19-LW-045 Full Length Final Report. Molecular Mechanisms of Bacterial Pathogenesis: Waging the Arms Race with Superbugs

Abstract

As the current global pandemic makes abundantly clear, we need a better understanding of infectious disease to safeguard human health, the economy and global security. Modern omics techniques hold the promise of providing a comprehensive understanding of the molecular mechanisms of life, including causes of pathogenesis from infectious disease at the molecular level, but we there is a serious gap in annotation of gene function. For as much as half of the genes and gene products encoded in genomes the molecular and/or cellular function is unknown or only partially understood. Recent innovations in fluorescence microscopy for live cell imaging and genetic engineering make it possible to determine the temporal correlation between molecular events, such as a gene being expressed due to host-pathogen interaction, and cellular events, such as bacterial invasion of immune cells. This is turn allows us to gain new insight as to the molecular and cellular role of individual genes and will enable the discovery and validation of new molecular mechanisms essential for infectious disease. Knowing the molecular mechanisms of disease processes will provide new therapeutic targets or novel countermeasure strategies. We aimed to develop a lattice light sheet fluorescence microscope as a unique resource at LLNL for long time course live cell imaging experiments; to develop the reagents and cell lines needed to monitor molecular events during the course pathogenic bacteria infecting mammalian immune cells; and to demonstrate that we could capture molecular events during an infection. We fully commissioned the LLNL lattice light sheet microscope and conducted initial proof of principle imaging experiments on mammalian immune cells and pathogenic bacteria. It is clear from the experience gained that long time course live cell imaging has tremendous potential to help elucidate molecular mechanisms of host-pathogen interactions and to help annotate gene function, which would establish a basis for new countermeasures. It is also clear that if live cell imaging is to realize its full potential new data processing and analysis tools will need to be developed to facilitate analysis of molecular events within cells; new sample chambers and stages could facilitate studies with a wider range of cell and tissue types; and alternative molecular tagging methods need to be explored to enable more facile engineering of cells labeled with molecular specificity.