点击显示 收起
Department of Molecular and Cell Biology and School of Public Health, University of California, Berkeley
Anatomic Pathology Service, Veterinary Medical Teaching Hospital, University of California, Davis
Listeria monocytogenes causes foodborne outbreaks that lead to infection in human and other mammalian fetuses. To elucidate the molecular and cellular mechanisms involved in transplacental transmission, we characterized placental-fetal infection in pregnant guinea pigs inoculated with wild-type (wt) or mutant L. monocytogenes strains. The wt strain increased in number in the placenta by >1000-fold during the first 24 h after inoculationan increase that was unparalleled in other maternal organs. The ActA- mutant, which is impaired in cell-to-cell spread and attenuated in maternal organs, increased in the placenta by a similar amount, although, in fetal infection, the number of ActA- mutant bacteria was 100-fold lower, compared with that of the wt strain. Furthermore, a mutant impaired in vacuolar escape was rapidly eliminated from maternal organs but persisted in the placenta. We concluded that cell-to-cell spread facilitates maternal-to-fetal transmission. Furthermore, the placenta provides a protective niche for growth of L. monocytogenes.
Listeria monocytogenes is a ubiquitous, rapidly growing, facultative, intracellular, gram-positive bacterium. Infection of humans and animals has been traced to contaminated foods and can lead to serious, often fatal disease. In humans, disease is most common in pregnant women, newborns, and immunocompromised individuals [1]. Pregnant women have an estimated 17-fold increased incidence of disease [2], compared with that in other individuals, and usually develop a nonspecific febrile illness, which can lead to a placental-fetal infection that results in spontaneous abortion, premature labor, stillbirth, or neonatal sepsis and meningitis [3, 4]. Despite significant fetal and neonatal morbidity and mortality due to vertical transmission of L. monocytogenes, little is known about its underlying molecular and cellular mechanisms. We previously developed a pregnant guinea pig model of listeriosis that mimics human disease [5]. The structural similarities between the guinea pig placenta and the human placenta make our model an attractive one for elucidation of the roles that important virulence factors play in vivo [6].
The biological processes of intracellular growth and the bacterial determinants of the pathogenicity of L. monocytogenes were extensively studied during the past decade [7, 8]. Bacterial cell-wall surface proteins called internalins (Inls) promote bacterial adherence and internalization into nonphagocytic host cells [9]. InlA and InlB are the best characterized Inls, and they bind to E-cadherin and c-Met-tyrosine kinase, respectively [10, 11]. After internalization, the bacterium escapes from the vacuole into the cytoplasm. This escape is largely mediated by a pore-forming virulence factor called listeriolysin O (LLO) [7, 8]. Once it is in the cytoplasm, the bacterium multiplies rapidly. ActA, a bacterial surface protein, induces polymerization of host cell actin filaments, which enable L. monocytogenes to migrate to the host cell periphery and into cell-wall protrusions. These pseudopodia are engulfed and ingested by neighboring cells, in which the life cycle begins anew [1214]. Therefore, L. monocytogenes can infect cells by 2 different mechanisms: direct invasion and cell-to-cell spread.
The roles that Inls play in virulence have yet to be fully explained. Although InlB is known to play a role in hepatic infection [15, 16], the mouse model, which has been used for decades in the study of listerial pathogenesis, is not a good model for use in the evaluation of the role of InlA, because there is a single amino-acid substitution in murine E-cadherin, compared with human E-cadherin. This structural difference significantly decreases the affinity of InlA to murine E-cadherin [17, 18]. Human and guinea pig E-cadherin interact equally well with InlA, and there is evidence that InlA plays a role in the crossing of the intestinal barrier in guinea pigs, humans, and transgenic mice that express human E-cadherin in the intestinal epithelium [19, 20].
