Using experimental evolution to create phage therapy agents to target the Horse Chestnut bleeding canker pathogen, Pseudomonas syringae pv. aesculi

在英国，一种出血性溃疡病正在困扰着大约47万棵具有生态和社会重要性的欧洲七叶树。该疾病是由细菌病原体，假单胞菌pv。七叶树（Pae），其感染树干和树枝中的韧皮部和木质部导管，当树木分泌物从感染部位流出时引起“出血”（绿色等人，2009 Plant Pathology 58，731-744）;其可最终导致树木死亡，从而造成另一种“荷兰榆树”灾难的可能性。噬菌体具有巨大的治疗细菌性疾病的潜力，因为它具有很大的多样性和在实验室中进化出多种基因型以作为多基因型混合物使用的能力。噬菌体疗法也可以用作预防性应用，以充当感染的进一步屏障。我们建议抽样调查：分析多样性;进化噬菌体与其主机创建新的基因型;分析噬菌体治疗树木的功效。目标1：采样和多样性为了对环境中的Pae菌进行采样，将从健康和患病的树木组织以及周围的土壤中分离出Pae菌，并将其以半固体覆盖物的形式接种在琼脂平板上的Pae草坪上：将通过其菌斑形态来识别裂解菌。平板计数将确定感染大肠杆菌的溶菌群的丰度。将使用显微镜和分子技术对裂解酶进行表征和比较。目标2：实验进化（两个阶段）第1阶段-进行大筛选以评估分离噬菌体的协同进化潜力。每种独特的噬菌体分离株的三个重复群体（如果>50，则使用分离株的代表性子样品）和Pae将通过分批培养（每48小时将1%的每个群体转移到新鲜的微观世界中）在微观世界（含有6 ml KB液体培养基的30 ml玻璃通用物）中繁殖12次转移。将每第2个转移群体样品在-80 ℃下在20%甘油中冷冻，并通过氯仿处理分离噬菌体样品并在4 ℃下储存。每4次转移，我们将使用标准时移协议测量共同进化（Brockhurst 2003 Ecol Lett 6：975-979）。细菌-噬菌体协同进化的特征是随着时间的推移，感染性和耐药性范围越来越广。因此，如果在重复群体中并且对于多个时间步，噬菌体感染性和细菌抗性随时间的斜率为正，则将定义为显著的协同进化。表现出最大共同进化的5个物种将被选择用于进一步的实验。阶段2 -进行两种类型的长期选择实验：[1]共同进化，选择增加的噬菌体宿主范围（即，可以感染广泛的细菌基因型的噬菌体），将如上所述进行实验50次转移，将使用标准时移实验来评估共同进化的进展;[2]在选择高宿主利用率的固定细菌基因型上进行传代，通过分批培养进行实验，由此在每次转移时仅将噬菌体从先前的微观世界转移到新的微观世界，而这些细菌被从实验室储备中生长出来的祖先基因型所取代。在50次转移后，通过进行一步生长曲线实验以确定关键的生活史参数（即吸附速率、裂解时间、爆发大小），在进化的噬菌体系中评估对选择的响应。目标三：噬菌体疗法在树木中的功效将使用在CASE合作伙伴BTE提供的受控环境生长室中生长的树木进行。使用不同的施用模式（喷雾和高压注射）将不同滴度的单一和混合噬菌体接种物施用于感染和预感染的树，以确定噬菌体作为预防剂以及治疗剂的效率。

A bleeding canker disease epidemic is afflicting the circa 470,000 ecologically and socially important European Horse Chestnut (Aesculus hippocastanum) trees in Britain. The disease is caused by the bacterial pathogen, Pseudomonas syringae pv. aesculi (Pae), which infects phloem and xylem vessels in the trunk and branches, causing 'bleeding' as tree exudates flow from infected sites (Green et al. 2009 Plant Pathology 58, 731-744); it can eventually lead to tree death thus posing the potential for another 'Dutch Elm' disaster. Bacteriophages have enormous potential for treating bacterial disease because of the large diversity and the ability to evolve multiple genotypes in the laboratory to use as multi-genotype mixtures. Phage therapies could also be used as a prophylactic application to act as a further barrier to infection. We propose to sample phages to: analyse diversity; evolve phage with its host to create new genotypes; analyse the efficacy of phage in treating trees. Objective 1: Sampling and diversity To sample environmental Pae phages, phages will be isolated from healthy and diseased tree tissue and from the surrounding soil and plated onto Pae lawns in semi-solid overlays on agar plates: lytic phages will be identified by their plaque morphology. Plate counts will determine the abundance of Pae-infecting lytic phages. Lytic phages will be characterised and compared using microscopy and molecular techniques. Objective 2: Experimental evolution (two stages) Stage 1 - Perform a large screen to assess the coevolutionary potential of isolated phages. Three replicate populations of each unique phage isolate (if >50 a representative subsample of isolates will be used) and Pae will be propagated in microcosms (30ml glass universals containing 6ml of KB liquid medium) by batch culture (1% of each population will be transferred to a fresh microcosm every 48 hours) for 12 transfers. Every 2nd transfer population samples will be frozen at -80C in 20% glycerol, and phage samples will be isolated by treatment with chloroform and stored at 4C. Every 4th transfer we will measure coevolution using a standard time-shift protocol (Brockhurst 2003 Ecol Lett 6:975-979). Bacteria-phage coevolution is characterised by the evolution of increasingly broad infectivity and resistance ranges through time. Therefore, significant coevolution will be defined if in replicate populations and for multiple timesteps there is a positive slope of phage infectivity and bacterial resistance with time. The 5 phages that display the greatest coevolution will be selected for further experiments. Stage 2 - Perform two types of long-term selection experiment: [1] coevolution, which selects for increased phage host range (i.e. phage that can infect a wide range of bacterial genotypes), experiments will be performed as described above for 50 transfers, standard time shift experiments will be used to assess the progress of coevolution; [2] passaging on a fixed bacterial genotype, which selects for high rates of host exploitation, experiments will be performed by batch culture whereby at each transfer only the phage is transferred from the previous to the new microcosm, and the bacteria are replaced by the ancestral genotype grown up from the laboratory stock. After 50 transfers the response to selection will be assessed in evolved phage lines by performing one-step growth curve experiments to determine key life history parameters (i.e. adsorbtion rate, lysis time, burst size). Objective 3: Therapy trials The efficacy of phage therapies in trees will be done using trees grown in controlled environment growth rooms supplied by the CASE partner, BTE. Different titers of single and mixed phage inocula will be applied to infected and pre-infected trees using different modes of application (spray and high pressure injection) to determine the efficiency of phage as a prophylactic as well as a therapy.