Isolation of new phage enzymes to kill B. anthracis

描述(申请人提供)：控制环境中炭疽病宿主的发生和持续的生态因素仍然不清楚。长期以来，人们认为炭疽杆菌的生长或营养形态不能在动物宿主之外存活，必须立即分化为休眠的内孢子，这一观点缺乏直接证据的支持。在我们的研究中，我们发现了炭疽杆菌一种更具活力的生活方式，在这种生活方式中，接触环境中的噬菌体深刻地改变了营养体和孢子体的长期生存能力。利用从不同环境中分离的一系列新的噬菌体，我们发现炭疽杆菌稳定的溶原菌经历了一个溶源转化过程，这与其产孢子、产生胞外多糖、形成生物膜、在土壤中生存和在蚯蚓肠道定居的能力的重大变化有关。因此，对于炭疽杆菌来说，噬菌体能够替代黯淡的孢子形成前景，并表明在爆发周期之间处于一个重要的环境阶段。在这里，我们试图扩大我们对炭疽杆菌溶原性转化的分析，以了解噬菌体引起这些变化的机制。初步证据表明，炭疽杆菌的溶原性转换是通过一种新的机制发生的，其中噬菌体编码的西格玛因子驱动细菌编码的表型的表达。为了追求这些发现，我们将首先使用一系列遗传方法来鉴定由六个已知环境噬菌体和炭疽杆菌噬菌体增强型元基因组文库的成分编码的溶原原转换因子。我们还将通过各种基因技术、转录研究和突变构建来鉴定至少两种溶原表型-生物膜形成和蚯蚓定植-的噬菌体诱导的炭疽杆菌编码的效应物。通过这种方式，我们打算研究炭疽杆菌前驱体可以驱动与环境生存相关的新表型的阐述的机制。作为这项工作的一部分，我们还将确定溶原菌是否会改变炭疽杆菌的毒力。最后，我们将确定噬菌体在炭疽杆菌中是如何存在的(以质粒或整合原噬菌体的形式)，以及它们的存在如何影响毒力质粒的维持和水平转移进出该病原体。如果我们要制定预防炭疽杆菌感染的策略，这些发现对炭疽杆菌生活史及其进化、维持和转移其致病表型的能力以及对受感染动物以外的环境的反应能力的影响是重要的。最终，如果我们能够了解病毒是如何帮助病原体适应寄主外的生活的，那么我们可能不仅能够利用这些机制来控制炭疽杆菌的毒力，而且还能够控制其他具有延长土相的A类生物制剂的毒力，如鼠疫耶尔森氏菌和图拉氏方济各氏菌，它们也具有广泛的环境噬菌体系统。 公共卫生相关性：我们首次发现炭疽杆菌炭疽杆菌可以在受感染的宿主外以长期、无孢子的形式存活，并由于受到环境噬菌体的感染而在新的利基环境中生存。这一新信息现在将使我们能够检查在其他A类生物制剂中发生的类似情况，这些生物制剂的土壤相延长，如鼠疫耶尔森氏菌(鼠疫制剂)和方济各氏菌图拉氏菌(图拉热制剂)。这一最新信息改变了我们对这些病原体多功能性的看法，使我们能够设计出创新的策略，在环境和感染期间控制它们。

DESCRIPTION (provided by applicant): Ecological factors governing the occurrence and persistence of anthrax reservoirs in the environment remain obscure. A long-held belief that the growing, or vegetative, form of B. anthracis does not survive outside its animal host and must immediately differentiate into a dormant endospore is poorly supported by direct evidence. In our studies, we have discovered a far more dynamic lifestyle for B. anthracis in which exposure to environmental bacteriophage profoundly alters the long-term survival capacities of both vegetative and spore forms. Using a collection of novel bacteriophages isolated from a variety of environments, we showed that stable lysogens of B. anthracis undergo a process of lysogenic conversion that is associated with major changes in their capacity to sporulate, produce exopolysaccharide, form biofilms, survive in the soil, and colonize the earthworm gut. Thus, for B. anthracis, bacteriophages enable alternatives to the bleak prospect of sporulation and indicate an important environmental phase between outbreak cycles. Here, we seek to expand our analysis of lysogenic conversion with B. anthracis to understand the mechanism by which bacteriophages induce these changes. Preliminary evidence suggests that lysogenic conversion in B. anthracis occurs by a novel mechanism in which phage-encoded sigma factors drive the expression of bacterial-encoded phenotypes. To pursue these findings, we will first use a series of genetic methods to identify the lysogen-converting factors encoded by six known environmental phages and by constituents of B. anthracis phage-enhanced metagenomic libraries. We will also identify the phage-induced, B. anthracis- encoded effectors of at least two lysogen phenotypes - biofilm formation and earthworm colonization - through a variety of genetic techniques, transcription studies, and mutant constructions. In this manner we intend to study the mechanism by which prophages of B. anthracis can drive the elaboration of novel phenotypes related to environmental survival. As part of this work, we will also determine whether lysogeny alters the virulence of B. anthracis. Finally, we will determine how phages exist in B. anthracis (as plasmidial or integrated prophage forms) and how their presence affects virulence plasmid maintenance and horizontal-transfer into and out of this pathogen. The implications of these findings with respect to the B. anthracis lifecycle and its ability to evolve, maintain and transfer its pathogenic phenotype, and respond to environments other than an infected animal are important if we are to devise strategies to prevent infection by this pathogen. Ultimately, if we can understand how viruses help pathogens adapt to life outside their host, then we may be able to use these mechanisms to control not only B. anthracis virulence, but that of other Category A biological agents with extended soil phases, like Yersinia pestis and Francisella tularensis, which also have extensive environmental phage systems. PUBLIC HEALTH RELEVANCE: We have discovered for the first time that the anthrax agent Bacillus anthracis can survive outside the infected host in long-lived, non-spore forms and in novel niches as a result of being infected by environmental bacteriophages. This new information will now enable us to examine similar occurrences in other Category A biological agents with extended soil phases, like Yersinia pestis (the plague agent) and Francisella tularensis (the tularemia agent). This latest information changes our thinking about the versatility of these pathogens, allowing us to devise innovative strategies to control them both in the environment and during infection.