关,吞噬细胞中的细菌复制,细胞内表型异质性,和生物膜的形成。