Bacteriolytic phage enzymes as novel antibacterials against Yersinia pestis

溶菌噬菌体酶作为针对鼠疫耶尔森菌的新型抗菌剂

• 批准号: 7626322
• 负责人: IAN J MOLINEUX
• 金额: $ 29.2万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7626322
• 关键词: Animal Model; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacterial Infections; Bacteriophage T7; Bacteriophages; Bdellovibrio; Bioterrorism; Cell Wall; Cells; Centers for Disease Control and Prevention (U.S.); Chemicals; Complex; Cytolysis; DNA; Development; Diagnostic; Diagnostic Reagent; Drug Formulations; Enzymes; Extended Family; Future; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Health; Human; Immune response; In Vitro; Infection; LytA enzyme; Lytic; Membrane; Membrane Proteins; Modeling; Mus; N-Acetylmuramoyl-L-alanine Amidase; Nosocomial Infections; O Antigens; Organism; Parasites; Penetration; Peptide Hydrolases; Phage Display; Plague; Protease Domain; Proteins; Reagent; Reporting; Resistance; Safety; Staging; Structure; Surface; Technology; Temperature; Testing; Therapeutic; Virion; Work; Yersinia pestis; bacterial resistance; base; biodefense; capsule; direct application; endopeptidase B; enzyme substrate; improved; in vivo; killings; lysin; macromolecule; mouse model; mutant; novel; novel strategies; pathogen; periplasm; pre-clinical; prevent; prophylactic; public health relevance; receptor; research study; resistant strain

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.

描述（由申请人提供）：迫切需要新型抗菌剂。许多物种已经对当前的抗生素具有抗药性，并且在生物恐怖攻击中可能使用精选剂和其他细菌。特别是，已知某些鼠疫的耶尔森氏菌分离株已知，鼠疫的病因是对常用抗生素的多种耐药性决定因素。噬菌体编码的细菌酶是有望治疗多种细菌感染的有前途的试剂，因为它们的作用机理与抗生素的作用机理不同。最近的几份报告已经评估了噬菌酶在局部使用和全身使用的潜力，但是最近仅通过治疗方法成功地对革兰氏阳性细菌进行了活跃的脂蛋白。但是，这些酶对革兰氏阴性细菌并不活跃，革兰氏阴性细菌造成了大多数医院感染。革兰氏阴性细菌的外膜对大分子不可渗透。我们建议基于能够裂解革兰氏阴性细菌的噬菌体开发新型抗菌剂。在这些细菌上生长的噬菌体显然开发了穿透外膜的机制。我们将使用耶尔森氏菌作为初始模型生物，因为它是粗糙的，因此比大多数主要的革兰氏阴性病原体具有不太复杂的外膜。噬菌体？xa1122，已知会在几乎所有Y. Pestis菌株上生长，与Coliphage T7密切相关，而在Y. pestis上生长良好的T7突变体可用。我们建议纯化噬菌体编码的毛细血管酶，并在治疗期间在噬菌体病毒座上显示它们，以维持酶的局部浓度较高。壁画酶将与两种蛋白一起使用，以促进进入细胞壁：T7 GP14从感染病毒体中喷出，并在外膜上制造通道，而Bdellovibrio capteriovorus表面蛋白CAE77837则用于侵入革兰氏病宿主。这两种蛋白质都将被纯化，CAE77837也将作为一个约100个残基蛋白酶结构域，将显示在T7病毒体上。蛋白质或常见的外膜剂量化学物质将测试其与毛刺酶协同作用的能力。我们的长期目标是开发可用于革兰氏阴性感染的新型抗菌剂。我们将使用鼠疫感染的鼠类模型，在预防性和治疗性治疗中，在预防性和治疗性治疗中，在体外对Y. pestis的结合的细菌和细菌性活性的条件进行优化。公共卫生相关性本提案中描述的工作将对使用噬菌体编码的裂解酶的可行性以及外膜均与外膜均与针对Y. pestis的新型抗菌剂作为模型的革兰氏阴性细菌的可行性。这类酶已被证明对革兰氏阳性细菌具有活性，但革兰氏阴性剂的外膜可阻止其直接应用。在初步研究中，我们表明，噬菌体感染的原油裂解物具有杀死和裂解的鼠疫。鼠疫作为模型生物的选择主要是基于其不完整的核心LP和最小胶囊。先验的是，基于噬菌体酶的抗菌配方的发展预计与含有完整的LPS和O-抗原的细菌相比，Y. pestis的问题较小。但是，如果我们可以在小鼠模型中证明针对Y. pestis的预防性和/或治疗活性，则未来的研究将把技术扩展到其他革兰氏阴性病原体。我们允许噬菌体进入革兰氏阴性细菌细胞壁的方法是制作一种配方，该配方还包含常见的膜污染化学物质，即在感染开始时跨膜的噬菌体病毒素蛋白，或者是b。clacteriovorusb。clacteriovorus的表面肽酶，该通道的范围为perifliply的范围，该通道是孔子孔的孔子。因此，我们要求“辅助试剂”促进噬菌体进入细菌细胞壁。我们还提议测试CDC用作诊断试剂的Pestis Phage？XA1122，因为它能够针对Pestis感染提供预防性和/或治疗益处。这些体内研究将平行使用鼠疫感染的小鼠模型涉及裂解蛋白的研究。