The placental-fetal barrier in the monohemochorial placenta of humans and guinea pigs consists of a single layer of fetally derived trophoblasts [6, 21]. We and others have shown elsewhere that the invasion of primary human trophoblasts in vitro is mediated by InlA [5, 22]. Furthermore, Lecuit et al. have shown that infection of syncytiotrophoblasts in human placental explants with a L. monocytogenes strain deficient in InlA reduced the number of bacteria by 10-fold, compared with that in infection with wild-type (wt) L. monocytogenes [22]. In humans, epidemiological data have shown that 100% of the L. monocytogenes strains isolated in pregnancy-associated and focal infections, 98% of the strains isolated in central nervous system (CNS) infections, and 93% of the strains isolated in bloodstream infections expressed full-length InlA. In contrast, only 65% of the strains isolated from food products expressed full-length InlA [20]. These data are consistent with experimental evidence suggesting that InlA plays a role in the crossing of the intestinal barrier [19]. Because the crossing of the intestinal barrier is a prerequisite for placental infection in naturally occurring listeriosis, it cannot be concluded from the available epidemiological data that InlA mediates the crossing of the placental-fetal barrier in humans. Furthermore, we have shown elsewhere that InlA is not important for the crossing of the placental-fetal barrier in the pregnant guinea pig model of listeriosis [5].
We therefore decided to examine the role that cell-to-cell spread plays in trophoblast infection and vertical transmission. The ActA- mutant, a L. monocytogenes strain deficient in ActA, is impaired in its ability to spread from cell to cell. In vitro studies have shown that a single J774 cell initially infected with a single ActA- bacterium harbors 250500 bacteria at 8 h after infection [23]. Subsequent bacterial growth results in lysis of the host cell and necrotic death. In an animal model of infection, cell lysis would result in the exposure of extracellular bacteria to host defense mechanismsspecifically neutrophils, macrophages, and the humoral immune response. Indeed, cell-to-cell spread has been shown to be important for virulence. In the murine model of listeriosis, an ActA- mutant strain of L. monocytogenes is 1000-fold less virulent than is the wt strain [23]. In the present study, we compare placental-fetal infection with the ActA- mutant and wt L. monocytogenes in the pregnant guinea pig model of listeriosis. We show that cell-to-cell spread facilitates maternal-to-fetal transmission. Although it is attenuated in maternal organs, the ActA- mutant seems to grow well in the placenta. Furthermore, a L. monocytogenes strain deficient in vascuolar escape is rapidly eliminated in maternal organs but is able to persist, and even to grow, in the placenta. Our results demonstrate that the mammalian placenta provides a unique protective environment for L. monocytogenes.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
L. monocytogenes 10403S (erythromycin-sensitive wt) [24] and the following strains derived from it were used: DP-L3903 (erythromycin-resistant wt) [25], DP-L3078 (ActA-) [26], DP-L2161 (erythromycin-sensitive LLO-) [27], and DP-L4694 (erythromycin-resistant ActA-). DP-L4694 was generated from strain DP-L3078 by transduction of the gene for erythromycin resistance from strain DP-L3903 by use of phage U153, as described elsewhere [25, 28, 29]. All strains were propagated in brain-heart infusion (BHI) agar and broth (Becton Dickinson). For guinea pig studies, bacteria were grown in BHI broth to log phase, washed once with PBS, and resuspended in fresh BHI. Aliquots were frozen and stored at 80°C. On the day of infection, aliquots were thawed, diluted to a concentration of 1 : 5 in fresh BHI, and grown to log phase.
Guinea pig studies.
All guinea pigs were housed and handled in accordance with federal and institutional guidelines. The animal use committee at the University of California, Berkeley, approved the animal use protocol. Pregnant female Hartley outbred guinea pigs between days 25 and 45 of gestation were purchased from Simonsen Laboratories or Elm Hill Breeding Labs. Pregnant females were injected with L. monocytogenes into their foot veins between days 42 and 52 of gestation. Guinea pigs were premedicated with a subcutaneous injection of 0.05 mg/kg atropine (Phoenix Scientific) and then anesthetized with isoflurane (Baxter Healthcare Corporation) before inoculation with either 1 × 108 or 1 × 109 cfu of bacteria. The dose of 1 × 109 cfu was used when histologic examination was planned. Such a high dose was necessary for sufficient numbers of bacteria to be detected by immunohistochemistry. Because this dose caused clinical illness in the guinea pigs at 24 h after inoculation, a dose of 1 × 108 cfu was used for experiments with later time pointsin particular, the competitive index analysis. Guinea pigs were euthanized at specified time points after inoculation, and the organs were harvested and homogenized in 0.2% NP-40 (Biosciences). Serial dilutions were plated on Luria-Bertani (LB) agar plates (Becton Dickinson) and were incubated overnight at 37°C. The number of bacteria per organ was counted.
