Mechanism of heme regulation of a P. aeruginosa non-coding RNA

铜绿假单胞菌非编码RNA的血红素调节机制

• 批准号: 8477120
• 负责人: Amanda Gail Oglesby
• 金额: $ 10.5万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8477120
• 关键词: Affect; Affinity Chromatography; Anabolism; Bacteria; Binding; Binding Proteins; Biochemical; Biological Assay; Boxing; Burn injury; Cancer Patient; Cells; Complex; Contact Lenses; Cystic Fibrosis; DNA Sequence; Data; Development; Disease; Distal; Environment; Functional RNA; Gene Expression; Gene Proteins; Genes; Genetic; Genetic Transcription; Heme; Heme Iron; Human; Human body; Individual; Infection; Intergenic Sequence; Iron; Laboratories; Lead; Mediating; Metabolic Pathway; Modeling; Mutagenesis; Nosocomial Infections; Nucleotides; Nutrient; Oxidative Stress; Pathway interactions; Persons; Physiology; Play; Production; Proteins; Pseudomonas aeruginosa; RNA; RNA Sequences; Reading; Regulation; Role; Signaling Molecule; Source; Structure; System; Testing; Toxic effect; Transcription Elongation; Virulence; antimicrobial; antitermination; chemotherapy; hatching; heme a; heme biosynthesis; mutant; new therapeutic target; pathogen; promoter; quorum sensing; research study; rho; therapeutic target; uptake

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in compromised individuals, including neutropenic cancer patients and individuals with cystic fibrosis (CF). To establish successful infection, P. aeruginosa requires iron and employs several strategies for its acquisition, including the uptake of heme. Although required for survival, surplus iron or heme can lead to oxidative stress; thus, the ferric uptake regulator (Fur) protein regulates the uptake of these nutrients. In iron-replete environments, Fur blocks expression of genes required for iron and heme uptake, as well as two nearly identical genes encoding the PrrF1 and PrrF2 small regulatory RNAs (Wilderman et al., 2004). We previously showed that the PrrF RNAs negatively affect the expression of at least 50 genes, the products of which function in key metabolic pathways, and one of which profoundly affects production of quorum sensing factors (Oglesby et al., 2008). Consequently, the PrrF RNAs are capable of exerting wide-ranging effects on P. aeruginosa physiology and virulence. Heme is an abundant source of iron in the human body, and its acquisition by P. aeruginosa is hypothesized to play a significant role in infection. Because of the potentially toxic effects of heme, it is expected that a heme regulatory system coordinates expression of genes for heme uptake, degradation, and biosynthesis. With the substantial implications that heme has for virulence, identifying the mechanism(s) by which this compound acts as a signaling molecule will yield novel targets for therapeutic purposes. The PrrF RNAs in P. aeruginosa are encoded in tandem by two virtually identical genes, prrF1 and prrF2 (Fig. 1), while all other pseudomonads encode for the PrrF RNAs at distal loci. I and others in Dr. Vasil's laboratory showed that the prrF locus of P. aeruginosa encodes an additional, 325-nucleotide (nt) RNA, designated PrrH, which is repressed by heme as well as iron (Oglesby-Sherrouse & Vasil, 2010, Ochsner et al., 2000). Transcription of PrrH initiates at the 5' end of prrF1, proceeds through the prrF1-prrF2 intergenic sequence (95 nt), and terminates at the 3' end of prrF2 (Fig. 1). Thus, expression of prrH is dependent on read-through transcription at the prrF1 Rho-independent, or intrinsic, terminator. My data indicate PrrH regulates genes involved in heme biosynthesis (Oglesby- Sherrouse & Vasil, 2010) and virulence (Table 1), suggesting this tandem prrF organization imparts unique heme regulatory activities to P. aeruginosa. I hypothesize that a heme- regulated antiterminator (PHAT - Fig. 1) recognizes RNA sequence preceding or DNA sequence following the prrF1 terminator and relieves the hairpin structure of the nascent RNA, allowing stabilization of the transcription elongation complex (TEC) and continued transcription of the PrrH RNA. This proposal will determine the mechanism of prrH expression and heme regulation by I) determining the sequence requirements for expression and heme regulation of prrH and putative PrrH targets and II) identifying genes and proteins involved in expression and regulation of prrH.

