Isolaton of new phage enzymes to kill B. anthracis

描述(由申请人提供)：我们目前对炭疽杆菌和其他细菌生物武器的防御主要基于抗生素疗法的使用。考虑到这类武器中使用的细菌可能对当前的治疗性或预防性抗生素表现出自然发生或人工产生的耐药性，这一计划是有缺陷的。为此，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赖氨酸对感兴趣的细菌病原体，特别是炭疽杆菌提供快速和特异的杀灭作用。除了提供一种以前无法获得的杀菌方法外，噬菌体赖氨酸的主要吸引力在于，即使在进行了大量尝试后，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在粘膜表面和血液中定植的致病菌。这些酶是特定于酶来源的物种或菌株的，这表明这些酶可能只用于靶向杀灭病原菌，而对正常的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(即酰胺酶和胞壁酰胺酶)在它们的杀伤能力方面具有协同效应。在我们对炭疽杆菌特异的伽玛噬菌体PlyG噬菌体酶的研究中，我们表明这种酶能够在体外杀死炭疽杆菌，在两分钟内将108个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杆菌和炭疽杆菌密切相关的致命性攻击。由于这些酶的协同作用，这一应用程序旨在识别和开发炭疽杆菌的酶组合，这些酶攻击芽孢杆菌细胞壁的四个不同的键。这将确保更有效的杀灭行动，并减少对这些酶产生抗药性的可能性。噬菌体酶将从环境中发现的噬菌体和溶原炭疽杆菌的噬菌体中分离出来。然后将对这些酶的特异性进行表征、纯化并在体外和体内系统中使用，以确定疗效。因为这些酶可能是抵抗抗药性炭疽杆菌攻击的一条重要防线，如果有一些酶可供我们使用，可能会在必要时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.