Yogi Berra, Forrest Gump, and the discovery of Listeria actin comet tails

摘要

In 1988, eminent cell biologist Lew Tilney and newly appointed Assistant Professor of Microbiology Dan Portnoy met at a picnic and initiated a collaboration that led to a groundbreaking paper published in Journal of Cell Biology entitled “Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes .” The paper has been cited more than 800 times, the most of any publication in the careers of both investigators. Using an electron microscope from the Sputnik era, they assembled a stunning collection of micrographs that illustrated how L. monocytogenes enters the host cell and exploits a host system of actin-based motility to move within cells and into neighboring cells without leaving the host cell cytosol. This research captured the imagination of cell biologists and microbiologists alike and led to novel insights into cytoskeletal dynamics. Here, Portnoy provides a retrospective that shares text from the original submission that was deleted at the time of publication, along with reviewers' comments ranging from “It is really just a show and tell paper and doesn';t have any meat” to “the finding will have major impact in cell biology and in medicine. Potentially, the paper will be a classic.” In 1988, I arrived at the University of Pennsylvania as Assistant Professor of Microbiology. My primary research focus was on intra-cellular pathogens, which then, as now, are responsible for an enormous amount of morbidity and mortality worldwide. The research began during my final year of postdoctoral training at the Rockefeller University and during two subsequent years as an Instructor at Washington University. In St. Louis, we developed quantitative assays to examine the interaction of bacteria and cultured cells, which years later would be referred to as the “bread and butter” of the Portnoy lab. Although Listeria monocytogenes was obscure to most cell biologists, and frankly scared many of them, it had been extensively studied for 25 years in a murine model of cell-mediated immunity ( Unanue, 1997 ) and is an important food-borne pathogen ( Farber and Peterkin, 1991 ). However, in 1986, virtually nothing was known about its determinants of pathogenesis or the cell biology of infection, and there was no genetic system to speak of. The first goal was to sort out the nuts and bolts of L. monocytogenes pathogenesis, then merge this information with immunological studies and ultimately apply this knowledge to generate vaccines that would be protective against intracellular pathogens. Prior to my move to the University of Pennsylvania, we did know a few things. We knew that L. monocytogenes replicated (doubling time of ～40 min) as rapidly in mammalian cells as in rich bacterial broth and grew in most, if not all, adherent mammalian cells. We also knew that a secreted pore-forming hemolysin called listeriolysin O (LLO) was required for L. monocytogenes intracellular growth, and there was evidence that its role was to allow internalized bacteria to escape from a phagosome into the host cell cytosol ( Gaillard et al. , 1987 ; Portnoy et al. , 1988 ). By simply observing stained cells infected with L. monocytogenes , it was obvious that the bacteria spread directly from cell to cell, even in the presence of gentamicin at levels that killed extracellular bacteria. Remarkably, a single cell could be infected, and by 8 h, 10 cells were infected. The first paper to demonstrate cell-to-cell spread was published in 1986 by Ed Havell while he was studying the interferon response to infection ( Havell, 1986 ). In addition, Chihiro Sasakawa at the University of Tokyo had identified a locus in Shigella flexneri essential for cell-to-cell spread ( Makino et al. , 1986 ). One of the first ideas to provide a possible explanation for cell-to-cell spread was suggested to me by Joel Swanson, then Assistant Professor at Harvard and a long-time friend and colleague whose lab was next door when we were postdoctoral fellows at Rockefeller University. Joel posited that spreading might require microtubules and recommended that I examine the effects of nocodozole. Although nocodozole caused the cells to round up, the bacteria still spread cell to cell. Larry Hale, who worked on S. flexneri at Walter Reed Army Institute of Research, offered the first substantial clue that led to the discovery by Tilney and Portnoy. Larry told me that spreading of S. flexneri was blocked by cytochalasin D, a chemical inhibitor of actin polymerization ( Pal et al. , 1989 ). Sure enough, cytochalasin D, at remarkably low concentrations, completely blocked the capacity of L. monocytogenes to spread within an infected cell; the bacteria grew as cytosolic microcolonies. Next, I heard through the grapevine that Philippe Sansonetti from the Pasteur Institute presented evidence at a Gordon Conference that intracellular S. flexneri were coated in filamentous actin, whereas mutants defective in cell-to-cell spread did