A competitive index analysis was performed as described elsewhere [25]. Briefly, the erythromycin-sensitive wt 10403S strain was compared with the erythromycin-resistant ActA- strain DP-L4694. The strains were mixed at a 1 : 1 ratio. Guinea pigs were injected with a total infectious dose of 1 × 108 cfu of bacteria. Guinea pigs were euthanized at 33 h after inoculation, and the organs were harvested and homogenized in 0.2% NP-40. Serial dilutions were plated on LB agar plates and on BHI agar plates containing erythromycin (Sigma) at a concentration of 2 g/mL. The plates were incubated overnight at 37°C, and erythromycin-sensitive and erythromycin-resistant colonies were counted. The competitive index was determined by calculation of the ratio of erythromycin-resistant to erythromycin-sensitive colonies.
Histologic analysis.
Tissue was fixed in 10% buffered formalin phosphate (Fisher Scientific) and processed by routine methods to provide paraffin wax sections (4 m), which were stained with hematoxylin-eosin. Immunohistochemistry was performed at the California Animal Health and Food Safety Laboratory in Davis, California, using a rabbit anti-Listeria primary antibody (Difco Laboratories) and a peroxidase detection kit (Vector Laboratories). Examination of stained placental sections was performed by microscopy, and examiners were blinded to the identity of the infecting strains.
RESULTS
Early events during L. monocytogenes infection in the pregnant guinea pig.
We intravenously (iv) inoculated pregnant guinea pigs between days 42 and 52 of gestation with 1 × 109 cfu of the wt strain. This gestational age corresponds with the late second/early third trimester of pregnancy. For histologic analysis, a sufficient number of bacteria had to be present in the placenta to allow for localization of organisms during the early stages of infection, specifically the first 24 h after inoculation. Thus, it was necessary to use a relatively high inoculum, which typically causes lethal disease in 23 days.
At 30 min after inoculation, the number of bacteria in the placenta was between 1000-fold and 10,000-fold lower than that in the maternal liver and spleen and between 10-fold and 100-fold lower than that in the maternal lungs and kidneys (figure 1). We concluded that L. monocytogenes seeds the placenta initially with fewer bacteria than it does any of the other maternal organs. During the subsequent 24 h after inoculation, we observed a >1000-fold increase in the number of L. monocytogenes in the placentaan increase unparalleled in any other maternal organ we tested (figure 1). This increase in the number of bacteria in the placenta could be due to bacterial growth or influx. Influx could occur in the form of extracellular bacteria or infected maternal cellsspecifically, macrophages. If the increase was solely due to bacterial growth, the predicted generation time of L. monocytogenes in the guinea pig placenta at 610 h after inoculation would be 70 min (figure 1A).
L. monocytogenes impaired in cell-to-cell spread.
We iv inoculated pregnant guinea pigs with 1 × 109 cfu of the ActA- mutant. In maternal organs, the ActA- mutant was attenuated, compared with the wt strain (figure 1BE). The ActA- mutant was either unable to grow (in maternal lungs [figure 1D] and kidneys [figure 1E]) or began to decrease in number at 10 h after inoculation (in maternal liver [figure 1B] and spleen [1C]). In the placenta, in contrast, the ActA- mutant increased in number by 1000-fold during the first 24 h after inoculation, which was similar to the growth of the wt strain (figure 1A).
Histologic examination of placental sections.
Histologic examination was performed on placental sections taken from guinea pigs at 0.5, 6, 10, and 24 h after inoculation with 1 × 109 cfu of either the wt strain or the ActA- mutant. Fewer than 5 bacteria per placental section were observed at 0.5, 6, and 10 h after inoculation. At these early time points, no difference in the number of bacteria was detected between guinea pigs inoculated with the wt strain and those inoculated with the ActA- mutant. Placental lesions consisting of neutrophils, macrophages, and necrotic trophoblasts were observed in both groups of guinea pigs at 24 h after inoculation. Lesions were most frequent and severe in the ascending and radial maternal arterial main lacunae. These vascular structures are lined by endovascular trophoblasts and are the primary arterial blood supply to the placenta from the maternal circulation. Bacteria were present in variable numbers and appeared to be extracellular, inside of neutrophils and macrophages, and occasionally inside of trophoblasts (figure 2). In one-half of the guinea pigs infected with the ActA- mutant, we found, in the labyrinth region, small clusters of trophoblasts that were filled with bacteria (figure 2B). This visually demonstrates the effect that impaired cell-to-cell spread has and shows that L. monocytogenes grows well inside of trophoblasts in vivo.
