Bacteriolytic phage enzymes as novel antibacterials against Yersinia pestis

描述（由申请人提供）：迫切需要新型抗菌剂。许多物种已经对目前的抗生素产生了抗药性，并且在生物恐怖袭击中可能会使用选择剂和其他细菌。特别是，鼠疫耶尔森氏菌（鼠疫的病原体）的一些分离株已知对常用抗生素具有多种抗性决定簇。噬菌体编码的溶菌酶是治疗多种细菌感染的有前途的试剂，因为它们的作用机制不同于抗生素。最近的几份报告已经评估了噬菌体酶用于局部和全身使用的潜力，但最近只有对革兰氏阳性细菌有活性的溶素在治疗上被成功地测试。然而，这些酶对革兰氏阴性菌没有活性，革兰氏阴性菌是大多数医院感染的原因。革兰氏阴性菌的外膜对大分子是不可渗透的。我们建议开发基于能够裂解革兰氏阴性菌的噬菌体酶的新型抗菌剂。在这些细菌上生长的噬菌体显然已经开发出穿透外膜的机制。我们将使用鼠疫耶尔森氏菌作为最初的模式生物，因为它是粗糙的，因此具有比大多数主要革兰氏阴性病原体更简单的外膜。噬菌体？XA 1122，已知其在几乎所有Y上生长并裂解。鼠疫菌株与大肠杆菌噬菌体T7关系密切，T7突变体在鼠疫菌上生长良好。鼠疫是可用的。我们建议纯化噬菌体编码的壁溶酶，并将它们展示在噬菌体病毒体上，以在治疗性治疗期间保持酶的高局部浓度。Muralytic酶将与两种蛋白质一起使用，以促进进入细胞壁：T7 gp 14从感染的病毒体中排出，并形成穿过外膜的通道，而噬菌蛭弧菌表面蛋白CAE 77837用于革兰氏阴性宿主的入侵。两种蛋白质都将被纯化，CAE 77837也作为约100个残基的蛋白酶结构域，将展示在T7病毒体上。蛋白质，或常见的外膜不稳定的化学物质，将被测试其与壁酶协同作用的能力。我们的长期目标是开发新的抗菌剂，可用于治疗革兰氏阴性菌感染。我们将在体外优化试剂组合对Y.鼠疫，然后测试最有效的制剂在体内，在预防性和治疗性治疗，使用鼠疫感染的鼠模型。公共卫生相关性本提案中描述的工作将提供使用噬菌体编码的裂解酶与外膜渗透物结合作为针对Y.鼠疫菌作为革兰氏阴性菌的模型。这类酶已被证明对革兰氏阳性菌有活性，但革兰氏阴性菌的外膜阻止了它们的直接应用。在初步研究中，我们已经表明噬菌体感染的粗裂解物能够杀死和裂解Y。鼠疫选择Y。鼠疫菌作为模式生物的预测主要基于其不完整的核心LPS和最小的包膜。先验地，基于噬菌体酶的抗菌制剂的开发预期对于Y.鼠疫杆菌比含有完整LPS和O-抗原的细菌。然而，如果我们能证明对Y.鼠疫在小鼠模型中，未来的研究将扩展该技术到其他革兰氏阴性病原体。我们使噬菌体溶素进入革兰氏阴性细菌细胞壁的方法是制备一种制剂，该制剂还含有常见的膜去稳定化化学物质、在感染开始时形成穿过外膜的通道的噬菌体病毒体蛋白或B的表面内肽酶。噬菌菌体，其钻入目标革兰氏阴性细胞的周质中。因此，我们要求“辅助试剂”促进噬菌体溶素接近细菌细胞壁。我们还建议测试Y。鼠疫噬菌体XA 1122，被CDC用作诊断试剂，因为它能够提供针对Y的预防和/或治疗益处。鼠疫感染这些体内研究将使用鼠疫感染的小鼠模型与涉及溶素的研究平行。

DESCRIPTION (provided by applicant): There is an urgent need for novel antibacterial agents. Many species have become resistant to current antibiotics and there is the potential use of Select Agents and other bacteria in bioterrorist attacks. In particular, some isolates of Yersinia pestis, the causative agent of plague, are known to harbor multiple resistance determinants to commonly used antibiotics. Phage-encoded bacteriolytic enzymes are promising reagents to treat a wide range of bacterial infections because their mechanisms of action are different from those of antibiotics. Several recent reports have evaluated the potential of phage enzymes for both topical and systemic use, but only lysins active against Gram-positive bacteria have recently been successfully tested therapeutically. However, these enzymes are not active against Gram-negative bacteria, which are responsible for the majority of hospital infections. The outer membrane of Gram-negative bacteria is impermeable to macromolecules. We propose to develop novel antibacterial agents based on phage enzymes capable of lysing Gram-negative bacteria. Phages that grow on these bacteria have obviously developed mechanisms for penetrating the outer membrane. We will use Yersinia pestis as the initial model organism because it is rough and thus possesses a less complex outer membrane than most major Gram-negative pathogens. Phage ?XA1122, which is known to grow on and lyse virtually all Y. pestis strains, is closely related to coliphage T7, and T7 mutants that grow well on Y. pestis are available. We propose to purify the phage-encoded muralytic enzymes, and