PATHOGENESIS AND PREVENTION OF BACTERIAL CYSTITIS

该项目的广泛、长期目标是澄清在急性疾病中的作用 四种明确毒力因子(VFS)的细菌性膀胱炎 Coli，既可以单独使用，也可以相互组合使用，并开发出 干预预防膀胱炎--通过干预一人的功能 这些VF中。具体目标是：1)完善标准的小鼠模型 UTI用于膀胱炎的研究；2)使用分子方法进行体外研究 诱变和等位基因交换创造等基因VF缺陷突变体 一种毒力很强的野生型大肠杆菌尿毒症分离株；3)使用这些 在小鼠模型中确定同基因突变的贡献 特异性VF的膀胱炎单独或合并；4) 从天然来源中提纯四种VFS之一的抑制物(P 菌毛)；以及5)评估该抑制剂预防 大肠埃希菌与尿路感染患者上皮细胞的粘附性 并保护小鼠免受实验性膀胱炎的侵袭。 实验设计和方法。修改后的接种技术将是 用于避免非生理性膀胱输尿管反流和肾损伤 膀胱炎小鼠模型。自杀质粒系统将被用于 将诱变的VF决定簇导入强毒野生型大肠杆菌 用功能野生型等位基因来交换菌株。病毒的致命性 产生的等基因突变将在小鼠模型中进行比较，以揭示 各室颤及合并室颤在急性心肌梗死发病机制中的重要性 膀胱炎。鸽子含P1抗原的碳水化合物成分 将使用生化方法分离卵粘液样物质并检测其 1)阻止P菌毛大肠杆菌与人尿液黏附的能力 以及来自频繁尿路感染女性的阴道上皮细胞和2)至 保护小鼠免受P菌毛大肠杆菌引起的膀胱炎。 该项目与健康的相关性在于需要更好的方法来 预防急性细菌性膀胱炎，这是妇女的一个主要健康问题 对巨大的发病率和增加的医疗费用负有责任。 通过干扰E. Coli VFS将通过1)识别最具体的VFS来进一步 负责膀胱炎(这将有助于指导未来的发展 抗房颤干预)，以及2)开发负担得起和有效的 抗黏附素干预预防膀胱炎。

THE PROJECT'S BROAD, LONG TERM OBJECTIVES are to clarify the role in acute bacterial cystitis of four defined virulence factors (VFs) of Escherichia coli both, singly and in combination with one another, and to develop an intervention to prevent cystitis by interfering with the function of one of these VFs. SPECIFIC AIMS are: 1) to refine a standard mouse model of UTI for the study of cystitis; 2) to use molecular methods for in vitro mutagenesis and allelic exchange to create isogenic VF-deficient mutants of a virulent wild-type E. coli urosepsis isolate; 3) to use these isogenic mutants in the mouse model to determine the contribution to cystitis of specific VFs singly and in combination with one another; 4) to purify from natural sources an inhibitor of one of the four VFs (P fimbriae); and 5) to evaluate the ability of this inhibitor to prevent adherence of E. coli to epithelial cells from women with frequent UTI's and to protect mice from experimental cystitis. EXPERIMENTAL DESIGN AND METHODS. Modified inoculation techniques will be used to avoid nonphysiological vesicoureteral reflux and renal trauma in the mouse model of cystitis. A suicide plasmid system will be used to introduce mutagenized VF determinants into a virulent wild-type E. coli strain in exchange for functional wild-type alleles. The virulence of the resulting isogenic mutants will be compared in the mouse model to reveal the importance of each VF and of combined VFs in the pathogenesis of cystitis. The P1-antigen-containing carbohydrate component of pigeon ovomucoid will be isolated using biochemical methods and tested for its ability 1) to block the adherence of P fimbriated E. coli to human urinary and vaginal epithelial cells from women with frequent UTIs and 2) to protect mice from cystitis due to P fimbriated E. coli. THE HEALTH RELATEDNESS OF THE PROJECT lies in the need for better ways to prevent acute bacterial cystitis, a major health problem of women that is responsible for tremendous morbidity and increased health care costs. Efforts to protect women from bacterial cystitis by interfering with E. coli VFs will be furthered by 1) identifying the specific VFs most responsible for cystitis (which would help guide the future development of anti-VF interventions), and 2) developing an affordable and effective anti-adhesin intervention for the prevention of cystitis.