Cell-to-cell spread and transmission of L. monocytogenes.
We compared transmission of L. monocytogenes from mother to fetus in pregnant guinea pigs iv inoculated with 1 × 109 cfu of either the wt strain or the ActA- mutant. At 10 h after inoculation, fetal infection with L. monocytogenes was not detectable (data not shown). At 24 h after inoculation, infected livers were found in all of the fetuses in pregnant guinea pigs inoculated with the wt strain but in only 3 of the fetuses in pregnant guinea pigs inoculated with the ActA- mutant (figure 3A). To further quantify the virulence defect of the ActA- mutant in fetal infection, we performed a competitive index analysis with a 1 : 1 ratio of the erythromycin-sensitive wt strain to the erythromycin-resistant ActA- mutant. At 33 h after inoculation, we observed a 100-fold decrease in virulence of the ActA- mutant in fetal liver (figure 3B). In contrast, we observed a <10-fold decrease in virulence of the ActA- mutant in placenta, maternal liver, and maternal spleen. This indicates that cell-to-cell spread contributes to the crossing of the placental-fetal barrier.
L. monocytogenes impaired in vacuolar escape.
To evaluate whether the placental environment is particularly permissive for listerial growth, we iv inoculated pregnant guinea pigs with 1 × 109 cfu of the LLO- mutant. This strain is impaired in its ability to escape the vacuole and is absolutely avirulent in the murine model of listeriosis [30]. At 2448 h after inoculation, the LLO- mutant was eliminated rapidly from maternal organs, including liver, spleen, lungs, and kidneys (figure 4A). In contrast, the LLO- mutant was able to persist in the placenta and even exhibited a slight increase in number (figure 4B). Most important, these results indicate that L. monocytogenes cannot be effectively eliminated from the placental environment.
DISCUSSION
The results of the present study show that L. monocytogenes does not preferentially seed the guinea pig placenta in an iv model of inoculation. However, there was a >1000-fold increase in the number of bacteria in the placenta during the first 24 h after inoculation. Such an increase was not observed in any other maternal organ. In addition, L. monocytogenes strains that were impaired in their ability to spread from cell to cell or to escape the vacuole were able to grow or persist in the placenta, despite being attenuated in maternal organs. Although cell-to-cell spread is not essential for placental infection, we observed a decrease in the number of bacteria in fetal infection with the ActA- mutant, compared with infection with the wt strain.
In the present study, we focused on the role that cell-to-cell spread plays in vertical transmission. This seems relevant, because L. monocytogenes is a facultative, intracellular organism, and its intracellular lifestyle is important for virulence. In the natural setting of foodborne listeriosis, L. monocytogenes disseminates after it crosses the intestinal barrier. Dissemination most likely occurs via the hematogenous route. Indeed, maternal bacteremia is documented in 50% of all cases of listeriosis during pregnancy in humans [3, 31]. L. monocytogenes could disseminate extracellularly or inside host cellspossibly infected maternal macrophages. Infected monocytes have been implicated in the crossing of the blood-brain barrier and the development of CNS infection with L. monocytogenes [3234]. A similar mechanism has also been proposed for the dissemination of Salmonella typhimurium [35], another foodborne pathogen that can survive in mononuclear phagocytic cells and cause invasive systemic disease. Another example is dissemination of the intracellular parasite Toxoplasma gondii, which has been suggested to occur via infected immature dendritic cells [36]. Furthermore, the interaction of Plasmodium falciparuminfected red blood cells expressing erythrocyte membrane protein 1 with chondroitin sulfate A, as well as with hyaluronic acid, leads to the sequestration of infected erythrocytes in the placenta and has been shown to be important for the pathogenesis of placental malaria [37, 38]. Thus, cell-to-cell spread from infected maternal macrophages to trophoblasts might be a more relevant mechanism for trophoblast infection than is direct invasion by extracellular bacteria. However, we found that cell-to-cell spread was not essential for placental infection. A disadvantage of our pregnant guinea pig model is that we infected the animals by iv inoculation, which led to an initial seeding of the placenta by extracellular bacteria. We used this route because oral infection of pregnant guinea pigs with 1 × 109 cfu of the wt strain did not lead to placental infection (data not shown).
