The Beginning of the Modern Era A Personal Recollection

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I began working on L. monocytogenes in the fall of 1986, shortly after starting my own laboratory at Washington University in St Louis. I should mention that Pascale Cossart also entered the field about the same time (Mengaud et al. 1987) and remains a monumental presence contributing to all aspects of L. monocyto-genes pathogenesis. In 1986, basic research on L. monocytogenes was mostly in the realm of immunology while ignored by the bacterial pathogenesis community. I was influenced by a number of immunologists, including David Hinrichs in Oregon, Emil Unanue at Washington University, and Ed Havell and Robert North at Trudeau Institute, in the belief that L. monocytogenes pathogenesis might represent a fertile area of research. At that time, it was understood that L. monocytogenes is a facultative intracellular pathogen, but we knew virtually nothing about its cell biology of infection, bacterial determinants of pathogenesis, and lacked basic tools for genetic analysis. However, in 1986, two seminal papers were published: Havell (1986) showed that L. monocytogenes enters fibroblasts, spreads to neighboring cells, and induces high levels of type 1 interferon, while Philippe Sansonetti's lab showed that transposon mutagenesis could be used effectively in L. monocytogenes to isolate hemolysin-negative mutants (Gaillard et al. 1986). A second paper from Werner Goebel's lab was published the following year, also using a conjugative transposon to isolate hemolysin-negative mutants (Kathariou et al. 1987). I used similar approaches in my new lab and also isolated hemolysin-negative mutants (Portnoy et al. 1988). Our hypothesis was that the cholesterol-dependent hemolysin, listeriolysin O (LLO), was essential for intracellular growth; indeed, this is the case in the vast majority of cultured cells. However, as luck would have it, the first cell line that we examined was Henle 407 cells, and contrary to our hypothesis, LLO-minus mutants grew fine. Fortunately, we examined a number of primary and cultured cells and soon appreciated that infection of Henle 407 cells represented an exception to the rule that LLO is essential for escape from a vacuole. It turns out that LLO-minus mutants escape from a vacuole, grow, and spread cell-to-cell in human epithelial cells such as HeLa. Later, we and others showed that the broad range phospholipase C (PLC), PlcB, was necessary in these cell types (Marquis et al. 1995; Grundling et al. 2003), but in most cell types, LLO is essential while the PLCs also contribute. A couple of years later, we cloned and expressed LLO in Bacillus subtilis, and to our amazement, B. subtilis escaped from a vacuole and grew intracellularly (Bielecki et al. 1990). Thus, not only is LLO required to mediate escape, it was sufficient!

It did not take long to notice that L. monocytogenes grew readily inside of cultured cells and spread directly from cell to cell even in the presence of high levels of gentamicin. Remarkably, a single cell could be infected, and by 8 h there were 10 cells infected. A clue that led to our current understanding regarding the mechanism of cell-to-cell spread was provided to me by Larry Hale, who worked on Shigellae. Shigellae flexneri also spreads cell to cell and Larry told me that it could be blocked by cytochalasin D (Pal et al. 1989). Sure enough, cytochalasin D completely blocked the capacity of L. monocytogenes to spread cell to cell (Tilney and Portnoy 1989), implicating a role for actin polymerization. This observation was well known in the Shigella field, and Sansonetti's group showed that S. flexneri was coated with filamentous actin while mutants defective in cell-to-cell spread were not (Bernardini et al. 1989). Sasakawa's group in Japan made similar observations as well (Lett et al. 1989).

My lab moved to the University of Pennsylvania in 1988 where I began to collaborate with the larger-than-life actin cell biologist Lew Tilney. Using electron microscopy, Lew documented all of the stages currently associated with the cell biology of infection (Tilney and Portnoy 1989) (Figure 1.1.). A similar

Listeria Monocytogenes Cell Cycle

Figure 1.1. Stages in the intracellular life cycle of L. monocytogenes. Center. Cartoon depicting entry, escape from a vacuole, actin nucleation, actin-based motility, and cell-to-cell spread. Outside: Representative electron micrographs from which the cartoon was derived. LLO, PLCs, and ActA are all described in the text. Reproduced from The Journal of Cell Biology, 2002, 158:409-14; copyright 2002; The Rockefeller University Press.

Figure 1.1. Stages in the intracellular life cycle of L. monocytogenes. Center. Cartoon depicting entry, escape from a vacuole, actin nucleation, actin-based motility, and cell-to-cell spread. Outside: Representative electron micrographs from which the cartoon was derived. LLO, PLCs, and ActA are all described in the text. Reproduced from The Journal of Cell Biology, 2002, 158:409-14; copyright 2002; The Rockefeller University Press.

study was also published from the Sansonetti lab about the time (Mounier et al. 1990), followed by a study by Fred Southwick and Joe and Jean Sanger that used video microscopy to show movement of L. monocytogenes in cultured cells (Dabiri et al. 1990). These studies caught the attention of the cell biology community attracting a number of investigators previously interested in the actin cytoskeleton, including Julie Theriot and Matt Welch at Tim Mitchison's lab in UCSF, Jurgen Wehland in Germany, and Marie-France Carlier in France. Indeed, L. monocytogenes became a model system with which to study actin-based motility. Lew Tilney et al. (1990) postulated that these studies could lead to the discovery of the actin nucleator, and indeed, the L. monocytogenes ActA protein was later shown to activate the actin nucleation properties of the Arp2/3 complex (Welch et al. 1998). Julie Theriot and Tim Mitchison were the first to reconstitute listerial actin-based motility in cell extracts (Theriot et al. 1994), and Marie-France Carlier later reconstituted actin-based motility in vitro using purified components (Loisel et al. 1999).

In 1988, I read an article by David Baltimore urging the scientific community to consider it our responsibility to work on AIDS and/or AIDS vaccines. Since we had just learned that L. monocytogenes entered the host cell cytosol, it was obvious that L. monocytogenes might be an efficient vector for the induction of cell-mediated immunity, possibly leading to an AIDS vaccine. We thus embarked on a collaborative project with Yvonne Paterson at Penn where we showed that L. monocytogenes could be engineered to express and secrete a viral antigen resulting in the induction of antigen-specific CD8+ T cells in vivo (Ikono-midis et al. 1994). Other groups of investigators went on to show the efficacy of L. monocytogenes as a live vaccine vector (Goossens et al. 1995; Shen et al. 1995), and later that L. monocytogenes vaccines could be used therapeu-tically to treat tumors as well (Pan et al. 1995). Two biotech companies are currently embarking on clinical trials to test the safety and efficacy of these vaccines in humans, and there has been some progress using L. monocytogenes-based vectors for AIDS vaccines (Paterson and Johnson 2004). In this chapter, I will continue with a personal perspective, summarize the current status of the field, and discuss future prospects and unanswered questions.

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