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（申请人提供）：目前迫切需要新型抗菌药物。许多物种已经对目前的抗生素产生耐药性，并且在生物恐怖袭击中有可能使用Select Agents和其他细菌。特别是，鼠疫病原体鼠疫耶尔森菌的一些分离株已知对常用抗生素具有多种耐药决定因素。噬菌体编码的溶菌酶因其作用机制不同于抗生素而成为治疗多种细菌感染的有前途的试剂。最近的几份报告已经评估了噬菌体酶在局部和全身使用方面的潜力，但最近只有对革兰氏阳性细菌有活性的溶酶被成功地用于治疗。然而，这些酶对革兰氏阴性菌没有活性，而革兰氏阴性菌是导致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渗透的。我们建议以能够裂解革兰氏阴性菌的噬菌体酶为基础开发新型抗菌剂。在这些细菌上生长的噬菌体显然已经发展出穿透外膜的机制。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?已知能在几乎所有鼠疫杆菌菌株上生长并溶解的XA1122与噬菌体T7密切相关，而在鼠疫杆菌上生长良好的T7突变体是可用的。我们建议纯化噬菌体编码的水解酶，并将其展示在噬菌体病毒粒子上，以便在治疗过程中保持高浓度的酶。Muralytic酶将与两种蛋白质一起使用，以促进进入细胞壁：T7 gp14从感染的病毒粒子中排出并在外膜上形成通道，而Bdellovibrio bacteriovorus表面蛋白CAE77837用于入侵革兰氏阴性宿主。这两种蛋白都将被纯化，CAE77837也将作为约100个残基蛋白酶结构域显示在T7病毒粒子上。这些蛋白质，或常见的破坏外膜稳定的化学物质，将被测试它们与水解酶协同作用的能力。我们的长期目标是开发可用于治疗革兰氏阴性感染的新型抗菌剂。我们将优化试剂组合在体外对鼠疫杆菌的溶菌和杀菌活性条件，然后利用鼠疫感染小鼠模型在体内测试最有效的预防和治疗制剂。本提案中描述的工作将全面评估使用噬菌体编码的裂解酶与外膜渗透剂作为新型抗菌剂直接对抗作为革兰氏阴性细菌的鼠疫杆菌的可行性。这类酶已被证明对革兰氏阳性细菌有活性，但革兰氏阴性细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鼠疫杆菌。鼠疫杆菌作为模式生物的选择主要取决于其不完整的核心LPS和最小的胶囊。先验地，基于噬菌体酶的抗菌制剂的开发对鼠疫杆菌的问题要小于对含有完整LPS和o抗原的细菌的问题。然而，如果我们能够在小鼠模型中证明对鼠疫杆菌的预防和/或治疗活性，未来的研究将把这项技术扩展到其他革兰氏阴性病原体。我们让噬菌体溶酶进入革兰氏阴性细菌细胞壁的方法是制作一种配方，该配方也含有常见的破坏膜稳定的化学物质，即在感染开始时形成穿过外膜通道的噬菌体病毒蛋白，或B. bacteriovorus的表面内肽酶，该酶会进入目标革兰氏阴性细胞的周质。因此，我们要求“辅助试剂”促进噬菌体溶酶进入细菌细胞壁。我们还提议测试鼠疫杆菌噬菌体？XA1122被CDC用作诊断试剂，因为它能够提供预防和/或治疗鼠疫杆菌感染的益处。这些体内研究将与使用鼠疫感染小鼠模型的溶酶研究平行。