Vol. 59, No. 11
INFECTION AND IMMUNITY, Nov. 1991, p. 4286-4290 0019-9567/91/114286-05$02.00/0 Copyright © 1991, American Society for Microbiology
Listeriolysin as a Virulence Factor in Listeria monocytogenes Infection of Neonatal Mice and Murine Decidual Tissue DIANNE B. McKAYt AND CHRISTOPHER Y. LU* Division of Nephrology, Department of Internal Medicine, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8856 Received 18 June 1991/Accepted 30 August 1991
Listeriolysin is a 60-kDa protein which allows the growth of Listeria monocytogenes in macrophages and other cells and has been shown to be a virulence factor in Listeria infections of adult mice. However, the neonate and fetoplacental unit are major populations susceptible to listeriosis. Recent data indicate that macrophage and T-cell functions are markedly inhibited in these young mice, and the virulence of listeriolysin-negative (HLY-) and listeriolysin-positive (HLY+) Listeria cells in the setting of such inhibited macrophage and T-cell functions has not previously been examined. We now compare CNL 85/162, a transposon-induced, HLYListeria strain, and CNL 85/163, a spontaneous HLY+ revertant. We found that all 18 neonates injected with CNL 85/163 (HLY+) died within 12 days after an injection of 104 Listeria cells per mouse. In contrast, all 16 neonates injected with 1,000 times more CNL 85/162 (HLY-) cells survived more than 14 days. Three days after injection, growth of CNL 85/163 (HLY+) in the internal organs was more than 5 log greater than that of CNL 85/162 (HLY-). We also found that CNL 85/162 (HLY-) did not proliferate well in decidual tissue, which is a major component of the placental region. Our studies indicate that HLY- bacteria are not virulent in the neonate and the fetoplacental unit despite the inhibited immune functions at these sites.
Although Listeria monocytogenes is an uncommon adult pathogen, it is the third most common cause of bacterial meningitis in neonates. Furthermore, prenatal Listeria infections cause abortion, stillbirth, and a devastating septic illness, granulomatosis infantisepticum (36). There is a growing body of evidence that listeriosis may be caused by contaminated food (for example, see references 25 and 36). However, the development of regulatory guidelines to prevent food-borne listeriosis is difficult because of outstanding questions concerning which strains of this common bacteria are virulent. In model systems using adult mice, production of listeriolysin is an important characteristic of virulent Listeria strains (13). This 60-kDa protein allows the intracellular growth of Listeria cells in macrophages and other cells by lysing phagolysosomes (4, 12, 14, 30). These observations suggest that the ability to produce this lysin will differentiate pathogenic from nonpathogenic Listeria strains. However, the listeriolysin virulence studies mentioned above employed adult rather than perinatal mice. Previous studies from this laboratory demonstrated that macrophage functions are profoundly inhibited in the murine neonate and decidua basalis, where the fetal placenta is anchored in the maternal uterus (21, 23, 24, 32). Macrophages are an important effector cell in the host defense against Listeria infection (17), and these studies raise the possibility that in the setting of such inhibited macrophage functions, lysin-negative Listeria strains are pathogenic. This is an important question because the neonate and fetoplacental unit are the human populations most susceptible to infection. Furthermore, additional virulence factors in listeriosis have recently been proposed (5, 10, 15, 19, 20, 26, 27) and may conceivably have greater importance than listeriolysin during the perinatal period. In this report, we compare the virulence of listeriol-
