INTRODUCTION

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The genus Listeria consists of a group of gram-positive bacteria of low G+C content closely related to Bacillus, Clostridium, Enterococcus, Streptococcus, and Staphylococcus. Listeria spp. are facultative anaerobic rods of 0.4 by 1 to 1.5 µm that do not form spores, have no capsule, and are motile at 10 to 25°C (98, 552, 579). Listeria spp. are isolated from a diversity of environmental sources, including soil, water, effluents, a large variety of foods, and the feces of humans and animals. The natural habitat of these bacteria is thought to be decomposing plant matter, in which they live as saprophytes. Domesticated ruminants probably play a key role in the maintenance of Listeria spp. in the rural environment via a continuous fecal-oral enrichment cycle (178, 318, 420, 559, 682, 687, 692). The genus Listeria currently includes six species: L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi. Two of these species, L. monocytogenes and L. ivanovii, are potentially pathogenic. The infectious disease caused by these bacteria is known as listeriosis. L. monocytogenes causes serious localized and generalized infections in humans and a variety of other vertebrates, including domesticated and wild birds and mammals. The official discovery of Listeria microorganisms dates back to 1924, when E. G. D. Murray, R. A. Webb, and M. B. R. Swann isolated L. monocytogenes as the etiological agent of a septicemic disease affecting rabbits and guinea pigs in their laboratory at Cambridge in England (458). The first cases of human listeriosis were reported in 1929 in Denmark (481). However, the first recorded culture of L. monocytogenes dates from 1921, with the bacterium isolated in France by Dumont and Cotoni from a patient with meningitis (159, 604). L. ivanovii (formerly known as L. monocytogenes serotype 5) was first isolated in Bulgaria in 1955 from lambs with congenital listeriosis (299). Human cases of L. ivanovii infection are rare (116), the vast majority of reported isolations of this species being from abortions, stillbirths, and neonatal septicemias in sheep and cattle (4, 90, 134, 529, 610, 693). A third species, L. seeligeri, is considered nonpathogenic (555), although it has been implicated in at least one case of human listeriosis (556).

For many years, clinical Listeria isolates were a mere laboratory rarity, and the epidemiology of the disease was an unresolved mystery (603, 604). However, at the end of the 1970s and the start of the 1980s, the number of reports on Listeria isolations begun to increase, and from 1983 onwards, a series of epidemic outbreaks in humans in North America and Europe clearly established listeriosis as an important food-borne infection (46, 180, 386, 421, 572). The foods most frequently implicated are soft cheeses and dairy products, pâtés and sausages, smoked fish, salads, "delicatessen," and in general industrially produced, refrigerated ready-to-eat products that are eaten without cooking or reheating (176, 425-427, 551, 572). In ruminants, Listeria infection is transmitted by consumption of spoiled silage, in which these bacteria multiply readily, resulting in herd outbreaks (178, 666, 671, 693). Listeria organisms are widely disseminated in the rural environment and, consequently, contaminate the raw materials used in the preparation of industrially processed foods and the production plants as well (237). These bacteria are well equipped to survive food-processing technologies. For example, they tolerate high concentrations of salt and relatively low pHs, and worst of all, they are able to multiply at refrigeration temperatures (145, 362, 393, 426). This makes Listeria microorganisms a serious threat to food safety and ranks them among the microorganisms that most concern the food industry.

Researchers in immunology were interested in L. monocytogenes long before its importance as a risk to public health and food safety was recognized, because an infection highly reminiscent of human listeriosis was easily reproducible in laboratory rodents and protection could be transferred in syngeneic mice through spleen cells. Since the pioneering work of Mackaness in the early 1960s, which demonstrated that L. monocytogenes is able to survive and multiply in macrophages, this bacterium has been used in immunological research as a prototype intracellular parasite (401). For decades, the experimental model of Listeria infection in the mouse has made a significant contribution to our understanding of the cellular immune response (615). For example, key concepts such as the inability of antibodies to protect against infections produced by intracellular pathogens, the importance of activated macrophages in the elimination of intracellular parasites, and that the T cell is the macrophage-activating element required for cell-mediated immunity were established based on studies with the murine model of listeriosis (405, 406, 443, 478, 479).

