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

描述（由申请人提供）：铜绿假单胞菌是一种机会性病原体，可在受损个体中引起严重感染，包括中性粒细胞减少的癌症患者和囊性纤维化（CF）患者。为了建立成功的感染，铜绿假单胞菌需要铁，并采用几种获取铁的策略，包括摄取血红素。虽然是生存所必需的，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）调节这些营养物质的摄取。在富含铁的环境中，皮毛阻断了铁和血红素摄取所需基因的表达，以及编码PrrF1和PrrF2小调控rna的两个几乎相同的基因（Wilderman et al., 2004）。我们之前的研究表明，PrrF rna会对至少50个基因的表达产生负面影响，这些基因的产物在关键的代谢途径中起作用，其中一个基因会深刻影响群体感应因子的产生（Oglesby等人，2008）。因此，PrrF rna能够对铜绿假单胞菌的生理和毒力产生广泛的影响。血红素是人体铁的丰富来源，据推测铜绿假单胞菌获得血红素在感染中起重要作用。由于血红素的潜在毒性作用，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，确定这种化合物作为信号分子的机制将产生新的治疗靶点。铜绿假单胞菌中的PrrF rna是由两个几乎相同的基因prrF1和prrF2串联编码的（图1），而所有其他假单胞菌在远端位点编码PrrF rna。我和Vasil博士实验室的其他人表明，P. aeruginosa的prrF位点编码另外一种325个核苷酸（nt）的RNA，称为PrrH，它被血红素和铁抑制（Oglesby-Sherrouse & Vasil, 2010, Ochsner et al., 2000）。PrrH的转录从prrF1的5‘端开始，通过prrF1-prrF2基因间序列进行（95 nt），并在prrF2的3’端终止（图1）。因此，prrH的表达依赖于prrF1 rho非依赖性或内在终止端的读透转录。我的数据表明，PrrH调节参与血红素生物合成的基因（Oglesby- Sherrouse & Vasil, 2010）和毒力（表1），这表明这种串联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物（PHAT -图1）识别prrF1终止物之前的RNA序列或之后的DNA序列，并减轻新生RNA的发夹结构，允许转录延伸复合体（TEC）的稳定和PrrH RNA的持续转录。本提案将通过确定prrH表达和血红素调控的序列要求和推测的prrH靶点，以及鉴定参与prrH表达和调控的基因和蛋白，来确定prrH表达和血红素调控的机制。

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.