E. coli O157:H7 genes induced during human infection

大肠杆菌 O157：人类感染期间诱导的 H7 基因

• 批准号: 6776744
• 负责人: INDIRA T KUDVA
• 金额: $ 34.8万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6776744
• 关键词: Escherichia coli 0157:H7; Escherichia coli infections; affinity chromatography; bacteria infection mechanism; bacterial antigens; bacterial genetics; bacterial toxins; enzyme linked immunosorbent assay; gene expression; genetic library; hemolytic anemia; host organism interaction; human tissue; nucleic acid sequence; polymerase chain reaction; virulence

DESCRIPTION (provided by applicant): Microbial genes expressed exclusively during infection confer unique attributes to the organism. They are usually induced in response to specific signals encountered within the host and may encode factors that help a pathogen adapt to the host environment, establish itself in its niche, and cause disease. Consequently, such virulence genes are suitable targets for the development of diagnostic assays, prophylactic and therapeutic options against disease caused by that pathogen. Here, we propose the identification of Escherichia coli O157:H7 (O157) genes expressed only during human infection, using a recently described novel technique termed In Vivo-lnduced Antigen Technology (IVIAT). This technique involves the sequential adsorption of convalescent sera from a patient who had hemolytic uremic syndrome (HUS) against whole cells, cell-lysates, and heat-denatured cell-lysates of the cognate pathogen grown under standard laboratory conditions. The adsorption process selectively removes antibodies against antigens expressed during in vitro growth, and enriches for those antibodies against in vivo-expressed antigens. Here, we will pool convalescent sera from three patients who recovered from HUS, adsorb them as described above against whole cells, cell lysates and heat-denatured cell lysates of three O157 strains isolated from the same patients who were the source of convalescent sera. The adsorbed convalescent serum pool will then be used to screen three individual O157 DNA expression libraries made from genomic DNA of the O157 strain isolated from the each of the above three patients. The genes contained in reactive clones will be identified by BLAST, against the genomes of O157 EDL 933 and Sakai strains, and other DNA sequences available in the National Center for Biotechnology Information (NCBI) non-redundant database and The Institute for Genomic Research (TIGR) database. Because of time and budgetary constraints, we will focus on only two or three of the IVIAT antigens. Previously unidentified antigens encoded by genes located on genomic sequences unique to O157 that are not transcribed during in vitro growth, will be purified via nickel affinity chromatography. Antibody responses against such antigens will be quantified in individual convalescent phase serum from the three patients described above. Ideal antigens for further study are those that are the target of robust antibody responses across all three patients. Such antigens (pending further studies) may help in the development of prophylactic options and could potentially serve as markers of ongoing or recent O157 disease in stool specimens. Broad, robust antibody responses against such antigens, could also serve as the basis for the development of serological diagnostic assays for recent O157 infection.

描述（由申请人提供）：在感染过程中专门表达的微生物基因赋予生物体独特的属性。它们通常是响应宿主内遇到的特定信号而被诱导的，并且可能编码帮助病原体适应宿主环境、在其生态位中建立自己并引起疾病的因子。因此，此类毒力基因是开发针对该病原体引起的疾病的诊断测定、预防和治疗选择的合适靶标。在这里，我们建议使用最近描述的称为体内诱导抗原技术（IVIAT）的新技术来鉴定仅在人类感染期间表达的大肠杆菌 O157:H7 (O157) 基因。该技术涉及对标准实验室条件下生长的同源病原体的全细胞、细胞裂解物和热变性细胞裂解物连续吸附溶血性尿毒症综合征 (HUS) 患者的恢复期血清。吸附过程选择性地去除针对体外生长期间表达的抗原的抗体，并富集针对体内表达的抗原的抗体。在这里，我们将汇集三名 HUS 康复患者的恢复期血清，按照上述方法将其吸附到从作为恢复期血清来源的同一患者中分离出的三种 O157 菌株的全细胞、细胞裂解物和热变性细胞裂解物中。然后，吸附的恢复期血清池将用于筛选三个单独的 O157 DNA 表达文库，这些文库由从上述三名患者中的每一位分离的 O157 菌株的基因组 DNA 制成。反应性克隆中包含的基因将通过 BLAST 与 O157 EDL 933 和 Sakai 菌株的基因组以及国家生物技术信息中心 (NCBI) 非冗余数据库和基因组研究所 (TIGR) 中提供的其他 DNA 序列进行鉴定。 ） 数据库。由于时间和预算限制，我们将仅关注其中的两到三种 IVIAT 抗原。先前未鉴定的抗原由位于 O157 独特的基因组序列上的基因编码，这些基因在体外生长期间不会转录，将通过镍亲和层析进行纯化。针对这些抗原的抗体反应将在上述三名患者的恢复期血清中进行定量。进一步研究的理想抗原是那些在所有三名患者中都能产生强烈抗体反应的目标抗原。此类抗原（有待进一步研究）可能有助于制定预防方案，并有可能作为粪便标本中正在进行或近期 O157 疾病的标记。针对此类抗原的广泛而强大的抗体反应也可以作为开发最近 O157 感染的血清学诊断测定的基础。