Inhibition of GBS Carriage by Engineered Lactobacilli

描述(申请人提供)：B组链球菌(GBS)是我国新生儿严重细菌感染的主要原因。出生时患有早发性GBS感染的婴儿的细菌来源是他们母亲的产道。这项申请描述了一种全新的方法来预防或消除GBS的阴道携带，从而减少新生儿GBS感染的发生率。其目标是通过基因工程，在阴道中产生一种正常的共生生物--乳杆菌，以分泌一种专门防止GBS生长的物质。所选择的物质是一种GBS噬菌体裂解素，它能降解GBS的细胞壁。第一个特异性目的是克隆并在大肠杆菌中表达GBS噬菌体裂解素，并检测纯化的重组酶的杀菌活性。纯化的溶酶对GBS和各种其他细菌的杀灭能力将首先在体外进行研究。然后，将确定经阴道注入这种酶是否会消除经阴道定居的小鼠的GBS。第二个特定目的是确定GBS噬菌体裂解酶的基本生化性质，包括鉴定它所属的酶类以及特定肽聚糖裂解所需的细胞壁成分(S)的身份。第三个具体目标是设计一种乳杆菌来分泌GBS噬菌体裂解素。这将首先使用包含从转化的乳杆菌高效分泌溶素所需的所有元素的质粒构建来完成。然后，为了克服固有的质粒不稳定性，同时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的染色体中。最终的具体目标是确定用工程乳杆菌对小鼠的阴道定植是否会阻止GBS建立持久的定植。此外，还将确定经阴道接种能够分泌GBS噬菌体裂解素的乳杆菌是否会导致GBS从先前定居在这种微生物中的小鼠的阴道中清除。这项拟议的研究可能会导致开发一种有效的新方法，长期抑制GBS阴道定植，即使女性可能会反复接触这种细菌。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，这种方法也可能用于预防其他生殖器、口腔和肠道病原体，特别是那些似乎没有形成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.