ysin-positive (HLY+) and -negative (HLY-) bacteria in neonatal mice and in decidual tissue of the placental region. The CNL 85/162 strain of L. monocytogenes has a single copy of the conjugative transposon Tn1545 inserted into the open reading frame of the listeriolysin gene and therefore does not produce active listeriolysin (HLY-) (6, 13). The Tn1S45 transposon contains kanamycin, tetracycline, and erythromycin resistance genes. CNL 85/163 is a spontaneous revertant which has lost the transposon and does produce listeriolysin (HLY+). Only the latter HLY+ listeria cells are virulent in adult mice (13). Both strains were originally derived by Gaillard and associates (13) and were the gift of Kirk Ziegler, Emory University, Atlanta, Ga. The bacteria were stored frozen in 20% glycerol in phosphate-buffered saline at -70°C. Aliquots of bacteria were thawed, diluted to an appropriate concentration with sterile phosphate-buffered saline, and plated on Trypticase soy agar with 5% sheep blood plates (BBL Microbiology Systems, Cockeysville, Md.) to confirm the number of viable bacteria as well as differentiate HLY+ and HLY- colonies. The experiments for Figures 1 and 2 were designed to determine whether HLY- Listeria cells were virulent in the neonate. CNL 85/162 (HLY-) or CNL 85/163 (HLY+) cells were inoculated into the peritoneal cavities of 4-day-old neonates. The neonates were the progeny of B10.A SgSn females and BALB/c By males from Jackson Laboratories, Bar Harbor, Maine. Although the dose of HLY- Listeria cells exceeded that of the HLY+ strain by 1,000-fold, Fig. 1 shows that the HLY- strain was not virulent throughout the 14-day period of observation. In contrast, all neonates inoculated with HLY+ Listeria cells died. Three days after the above-described inoculation of HLY+ or HLY- Listeria cells, four neonates from each group were sacrificed. The heart, lung, liver, and spleen from each neonate were homogenized together in 0.005% Triton X-100 in phosphate-buffered saline and then plated on Trypticase soy agar-blood plates. Colonies of HLY+ cells were distinguished from HLY- bacteria by the presence of
* Corresponding author. t Present address: Renal Division, Brigham and Women's Hospital, Boston, MA 02115.
4286
VOL. 59, 1991
NOTES
4287
1100-
90
-C-- HLY+ 70-
HLY-
-J
60-
5: a: C')
Iz w
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40 -
CL 30 20-
10 0 1
2
3
4
5
6
7
8
9
10
1 1
12
13
14
DAYS POST LISTERIA INOCULATION
FIG. 1. Percent survival of neonates receiving an intraperitoneal injection of Listeria cells on day 4 after birth. HLY+, 104 L. monocytogenes CNL 85/163 cells per neonate (n = 16); HLY-, 107 L. monocytogenes CNL 85/162 cells per neonate (n = 18).
hemolysis around colonies of the former. Figure 2 shows that neonates injected with HLY+ Listeria cells had 4 orders of magnitude more bacteria than neonates injected with HLY- Listeria cells, even though the HLY- inocula were 1,000 times larger than the HLY+ inocula. Listeria infection is particularly devastating in the placental region, where macrophage and T-cell functions are profoundly inhibited (31, 32). We have previously reported that such inhibition results in the unrestrained infection of this region. Listeria cells initially enter and proliferate in the decidua basalis underlying the placental disc (32). This is the most profoundly infected site of the placental region. Maternal decidual stromal cells are a major constituent of this region (33), and we have demonstrated that these cells and their associated extracellular matrix are responsible for inhibiting macrophage functions both in vivo and in vitro (32, 34, 35). Our next experiments were designed to determine whether HLY- Listeria cells were virulent in decidual tissue. As discussed below, we employed the deciduoma-ofpseudopregnancy model because of its similarities to the decidua basalis of true pregnancy and because it allowed us to inject defined numbers of HLY+ and HLY- Listeria cells directly into the uterus, a maneuver which was technically not possible in true pregnancy. L. monocytogenes injected into the uterine horns of mice made pseudopregnant by hormone treatment causes the formation of deciduomas (35). Such deciduomas are similar to the decidua basalis of true pregnancy in that maternal decidual cells are a major component (1, 2, 28, 29, 37, 38). Macrophage antilisteria re-
LuJ -I
-
HLY+
HLY-
Loglo bacterial titers of neonatal mice receiving an intraperitoneal injection of either HLY+ or HLY- Listeria cells on day 4 after birth. HLY+, 104 L. monocytogenes CNL 85/163 cells per neonate; HLY-, 107 L. monocytogenes CNL 85/162 cells per neonate. Three days after inoculation, the mice were sacrificed and bacterial growth in an organ block containing heart, lungs, liver, and spleen was determined. The values shown are the means and standard errors from four animals. FIG. 2.
5
4
3.1
2
NOTES
4288
INFECT. IMMUN.
