The Maverick Bacterium

Whether it’s powering through the cytoplasm leaving a trail of polymerized actin, activating an arsenal of virulence factors through changes in RNA structure, or storing the code for RNA transcripts on the wrong side of DNA, Listeria makes up its own rules for survival.

© Hans Ackermann / Visuals Unlimited / Corbis

After several years at the Pasteur Institute working on protein structure and DNA-protein interactions, I had the chance in the mid-1980s to change projects and start studying bacterial pathogens. With my colleague Brigitte Gicquel, I identified two models to work on: the bacterium that causes tuberculosis, a disease that infects about 9 million people per year, or Listeria, a bacterium that causes disease in some 2,500 people in the United States annually, with only about 500 deaths per year. I chose Listeria.

To me, it seemed a perfect model organism. Unlike Mycobacterium tuberculosis...

By 1989, Lewis Tilney and Dan Portnoy discovered the cause of Listeria’s jet-powered movement in the cell. It wasn’t due to its flagellum. The whiplike organelle is a major propellant used in the environment before Listeria enters the body via infected food, but is shed once the bacterium enters a host’s warm gut. By comparing the rate of actin polymerization in the host cell against the bacteria’s distance traveled, researchers surmised that Listeria was charging through the cell by rapidly building a stepladder of actin proteins underneath it, rung by rung.

Following these discoveries, there was a race to find the gene that enabled Listeria to polymerize actin. But it wasn’t the problem I was focusing on. My lab was working to identify the genes responsible for the bacterium’s virulence. I had created a panel of Listeria mutants, and with my colleague Edith Gouin, I was screening for clones lacking the enzyme phospholipase. I suspected the enzyme was involved in virulence by helping the bacterium lyse the membranes it crosses. Upon finding such a mutant, it became rapidly clear that the bacterium was dramatically attenuated in other ways. When my postdoc Christine Kocks infected cells with this mutant, it was unable to grow an actin tail and shuttle around the cytoplasm. Without the aim of participating in the race, it looked as though we had won! Indeed, the gene mutation mapped to the gene responsible for the actin-based motility. It was part of an operon that also contained the phospholipase gene, explaining why both genes were affected by the mutation. We named the new gene ActA,1 for Actin gene A, because I assumed that such an interesting and unique process would require the activity of multiple genes that we would find later. Remarkably, there is still only one gene controlling this bacterium’s unique property.

Listeria appears to don a cape made of the host cell’s actin and fly around like a mini supervillain.

By chasing answers to the questions that struck my curiosity, I’ve let Listeria lead me into new fields of biology, such as, in recent years, noncoding RNA and RNA-mediated regulation. Like making use of the host cell’s actin to power its movement within the cell, this unusual bacterium has developed very clever methods for utilizing nontranslated RNA transcripts.

acteria exploit every possible component of their host cell’s defenses for their own profit. They hide within cells, hijack receptors, and interfere with regulatory pathways. Listeria is especially adaptable, being as happy to grow in the fridge or in soil at 4°C as it is in a human body at 37°C. In its host, it quickly adapts to an oxygen-deprived environment and becomes a pathogen. It is this transition between saprophytic—living on decaying vegetation—and pathogenic—the ability to cause disease—that became increasingly interesting to me.

Since 1986 we had been studying virulence using molecular biology, biochemistry, and infection of tissue cultured cells. We had also used mice that we infected intravenously, but the model was quite artificial, as Listeria doesn’t naturally infect mice. (Its tropism is limited to humans and farm animals such as cows and sheep.) We discovered that the human version of the receptor E-cadherin, an adhesion molecule used to maintain the junctions between epithelial cells, was involved in bacterial entry. It acted as a receptor for the Listeria protein called internalin A (InlA), forcing those host cells that produce E-cadherin to engulf the bacterium. While E-cadherin is expressed by most animals, just one amino acid sequence variation between human and mouse E-cadherin is enough to ensure that mice are resistant to oral Listeria infection.

Listeria's bag of tricks
Once inside the gut, Listeria expresses internalin, an adhesion molecule that attaches to the host's E-cadherin 1. The bacterium becomes engulfed by the host membrane 2. Escaping the vesicle, Listeria replicates 3, with each new bacterium becoming surrounded by an actin cloud 4. The Listeria then shuttles through the cell by forming a comet of actin filaments, eventually punching through the membrane of a neighboring cell 5. in which it is engulfed and repeats steps 3 through 5.

To get around this issue, together with my colleagues Marc Lecuit and Charles Babinet, I created a relevant animal model by expressing the human E-cadherin in a mouse intestine.2 This was the first-ever use of humanized mice to study a bacterial disease, and it has proven to be a powerful method. We can infect the mouse orally to mimic the human path of infection, and have been using it to study how the bacterium crosses the intestinal barrier. We recently improved our model by creating a mouse that expresses human E-cadherin in place of mouse E-cadherin, and have used it to investigate how Listeria crosses the placental barrier.

As we identified more of Listeria’s virulence factors, we noticed that they were only expressed at 37°C—body temperature. At room temperature, none of the virulence genes were expressed. We hypothesized that a switch, or transcription factor that regulates the major virulence genes, might be regulated by temperature.

