Role of Bacteroides fragilis new hemolysins in experimental infection.

描述(申请人提供)：脆弱类杆菌是从厌氧感染中分离出的最常见的条件致病菌。脆弱芽孢杆菌致病成功的原因尚不完全清楚，但衣壳多糖、黏附和神经氨酸酶等毒力因子起着重要作用。尚未确定的潜在毒力因子是脆弱芽孢杆菌产生溶血素/溶血素的能力。尽管一些临床分离株在血琼脂平板上表现出溶血表型，但到目前为止，这种厌氧菌还没有表现出溶血素/细胞溶血素的特征。对脆弱芽孢杆菌全基因组序列的分析表明，存在与细菌溶血素同源的[10]个基因，支持溶血素可能参与脆弱芽孢杆菌致病作用的观点。溶血素是一种细胞毒性蛋白质，由原核生物和真核生物等多种生物产生。尽管它们因其溶解红细胞的能力而命名，但许多对其他类型的细胞也是有毒的。微生物溶血素被认为是一种毒力因子，能够杀死传入的白细胞，从而为入侵微生物提供优势。这可能会通过削弱宿主的免疫反应和获得营养来提高存活率。尽管有大量与溶血素同源的基因，但还没有研究确定溶血素/溶血素在脆弱芽孢杆菌致病中的作用和功能。因此，本研究项目的长期目标是验证溶血素/细胞溶血素是脆弱芽孢杆菌致病因子的假说。在本研究中，我们建议开展研究，以鉴定和鉴定脆弱芽胞杆菌的溶血活性基因，并描述溶血素在实验性感染和中性粒细胞毒性中的作用。为实现这些目标，将实现以下目标。[目的1.描述脆弱芽孢杆菌溶血素在实验性感染中的作用。]A)我们将通过Real-Time RT-PCR检测溶血素基因在体内的表达。B)描绘对溶血素的免疫反应，作为评估其在体内表达的一种手段。C)阐明溶血素在腹内脓肿模型中的作用。D)确定溶血素对中性粒细胞的毒性。[目的2.完成脆弱芽孢杆菌溶血素的分析。]A)我们将完成对脆弱芽孢杆菌溶血素的溶血/溶细胞活性的初步分析。B)我们将完成溶血素突变体的构建。

DESCRIPTION (provided by applicant): Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infections. The success of B. fragilis pathogenicity is still not completely understood but virulence factors such as capsular polysaccharides, adherence, and neuraminidase play an important role. Potential virulence factor that has yet to be characterized is the ability of B. fragilis to produce hemolysins/cvtolysins. To date, no hemolysin/cytolysin has been characterized in this anaerobic pathogen despite the fact that some clinical isolates show a hemolytic phenotype on blood agar plates. Analysis of the complete B. fragilis genome sequence revealed the presence of [ten] genes homologous to bacterial hemolysins supporting our idea that hemolysins might be involved in the pathogenicity of B. fragilis. Hemolysins are cytotoxic proteins produced by many organisms ranging from prokaryotes to eukaryotes. Though they are named for their capacity to lyse erythrocytes, many are toxic to other cell types as well. Microbial hemolysins are considered to be virulence factors that provide an advantage to invading microorganisms by their ability to kill incoming leukocytes. This may enhance survival by weakening the host immune response and gaining access to nutrients. Despite this extended number of genes homologous to hemolysins, no study has characterized the role and function of hemoiysins/cytolysins in B. fragilis pathogenicity. Thus, the long-term goal of this research project is to test the hypothesis that hemolysins/cytolysins are virulence factors in B. fragilis pathogenicity. In the present study we propose to initiate studies to identify and characterize the genes conferring hemolytic activity in B. fragilis and delineate the role of hemolysins in experimental infection and neutrophil toxicity. To accomplish these objectives, the following aims will be pursued. [Aim 1. Delineate the role of B. fragilis hemolysins in experimental infections.] A) We will assess the expression of hemolysin genes in vivo by Real-Time RT-PCR. B) Delineate the immune response to hemolysins as a mean to evaluate their expression in vivo. C) Elucidate the role of hemolysins in an intra-abdominal abscess model. D) Determine the toxicity of hemolysins to PMNs. [Aim 2. Complete the analysis of B. fragilis hemolysins.] A) We will complete the initial analysis of the hemolytic/cytolytic activity of B. fragilis hemolysins. B) We will complete the construction of hemolysin mutants.