Revealing Molecular and Cellular Mechanisms of Microbial Pathogenesis Through Multiscale Dynamic Imaging

Project Overview

There is a critical need to develop more rapid countermeasures to combat infectious disease for public health, more specifically for the growing threat of antimicrobial resistanceand the ominous threat of future pandemics. Modern medical science responded to the COVID-19 pandemic in record time, and yet we suffered more than 1M dead and an estimated >$16T in economic losses in the US alone. The accelerated response to COVID-19 was due to new vaccine technology, but was also made possible by existing knowledge generated by decades of research on related pathogens, SARS and MERS.

The audacious objective of this project was to establish a new paradigm for countermeasure target validation, providing a new approach to addressing a major hurdle in combating infectious disease and thereby accelerating countermeasure development. The new paradigm for target validation is using high temporal resolution, long time course bioimaging, enabled by lattice light sheet microscopy (LLSM), with molecule specific fluorescent probes attached to macromolecules of interest. Live cell imaging with molecular specificity allows us to observe onset of production and subcellular localization of specific macromolecules in situ. This in turn allows the temporal correlation of onset of production and subcellular localization with stages of progression of pathogenesis with unprecedented temporal precision; a high correlation is strong validation that a biomolecule of interest is involved in pathogenesis.

This new paradigm could be applied to many different pathogens, but as a demonstration case was applied to validating two putative virulence factors (biomacromolecules encoded in a pathogen genome and contributing to the pathogen's ability to cause disease) in Francisella Novicida, a surrogate for the biothreat agent Francisella Tularensis. The two putative virulence factors were identified at LLNL in previous work. The major accomplishments of the project were to assemble the interdisciplinary team to execute three main thrusts of the project (instrument/capability development, 4D data visualization and analysis, and molecular mechanisms of host-pathogen interaction); commission the custom built LLSM at Lawrence Livermore National Laboratory (LLNL); implement data management, visualization, and analysis capabilities; complete proof-of-principle experiments that LLSM can be used to track individual cells during host-pathogen interaction studies over long time coarse (>12 hours) and observe protein specific tags in situ; establish new collaborations (nascent and newly awarded) in the host-pathogen application space but also in other life science research areas (notably neurodegenerative disease and bioengineering). We also generated important preliminary results that establish the foundation for several applications of NIH, DOE, DOD, and ARPA-H grants.

Mission Impact

This project aligns well with the NNSA bioassurance, LLNL bioresilience MFA, and broad biosecurity thrusts. This project could also provide foundational knowledge that would inform molecular signatures or assays to identify the presence of biological threat agents. This project is aligned with the recently announced NNSA bioassurance program in that it uses NNSA's labs unique science, engineering, and technology capabilities. This project can help promote the growing bioeconomy in at least two ways; first by forming countermeasure strategies which would in turn utilize biomanufacturing, and secondly by strengthening biodefense, both in the form of new countermeasure strategies against known biological threats and in the form of rapid response to newly emerging biological threats. 

Publications, Presentations, and Patents

X. Yi, H. Miao, J.K.Y.Lo,  M.M. Elsheikh, T.H. Lee, C. Jiang, Y. Zhang, B.W. Segelke, K.W. Overton, P.T. Bremer, and T.A. Laurence. "Tailored approach to study Legionella infection using a lattice light sheet microscope" (LLSM). (2022) , Biomedical Optics Express, 13(8), pp.4134-4159.

Brent W. Segelke. "Functional Annotation from Structural Homology." Microbial Systems Biology, pp. 215-257. Humana, New York, NY, 2022.

Sonny Li, "Shockwave Enhanced Ablation" (Presentation,115th European Workshop on Laser Ablation Bern, Switzerland, July 2022).

Xiyu Yi,, Haichao Miao, Jacky Kai-Yin Lo, Wes Overton, Tek Hyung Lee, Maher M. Elsheikh, Dante Paul Ricci, Dan McFarland Park, Peet-Timo Bremer, Brent W. Segelke, Ted A. Laurence, "Commissioning a home-built Lattice Light Sheet Microscope for the study of Legionella infection" (Presentation, 66th Biophysical Society Annual Meeting, San Francisco CA, Feb 19-23, 2022).