also to display them on phage virions in order to maintain high local concentrations of the enzyme during therapeutic treatment. Muralytic enzymes will be used with two proteins to promote access to the cell wall: T7 gp14 is ejected from infecting virions and makes a channel across the outer membrane, and the Bdellovibrio bacteriovorus surface protein CAE77837 is used for invasion of Gram-negative hosts. Both proteins will be purified, CAE77837 also as a ~100 residue protease domain that will be displayed on T7 virions. The proteins, or common outer membrane-destabilizing chemicals, will be tested for their ability to act synergistically with the muralytic enzymes. Our long-term goal is to develop novel antibacterial agents that can be used therapeutically for Gram-negative infections. We will optimize conditions for bacteriolytic and bacteriocidal activity of combinations of the reagents in vitro against Y. pestis, and then test the most effective formulations in vivo, in both prophylactic and therapeutic treatments, using a murine model of plague infection. PUBLIC HEALTH RELEVANCE The work described in this proposal will provide a thorough assessment of the feasibility of using phage-encoded lytic enzymes, in conjunction with outer membrane permeants, as novel antibacterial agents directed against Y. pestis as a model Gram-negative bacterium. This class of enzymes has been demonstrated to be active against Gram-positive bacteria but the outer membrane of Gram-negatives prevents their direct application. In preliminary studies we have shown that phage-infected crude lysates are competent for killing and lysing Y. pestis. The choice of Y. pestis as a model organism is predicated mainly on its incomplete core LPS and minimal capsule. A priori, development of an antibacterial formulation based on phage enzymes is expected to be less problematic with Y. pestis than with bacteria containing a complete LPS and O-antigen. However, if we can demonstrate prophylactic and/or therapeutic activity against Y. pestis in a mouse model, future studies will extend the technology to other Gram-negative pathogens. Our approach to allowing the phage lysins access to the Gram-negative bacterial cell wall is to make a formulation that also contains either common membrane-destabilizing chemicals, the phage virion protein that forms a channel across the outer membrane at the initiation of infection, or the surface endopeptidase of B. bacteriovorus, which bores its way into the periplasm of a target Gram-negative cell. We are therefore asking that the "accessory reagents" promote access of the phage lysins to the bacterial cell wall. We are also proposing to test Y. pestis phage ?XA1122, used by the CDC as a diagnostic reagent, for its ability to provide prophylactic and/or therapeutic benefits against Y. pestis infections. These in vivo studies will parallel those involving lysins using the mouse model of plague infection.