In addition to the emergence of L. monocytogenes as a major food-borne pathogen, the 1980s also marked the start of investigations into the molecular mechanisms underlying Listeria virulence. The attention of researchers was first drawn to hemolytic activity, classically considered a virulence marker because it was present in the pathogenic species but not (with the exception of L. seeligeri) in the nonpathogenic species. These studies led between 1986 and 1989 to the discovery of the hemolysin gene, hly, and to elucidation of the key role that hemolysin plays in escape from destruction inside phagosomes, a prerequisite for intracellular bacterial proliferation. So, a decade ago, hemolysin became not only the first Listeria virulence factor to have its gene characterized, but also the first bacterial gene product to which a function critical to the survival of a parasite within the cells of the eukaryote host was attributed.

L. monocytogenes has since become not only an important paradigm for immunological investigation but also an important model system for analysis of the molecular mechanisms of intracellular parasitism (107). The identification of the hly gene was rapidly followed by a succession of discoveries resulting in complete characterization of the genetic locus to which this gene maps. This locus is a 9-kb virulence gene cluster that is involved in functions essential to intracellular survival. The internalin locus, inlAB, was also identified and characterized at this time (193). This locus encodes the first invasin described in a gram-positive bacterium, implicated in internalization by cells that are not usually phagocytic, such as epithelial and endothelial cells and hepatocytes. The progress made in the molecular characterization of listerial virulence factors during this period was summarized in a minireview published in 1992 (517). Many advances have since been made that have significantly increased our understanding of the molecular mechanisms of Listeria intracellular parasitism. The sequencing of the genomes of L. monocytogenes and L. innocua is currently finished (European Listeria Genome Consortium, unpublished data), with the expected outcome that very soon the panorama of research into the molecular pathogenesis of Listeria spp. will change dramatically.

The aim of this article is to review the knowledge gathered during the pregenomic era of Listeria research about the pathogenesis of listeriosis and the molecular virulence determinants involved. Readers interested more specifically in immunopathogenesis and the immune response should consult reference 497a. Those interested in issues related to taxonomy, clinical diagnosis and disease management, molecular typing, epidemiology, ecology, and food safety are also referred to other publications (145, 176, 289, 395, 550, 551, 573, 594, 605). For a historical perspective on Listeria spp. and listeriosis, we recommend the book Listeriosis by the pioneer listeriologist Heinz P. R. Seeliger (602) and the classical review, "Listeria monocytogenes and listeric infections," by Gray and Killinger (238).

PATHOPHYSIOLOGY OF LISTERIA INFECTION

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Clinical Features

The clinical signs of L. monocytogenes infection are very similar in all susceptible hosts. Two basic forms of presentation can be distinguished: perinatal listeriosis and listeriosis in the adult patient. In both instances, the predominant clinical forms correspond to disseminated infection or to local infection in the central nervous system (CNS). Listeriosis is usually a very severe diseasein fact, one of the most deadly bacterial infections currently knownwith a mean mortality rate in humans of 20 to 30% or higher despite early antibiotic treatment (422, 423, 554, 594).

