Augmenting innate immunity to combat nasal carriage of Staphylococcus aureus

 描述(申请人提供)：金黄色葡萄球菌(SA)鼻腔携带是人类的一种常见疾病，是传播这种病原体的重要宿主，在美国每年导致的死亡人数超过艾滋病毒/艾滋病。住院前使用抗生素使金黄色葡萄球菌鼻部去克隆，可显著降低医院感染的发生率，这有力地表明减少或消除金黄色葡萄球菌鼻腔携带将直接导致金黄色葡萄球菌临床感染的减少。外用抗生素如莫匹罗星可以有效地消除金黄色葡萄球菌鼻腔携带，但重复使用会导致细菌耐药性。我们提出了一种新的方法，通过提高鼻子对SA的天然免疫力来减少SA的鼻腔携带。为了实现这一目标，我们将恢复被称为“逆周期蛋白”的环状抗菌肽的生产，这种蛋白在进化上在人类基因组中是沉默的。虽然一些非人类灵长类动物会产生逆转录周期素样的多肽，但人类的逆周期蛋白基因含有一个提前终止密码子，阻止了这些多肽的翻译。基于包含在其他完整的逆周期蛋白基因中的遗传信息，我们首先合成了逆周期蛋白，并发现逆周期素肽对甲氧西林耐药金黄色葡萄球菌(MRSA)菌株和所有其他被测试的金黄色葡萄球菌都具有很强的活性，并且可以与至少一种参与逆周期蛋白生物合成的前体多肽协同作用。在相关研究中，我们使用氨基糖苷类化合物恢复了宫颈阴道细胞和组织中逆转录周期素肽的自然产生，氨基糖苷类药物抑制了逆转录周期蛋白的提前终止密码子，从而产生了可以保护人类免疫缺陷病毒感染的天然逆转录周期素肽。我们现在建议使用一种名为PTC124的新一代非抗生素直读剂来纠正鼻黏膜中的“逆周期蛋白缺乏症”，初步的免疫细胞化学研究表明，这种药物可以在鼻上皮细胞中产生逆周期蛋白。根据我们的初步研究，我们假设PTC124将恢复鼻黏膜中逆转录周期素的自然产生，并且前体和成熟形式将协同工作，以提供对SA定植的持续保护。目的1评价PTC124诱导人鼻腔上皮细胞产生抗金黄色葡萄球菌逆转录周期蛋白的能力。我们将证实，经PTC124处理的人鼻上皮细胞表达具有抗SA活性的完整的逆周期素肽，确定足以保护鼻黏膜免受SA定植的逆周期素浓度，并检测PTC124单独处理能维持逆周期素介导保护多久。然后，我们将探索产生哪些成熟的和前体的逆转录周期蛋白，以及它们在细胞内和细胞外的位置。虽然有相当多的证据表明，过早终止密码子和正常密码子终止的机制有很大不同，而且抑制剂不应该针对正常终止密码子，但Aim 2将验证PTC124不会对鼻黏膜上皮细胞产生非靶向影响。使用PTC124等药物来增强鼻腔天然免疫，可能会减少鼻腔携带以及随后由SA传播和感染的发生率。

 DESCRIPTION (provided by applicant): Staphylococcus aureus (SA) nasal carriage is a common condition in humans that acts as an important reservoir for spreading this pathogen, which causes more deaths each year in the U.S. than HIV/AIDS. Using antibiotics to decolonize the nose of SA, prior to hospitalization, significantly reduces the incidence of nosocomial infection, strongly suggesting that reducing or eliminating nasal carriage of SA would directly lead to a reduction in SA clinical infections. Topical antibiotics such as mupirocin can effectivel rid SA nasal carriage, but repetitive use leads to bacterial resistance. We are proposing a novel approach to reduce nasal carriage of SA, by improving upon the nose's natural immunity against SA. To accomplish this goal, we will restore the production of cyclic antimicrobial peptides called "retrocyclins", which have been evolutionarily silenced in the human genome. While some nonhuman primates produce retrocyclin-like peptides, human retrocyclin genes harbor a premature termination codon that blocks translation of these peptides. Based on the genetic information contained within the otherwise intact retrocyclin genes, we first recreated retrocyclins synthetically, and discovered that retrocyclin peptides are potently active against methicillin-resistant SA (MRSA) strains and all other SA isolates tested, and can work in concert with at least one precursor peptide involved in retrocyclin biosynthesis. In related studies, we restored the natural production of retrocyclin peptides in cervicovaginal cells and tissues using aminoglycosides, which suppressed retrocyclin's premature termination codon, resulting in natural retrocyclin peptides that could protect against HIV infection. We now propose to correct "retrocyclin deficiency" in the nasal mucosa using a next- generation, non-antibiotic, read-through agent called PTC124, which pilot immunocytochemical studies indicate can produce retrocyclins in nasal epithelia. Based on our preliminary studies, we hypothesize that PTC124 will restore the natural production of retrocyclins in the nasal mucosa, and the precursor and mature forms will work in concert to confer sustained protection against SA colonization. Aim 1 will assess the ability of PTC124 to induce anti-SA retrocyclins in human nasal epithelia. We will confirm that human nasal epithelia, treated with PTC124, express intact retrocyclin peptides active against SA, determine the concentration of retrocyclins that is sufficient to protect nasal mucosa from SA colonization, and examine how long a single treatment of PTC124 can maintain retrocyclin-mediated protection. We will then explore which mature and precursor retrocyclins are produced, and where they reside subcellularly and extracellularly. Although there is considerable evidence that mechanisms underlying premature and normal codon termination differ substantially, and suppressing agents should not target normal termination codons, Aim 2 will verify that PTC124 does not induce off-target effects to nasal epithelia. Using agents such as PTC124 to bolster nasal innate immunity would likely reduce the incidence of nasal carriage, and subsequent transmission and infection by SA.