Isolaton of new phage enzymes to kill B. anthracis

描述（由申请人提供）：我们目前对炭疽芽孢杆菌和其他细菌生物武器的防御主要基于抗生素治疗的使用。考虑到用于此类武器的细菌可能对目前的治疗性或预防性抗生素表现出自然产生的或经过改造的耐药性，这一计划存在缺陷。因此，我们目前正在开发一类新型抗菌剂。我们的系统是基于使用噬菌体溶酶来提供对感兴趣的细菌病原体的快速和特异性杀伤作用，特别是炭疽芽孢杆菌。除了提供一种以前无法获得的杀死细菌的方法外，噬菌体溶酶的主要吸引力在于，即使经过广泛的尝试，也无法检测到细菌对其作用的耐药性。我们的实验室是第一个使用这些酶的纯化形式来杀死粘膜表面和血液中的定植致病菌的实验室。这些酶对其衍生的物种或菌株具有特异性，表明这些酶可能仅用于靶向杀死致病菌，而对正常菌群细菌几乎没有影响。在这些研究中，我们发现具有两种不同细胞壁键特异性的酶（即酰胺酶和酶酰胺酶）在其杀伤能力方面具有协同作用。在我们对炭疽杆菌特异性γ噬菌体的PlyG噬菌体酶的研究中，我们发现这种酶能够在体外杀死炭疽杆菌，在两分钟内将108种细菌减少到无菌状态。在体内，我们能够保护动物免受与炭疽芽孢杆菌密切相关的芽孢杆菌和炭疽芽孢杆菌的致命攻击。由于这些酶具有协同作用，本应用程序旨在鉴定和开发一种针对炭疽芽孢杆菌的酶组合，该酶组合可以攻击芽孢杆菌细胞壁上的四种不同键。这将确保更有效的杀灭行动，并减少对这些酶产生耐药性的可能性。噬菌体酶将从环境中发现的噬菌体和噬菌体溶解菌中分离出来。然后，这些酶将被表征为它们的特异性，纯化并在体外和体内系统中使用，以确定功效。因为这些酶可能是抵御耐药炭疽杆菌攻击的重要防线，所以如果有必要，我们可以使用一些酶来更好地决定它们的使用。

DESCRIPTION (provided by applicant): Our current defense against Bacillus anthracis, and other bacterial bioweapons, is based primarily on the use of antibiotic therapies. This plan is flawed considering the possibility that the bacteria used in such weapons may express naturally occurring or engineered resistances to current therapeutic or prophylactic antibiotics. For this reason we are currently developing a novel class of antibacterial agents. Our system is based on the use of phage lysins to provide a rapid and specific killing action against bacterial pathogens of interest, in particular B. anthracis. In addition to offering a previously unavailable method of bacterial killing, phage lysins are primarily attractive in that bacterial resistance to their action cannot be detected, even after extensive attempts. Our laboratory is the first to use these enzymes in their purified form to kill colonizing pathogenic bacteria on mucous membrane surfaces and in blood. The enzymes are specific for the species or strain from which the enzymes were derived, indicating that these enzymes may be used for targeted killing of only the pathogenic bacterium with little to no effect on normal flora bacteria. During these studies we discovered that enzymes with two different specificities for cell wall bonds (i.e., amidase and muramidase) have a synergistic effect in their killing capacity. In our studies with the PlyG phage enzyme from the gamma phage that is specific for B. anthracis, we show that this enzyme is able to kill anthrax bacilli in vitro, reducing 108 bacteria to sterility in two minutes. In vivo, we are able to protect animals from lethal challenge with both a closely related bacillus to B. anthracis as well as B. anthracis. Because of the synergistic effects in these enzymes, this application is designed to identify and develop a combination of enzymes for B. anthracis that attack the four different bonds in the bacillus cell wall. This will ensure a more efficient killing action as well as reduce the possibility of the development of resistance to these enzymes. Phage enzymes will be isolated from phage found in the environment and phage lysogenizing B. anthracis. These enzymes will then be characterized as to their specificity, purified and used in both in vitro and in vivo systems to determine efficacy. Because these enzymes may be an important line of defense against an attack with drug-resistant B. anthracis, having a number of enzymes at our disposal may allow for better decisions as to their use if necessary.