Fetomaternal and neonatal listeriosis. This form of presentation mainly results from invasion of the fetus via the placenta and develops as chorioamnionitis. Its consequence is abortion, usually from 5 months of gestation onwards, or the birth of a baby or stillborn fetus with generalized infection, a clinical syndrome known as granulomatosis infantiseptica and characterized by the presence of pyogranulomatous microabscesses disseminated over the body and a high mortality (336a) (Fig. 1). The infection is usually asymptomatic in the mother or may present as a mild flu-like syndrome with chills, fatigue, headache, and muscular and joint pain about 2 to 14 days before miscarriage. Less frequently (10 to 15% of perinatal cases), late neonatal listeriosis is observed. It generally occurs 1 to 8 weeks postpartum and involves a febrile syndrome accompanied by meningitis and, in some cases, gastroenteritis and pneumonia. It presumably results from aspiration of contaminated maternal exudates during delivery, although hospital-acquired cases involving horizontal transmission by fomites or medical personnel in neonatal units have been reported (177, 554, 628). The mortality of late-onset neonatal listeriosis is lower (10 to 20%), but like early-onset neonatal listeriosis, it may have sequelae such as hydrocephalus or psychomotor retardation (173). L. monocytogenes is one of the three principal causes of bacterial meningitis in neonates (391, 554, 594, 645).

View larger version (91K): [in this window] [in a new window]   FIG. 1.   Clinical and pathological characteristics of Listeria infection in animals and humans. (A to D) Neuromeningeal listeriosis in sheep. (A) Typical aspect of circling disease, first described by Gill in 1933 in New Zealand (215, 216) and the most characteristic clinical manifestation of listeriosis in ruminants; the syndrome is characterized by involuntary torticollis and walking aimlessly in circles as a result of brainstem lesions. (B) In a further step of the infectious process, animals lie on the ground with evident signs of uncoordination (paddling movements) and cranial nerve paralysis (strabismus, salivation, etc.). (C) Section of the medulla oblongata of a sheep with listerial rhombencephalitis, showing clear inflammatory lesions in the brain tissue. (D) Microscopic preparation of the brainstem showing extensive parenchymal inflammatory infiltration and typical perivascular cuffing (one is indicated by an arrowhead) (panels C and D copyright M. Domingo, Barcelona, Spain). (E and F) Stillborn with generalized L. monocytogenes infection, a clinical condition also known as granulomatosis infantiseptica and characterized by the presence of military-disseminated pyogranulomatous lesions on the body surface (E) and internal organs (F, liver of the fetus in E) and a high mortality. (G and H) Extensive pyogranulomatous hepatitis in a lamb experimentally infected with L. ivanovii, with multiple necrotic foci in the liver surface (G) and the parenchyma (H, hematoxylin/eosin-stained cut from the liver in G, showing two subcapsular pyogranulomes [magnification, ×60]).

Listeriosis in adults. The listerial infection most frequently reported in nonpregnant adults is that affecting the CNS (55 to 70% of cases). Pure meningeal forms are observed in some cases, but infection normally develops as a meningoencephalitis accompanied by severe changes in consciousness, movement disorders, and, in some cases, paralysis of the cranial nerves (Fig. 1). The encephalitic form, in which Listeria organisms are isolated with difficulty from the cerebrospinal fluid (CSF), is common in animals (Fig. 1A to D) but rare in humans (see below). Its course is usually biphasic, with an initial subfebrile phase lasting 3 to 10 days in which there may be headache, vomiting, visual disorders, and general malaise, followed in a second phase by the onset of severe signs of rhombencephalitis. The mortality rate for CNS infection is around 20% but may be as high as 40 to 60% if associated with concurrent, underlying debilitating disease. It has been estimated that L. monocytogenes accounts for 10% of community-acquired bacterial meningitis. Due to effective vaccination against Haemophilus influenzae, L. monocytogenes is now the fourth most common cause of meningeal infection in adults after Streptococcus pneumoniae, Neisseria meningitidis, and group B streptococci. However, in certain high-risk groups, such as cancer patients, L. monocytogenes is the most common cause of bacterial meningitis (391, 473, 593). Another frequent form of listeriosis (in some series of patients reported, even more frequent than CNS infection) is bacteremia or septicemia (15 to 50% of cases), with a high mortality rate (up to 70%) if it is associated with severe underlying debilitating conditions (391) (see below). There are other atypical clinical forms (5 to 10% of cases), such as endocarditis (the third most frequent form), myocarditis, arteritis, pneumonia, pleuritis, hepatitis, colecystitis, peritonitis, localized abscesses (e.g., brain abscess, which accounts for about 10% of CNS infections by Listeria spp.), arthritis, osteomyelitis, sinusitis, otitis, conjunctivitis, ophthalmitis, and, in cows, mastitis (48, 176, 199, 201, 390, 391, 395, 422-424, 550, 628).

