Periodic Reporting for period 2 - DynaMO_TB (Spatiotemporal regulation of localization and replication of M. tuberculosis in humanmacrophages) Reporting period: 2020-03-01 to 2021-08-31 Summary of the context and overall objectives of the project Tuberculosis (TB) caused by the bacterial pathogen M. tuberculosis (Mtb) remains one of the deadliest infectious diseases with over a billion deaths in the past two hundred years (Paulson, 2013). TB causes more deaths worldwide than any other single infectious agent, with 10.4 million new cases and close to 1.7 million deaths in 2017. The obstacles that make TB hard to treat and eradicate are intrinsically linked to the intracellular lifestyle of Mtb. Mtb needs to replicate within human cells to disseminate to other individuals and cause disease. However, we still do not completely understand how Mtb manages to survive within eukaryotic cells and why some cells are able to eradicate this lethal pathogen. In this project, we are developing cutting-edge imaging technologies, such as live cell imaging, super resolution microscopy and correlative live cell 3D- electron microscopy, capable of imaging and quantifying Mtb localisation and replication. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far With the help of these innovative technologies we started to define the dynamic interactions between the different populations of Mtb and intracellular organelles and define the sites of Mtb replication and growth restriction. The development of this in vitro system to monitor bacterial replication in real time is being adapted to be used in HCS to target either host (Host directed therapies, HDT) or Mtb (antibiotic discovery). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This novel approach will also open up a major new “assay development space” by broadening the range of analysis strategies that drive novel cell-based assay designs for drug discovery. The understanding of these early stages of disease could also impact the direction and the design of future vaccines and drugs, as efficacy may be affected by the localization of bacteria at the time of treatment. Altogether, this project aims to identify the intracellular sites that allow or restrict Mtb replication in humans, thus opening new avenues for a deeper understanding of human tuberculosis and facilitating the development of vaccines and antibiotics. Mtb infecting macrophages
