Inhibition of GBS Carriage by Engineered Lactobacilli

描述（由申请人提供）：B群链球菌（GBS）是该国新生儿严重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其母亲的产道。本应用描述了一种全新的方法来预防或消除阴道携带的GBS，从而减少新生儿GBS感染的发生率。其目标是通过基因工程改造阴道中存在的一种正常共生生物，即乳酸杆菌，使其分泌一种物质，专门防止GBS的生长。所选择的物质是一种GBS噬菌体裂解素，可以降解GBS的细胞壁。第一个具体目的是在大肠杆菌中克隆和表达GBS噬菌体裂解酶，并评价纯化后的重组酶的杀菌活性。纯化后的溶酶杀灭GBS和其他多种细菌的能力将首先在体外进行研究。然后将确定阴道注射这种酶是否能消除经阴道定植的小鼠体内的GBS。第二个具体目标是确定GBS噬菌体裂解素的基本生化特性，包括鉴定其所属的酶类和鉴定特定肽聚糖裂解所需的细胞壁成分。第三个具体目标是设计一种乳酸菌分泌GBS噬菌体溶酶。这将首先使用含有从转化的乳酸菌中有效分泌溶酶所需的所有元素的质粒结构来完成。然后，为了克服质粒固有的不稳定性，也为了消除抗生素选择标记，将溶酶基因分泌盒整合到乳酸菌的染色体中。最后的具体目的是确定是否阴道定植与工程乳酸菌的小鼠将阻止GBS建立持久定植。此外，还将确定阴道接种分泌GBS噬菌体裂解素的乳酸菌是否会导致先前定植过GBS的小鼠阴道清除GBS。拟议的研究可能会导致开发一种有效的新方法来长期抑制GBS阴道定植，即使女性可能会反复暴露于细菌。这种方法不太可能干扰正常的细菌菌群，应该是非常安全的。如果成功，这种方法也可以用于防止其他生殖器、口腔和肠道病原体，特别是那些没有有效粘膜免疫的病原体。

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.