The pathophysiology of Listeria infection in humans and animals is still poorly understood. Most of the available information is derived from interpretation of epidemiological, clinical, and histopathological findings and observations made in experimental infections in animals, particularly in the murine model. As contaminated food is the major source of infection in both epidemic and sporadic cases (176, 512), the gastrointestinal tract is thought to be the primary site of entry of pathogenic Listeria organisms into the host. The clinical course of infection usually begins about 20 h after the ingestion of heavily contaminated food in cases of gastroenteritis (118), whereas the incubation period for the invasive illness is generally much longer, around 20 to 30 days (386, 542). Similar incubation periods have been reported in animals for both gastroenteric and invasive disease (138, 219, 671).

Ingestion of L. monocytogenes is likely to be a very common event, given the ubiquitous distribution of these bacteria and the high frequency of contamination of raw and industrially processed foods. However, the incidence of human listeriosis is very low, normally around 2 to 8 sporadic cases annually per million population in Europe and the United States (176, 301, 554, 649). Higher incidence rates have been reported for sporadic listeriosis (for example, 10.9 cases per million population per year in Barcelona, Spain [476], and 14.7 in France [234]), but these are exceptional. In epidemic situations, the incidence in the target population increases by a factor of 3 to 10 (46, 386, 587, 599). Thus, L. monocytogenes seems to have a lower pathogenic potential than other food-borne pathogens. This is consistent with the relatively high 50% lethal dose (LD₅₀) values reported for mice infected experimentally by the oral (10⁹ [18, 485]) or parenteral (10⁵ to 10⁶ [18, 333, 553]) routes. The minimum dose required to cause clinical infection in humans has not been determined, but the large numbers of L. monocytogenes bacteria detected in foods responsible for epidemic and sporadic cases of listeriosis (typically 10⁶) suggest that it is high. However, these data should be interpreted with caution, given the long incubation period of invasive listeriosis and the time normally elapsed between diagnosis and analysis of the food eaten, during which Listeria organisms can have multiplied in the patient's refrigerator. It therefore cannot be excluded that low doses may be able to cause infection, at least in immunosuppressed persons, old people, and pregnant women. Indeed, levels of contamination as low as 10² to 10⁴ L. monocytogenes cells per g of food have been associated with listeriosis in humans (176, 425). Obviously, the infectious dose may vary depending upon the pathogenicity and virulence of the L. monocytogenes strain involved and the host risk factors.

Pathogenicity of L. monocytogenes. Heterogeneity in the virulence of L. monocytogenes has been observed in several in vivo (mice) and in vitro (cell culture) studies (68, 129, 550, 663), but in most cases a clear correlation between the level of virulence and the origin or type characteristics of the strain could not be established. However, a recent study has found statistically significant differences in virulence between strains of food and clinical origin, the latter having slightly lower lethal doses (477). On the other hand, there is ample circumstantial evidence for an association between antigenic composition and pathogenicity in Listeria spp. The clearest example is provided by the L. ivanovii-specific serovar 5, which is recovered almost exclusively from ruminants, especially sheep. In these animals, serovar 5 strains cause perinatal infections but not encephalitis, the most typical clinical manifestations of ovine listeriosis (134, 397, 606, 610, 693) (Fig. 1A to D). Further evidence comes from the fact that only 3 of the 12 known serovars of L. monocytogenes, 1/2a, 1/2b, and 4b, account for more than 90% of human and animal cases of listeriosis (176, 397, 594), although other serovars, such as 1/2c, are often found as food contaminants (172, 176). Among the listeriosis-associated serovars, 4b strains cause over 50% of listeriosis cases worldwide, but strains of antigenic group 1/2 (1/2a, 1/2b, and 1/2c) predominate in food isolates (52, 172, 550, 557, 591). This suggests that serovar 4b strains are more adapted to mammalian host tissues than strains from serogroup 1/2. A phenotypically and genomically closely related group of serovar 4b isolates was found to be responsible for major outbreaks of food-borne human listeriosis in California in 1985 (386), Switzerland from 1983 to 1987 (46), Denmark from 1985 to 1987 (580), and France in 1992 (233), supporting the notion that some clones of L. monocytogenes may be particularly pathogenic (302, 510).