SPLE EN
RT UTERUS
LFT UTERUS
FIG. 3. Loglo bacterial titers of pseudopregnant mice receiving HLY+ Listeria cells in the left uterine horn and HLY- Listeria cells in the right uterine horn. HLY+, 5 x 10' CNL 85/163 cells (solid bars); HLY-, 5 x 104 CNL 85/162 cells (open bars). HLY+ Listeria colonies were distinguished from HLY- colonies by the presence of hemolysis around the former on Trypticase soy agar-blood plates. Mice were sacrificed 3 days after receiving Listeria inoculation. The values shown are the means and standard errors from five mice.
sponses are inhibited in the deciduoma of pseudopregnancy in a manner exactly analogous to that of the decidua basalis of true pregnancy (35). Deciduomas were induced by methods previously published by our laboratory (35). Briefly, 1 week before inducing deciduoma, female B10.A x BALB/c mice 2 months old, were primed with CNL 85/163 (HLY+) by injection of 104 bacteria into the lateral tail veins. One week later, the animals were anesthetized with tribromoethanol (Avertin) and bilateral oophorectomies were performed. Six days after the oophorectomies, subcutaneous hormone injections were begun. Each animal received 0.1 mg of estrogen daily for 3 days, rested for 2 days without hormone injections, and then was injected with a mixture of estrogen (10 ng) and progesterone (1 mg) for daily 3 more days. Six hours following the third estrogen-progesterone injection, the animals were anesthetized and each uterine horn was injected with 100 ,ul of the appropriate Listeria strain. The mice continued to receive progesterone injections for the next 3 days. The morning following the last injection, the mice were sacrificed and the tissues were analyzed for Listeria infection as described above. For Fig. 3, the left uterine horns of pseudopregnant mice were injected with HLY+ Listeria cells and the right uterine horns of the same mice were injected with 100 times more HLY- Listeria cells. After 3 days, Listeria growth in the individual uterine horns and spleens was determined. HLY+ and HLY- Listeria cells were distinguished by the presence or absence of hemolysis on Trypticase soy agar-blood plates. Note that there was a spread of HLY+ Listeria cells from the initial site of injection in the left uterine horn to the spleen and right uterine horn. There was no detectable growth of HLY- Listeria cells. For Fig. 4, pseudopregnant mice were injected in both uterine horns with the same Listeria strain. One group of mice received HLY+ Listeria cells; the other received
A) HLY+ 7
B) HLY7
a a
N PZ
N5 13 4
3
44
I"
N
N 4 N
3 2
U
I
1
0
0 SPLEEN
UTERUS
SPLEEN
UTERUS
FIG. 4. Loglo bacterial titers in pseudopregnant mice receiving either HLY+ or HLY- Listeria cells into their uterine horns. Each mouse received only one Listeria strain. (A) HLY+, 5 x 102 CNL 85/163 cells injected into each uterine horn; (B) HLY-, 5 x 104 CNL 85/162 cells. Mice were sacrificed 3 days after receiving the Listeria injection. The values shown are the means and standard errors from three mice.
VOL. 59, 1991
HLY- Listeria cells. As a precaution, the latter mice also received kanamycin sulfate to prevent growth of spontaneous revertants which had lost the transposon and its antibiotic resistance gene (3). In contrast to the Fig. 3 experiment, each individual mouse received only one Listeria strain. There were 100 to 1,000 times more Listeria cells in the spleens and uteri of mice receiving intrauterine HLY+ bacteria (Fig. 4A) than in those of mice receiving HLYbacteria (Fig. 4B), even though the latter mice received 100 times more Listeria cells. Thus, there was 1,000-fold growth of the HLY+ Listeria cells from the 1,000 bacteria initially injected into the uteri. We have previously performed detailed immunohistochemical studies of HLY+ Listeria growth in decidual tissues (32, 35). Listeria cells were found in both extra- and intracellular sites. At early stages of infection, inflammatory cells were not present and any intracellular bacteria were in decidual cells. In contrast, the number of HLY- Listeria cells in the uteri decreased to 1/100 of the initial 10,000 bacteria injected. Altogether, the data in Fig. 3 and 4 indicate that HLYListeria cells were not virulent even when injected directly into decidual tissue, a site at which macrophage functions are profoundly inhibited. In conclusion, macrophages play a major role in the host defense against Listeria infection. Recent data from our laboratory indicate that macrophage functions are inhibited in the murine neonate and placental region by two entirely different mechanisms. In the neonate, in contrast to the adult, concentrations of a-fetoprotein (8) and nonesterified docosahexaenoic acid (9) in serum are high. These molecules inhibit the ability of macrophages to be activated by gamma interferon (7, 11, 22) and thus inhibit an important step in the macrophage response against L. monocytogenes (17). Despite