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

描述(申请人提供)：铜绿假单胞菌是一种机会性病原体，可导致严重感染患者，包括中性粒细胞减少的癌症患者和囊性纤维化患者。为了建立成功的感染，铜绿假单胞菌需要铁，并采取几种策略来获得铁，包括吸收血红素。虽然是生存所必需的，但多余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吸收。在铁充足的环境中，毛皮阻止铁和血红素吸收所需基因的表达，以及两个编码PrrF1和PrrF2小调控RNA的几乎相同的基因的表达(Wilderman等人，2004年)。我们以前发现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的代谢途径中发挥作用，其中一个基因深刻地影响群体感应因子的产生(Oglesby等人，2008年)。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。血红素是人体内丰富的铁源，铜绿假单胞菌对它的获取被认为在感染中起着重要作用。由于血红素的潜在毒性作用，预计血红素调节系统将协调血红素吸收、降解和生物合成的基因表达。由于血红素对毒力具有实质性的影响，确定这种化合物作为信号分子的机制(S)将产生用于治疗目的的新靶点。铜绿假单胞菌中的PrrF RNAs由两个几乎相同的基因prrF1和prrF2(图1)串联编码，而所有其他假单胞菌则编码远端的PrrF RNAs。我和Vasil博士实验室的其他人发现，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，命名为PrrH，它被血红素和铁抑制(Oglesby-Sherrouse&Vasil，2010，Ochsner等人，2000)。PrrH的转录开始于prrF1的5‘端，通过prrF1-prrF2基因间序列(95nt)进行，在prrF2的3’端终止(图1)。因此，prrH的表达依赖于prrF1 Rho非依赖的或内在的终止子的通读转录。我的数据表明PrrH调控与血红素生物合成(Oglesby-Sherrouse&Vasil，2010)和毒力相关的基因(表1)，这表明这个串联的prrF组织赋予铜绿假单胞菌独特的血红素调控活性。我假设，一个受血红素调节的抗终止子(图1)识别prrF1终止子之前的RNA序列或prrF1终止子之后的DNA序列，并解除新生RNA的发夹结构，使转录延伸复合体(TEC)稳定和PrrH RNA继续转录。这一建议将通过以下方式确定prrH表达和血红素调控的机制：i)确定prrH和可能的prrH靶标的表达和血红素调控的序列要求；ii)识别参与prrH表达和调控的基因和蛋白质。

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.