Host risk factors. Host susceptibility plays a major role in the presentation of clinical disease upon exposure to L. monocytogenes. Thus, most listeriosis patients have a physiological or pathological defect that affects T-cell-mediated immunity. This justifies the classification of L. monocytogenes as an opportunisitic pathogen. The groups at risk for listeriosis are pregnant women and neonates, the elderly (55 to 60 years and older), and immunocompromised or debilitated adults with underlying diseases. Listeriosis in nonpregnant adults is associated in most cases (>75%) with at least one of the following conditions: malignancies (leukemia, lymphoma, or sarcoma) and antineoplastic chemotherapy, immunosuppresant therapy (organ transplantation or corticosteroid use), chronic liver disease (cirrhosis or alcoholism), kidney disease, diabetes, and collagen disease (lupus) (176, 423, 551, 554, 594).

(i) Crossing the intestinal barrier. Before reaching the intestine, the ingested Listeria organisms must withstand the adverse environment of the stomach (Fig. 2). Oral infective doses are lower for cimetidine-treated experimental animals than for untreated animals (586a), and the use of antacids and H2-blocking agents has been reported to be a risk factor for listeriosis (286, 592). This indicates that gastric acidity may destroy a significant number of the Listeria organisms ingested with contaminated food.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Schematic representation of the pathophysiology of Listeria infection. See text for details.

(ii) Multiplication in the liver. The Listeria organisms that cross the intestinal barrier are carried by the lymph or blood to the mesenteric lymph nodes, the spleen, and the liver (411, 523) (Fig. 2). As stated above, this initial step of host tissue colonization by L. monocytogenes is rapid. The unusually long incubation period required by L. monocytogenes for the development of symptomatic systemic infection after oral exposure in relation to that for other food-borne pathogens is therefore puzzling and indicates that listerial colonization of host tissues involves a silent, subclinical phase, many of the events and underlying mechanisms of which are unknown.

(iii) Colonization of gravid uterus and fetus. Abortion and stillbirth due to Listeria spp. have been reproduced experimentally by intravenous, oral, and respiratory inoculation in naturally susceptible gestating animal hosts, such as sheep, cattle, rabbits, and guinea pigs, as well in pregnant mice and rats (1, 238, 239, 329, 450, 487, 488, 586). This shows that L. monocytogenes gains access to the fetus by hematogenous penetration of the placental barrier (Fig. 2). In pregnant mice, the blood-borne bacteria first invade the decidua basalis and then progress to the placental villi, where they cause diffuse inflammatory infiltration and necrosis (1, 535, 536). Macrophages appear to be excluded from the murine placenta, neutrophils acting as the main antilisterial effector cell population (251a). Using homozygous mutant mice, it has been shown recently that colony-stimulating factor-1 is required for the recruitment of neutrophils to the infectious foci in the decidua basalis. This occurs via induction of neutrophil chemoattractant synthesis by the trophoblast (251a). In humans, placental infection is characterized by numerous microabscesses and focal necrotizing villitis (502, 582). Colonization of the trophoblast layer followed by translocation across the endothelial barrier would enable the bacteria to reach the fetal bloodstream, leading to generalized infection and subsequent death of the fetus in utero or to premature birth of a severely infected neonate with miliary pyogranulomatous lesions (the above-mentioned granulomatosis infantiseptica) (Fig. 1E and F).

