Research Projects: Bacterial Pathogens


Bacterial pathogens, such as Listeria, Shigella and enteropathogenic E. coli, cause significant human disease by subverting host cell functions. To escape detection by the host immune system, these particular pathogens commandeer the host cell’s cytoskeleton to enhance both uptake and invasion of neighboring uninfected cells. By understanding how pathogens co-opt cellular processes, we might better understand normal host cell functions as well as exploit them for cell/tissue engineering and gene therapy. Combining novel optical tools with established molecular and cellular techniques, research projects focus on two broad questions (all bacterial strains, including mutants, provided by collaborators):

1) How do pathogenic bacteria enhance their uptake? Pathogenic bacteria have evolved exquisite mechanisms to enhance their uptake and subsequent vacuolar/lysosomal escape into cytoplasm. Understanding these tricks could aid therapeutic delivery of nanoparticles containing drugs or DNA into cells. Using LTM in the macrophage-like D. discoideum, we are the first to resolve the fast kinetics (<10s) and forces of nanoparticle uptake in living cells. In subsequent studies, we plan to use optical tweezers to micromanipulate both bacteria and particles onto cells, thus precisely initiating particle uptake. Subsequent monitoring using both real-time fluorescence microscopy and LTM will quantify their uptake kinetics and forces. By comparing wild type and mutant bacteria, we should be able to identify the factors that control uptake. Because most candidate proteins have been cloned, we can attach these factors onto particles and determine if their uptake is also enhanced. Listeria, Shigella, and enteropathogenic E. coli all use different mechanisms of entry and would provide complementary information.

2) How does actin polymerization push bacteria? Once inside cellular cytoplasm, bacteria recruit host factors to polymerize an actin “rocket” and push bacteria into neighboring uninfected cells. Mutants that cannot generate rockets are no longer pathogenic. Even though actin polymerization has been implicated in many essential cellular functions, its mechanism of force-generation remains mysterious. Using Listeria monocytogenes, our LTM data showed that existing molecular models, including the Brownian ratchet model, are flawed, if not wrong. In the absence of credible models, we are in a unique position to explain how actin polymerization pushes the edges of cells, as well as pushes bacteria. Additional studies using LTM and mutant bacteria, both in host cells and in biochemically reconstituted systems, will provide new data to form newer models.