Isolation of new phage enzymes to kill B. anthracis

描述（由申请人提供）：控制环境中炭疽宿主发生和持续存在的生态因素仍然不清楚。一种长期以来的观点认为，生长或营养形式的B。炭疽不能在其动物宿主之外存活，必须立即分化成休眠的内生孢子，这一点没有得到直接证据的支持。在我们的研究中，我们发现了B更有活力的生活方式。炭疽病，其中暴露于环境噬菌体深刻地改变了营养体和孢子形式的长期生存能力。利用从各种环境中分离的新型噬菌体，我们发现B.炭疽菌经历溶原性转化过程，这与它们形成孢子、产生胞外多糖、形成生物膜、在土壤中存活和定殖肠的能力的重大变化有关。因此，对于B.在炭疽病中，噬菌体使孢子形成的暗淡前景成为替代方案，并表明爆发周期之间的一个重要环境阶段。在这里，我们试图扩大我们的分析溶原性转换与B。炭疽菌，以了解噬菌体诱导这些变化的机制。初步证据表明B.炭疽通过一种新的机制发生，其中噬菌体编码的σ因子驱动细菌编码的表型的表达。为了进一步研究这些发现，我们将首先使用一系列遗传学方法来鉴定由六种已知环境因子和B组分编码的溶原转化因子。炭疽菌噬菌体增强的宏基因组文库。我们还将鉴定噬菌体诱导的，B。炭疽编码的至少两种溶原表型的效应物-生物膜形成和细菌定植-通过多种遗传技术、转录研究和突变体构建。以这种方式，我们打算研究B.炭疽病可以促使与环境生存有关的新表型的形成。作为这项工作的一部分，我们还将确定溶原性是否改变了B的毒力。炭疽病最后，我们将确定如何在B中存在。炭疽菌（作为质粒或整合的原噬菌体形式），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这种病原体。这些发现对B.如果我们要设计出预防这种病原体感染的策略，炭疽的生命周期及其进化、维持和转移其致病性表型的能力以及对感染动物以外的环境的反应是重要的。最终，如果我们能够了解病毒如何帮助病原体适应宿主之外的生活，那么我们也许能够利用这些机制来控制的不仅仅是B。炭疽菌的毒性，但其他A类生物制剂的扩展土壤阶段，如鼠疫耶尔森氏菌和土拉弗朗西斯菌，也有广泛的环境噬菌体系统。

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.