(iv) Invasion of the brain. In humans, CNS infection by Listeria spp. presents primarily in the form of meningitis. This meningitis, however, is often associated with the presence of infectious foci in the brain parenchyma, especially in the brain stem (391, 473), suggesting L. monocytogenes has a tropism for nerve tissue. The neurotropism and special predilection of L. monocytogenes for the rhombencephalon are shown most clearly in ruminants, in which listerial CNS infection, in contrast to the situation in humans, develops mainly as primary encephalitis. In these animals, infectious foci are restricted to the pons, medulla oblongata, and spinal cord (Fig. 1C). Although there is inflammatory lymphocyte or mononuclear cell infiltration of the meninges, this condition occurs as an extension of the brain process, and macroscopic lesions may not even be evident or may be restricted to basal areas, midbrain, and cerebellum. Unilateral cranial nerve paralysis is a characteristic of listerial rhombencephalitis in ruminants, leading to the well-known circling disease syndrome (92, 103, 215, 216, 312, 534, 666, 693) (Fig. 1A and B). In humans, primary nonmeningeal brain infection is seldom observed. However, as in ruminants, it develops as cerebritis involving the rhombencephalon (15, 20, 69, 390, 391, 473).

Hypothetical scenario. From the information presented above, the following hypothetical scenario for the pathogenesis of listeriosis can be proposed. Clinical outcome of Listeria infection depends on three major variables: (i) the number of bacteria ingested with food, (ii) the pathogenic properties of the strain, and (iii) the immunological status of the host. In immunocompetent individuals with no predisposing conditions, ingestion of low doses of L. monocytogenes will probably have no effect other than the development or boosting of antilisterial protective immunity. In contrast, oral exposure to large doses is likely to result in an episode of gastroenteritis and fever and, depending on the virulence of the strain, possible invasive disease. Immunocompromised and debilitated individuals, however, cannot mount an immune response strong enough to control bacterial proliferation in the liver, the primary target organ of L. monocytogenes, and are therefore susceptible to invasive disease following the ingestion of a lower inoculum. Inefficiently restricted growth of L. monocytogenes in the hepatocytes in these individuals is likely to result in an increase in the critical mass of bacteria and their release into the bloodstream. The ensuing prolonged bacteremia will result in local infections in secondary target organs (particularly the brain and placenta) or in septicemic disease in severely immunocompromised hosts. Figure 2 summarizes the key steps of the pathophysiology of Listeria infection.

In addition to multiplying within macrophages, Listeria organisms are invasive pathogens that can induce their own internalization in various types of cell that are not normally phagocytic. These include epithelial cells (194, 435, 518), fibroblasts (359, 641), hepatocytes (149, 242, 701), endothelial cells (157, 245, 498), and various types of nerve cell, including neurons (151). Listeria organisms have been also shown to be taken up by and survive within dentritic cells (253, 345). In the interior of all cells that L. monocytogenes is able to penetrate, whether macrophages or nonprofessional phagocytes, it develops an intracellular life cycle with common characteristics (Fig. 3).

