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

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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.