these inhibitors, we now find that listeriolysin is still required for virulence in neonatal infections. Listeriolysin is also required in the placental region, where macrophage functions are inhibited by a different mechanism. The decidua basalis is where the placenta is anchored in the maternal uterus. Infection at this site is a complex event because it involves not only the pathogen and the host defenses but also the maternal-fetal immunologic relationship. The latter may be considered an allograft-host relationship because of the paternal antigens of the fetus and placenta (see reviews in references 16 and 18). Macrophage functions are profoundly inhibited in the decidua basalis, perhaps to prevent maternal antifetal immunologic responses. We have previously shown that solid-phase molecules embedded in these decidual tissues inhibit macrophage functions (31, 32, 34, 35). Similar decidual tissue is present in the deciduoma of pseudopregnancy. We now report that despite the profoundly inhibited macrophage functions in the decidua and the neonate, listeriolysin is still required for virulence. One explanation of this is that listeriolysin is required for virulence because it allows the bacteria to survive within macrophages and other cells (4, 12, 14, 30). Since listeriolysin functions best at pH 5, which is found within phagolysosomes, but minimally at pH 7, which is found in the extracellular environment, it is unlikely to contribute to bacterial growth outside of cells. Listeriosis may be transmitted by contaminated food (25, 36). The development of regulatory guidelines to prevent such infections will require identification of virulence factors which differentiate pathogenic from nonpathogenic strains of this common bacterium. To our knowledge, this report is the first to demonstrate that production of listeriolysin is a virulence factor in infection of the most susceptible popula-
NOTES
4289
tions, the neonate and decidual tissue of the fetoplacental unit. This work was supported by grants from the March of Dimes and NIH RO1-HD24797 and RO1 DK43634. Dianne B. McKay is supported by an institutional postdoctoral fellowship grant from the NIH (2T32-DK-07527). Christopher Y. Lu is the recipient of an NIH Research Career Development Award (K04-HD00862). We thank Miguel A. Vazquez for critically reviewing the manuscript. REFERENCES 1. Bell, S. C. 1983. Decidualization: regional differentiation and
associated function. Oxford Rev. Reprod. Biol. 5:220-271. 2. Bell, S. C. 1985. Comparative aspects of decidualization in rodents and human: cell types, secreted products and associated function, p. 71-132. In R. G. Edwards, J. M. Purdy, and P. C.
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Steptoe (ed.), Implantation of the human embryo. Academic Press, Inc., London. Berche, P., J. L. Gaillard, and P. J. Sansonetti. 1987. Intracellular growth of Listeria monocytogenes as a prerequisite for in vivo induction of T cell-mediated immunity. J. Immunol. 138: 2266-2271. Bielecki, J., P. Youngman, P. Connelly, and D. A. Portnoy. 1990. Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells. Nature (London) 345:175-176. Camilli, A., H. Goldfine, and D. A. Portnoy. 1991. Listeria monocytogenes mutants lacking phosphatidylinositol-specific phospholipase C are avirulent. J. Exp. Med. 173:751-754. Cossart, P. 1988. The listeriolysin 0 gene: a chromosomal locus crucial for the virulence of Listeria monocytogenes. Infection 16(Suppl. 2):S157-S159. Crainie, M., A. Semeluk, K. C. Lee, and T. G. Wegmann. 1989. Regulation of constitutive and lymphokine induced Ia expression by murine alpha-fetoprotein. Cell. Immunol. 118:41-52. Crandall, B. F. 1981. Alpha-fetoprotein: a review. Crit. Rev. Clin. Lab. Sci. 15:127-185. Delorme, J., C. Benassayag, N. Christeff, G. Vallette, L. Savu, and E. Nunez. 1984. Age-dependent responses of the serum non-esterified fatty acids to adrenalectomy and ovariectomy in developong rats. Biochim. Biophys. Acta 792:6-10. Domann, E., M. Leimeister-Wachter, W. Goebel, and T. Chakraborty. 1991. Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene. Infect. Immun. 59:65-72. Dustin, L. B., R. Soberman, C. M. Shea, and C. Y. Lu. 1990. Docosahexaenoic acid, a constituent of rodent fetal serum and fish oil diets, inhibits acquisition of macrophage tumoricidal function. J. Immunol. 144:4888-4897. Gaillard, J.-L., P. Berche, J. Mounier, S. Richard, and P. Sansonetti. 1987. In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect. Immun. 55:2822-2829. Gaillard, J. L., P. Berche, and P. Sansonetti. 1986. Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect. Immun. 52:50-55. Geoffroy, C., J.-L. Gaillard, J. E. Alouf, and P. Berche. 1987. Purification, characterization, and toxicity of the sulfhydrylactivated hemolysin listeriolysin 0 from Listeria monocytogenes. Infect. Immun. 55:1641-1646. Geoffroy, C., J. Raveneau, J.-L. Beretti, A. Lecroisey, J.-A.