In our pregnant guinea pig model, we observed a 100-fold decrease in the number of bacteria in fetal infection with the ActA- mutant, compared with that in fetal infection with the wt strain. These results suggest that cell-to-cell spread plays a role in vertical transmission. A potential explanation for this finding is the large size of the syncytiotrophoblasts, compared with that of macrophages. In pregnant guinea pigs infected with the wt strain, the trophoblasts contained only a small number of bacteria. In contrast, the ActA- mutant appeared to reach high densities inside the trophoblasts before being able to infect neighboring cells (figure 2). This could decrease the efficiency of spread to fetal tissues.
Once the placenta was infected, we found a >1000-fold increase in the number of bacteria, which could be due to growth or influx of bacteria. The generation time of L. monocytogenes in a variety of guinea pig, murine, and human cell lines and primary cells is 60 min [30]. In comparison, if the increase of L. monocytogenes in the guinea pig placenta is solely due to bacterial growth, the generation time would be 70 min. This would lead to the conclusion that L. monocytogenes proliferates virtually unrestricted in the placental environment. In addition, in the placenta, we observed an increase in the number of LLO- mutant, which could be due to extracellular or intracellular bacterial growth. The latter could occur if the LLO- mutant could escape from the phagolysosome because of the activity of phospholipases, which contribute to vacuolar escape in some cell types [39]. Consistent with this hypothesis is evidence that the LLO- mutant escapes to a limited degree from the vacuole in the human choriocarcinoma cell line BeWo (data not shown). Our observations that L. monocytogenes mutants that are attenuated in maternal organs are able to increase in number or persist in the placenta suggest that growth, at least, contributes to the observed increase in the number of bacteria in the placenta.
The unique immunological condition of pregnancy is a possible explanation for why the placenta provides a protective environment for the growth of L. monocytogenes. The maternal immune system faces the double task of preventing the rejection of the semi-allogeneic fetus and protecting the mother and the fetus against infection. How these tasks are accomplished simultaneously remains to be fully explained. Suppression of maternal T cellmediated immunityfor example, by induction of apoptosis in T cells via secretion of HLA-G and Fas ligand [40, 41] and by starvation of T cells for the essential amino acid tryptophan via production of indoleamine 2,3-dioxygenase [42]most likely contributes to maternal immunological tolerance of the fetus. Changes in maternal cell-mediated immunity may increase the importance of the innate immune response for the defense against infection [43]especially against intracellular pathogens. Indeed, cells of the innate immune response are strongly represented at the maternal-fetal interface. The predominant population of leukocytes in the hemochorial placenta of humans and rodents consists of nonantigen-specific NK cells and macrophages [4446]. The interaction between macrophages and NK cells is critical during the early stages of listeriosis [47, 48]. However, the roles that uterine macrophages and NK cells play in protection against vertical transmission of L. monocytogenes are unknown.
In human tissues, uterine NK cells comprise 30%40% of the total cells in first trimester decidua [44]. Notably, interleukin-15deficient mice, which are depleted of NK cells, are not more susceptible to placental infection with L. monocytogenes than are wt mice [49], which suggests a negligible role for NK cells in the defense against L. monocytogenes, at least in murine placental tissues.
