Scientists at the Institut Pasteur have discovered exactly how the pathogen, Listeria monocytogenes penetrates the body to sicken and sometimes kill vulnerable individuals who eat contaminated foods.
The discovery, reported in the 1 June issue of the journal Science, provides fundamental new information on Listeria. Researchers cautioned that their work doesn't promise an antidote or new treatments for Listeria infection, called listeriosis.
But, the molecule that helps Listeria attack the digestive tract and cross the intestine may ultimately suggest a carrier for delivering various medicines directly to intestinal cells, or to deeper tissues. Thus, the work could prove useful for gene therapy efforts.
"This is a very tricky bacterium," said Pascale Cossart, a professor at the Institut Pasteur in Paris. "For the moment, most people are reasonably safe because the bacterium is sensitive to antibiotics. If it becomes resistant, as we have seen with other bacteria, then it will be critical to know precisely how the infection proceeds in order to design new therapeutic strategies."
A bacterium commonly present in soil, vegetation and sewage, Listeria can contaminate raw vegetables and "ready-to-eat" or processed foods requiring refrigeration, such as soft cheeses, pate and some processed meats.
Listeriosis primarily threatens developing fetuses, newborns, older adults, and those with weakened immune systems, causing meningitis and death among some 30 percent of these patients. In healthy adults, however, symptoms are typically far less serious, ranging from gastrointestinal illness to mild fever and headache.
To cause infection through contaminated food, Listeria must move through the stomach and then cross the intestine to enter the bloodstream, where it disseminates to the central nervous system and the placenta. But, the molecular events that allow Listeria to cross the intestinal barrier have remained a mystery-until now.
In previous studies, Cossart and her colleagues-including Jean Louis Gaillard, Jerôme Mengaud and Marc Lecuit-had shown that a protein on Listeria's surface called "internalin" interacts with a receptor, E-cadherin found on cultured cells derived from the intestine. Similar to a key-and-lock system, internalin interacts with E-cadherin to promote the entry of Listeria into intestinal cells. Listeria can then cross the intestine and pass into the bloodstream, scientists said.
Studying this mechanism in animals proved difficult, however, because rats and mice produce a form of cadherin that doesn't bind with internalin. Fortunately, guinea pigs generate a suitable version of the protein, making these animals a useful model for studying oral Listeria infection. The difference between the two forms of cadherin, Cossart explained, is a single amino acid in the 16th position of cadherin's molecular chain.
For their latest Science study, Lecuit et al.(Cossart's team in collaboration with transgenesis expert Charles Babinet and other scientists at the Institut Pasteur), investigated oral infection mechanisms within guinea pigs and transgenic mice, which had been genetically modified to express the human version of cadherin in the intestine. Animals received oral doses of both wild-type Listeria, as well as a mutant, internalin-free version of the bacterium. Wild Listeria, featuring the internalin protein, proved fatal in most cases, whereas the mutant strain caused no deaths and failed to cross the intestine and invade organ tissues.
Thus, the French investigation identifies a "virulence factor" or molecular mechanism by which Listeria causes disease, thereby addressing a key recommendation of a preliminary report on Listeria, prepared by the FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization).*
"Internalin should now be considered as a virulence factor as important as the other L. monocytogenes virulence factors best characterized so far," the Science paper concludes. Other factors known to play a role in listeriosis include, for example, a pore-forming toxin, listeriolysin O (LLO), which helps Listeria escape from the vacuole, the fluid-filled cavity that surrounds the bacterium after its entry into the cell. A pair of enzymes, PI-PLC and lecithinase, also help free Listeria from the vacuole. Yet another factor, ActA, interacts with actin, a cellular protein, to propel the bacterium from cell to cell.
"A number of questions still must be answered," Cossart noted. Most importantly, scientists want to know exactly how internalin manages to find E-cadherin-a relatively inaccessible protein within the "brush border" between two tightly packed intestinal cells (enterocytes). Cossart's team speculates that cell-cell junctions may open briefly during the renewal phase of the enterocytes' three-day life cycle. It's also possible that the bacterium destabilizes the intestinal barrier, perhaps in tandem with molecular co-conspirators, thus providing access to E-cadherin.
Though its' precise molecular path to E-cadherin remains elusive, Cossart said: "It's clear that internalin targets enterocytes." Consequently, "In the future, it may be possible to use internalin as a vehicle, for driving different therapeutic agents to the enterocytes, and across the intestine."
For now, scientists hope to learn how Listeria crosses other barriers within the human body to attack the brain and infect unborn children. Cells expressing E-cadherin are found in the blood-brain and placental barriers, Cossart said, but internalin's activity in those environments has not yet been investigated.
These results, the culmination of a long-term project, illustrate how apparently reductionist approaches may help generate highly relevant animal models
Transgenic mice used in the French study are likely to assist scientists in better understanding a range of oral infection events. In addition, the Science findings set the stage for new studies of the body's immunological response to infection.
A March 2001 consumer advisory, issued by the U.S. Food and Drug Administration (FDA), notes that perishable items that are pre-cooked or ready-to-eat should be consumed as quickly as possible to minimize the risk of Listeria infection. The advisory also recommends cleaning refrigerators regularly and using a thermometer to maintain foods at 40 degrees Fahrenheit or colder. Further, the FDA suggests that pregnant women, older adults, and those with weakened immune systems should avoid certain foods-such as hot dogs and luncheon meats, unless they are reheated until steaming hot; as well as certain soft cheeses, refrigerated pates or meat spreads, or refrigerated smoked seafood, unless contained in a cooked dish.
The Science paper was prepared at the Institut Pasteur by Pascale Cossart's team (Unité des Interactions Bactéries Cellules), and Charles Babinet 's Unité de Biologie du Développement, Institut Pasteur ,URA CNRS; together with Jean Lefort of Unité de Pharmacologie Cellulaire; Michael Huerre of Unité d'Histopathologie; and Pierre Gounon of Station Centrale de Microscopie Electronique.
· * REFERENCE: "Joint FAO/WHO Activities on Risk Assessment of Microbiological Hazards in Foods Risk Assessment: Listeria monocytogenes in ready-to-eat foods." Preliminary report prepared for Joint FAO/WHO Expert Consultation on Risk Assessment of Microbiological Hazards in Foods, FAO Headquarters, Rome, Italy, 17-21 July 2000. Prepared by Robert Buchanan, U.S. Food and Drug Administration, and Roland Lindqvist, National Food Administration, Sweden. See < http://www.who.int/fsf/mbriskassess/> and then select "Hazard identification, hazard characterization of Listeria monocytogenes in ready-to-eat foods."
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