Vasquez-Boland, J. E. Alouf, P. Berche. 1991. Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes. Infect. Immun. 59:2382-2388. 16. Gill, T. J., HI, and T. G. Wegmann. 1987. Immunoregulation and fetal survival. Oxford University Press, New York. 17. Hahn, H., and S. H. E. Kaufmann. 1981. The role of cellmediated immunity in bacterial infections. Rev. Infect. Dis. 3:1221-1250. 18. Hunziker, R. D., and T. G. Wegmann. 1986. Placental immunoregulation. Crit. Rev. Immunol. 6:245-285.
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19. Kathariou, S., J. Rocourt, H. Hof, and W. Goebel. 1988. Levels of Listeria monocytogenes hemolysin are not directly proportional to virulence in experimental infections of mice. Infect. Immun. 56:534-536. 20. Leimeister-Wachter, M., E. Domann, and T. Chakraborty. 1991. Detection of a gene encoding a phosphatidylinositol-specific phospholipase C that is co-ordinately expressed with listeriolysin in Listeria monocytogenes. Mol. Microbiol. 5:361-366. 21. Lu, C. Y., E. G. Calamai, and E. R. Unanue. 1979. A defect in the antigen-presenting function of macrophages from neonatal mice. Nature (London) 282:327-329. 22. Lu, C. Y., P. S. Changelian, and E. R. Unanue. 1984. Alphafetoprotein inhibits macrophage expression of Ia antigens. J. Immunol. 132:1722-1727. 23. Lu, C. Y., R. W. Redline, L. B. Dustin, D. B. McKay, and C. M. Shea. 1991. Inhibition of macrophage and T-lymphocyte functions in the placenta and decidua during listeriosis: implications for tolerance of the fetoplacental allograft p. 141-167. In T. G. Wegmann, T. J. Gill III, and E. Nisbet-Brown (ed.), Molecular and cellular immunobiology of the maternal fetal interface. Oxford University Press, New York. 24. Lu, C. Y., and E. R. Unanue. 1985. Macrophage ontogeny: implications for host defence, T lymphocyte differentiation, and the acquisition of self-tolerance. Clin. Immunol. Allergy 5:253269. 25. Mascola, L., F. Sorvillo, J. Neal, K. Iwakoshi, and R. Weaver. 1989. Surveillance of listeriosis in Los Angeles County, 19851986. A first year's report. Arch. Int. Med. 149:1569-1572. 26. Mengaud, J., C. Braun-Breton, and P. Cossart. 1991. Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor? Mol. Microbiol. 5:367-372. 27. Mengaud, J. C. Geoffroy, and P. Cossart. 1991. Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases. Infect. Immun. 59:1043-1049. 28. Nieder, G. L., and G. R. Macon. 1987. Uterine and oviducal
29. 30. 31. 32.
33.
34.