View larger version (126K): [in this window] [in a new window]   FIG. 3.   Stages of listerial intracellular parasitism. (A) Scheme of the intracellular life cycle of pathogenic Listeria spp. (reproduced from reference 655 with permission of the Rockefeller University Press). In anticlockwise order: entry into the host cell by induced phagocytosis, transient residence within a phagocytic vacuole, escape from the phagosome into the cytoplasm, cytosolic replication and recruitment of host cell actin onto the bacterial surface, actin-based motility, formation of pseudopods, phagocytosis of the pseudopods by neighboring cells, formation of a double-membrane phagosome, escape from this secondary phagosome, and reinitiation of the cycle. (B to H) Scanning and transmission electron micrographs of cell monolayers infected with L. monocytogenes (B) Numerous bacteria adhering to the microvilli of a Caco-2 cell (30 min after infection). (C) Two bacteria in the process of invasion (Caco-2 cell, 30 min postinfection). (D) Two intracellular bacteria soon after phagocytosis, still surrounded by the membranes of the phagocytic vacuole (Caco-2 cell, 1 h postinfection). (E) Intracellular Listeria cells free in the host cell cytoplasm after escape from the phagosome (Caco-2 cell, 2 h postinfection). (F) Pseudopod-like membrane protrusion induced by moving Listeria cells, with the bacterium being evident at the tip (brain microvascular endothelial cell, 4 h postinfection; taken from reference 245 with permission). (G) Section of a pseudopod-like structure in which a thin cytoplasmic extension of an infected cell is protruding into a neighboring noninfected cell (notice that the protrusion is covered by two membranes) (Caco-2 cell, 4 h postinfection). (H) Bacteria in a double-membrane vacuole formed during cell-to-cell spread (Caco-2 cell, 4 h postinfection).

The cycle begins with adhesion to the surface of the eukaryote cell and subsequent penetration of the bacterium into the host cell. The invasion of nonphagocytic cells involves a zipper-type mechanism, in that the bacterium gradually sinks into diplike structures of the host cell surface until it is finally engulfed. During this process, the target cell membrane closely surrounds the bacterial cell and does not form the spectacular local processes or membrane ruffles characteristic of invasion by Salmonella and Shigella spp. (147, 322, 358, 435, 642) (Fig. 3B and C). The structures, mechanisms, and signal transduction cascades involved in the interaction between the bacterium and the cell during phagocytosis are only beginning to be elucidated.

The multisystemic nature of listerial infection indicates that L. monocytogenes probably recognizes a number of different eukaryotic receptors. The C3bi and C1q complement receptors have been reported to be involved in L. monocytogenes uptake by phagocytic cells (10, 115, 155, 156). L. monocytogenes is also efficiently internalized in the absence of serum, indicating that nonopsonic receptor-ligand interactions are also involved in host cell recognition by Listeria spp. (509). The eukaryote cell receptors used by Listeria spp. include the transmembrane glycoprotein E-cadherin (435), the C1q complement fraction receptor (61), the Met receptor for hepatocyte growth factor (HGF) (615a), and components of the extracellular matrix (ECM) such as heparan sulfate proteoglycans (HSPG) (12) and fibronectin (217). The macrophage scavenger receptor was shown to bind L. monocytogenes lipoteichoic acids and hence may also be involved in Listeria-macrophage interactions (160, 240). The bacterial ligands identified to date are all surface proteins, such as the internalins InlA and InlB, the actin-polymerizing protein ActA, and p60 (see below). Recently, several putative adhesion factors of L. monocytogenes have been identified by screening transposon mutants for defective attachment to eukaryotic cells. Among these putative adhesins are the surface protein Ami, an autolysin with a C-terminal cell wall-anchoring domain similar to InlB (60, 429, 445) (see below), and Lap, a 104-kDa surface protein involved in attachment to Caco-2 cells (581a). Also recently, a 24.6-kDa fibronectin-binding protein has been identified in L. monocytogenes by screening a genomic library in Escherichia coli for fibronectin binding (215). Bacterium-host cell interactions involving lectins (482) may also be involved in L. monocytogenes adhesion to eukaryotic cells (174). Indirect evidence has been obtained for an L. monocytogenes adhesin containing -D-galactose, involved in uptake by mouse CB1 dendritic cells (253) and human HepG22 hepatocarcinoma cells (114) upon recognition of a carbohydrate receptor at the eukaryotic cell surface. The presence in L. monocytogenes of lectin-like ligands mediating binding to D-glucosamine, L-fucosylamine, and p-aminophenyl--D-mannopyranoside moieties has also been indirectly demonstrated (110).

