Understanding Phage-Host Interactions to Improve Rapid Diagnosis of Early TB Infections in Humans

尽管世界卫生组织制定了全球结束结核病战略，但结核病仍被列为全球人类死亡的十大原因之一。控制这种疾病的策略的一个主要限制是目前的诊断测试不够充分。我们最近描述了一种新的快速检测牛分枝杆菌感染的方法(Actiphage)。最近我们报道，这也可以用来检测人类血液样本中的结核分枝杆菌，作为活动性疾病的早期指征。活性噬菌体测试使用的是一种专门感染分枝杆菌的噬菌体，并在病毒复制周期结束时将这些难以溶解的细菌裂解。这使得使用标准的基于PCR的分枝杆菌鉴定方法可以检测到低数量的细胞(每毫升血液10-20个)。尽管进行了广泛的研究，但细菌细胞表面的活化噬菌体受体仍然未知，但众所周知，培养条件可以影响噬菌体感染的效率。一个可能防止感染的未知因素是从人类样本中分离的分枝杆菌的形态转变为细胞壁缺陷的L--这种形式是标准显微镜染色无法检测到的。在人类中，L型与持续感染有关，可能是由于免疫识别发生了改变，问题是从患者的血液样本中提取的L型分枝杆菌能否通过噬菌体检测到。该项目将使用一系列方法学来尝试和更好地了解分枝杆菌与噬菌体的相互作用，包括产生分枝杆菌的生物发光和荧光衍生物，以使简单的显微镜确认L形式的存在。将使用活跃生长的分枝杆菌和L形式的分枝杆菌来研究噬菌体-宿主相互作用的动力学。此外，还将筛选转座子突变体文库，以确定噬菌体感染所需的基因。所有这些信息将被用来缩小候选受体基因的范围，然后使用标准的基因敲除技术来突变这些候选受体基因，以测试假定的受体的作用。该项目的最终目标将是利用所获得的知识来确定是否可以使用激活噬菌体方法或该方法的改进来检测人体血液样本中的L型，并进一步了解L型在人类感染中的作用。除了成为分枝杆菌基因操作方面的专家外，成功的候选人还将接受与病原体合作的培训，最高可达BS 3级。

Despite the WHO global End TB Strategy, Tuberculosis is still listed as one of the top 10 causes of human deaths worldwide. A major limitation of strategies to control this disease is that current diagnostic tests are inadequate. We have recently described a new rapid test for the detection of mycobacterial infections in cattle (Actiphage). Recently we reported that this can also be used to detect Mycobacterium tuberculosis in human blood samples as an early indication of active disease. The Actiphage test uses a bacteriophage that specifically infects Mycobacteria and breaks open these difficult to lyse bacteria at the end of the viral replication cycle. This allows low numbers of cells (10-20 per ml of blood) to be detected using standard PCR-based mycobacterial identification methods. Despite extensive study, the Actiphage receptor on the bacterial cell surface remains unknown, but it is known that culture conditions can affect the efficiency of phage infection. One unknown factor that may prevent infection is the morphological transition of mycobacteria isolated from human samples into cell-wall deficient L-forms that are not detected using standard microscopic stains. In humans the L-form is associated with persistent infection, perhaps due to altered immune recognition and the question remains whether L-form mycobacteria present in blood samples taken from patients can be detected by Actiphage. This project will use a range of methodologies to try and better understand the mycobacterium-phage interaction, including generating bioluminescent and fluorescent derivatives of Mycobacteria to allow simple microscopic confirmation of the presence of L-forms. Studies will be performed to interrogate the kinetics of phage-host interactions using both actively growing and L-form mycobacteria. In addition a library of Transposon mutants will be screened to identify genes required for phage infection. All of this information will be used to narrow down the candidate receptor genes which will then be mutated using standard gene knock out technology to test the role of the putative receptors. The ultimate aim of the project will be to use the knowledge gained to determine whether L-forms in human blood samples can be detected using the Actiphage method - or a modification of this method - and to provide further understanding of the role of L-forms in human infection. In addition to becoming expert in mycobacterial genetic manipulation, the successful candidate will gain training in working with pathogens up to BS level 3.