35. 36. 37. 38.
protein secretion during early pregnancy in the mouse. J. Reprod. Fertil. 81:287-294. O'Shea, J. D., R. G. Kleinfeld, and H. A. Morrow. 1983. Ultrastructure of decidualization in the pseudopregnant rat. Am. J. Anat. 166:271. Portnoy, D. A., P. Suzanne-Jacks, and D. J. Hinrichs. 1988. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167:1459-1471. Redline, R. W., and C. Y. Lu. 1987. Role of local immunosuppression in murine fetoplacental listeriosis. J. Clin. Invest. 79:1234-1241. Redline, R. W., and C. Y. Lu. 1988. Specific defects in the anti-listerial immune response in discrete regions of the murine uterus and placenta account for susceptibility to infection. J. Immunol. 140:3947-3955. Redline, R. W., and C. Y. Lu. 1989. Localization of fetal major histocompatibility complex (MHC) antigens and maternal leukocytes in the murine placenta: implications for understanding the regulation of maternal anti-fetal immune responses. Lab. Invest. 61:27-36. Redline, R. W., D. B. McKay, V. E. Papaioannou, C. M. Shea, and C. Y. Lu. 1990. Macrophage functions are regulated by the substratum of decidualized endometrial stromal cells. J. Clin. Invest. 85:1951-1958. Redline, R. W., C. M. Shea, V. E. Papaioannou, and C. Y. Lu. 1988. Defective anti-listerial responses in deciduoma of pseudopregnant mice. Am. J. Pathol. 133:485. Schlech, W. F., P. M. Lavigne, R. A. Bortolussi, et al. 1983. Epidemic listeriosis-evidence for transmission by food. N. Engl. J. Med. 308:302-306. Weitlauf, H. M., and M. Suda-Hartman. 1988. Changes in secreted uterine proteins associated with embryo implantation in the mouse. J. Reprod. Fertil. 84:539-549. Wewer, U. M., A. Damjanov, J. Weiss, L. A. Liotta, and I. Damjanov. 1986. Mouse endometrial stromal cells produce basement-membrane components. Differentiation 32:49.
INFECTION AND IMMUNITY, Nov. 1991, p. 4286-4290 0019-9567/91/114286-05$02.00/0 Copyright © 1991, American Society for Microbiology
Listeriolysin as a Virulence Factor in Listeria monocytogenes Infection of Neonatal Mice and Murine Decidual Tissue DIANNE B. McKAYt AND CHRISTOPHER Y. LU* Division of Nephrology, Department of Internal Medicine, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8856 Received 18 June 1991/Accepted 30 August 1991
Listeriolysin is a 60-kDa protein which allows the growth of Listeria monocytogenes in macrophages and other cells and has been shown to be a virulence factor in Listeria infections of adult mice. However, the neonate and fetoplacental unit are major populations susceptible to listeriosis. Recent data indicate that macrophage and T-cell functions are markedly inhibited in these young mice, and the virulence of listeriolysin-negative (HLY-) and listeriolysin-positive (HLY+) Listeria cells in the setting of such inhibited macrophage and T-cell functions has not previously been examined. We now compare CNL 85/162, a transposon-induced, HLYListeria strain, and CNL 85/163, a spontaneous HLY+ revertant. We found that all 18 neonates injected with CNL 85/163 (HLY+) died within 12 days after an injection of 104 Listeria cells per mouse. In contrast, all 16 neonates injected with 1,000 times more CNL 85/162 (HLY-) cells survived more than 14 days. Three days after injection, growth of CNL 85/163 (HLY+) in the internal organs was more than 5 log greater than that of CNL 85/162 (HLY-). We also found that CNL 85/162 (HLY-) did not proliferate well in decidual tissue, which is a major component of the placental region. Our studies indicate that HLY- bacteria are not virulent in the neonate and the fetoplacental unit despite the inhibited immune functions at these sites.
