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

尽管世卫组织制定了全球终止结核病战略，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，目前的诊断测试是不够的。我们最近描述了一种新的快速检测牛分枝杆菌感染（Actiphage）。最近我们报告说，这也可用于检测人体血液样本中的结核杆菌，作为活动性疾病的早期指征。Actiphage测试使用一种噬菌体，专门感染分枝杆菌，并在病毒复制周期结束时打破这些难以裂解的细菌。这允许使用标准的基于PCR的分枝杆菌鉴定方法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，但细菌细胞表面上的Actiphage受体仍然未知，但已知培养条件会影响噬菌体感染的效率。一个未知的因素，可能会防止感染的形态转变的分枝杆菌从人类样本分离到细胞壁缺陷的L型，不能检测到使用标准的显微镜染色。在人类中，L型分枝杆菌与持续感染有关，可能是由于免疫识别的改变，问题仍然是从患者血液样本中提取的L型分枝杆菌是否可以被Actiphage检测到。该项目将使用一系列方法来尝试更好地了解分枝杆菌-噬菌体相互作用，包括产生分枝杆菌的生物发光和荧光衍生物，以允许简单的显微镜确认L型的存在。将使用活跃生长的分枝杆菌和L型分枝杆菌进行研究以询问噬菌体-宿主相互作用的动力学。此外，将筛选转座子突变体文库以鉴定噬菌体感染所需的基因。所有这些信息将用于缩小候选受体基因，然后使用标准基因敲除技术对其进行突变，以测试推定受体的作用。该项目的最终目的是利用所获得的知识来确定是否可以使用Actiphage方法或该方法的修改来检测人类血液样本中的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的专家外，成功的候选人还将获得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.