Macrophages act as primary host cells for L. monocytogenes and critical effector cells that are essential for the defense against listeriosis [47]. Colony-stimulating factor 1 (CSF-1) is the major regulator of cells of the mononuclear phagocytic lineage [50, 51]. CSF-1 is synthesized to very high concentrations at the maternal-fetal interface by the uterine epithelium [5254]. In mouse peritoneal macrophages, CSF-1 stimulates macrophage phagocytic activity and intracellular growth of L. monocytogenes but not bactericidal activity [55, 56]. High concentrations of CSF-1 in the placenta could, therefore, shift the role of macrophages away from that of immune effector cells and toward that of primary host cells for L. monocytogenes. Contrary to this explanation are the results of a study on the effect that CSF-1 has on placental-fetal infection in a pregnant mouse model of listeriosis [57, 58]. The osteopetrotic mouse (Csf1op/Csf1op), which carries a null mutation in the CSF-1 gene, had higher levels of L. monocytogenes in placental-fetal tissues than did the heterozygous control mouse (+/Csf1op), because of a failure to recruit neutrophils to the placenta [58]. However, results in a pregnant mouse model of listeriosis might not readily reflect the situation in the pregnant guinea pig model. For unknown reasons, the placental-fetal unit of guinea pigsand humansis much more susceptible to infection than is that of mice. Differences in susceptibility are apparent for infection with a variety of pathogens, such as cytomegalovirus [59, 60] and T. gondii [61, 62], in addition to L. monocytogenes. Furthermore, the time course of infection may be another important factor. The present study focused on the first 24 h after inoculation, whereas increased susceptibility of the Csf1op/Csf1op mouse to placental infection started to become apparent at 24 h and was significant at 72 h after inoculation.
In summary, we show evidence that the placenta provides a protective niche for L. monocytogenes and that cell-to-cell spread facilitates transplacental fetal infection. Regardless of how the bacteria travel to the placenta, the results of the present study clearly show that the placenta is uniquely permissive of listerial growth.
Acknowledgments
We thank Lindsey Jennings, for assistance with the guinea pig inoculations; Laurel Lenz, for construction of Listeria monocytogenes strain DP-L4694; Stephen Griffey, for advice on the histologic examination of placental sections; and staff of the California Animal Health and Food Safety Laboratory, for performing immunohistochemistry staining.
References
1. Schlech WF III. Epidemiology and clinical manifestations of Listeria monocytogenes infection. In: Fischetti VA, ed. Gram-positive pathogens. Washington, DC: American Society for Microbiology Press, 2000:4739. First citation in article
2. Southwick FS, Purich DL. Intracellular pathogenesis of listeriosis. N Engl J Med 1996; 334:7706. First citation in article
3. Mylonakis E, Paliou M, Hohmann EL, Calderwood SB, Wing EJ. Listeriosis during pregnancy: a case series and review of 222 cases. Medicine (Baltimore) 2002; 81:2609. First citation in article
4. Teberg AJ, Yonekura ML, Salminen C, Pavlova Z. Clinical manifestations of epidemic neonatal listeriosis. Pediatr Infect Dis J 1987; 6:81720. First citation in article
5. Bakardjiev AI, Stacy BA, Fisher SJ, Portnoy DA. Listeriosis in the pregnant guinea pig: a model of vertical transmission. Infect Immun 2004; 72:48997. First citation in article
6. Leiser R, Kaufmann P. Placental structure: in a comparative aspect. Exp Clin Endocrinol 1994; 102:12234. First citation in article
7. Dussurget O, Pizarro-Cerda J, Cossart P. Molecular determinants of Listeria monocytogenes virulence. Annu Rev Microbiol 2004; 58:587610. First citation in article
8. Portnoy DA, Auerbuch V, Glomski IJ. The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity. J Cell Biol 2002; 158:40914. First citation in article
9. Cabanes D, Dehoux P, Dussurget O, Frangeul L, Cossart P. Surface proteins and the pathogenic potential of Listeria monocytogenes. Trends Microbiol 2002; 10:23845. First citation in article
10. Mengaud J, Ohayon H, Gounon P, Mege RM, Cossart P. E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell 1996; 84:92332. First citation in article
11. Shen Y, Naujokas M, Park M, Ireton K. InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase. Cell 2000; 103:50110. First citation in article