描述（由申请人提供）：铜绿假单胞菌是一种机会性病原体，可导致受损个体严重感染，包括中性粒细胞减少症癌症患者和囊性纤维化 (CF) 患者。为了成功感染，铜绿假单胞菌需要铁并采用多种策略来获取铁，包括吸收血红素。尽管是生存所必需的，但过量的铁或血红素会导致氧化应激；因此，铁摄取调节蛋白 (Fur) 可以调节这些营养物质的摄取。在铁充足的环境中，Fur 会阻断铁和血红素吸收所需基因的表达，以及编码 PrrF1 和 PrrF2 小调节 RNA 的两个几乎相同的基因的表达（Wilderman 等，2004）。我们之前表明，PrrF RNA 对至少 50 个基因的表达产生负面影响，这些基因的产物在关键代谢途径中发挥作用，其中之一深刻影响群体感应因子的产生（Oglesby 等，2008）。因此，PrrF RNA 能够对铜绿假单胞菌生理学和毒力产生广泛的影响。血红素是人体内丰富的铁来源，推测铜绿假单胞菌获取血红素在感染中发挥重要作用。由于血红素具有潜在的毒性作用，因此预计血红素调节系统可以协调血红素摄取、降解和生物合成的基因表达。鉴于血红素对毒力的重大影响，确定该化合物作为信号分子的机制将产生用于治疗目的的新靶点。铜绿假单胞菌中的 PrrF RNA 由两个几乎相同的基因 prrF1 和 prrF2 串联编码（图 1），而所有其他假单胞菌在远端基因座编码 PrrF RNA。我和 Vasil 博士实验室的其他人表明，铜绿假单胞菌的 prrF 基因座编码一个额外的 325 个核苷酸 (nt) RNA，称为 PrrH，它受到血红素和铁的抑制（Oglesby-Sherrouse & Vasil, 2010，奥克斯纳等人，2000）。 PrrH 的转录起始于 prrF1 的 5' 末端，通过 prrF1-prrF2 基因间序列（95 nt）进行，并终止于 prrF2 的 3' 末端（图 1）。因此，prrH 的表达依赖于 prrF1 Rho 独立终止子或内在终止子处的通读转录。我的数据表明 PrrH 调节参与血红素生物合成 (Oglesby-Sherrouse & Vasil, 2010) 和毒力 (表 1) 的基因，表明这种串联的 prrF 组织赋予铜绿假单胞菌独特的血红素调节活性。我假设血红素调节的抗终止子（PHAT - 图 1）识别 prrF1 终止子之前的 RNA 序列或之后的 DNA 序列，并解除新生 RNA 的发夹结构，从而稳定转录延伸复合物 (TEC) 并持续转录PrrH RNA。该提案将通过以下方式确定 prrH 表达和血红素调节的机制：I) 确定 prrH 表达和血红素调节的序列要求以及假定的 PrrH 靶标；II) 识别参与 prrH 表达和调节的基因和蛋白质。

项目成果

Iron-responsive bacterial small RNAs: variations on a theme.

• DOI: 10.1039/c3mt20224k
• 发表时间: 2013-04
• 期刊: Metallomics : integrated biometal science
• 作者: Oglesby-Sherrouse AG;Murphy ER
• 通讯作者: Murphy ER

Adaptation of iron homeostasis pathways by a Pseudomonas aeruginosa pyoverdine mutant in the cystic fibrosis lung.

囊性纤维化肺中铜绿假单胞菌pyoverdine突变体对铁稳态途径的适应。

• DOI: 10.1128/jb.01491-14
• 发表时间: 2014
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Nguyen,AngelaT;O'Neill,MauraJ;Watts,AnnabelleM;Robson,CynthiaL;Lamont,IainL;Wilks,Angela;Oglesby-Sherrouse,AmandaG
• 通讯作者: Oglesby-Sherrouse,AmandaG