TARGETABLE BACTERICIDAL PROTEINS TO SPECIFICALLY KILL CLOSTRIDIUM DIFFICILE BACTE

描述(由申请人提供)：这项提案和任何后续的第二阶段提案的最终目标是开发一种独特的蛋白质制剂，以预防那些高风险患者的艰难梭菌相关疾病，而不是等待治疗他们危险和昂贵的感染。在这个SBIR第一阶段项目中，我们的目标是确定口服铅的可行性，特别是靶向杀菌蛋白，以消除艰难梭菌的龋齿，而不会对肠道微生物区系造成意外的附带损害。领先的候选者被称为“扩散素”，杀死了28株BI/NAP1/027菌株中的27株；Lawley等人(2010)描述了艰难梭菌携带的小鼠模型。我们最近已经证明，另一种工程R型细菌素口服给兔子可以通过胃肠道作为一种特定的杀菌剂，杀死末端回肠和结肠中的另一种细菌病原体。因此，在提高这种先导重组蛋白在枯草杆菌中的实验室规模并进行初步的药理学和药效学研究以指导给药后，我们将评估Diffocin对C57BL/6小鼠的疗效，这些小鼠是艰难梭菌敏感株的无症状携带者和脱落者。如果Diffocins能消除携带艰难梭菌的小鼠，那么口服Diffocin对小鼠肠道微生物区系的影响将通过给药后正常和艰难梭菌携带者的粪便微生物组的核糖体分型来确定。 与公共卫生相关：艰难梭菌是一种可以驻留在人体肠道中的细菌，在共享肠道空间的健康细菌被抗生素破坏之前不会导致疾病。由于艰难梭菌通常对抗生素具有抗药性，它们可以繁殖，产生强有力的毒素，导致严重腹泻和潜在的致命性结肠炎。现在，这种感染在医院比MRSA更常见。我们已经发现、克隆并制造了一种蛋白质，它能特异性地杀死最有毒的艰难梭菌。我们打算确定这种药物，一种“扩散素”，当口服给药时，是否能够消除在人类感染的小鼠模型中无害地驻留在肠道中的艰难梭菌。如果是这样的话，我们计划开发这种杀菌蛋白作为一种试剂，在患者接受抗生素之前杀死患者体内存在的艰难梭菌，从而在不损害肠道健康细菌的情况下防止严重的反复感染。

DESCRIPTION (provided by applicant): The ultimate goal of this proposal and any subsequent phase II proposal is to develop a unique protein agent to prevent Clostridium difficile associated diseases in those patients at high risk rather than wait to treat their dangerous and costly infections. We aim in this SBIR phase I project to determine the feasibility of oral delivery of a lead, specifically targeted bactericidal protein to eliminate C. difficile cariage without untended collateral damage to the intestinal microbiota. The lead candidate, termed a "diffocin", kills 27 of a collection of 28 BI/NAP1/027 strains; and Lawley et al (2010) have described a mouse model of C. difficile carriage. We have recently shown that another engineered R-type bacteriocin administered orally to rabbits can transit the GI tract to act as a specific bactericidal agent killing another bacterial pathogen in the terminal ileum and colon. Thus, after improving the lab scale production of this lead recombinant protein in B. subtilis and conducting preliminary pharmacology and pharmacodynamic studies to guide dosing, we shall evaluate the efficacy of diffocins in C57Bl/6 mice that are asymptomatic carriers and shedders of a sensitive strain of C. difficile. If diffocins eliminate C. difficile from carrier mice, the efect of oral diffocins on the mouse intestinal microbiota will be determined by ribotyping the fecal microbiome of normal and C. difficile carrier mice after diffocin administration. PUBLIC HEALTH RELEVANCE: Clostridium difficile is a bacterium that can reside in human intestines and not cause disease until the healthy bacteria sharing the intestinal space are damaged by antibiotics. Because Clostridium difficile bacteria are usually resistant to antibiotics they can then proliferate, make potent toxins, and cause severe diarrhea and potentially lethal inflammation of the colon. Such infections are now more common in hospitals than MRSA. We have discovered, cloned and made a protein that specifically kills the most toxic form of Clostridium difficile bacteria. We intend to determine whether this agent, a "diffocin", when administered orally is capable of eliminating Clostridium difficile bacteria residing innocuously i the intestine of a mouse model of the human infection. If so, we plan to develop this bacteria-killing protein as an agent to kill Clostridium difficile present in patients before they receive antibiotics and thereby prevent the severe, recurring infections without damaging the healthy bacteria of the gut.