12. Gruenheid S, Finlay BB. Microbial pathogenesis and cytoskeletal function. Nature 2003; 422:77581. First citation in article
13. Welch MD, Mullins RD. Cellular control of actin nucleation. Annu Rev Cell Dev Biol 2002; 18:24788. First citation in article
14. Goldberg MB. Actin-based motility of intracellular microbial pathogens. Microbiol Mol Biol Rev 2001; 65:595626. First citation in article
15. Brockstedt DG, Giedlin MA, Leong ML, et al. Listeria-based cancer vaccines that segregate immunogenicity from toxicity. Proc Natl Acad Sci USA 2004; 101:138327. First citation in article
16. Gaillard JL, Jaubert F, Berche P. The inlAB locus mediates the entry of Listeria monocytogenes into hepatocytes in vivo. J Exp Med 1996; 183:35969. First citation in article
17. Lecuit M, Dramsi S, Gottardi C, Fedor-Chaiken M, Gumbiner B, Cossart P. A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes. EMBO J 1999; 18:395663. First citation in article
18. Schubert WD, Urbanke C, Ziehm T, et al. Structure of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin. Cell 2002; 111:82536. First citation in article
19. Lecuit M, Vandormael-Pournin S, Lefort J, et al. A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier. Science 2001; 292:17225. First citation in article
20. Jacquet C, Doumith M, Gordon JI, Martin PM, Cossart P, Lecuit M. A molecular marker for evaluating the pathogenic potential of foodborne Listeria monocytogenes. J Infect Dis 2004; 189:2094100. First citation in article
21. Kaufmann P, Davidoff M. The guinea-pig placenta. Adv Anat Embryol Cell Biol 1977; 53:591. First citation in article
22. Lecuit M, Nelson DM, Smith SD, et al. Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes: role of internalin interaction with trophoblast E-cadherin. Proc Natl Acad Sci USA 2004; 101:61527. First citation in article
23. Brundage RA, Smith GA, Camilli A, Theriot JA, Portnoy DA. Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells. Proc Natl Acad Sci USA 1993; 90:118904. First citation in article
24. Bishop DK, Hinrichs DJ. Adoptive transfer of immunity to Listeria monocytogenes: the influence of in vitro stimulation on lymphocyte subset requirements. J Immunol 1987; 139:20059. First citation in article
25. Auerbuch V, Lenz LL, Portnoy DA. Development of a competitive index assay to evaluate the virulence of Listeria monocytogenes actA mutants during primary and secondary infection of mice. Infect Immun 2001; 69:59537. First citation in article
26. Skoble J, Portnoy DA, Welch MD. Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility. J Cell Biol 2000; 150:52738. First citation in article
27. Jones S, Portnoy DA. Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O. Infect Immun 1994; 62:560813. First citation in article
28. Hodgson DA. Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes. Mol Microbiol 2000; 35:31223. First citation in article
29. Glomski IJ, Decatur AL, Portnoy DA. Listeria monocytogenes mutants that fail to compartmentalize listerolysin O activity are cytotoxic, avirulent, and unable to evade host extracellular defenses. Infect Immun 2003; 71:675465. First citation in article
30. Portnoy DA, Jacks PS, Hinrichs DJ. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med 1988; 167:145971. First citation in article
31. Siegman-Igra Y, Levin R, Weinberger M, et al. Listeria monocytogenes infection in Israel and review of cases worldwide. Emerg Infect Dis 2002; 8:30510. First citation in article
32. Drevets DA, Jelinek TA, Freitag NE. Listeria monocytogenes-infected phagocytes can initiate central nervous system infection in mice. Infect Immun 2001; 69:134450. First citation in article
33. Drevets DA. Dissemination of Listeria monocytogenes by infected phagocytes. Infect Immun 1999; 67:35127. First citation in article
34. Drevets DA, Leenen PJ, Greenfield RA. Invasion of the central nervous system by intracellular bacteria. Clin Microbiol Rev 2004; 17:32347. First citation in article
35. Vazquez-Torres A, Jones-Carson J, Baumler AJ, et al. Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes. Nature 1999; 401:8048. First citation in article
36. McKee AS, Dzierszinski F, Boes M, Roos DS, Pearce EJ. Functional inactivation of immature dendritic cells by the intracellular parasite Toxoplasma gondii. J Immunol 2004; 173:263240. First citation in article
