Julie A. Theriot

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Julie A. Theriot, Assistant Professor Dept. of Biochemistry, Beckman 400, Stanford University School of Medicine, Stanford, CA 94305-5307, Tel: (650) 723-6161, FAX: (650) 723-6783
Email: theriot@cmgm.stanford.edu

Cell biology of host-pathogen interactions. We study the interactions between infectious bacteria and the human host cell actin cytoskeleton. Listeria monocytogenes and Shigella flexneri are unrelated food-borne bacterial pathogens that share a common mechanism of invasion and actin-dependent intercellular spread in epithelial cells. Our studies fall into three broad areas: the biochemical basis of actin-based motility by these bacteria, the biophysical mechanism of force generation, and the evolutionary origin of pathogenesis.
Medical studies of bacterial diseases have traditionally focused on the behavior of the bacteria themselves and on the response of the host immune system to infection. However, recent advances in our understanding of the cell biology of host-pathogen relationships indicate that disease-causing bacteria have developed extraordinarily complex and subtle ways of communicating with the host. It is clear that infection is not a process performed solely by a bacterium, but rather an elaborately choreographed interaction between the bacterium and the host cell. Using a combination of videomicroscopy, biochemistry, and molecular genetics, we study the interactions between infectious bacteria and the human host cell cytoskeleton. By examining the mechanisms these bacteria use to communicate with the host cell cytoskeleton, we hope to identify new ways to interfere with the infection process, and arrive at a deeper understanding of the normal regulation of cytoskeletal shape changes and cell movement.
Listeria monocytogenes is a ubiquitous Gram-positive bacterium that can cause serious food-borne infections in pregnant women, newborns, and immunocompromised adults. Shigella flexneri is an unrelated Gram-negative bacterium, a causative agent of bacillary dysentery. Both grow directly in the cytoplasm of infected host cells, and move rapidly throughout the infected cell using a remarkable form of actin-based motility. Within a few hours after infection, host cell actin filaments initially form a dense cloud around the intracytoplasmic bacteria, and then rearrange to form a polarized "comet tail," which is associated with all moving bacteria. The comet tail is made up of short actin filaments crosslinked into a meshwork in which the majority of filaments have their barbed (rapidly growing) ends oriented toward the bacterium. We have demonstrated that new actin filament polymerization occurs only at the front of the tail, adjacent to the surface of the bacterium, and that polymerization occurs at the same rate as bacterial propulsion. Bacteria spread from cell to cell by moving into long membrane-bound protrusions that are phagocytosed by neighboring cells. We have found that a single bacterial surface protein is necessary and sufficient for motility in each organism; ActA in L. monocytogenes and IcsA in Shigella flexneri. Surprisingly, these two proteins share no primary sequence similarity, though their functions are essentially identical. Neither bacterial protein exerts any direct influence on polymerization of pure actin; both must induce comet tail formation and actin-based motility through interactions with other host cell factors.
We have reconstituted motility of both pathogens in cell-free cytoplasmic extracts. We are currently attempting to identify host cell factors that interact with the bacterial proteins so that we can reconstitute motility in a defined biochemical system. Since ActA and IcsA appear to act through distinct biochemical mechanisms, we hope to learn more about the motility process by exploring the behaviors of both pathogens than we would by studying either one alone. There does not appear to be a myosin or any other known motor protein involved in this form of actin-based motility. Instead, the force for bacterial movement seems to be derived from actin polymerization itself. We are using biophysical techniques to attempt to measure the force of actin polymerization directly, and hope to determine whether actin polymerization may also contribute to force generation at the leading edge of motile eukaryotic cells.

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