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。长期以来，人们一直认为生长或营养形式的炭疽芽孢杆菌不能在其动物宿主外存活，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，我们发现了炭疽芽孢杆菌更有活力的生活方式，在这种生活方式中，暴露于环境噬菌体会深刻地改变营养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，我们发现稳定的炭疽芽孢杆菌溶原经历了一个溶原转化过程，这与它们产孢、产生外多糖、形成生物膜、在土壤中存活和定植蚯蚓肠道的能力发生重大变化有关。因此，对于炭疽芽孢杆菌，噬菌体可以替代黯淡的产孢前景，并表明爆发周期之间的重要环境阶段。在这里，我们试图扩大我们与炭疽芽胞杆菌的溶原转化的分析，以了解噬菌体诱导这些变化的机制。初步证据表明，炭疽芽孢杆菌的溶原转化是通过噬菌体编码的sigma因子驱动细菌编码表型的表达的新机制发生的。为了实现这些发现，我们将首先使用一系列遗传方法鉴定由六种已知环境噬菌体和炭疽芽孢杆菌噬菌体增强宏基因组文库成分编码的溶菌原转化因子。我们还将通过各种遗传技术、转录研究和突变体构建，鉴定噬菌体诱导的、炭疽芽孢杆菌编码的至少两种溶原表型（生物膜形成和蚯蚓定植）的效应物。通过这种方式，我们打算研究炭疽芽孢杆菌的噬菌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，我们还将确定溶原性是否会改变炭疽杆菌的毒力。最后，我们将确定噬菌体如何在炭疽芽胞杆菌中存在（作为质粒或整合的噬菌体形式），以及它们的存在如何影响毒力质粒的维持和水平转移进入和离开这种病原体。如果我们要设计预防这种病原体感染的策略，这些发现对炭疽芽胞杆菌的生命周期及其进化、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的意义是重要的。最终，如果我们能够理解病毒如何帮助病原体适应宿主外的生活，那么我们可能不仅能够使用这些机制来控制炭疽芽孢杆菌的毒力，还可以控制其他具有延长土壤相的A类生物制剂，如鼠疫耶尔森氏菌和土拉弗朗西斯菌，它们也具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. 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.