During invasion, Listeria bacteria become engulfed within a phagocytic vacuole (194) (Fig. 3D). Little is known about the characteristics of the Listeria-containing vacuolar compartment, but the vacuoles become acidified soon after uptake (25). There is evidence that L. monocytogenes ensures its intravacuolar viability by preventing phagosome maturation to the phagolysosomal stage (11). Thirty minutes after entry, the bacteria begin to disrupt the phagosome membrane (194), and within 2 h, about 50% of the intracellular bacterial population is free in the cytoplasm (655) (Fig. 3E). This membrane disruption step is essential for listerial intracellular survival and proliferation (220) and is mediated by the hemolysin in combination with phospholipases (see below).

Once in the cytosol, bacteria multiply (Fig. 3E), with a doubling time of approximately 1 h (194, 518), i.e., approximately three times slower than in rich medium. In L. monocytogenes, intracellular growth does not involve the upregulation of known stress proteins (261) (see below), and auxotrophic mutants requiring aromatic amino acids or purines for growth in minimal medium grow inside cells at rates similar to those of the wild-type parent strain (413). This indicates that the cytoplasmic compartment is permissive for Listeria proliferation. However, three metabolic genes (purH, purD, and pyrE, involved in purine and pyrimidine biosynthesis) and an arginine ABC transporter (arpJ) have been shown to be induced within cells (336). Mutation of these genes had no major effect on intracellular proliferation and virulence (336), which can mean that certain nutrients are present in the host cell cytoplasm at concentrations that, although not limiting, are sufficiently low that L. monocytogenes has to upregulate certain metabolic genes to grow efficiently within cells. Recent experimental evidence indicates that pathogenic Listeria spp. may exploit hexose phosphates from the host cell cytoplasm for efficient intracellular growth (543) (see below). Evidence has also been recently presented that antimicrobial peptides from the host cell cytosol, such as ubiquicidin found in macrophages (283a), may play a role in restraining intracellular proliferation of L. monocytogenes. Nothing is known about the listerial mechanisms counteracting these inhibitory peptides.

Intracytoplasmic bacteria are immediately surrounded by a cloud of a fine, fuzzy, fibrillar material composed of actin filaments, which later (around 2 h postinfection) rearranges to form an actin tail of up to 40 µm at one of the poles of the bacterium (see Fig. 6B). This actin tail consists of two populations of cross-linked actin filaments, one formed by relatively long, axially arranged actin bundles and the other by shorter, randomly arranged filaments. The polar assembly of actin filaments propels the bacterial cell in the cytoplasm with a mean speed of 0.3 µm/s, in a fashion reminiscent of the "action-reaction" principle that governs rocket movement. Moving Listeria cells leave an evanescent trail of F-actin undergoing depolymerization at the distal end, the steady-state length of which is proportional to the speed of bacterial movement. This movement is random, so some bacteria eventually reach the cell periphery, come into contact with the membrane, and push it, leading to the formation of finger-like protrusions with a bacterium at the tip (Fig. 3F and G). These pseudopods penetrate uninfected neighboring cells and are in turn engulfed by phagocytosis, resulting in the formation of a secondary phagosome delimited by a double membrane (Fig. 3H), with the inner membrane originating from the donor cell (Fig. 3G). Bacteria escape rapidly (within 5 min) from the newly formed vacuole by dissolving its double membrane, reach the cytoplasm, and initiate a new round of intracellular proliferation and direct intercellular spread (117, 453, 549, 601, 652, 654-656) (Fig. 3A). In tissue culture, this infectious cycle is reflected in the formation of plaques in the cell monolayer (641, 672). Actin-based i