We found that the switch was actually relying on the structure of the messenger RNA for PrfA.3 Below 37°C, the terminal sequence of the RNA folds on itself in a hairpin structure that makes the start sequence inaccessible to ribosome binding and translation. As the temperature is increased, the RNA unfurls and is translated into the transcription factor protein PrfA that turns on the set of virulence genes (see graphic below). Together with my postdoc Jörgen Johansson, I called the RNA element in the beginning of the transcript an RNA thermosensor.3

By then, around 2002, small RNAs were emerging as important regulators of genes in both eukaryotes and prokaryotes. But our RNA thermosensor certainly wasn’t behaving like most small RNAs. It was more in line with what had been described as riboswitches—elements within messenger RNAs that form a hairpin loop, inhibiting translation. Until our discovery, all riboswitches were thought to be released by small molecules rather than by temperature.

What other tricks did Listeria have up its sleeve to ensure its survival inside a human host?

he more papers I read on the subject, the more I was intrigued by the power of RNA. By 2005, researchers had been publishing on the role of small RNAs in animals and plants, but there hadn’t been much published on small RNA in bacteria. My lab undertook a project to investigate Listeria’s noncoding RNA by analyzing the bacterium’s transcripts when it was in stationary phase (without food), when it was in growth phase, and when present in the blood in its pathogenic phase. We sought to understand which sequences were involved in regulating the bacterium’s behavior at the major turning points in its life cycle and to understand how it undergoes its switch from a harmless bacterium to a pathogenic one.

A thermosensor for virulence
The gene prfA acts as a master switch for Listeria's virulence genes. At low temperatures, when the bacterium lives on decaying vegetation, the start sequence of the PrfA mRNA transcript is locked in a hairpin structure. When the temperature increases to 37°C, body temperature, the hairpin buckles outwards, releasing the start sequence that a ribosome can bind to and then initiate the translation of the gene.

We characterized the transcripts using a tiling microarray, a DNA chip that maps all the transcripts produced by a bacterium, on both strands of the DNA. Because we had sequenced and then annotated the complete genome of Listeria several years earlier, we could map each operon of the genome and carefully identify the beginning and end of their sequences.

Our scan revealed at least 50 small RNAs that could be involved in gene regulation, two of which were virulence factors.4 While we and others had already reported some 20 small RNAs regulating Listeria genes—which our screen also captured—none had been found to control virulence.

The study of Listeria has been a gold mine for me and others in the field. Many questions we asked led to the discovery of important concepts and mechanisms.

What was most surprising was that there were several long RNA transcripts on the opposite side of strands that encoded for genes. If you have a protein-coding gene on one strand of DNA, you never look to the opposite strand. Now, we had identified these long antisense transcripts, although we didn’t quite know what most of them did. One of these antisense threads comprised a long 5’ non-coding region as well as a region encoding a transcriptional regulator of the flagellin gene.5 This discovery added a level of complexity to the already complex regulation of Listeria’s flagella.

Together with Johansson, now a group leader at Umeä University, I showed that riboswitches can act on upstream genes. Moreover, when a riboswitch is produced as a small transcript, instead of being degraded, it can act on the opposite strand of DNA in distant genes. Indeed, we were able to show that two riboswitch elements, SreA and SreB, can act also on the PrfA transcript by binding to the very start of its message. The SreA and SreB short transcripts form when S-adenosyl is in excess in the bacterium, in nutrient-rich conditions. These transcripts bind pfrA, impairing its translation. Together, these results show that prfA—and Listeria virulence in general—are heavily controlled by a series of sophisticated environmental cues, including temperature and nutrient availability.

The study of Listeria has been a gold mine for me and others in the field. Many questions we asked led to the discovery of important concepts and mechanisms. More than using clever tricks for getting into cells and spreading intracellularly, Listeria also modifies its metabolism by turning on genes that allow it to survive in the host. For example it can absorb its host’s sugars. In addition, it escapes the innate immune system by deacetylating its peptidoglycan—a component of the bacterial cell wall that normally triggers inflammation. It also takes over histone modifications and reprograms the infected host cell. Recently we even discovered proteins Listeria sends to the host nucleus to reprogram the cell. And there is now evidence that Listeria manipulates ubiquitin-like pathways to increase infection.

Listeria is a bacterium that has developed amazingly creative mechanisms for survival in diverse environments. I have never regretted my choice to study this microscopic miscreant.

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Pascale Cossart is a professor at the Pasteur Institute and an HHMI international research scholar.

1. C. Kocks et al., “L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein,” Cell, 68:521–31, 1992.
2. M. Lecuit et al., “A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier,” Science, 292:1722-5, 2001.
3. J. Johansson et al., “An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes,” Cell, 110:551–61, 2002.
4. A. Toledo-Arana et al., “The Listeria transcriptional landscape from saprophytism to virulence,” Nature, 459:950–56, 2009.
5. E. Loh et al., “A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes,” Cell, 139:770–79, 2009.

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