Inhibition of GBS Carriage by Engineered Lactobacilli

工程乳酸杆菌抑制 GBS 携带

• 批准号: 6699312
• 负责人: DAVID G PRITCHARD
• 金额: $ 21.75万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6699312
• 关键词: Lactobacillus; Streptococcus agalactiae; antibacterial agents; biotechnology; genetic manipulation

DESCRIPTION (provided by applicant): Group B Streptococci (GBS) are a major cause of serious neonatal bacterial infections in this country. The origin of the bacteria for babies born with early-onset GBS infections is the birth canal of their mothers. This application describes an entirely new approach for preventing or eliminating vaginal carriage of GBS, thereby reducing the incidence of neonatal GBS infections. The goal is to genetically engineer a normal commensal organism present in the vagina, a Lactobacillus, to secrete a substance that will specifically prevent the growth of GBS. The substance selected is a GBS phage lysin that degrades the cell walls of GBS. The first specific aim is to clone and express the GBS phage lysin in E. coli and assess the bacteriocidal activity of purified recombinant enzyme. The ability of the purified lysin to kill GBS and various other bacteria will be first studied in vitro. Then it will then be determined if vaginal instillation of the enzyme will eliminate GBS in vaginally colonized mice. The second specific aim is to determine basic biochemical properties of the GBS phage lysin, including identifying the enzyme class to which it belongs and the identity of the cell wall component(s) necessary for specific peptidoglycan cleavage. The third specific aim is to engineer a Lactobacillus to secrete the GBS phage lysin. This will be done initially using a plasmid construct containing all the elements necessary for efficient secretion of the lysin from the transformed Lactobacillus. Then, in order to overcome inherent plasmid instability and also to eliminate the antibiotic selection markers, the lysin gene secretion cassette will be integrated into the chromosome of the Lactobacillus. The final specific aim is to determine if vaginal colonization of mice with the engineered Lactobacillus will prevent GBS from establishing a persistent colonization. In addition, it will also be determined if vaginal inoculation with the lactobacillus engineered to secrete GBS phage lysin will result in the clearance of GBS from the vaginas of mice previously colonized with the organism. The proposed research may lead to the development of an effective new method for long-term inhibition of GBS vaginal colonization, even though women may be repeatedly re-exposed to the bacteria. The method is unlikely to disturb the normal bacterial flora and should be very safe. If successful, this approach might also be used to protect against other genital, oral, and intestinal pathogens, especially those to which no effective mucosal immunity appears to develop.

描述（由申请方提供）：B组链球菌（GBS）是该国严重新生儿细菌感染的主要原因。患有早发性GBS感染的婴儿的细菌来源是他们母亲的产道。本申请描述了一种用于预防或消除GBS阴道携带的全新方法，从而降低新生儿GBS感染的发生率。目标是通过基因工程改造阴道中存在的正常阴道生物体，即乳酸杆菌，以分泌一种专门防止GBS生长的物质。所选择的物质是降解GBS细胞壁的GBS噬菌体溶素。第一个具体目标是在大肠杆菌中克隆并表达GBS噬菌体溶素。大肠杆菌中的表达，并对纯化的重组酶的杀菌活性进行评价。将首先在体外研究纯化的溶素杀死GBS和各种其它细菌的能力。然后确定阴道滴注酶是否会消除阴道定殖小鼠中的GBS。第二个具体目标是确定GBS噬菌体溶素的基本生化性质，包括鉴定其所属的酶类别和特异性肽聚糖切割所需的细胞壁组分的身份。第三个具体目标是工程化乳杆菌以分泌GBS噬菌体溶素。这将首先使用含有从转化的乳杆菌有效分泌溶素所必需的所有元件的质粒构建体来完成。然后，为了克服固有的质粒不稳定性并且也为了消除抗生素选择标记，将溶素基因分泌盒整合到乳杆菌属的染色体中。最终的具体目标是确定用工程化的乳酸杆菌对小鼠进行阴道定殖是否会阻止GBS建立持久的定殖。此外，还将确定阴道接种经工程改造以分泌GBS噬菌体溶素的细菌是否会导致GBS从先前用该生物体定殖的小鼠的阴道中清除。这项研究可能会导致开发一种长期抑制GBS阴道定植的有效新方法，即使女性可能会反复再次暴露于细菌。该方法不太可能干扰正常的细菌植物群，应该是非常安全的。 如果成功的话，这种方法也可以用于保护其他生殖器，口腔和肠道病原体，特别是那些没有有效的粘膜免疫似乎发展。