PATHOGENESIS AND PREVENTION OF BACTERIAL CYSTITIS

该项目的广泛，长期的承诺是澄清在急性 大肠埃希氏菌四种确定毒力因子的细菌性膀胱炎 大肠杆菌，单独和相互结合，并开发一个 通过干预一个人的功能来预防膀胱炎 这些VF。具体目标是：1）完善标准小鼠模型， 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以产生同基因VF缺陷突变体 一种毒性野生型E.大肠杆菌尿脓毒症分离株; 3）使用这些 小鼠模型中的同基因突变体，以确定对 单独和彼此组合的特定VF的膀胱炎; 4） 从天然来源中纯化四种VF之一的抑制剂（P 菌毛）;和5）评价该抑制剂预防 E.大肠杆菌感染频繁UTI的女性上皮细胞 并保护小鼠免受实验性膀胱炎的侵害。 实验设计和方法.改良的接种技术将 用于避免非生理性膀胱输尿管反流和肾损伤， 膀胱炎的小鼠模型。自杀质粒系统将用于 将诱变VF决定簇引入毒性野生型E.杆菌 菌株交换功能性野生型等位基因。的毒力 将在小鼠模型中比较产生的同基因突变体，以揭示 每个VF和组合VF在发病机制中的重要性 膀胱炎鸽的含P1抗原的碳水化合物成分 卵类粘蛋白将使用生物化学方法分离并测试其 能力1）阻断P菌毛E.大肠杆菌致人尿 和来自频繁UTI的女性的阴道上皮细胞，以及2） 保护小鼠免受由P菌毛E.杆菌 该项目的健康性在于需要更好的方法来 预防急性细菌性膀胱炎，这是女性的主要健康问题， 造成巨大的发病率和增加的医疗保健费用。 通过干扰E. 大肠杆菌VFs将进一步通过1）识别特定的VFs最 负责膀胱炎（这将有助于指导未来的发展， 抗VF干预措施），以及2）制定负担得起的有效 抗粘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.