37. Fried M, Duffy PE. Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta. Science 1996; 272:15024. First citation in article
38. Beeson JG, Rogerson SJ, Cooke BM, et al. Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria. Nat Med 2000; 6:8690. First citation in article
39. Marquis H, Doshi V, Portnoy DA. The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells. Infect Immun 1995; 63:45314. First citation in article
40. Fournel S, Aguerre-Girr M, Huc X, et al. Cutting edge: soluble HLA-G1 triggers CD95/CD95 ligand-mediated apoptosis in activated CD8+ cells by interacting with CD8. J Immunol 2000; 164:61004. First citation in article
41. Makrigiannakis A, Zoumakis E, Kalantaridou S, et al. Corticotropin-releasing hormone promotes blastocyst implantation and early maternal tolerance. Nat Immunol 2001; 2:101824. First citation in article
42. Munn DH, Zhou M, Attwood JT, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998; 281:11913. First citation in article
43. Sacks G, Sargent I, Redman C. An innate view of human pregnancy. Immunol Today 1999; 20:1148. First citation in article
44. Hunt JS, Petroff MG, Burnett TG. Uterine leukocytes: key players in pregnancy. Semin Cell Dev Biol 2000; 11:12737. First citation in article
45. Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol 2002; 2:65663. First citation in article
46. Trundley A, Moffett A. Human uterine leukocytes and pregnancy. Tissue Antigens 2004; 63:112. First citation in article
47. Edelson BT, Unanue ER. Immunity to Listeria infection. Curr Opin Immunol 2000; 12:42531. First citation in article
48. Unanue ER. Inter-relationship among macrophages, natural killer cells and neutrophils in early stages of Listeria resistance. Curr Opin Immunol 1997; 9:3543. First citation in article
49. Barber EM, Pollard JW. The uterine NK cell population requires IL-15 but these cells are not required for pregnancy nor the resolution of a Listeria monocytogenes infection. J Immunol 2003; 171:3746. First citation in article
50. Stanley ER, Guilbert LJ, Tushinski RJ, Bartelmez SH. CSF-1a mononuclear phagocyte lineage-specific hemopoietic growth factor. J Cell Biochem 1983; 21:1519. First citation in article
51. Webb SE, Pollard JW, Jones GE. Direct observation and quantification of macrophage chemoattraction to the growth factor CSF-1. J Cell Sci 1996; 109:793803. First citation in article
52. Bartocci A, Pollard JW, Stanley ER. Regulation of colony-stimulating factor 1 during pregnancy. J Exp Med 1986; 164:95661. First citation in article
53. Pollard JW, Bartocci A, Arceci R, Orlofsky A, Ladner MB, Stanley ER. Apparent role of the macrophage growth factor, CSF-1, in placental development. Nature 1987; 330:4846. First citation in article
54. Pollard JW. Role of colony-stimulating factor-1 in reproduction and development. Mol Reprod Dev 1997; 46:5460; discussion 601. First citation in article
55. Cheers C, Hill M, Haigh AM, Stanley ER. Stimulation of macrophage phagocytic but not bactericidal activity by colony-stimulating factor 1. Infect Immun 1989; 57:15126. First citation in article
56. Denis M, Gregg EO. Identification of cytokines which enhance (CSF-1, IL-3) or restrict (IFN-gamma) growth of intramacrophage Listeria monocytogenes. Immunol Lett 1991; 27:23742. First citation in article
57. Guleria I, Pollard JW. Aberrant macrophage and neutrophil population dynamics and impaired Th1 response to Listeria monocytogenes in colony-stimulating factor 1-deficient mice. Infect Immun 2001; 69:1795807. First citation in article
58. Guleria I, Pollard JW. The trophoblast is a component of the innate immune system during pregnancy. Nat Med 2000; 6:58993. First citation in article
59. Medearis DN. Mouse cytomegalovirus infection III. Attempts to produce intrauterine infections. Am J Hyg 1964; 80:11320. First citation in article
60. Schleiss MR. Animal models of congenital cytomegalovirus infection: an overview of progress in the characterization of guinea pig cytomegalovirus (GPCMV). J Clin Virol 2002; 25(Suppl 2):S3749. First citation in article
61. Wright I. Transmission of Toxoplasma gondii across the guinea-pig placenta. Lab Anim 1972; 6:16980. First citation in article
62. Haumont M, Delhaye L, Garcia L, et al. Protective immunity against congenital toxoplasmosis with recombinant SAG1 protein in a guinea pig model. Infect Immun 2000; 68:494853. First citation in article