Although Listeria monocytogenes is an uncommon adult pathogen, it is the third most common cause of bacterial meningitis in neonates. Furthermore, prenatal Listeria infections cause abortion, stillbirth, and a devastating septic illness, granulomatosis infantisepticum (36). There is a growing body of evidence that listeriosis may be caused by contaminated food (for example, see references 25 and 36). However, the development of regulatory guidelines to prevent food-borne listeriosis is difficult because of outstanding questions concerning which strains of this common bacteria are virulent. In model systems using adult mice, production of listeriolysin is an important characteristic of virulent Listeria strains (13). This 60-kDa protein allows the intracellular growth of Listeria cells in macrophages and other cells by lysing phagolysosomes (4, 12, 14, 30). These observations suggest that the ability to produce this lysin will differentiate pathogenic from nonpathogenic Listeria strains. However, the listeriolysin virulence studies mentioned above employed adult rather than perinatal mice. Previous studies from this laboratory demonstrated that macrophage functions are profoundly inhibited in the murine neonate and decidua basalis, where the fetal placenta is anchored in the maternal uterus (21, 23, 24, 32). Macrophages are an important effector cell in the host defense against Listeria infection (17), and these studies raise the possibility that in the setting of such inhibited macrophage functions, lysin-negative Listeria strains are pathogenic. This is an important question because the neonate and fetoplacental unit are the human populations most susceptible to infection. Furthermore, additional virulence factors in listeriosis have recently been proposed (5, 10, 15, 19, 20, 26, 27) and may conceivably have greater importance than listeriolysin during the perinatal period. In this report, we compare the virulence of listeriol-
ysin-positive (HLY+) and -negative (HLY-) bacteria in neonatal mice and in decidual tissue of the placental region. The CNL 85/162 strain of L. monocytogenes has a single copy of the conjugative transposon Tn1545 inserted into the open reading frame of the listeriolysin gene and therefore does not produce active listeriolysin (HLY-) (6, 13). The Tn1S45 transposon contains kanamycin, tetracycline, and erythromycin resistance genes. CNL 85/163 is a spontaneous revertant which has lost the transposon and does produce listeriolysin (HLY+). Only the latter HLY+ listeria cells are virulent in adult mice (13). Both strains were originally derived by Gaillard and associates (13) and were the gift of Kirk Ziegler, Emory University, Atlanta, Ga. The bacteria were stored frozen in 20% glycerol in phosphate-buffered saline at -70°C. Aliquots of bacteria were thawed, diluted to an appropriate concentration with sterile phosphate-buffered saline, and plated on Trypticase soy agar with 5% sheep blood plates (BBL Microbiology Systems, Cockeysville, Md.) to confirm the number of viable bacteria as well as differentiate HLY+ and HLY- colonies. The experiments for Figures 1 and 2 were designed to determine whether HLY- Listeria cells were virulent in the neonate. CNL 85/162 (HLY-) or CNL 85/163 (HLY+) cells were inoculated into the peritoneal cavities of 4-day-old neonates. The neonates were the progeny of B10.A SgSn females and BALB/c By males from Jackson Laboratories, Bar Harbor, Maine. Although the dose of HLY- Listeria cells exceeded that of the HLY+ strain by 1,000-fold, Fig. 1 shows that the HLY- strain was not virulent throughout the 14-day period of observation. In contrast, all neonates inoculated with HLY+ Listeria cells died. Three days after the above-described inoculation of HLY+ or HLY- Listeria cells, four neonates from each group were sacrificed. The heart, lung, liver, and spleen from each neonate were homogenized together in 0.005% Triton X-100 in phosphate-buffered saline and then plated on Trypticase soy agar-blood plates. Colonies of HLY+ cells were distinguished from HLY- bacteria by the presence of
* Corresponding author. t Present address: Renal Division, Brigham and Women's Hospital, Boston, MA 02115.
4286
VOL. 59, 1991
NOTES
4287
1100-
90
-C-- HLY+ 70-
HLY-
-J
60-
5: a: C')
Iz w
C,
40 -
CL 30 20-
10 0 1
2
3
4
5
6
7
8
9
10
1 1
12
13
14
DAYS POST LISTERIA INOCULATION
FIG. 1. Percent survival of neonates receiving an intraperitoneal injection of Listeria cells on day 4 after birth. HLY+, 104 L. monocytogenes CNL 85/163 cells per neonate (n = 16); HLY-, 107 L. monocytogenes CNL 85/162 cells per neonate (n = 18).
hemolysis around colonies of the former. Figure 2 shows that neonates injected with HLY+ Listeria cells had 4 orders of magnitude more bacteria than neonates injected with HLY- Listeria cells, even though the HLY- inocula were 1,000 times larger than the HLY+ inocula. Listeria infection is particularly devastating in the placental region, where macrophage and T-cell functions are profoundly inhibited (31, 32). We have previously reported that such inhibition results in the unrestrained infection of this region. Listeria cells initially enter and proliferate in the decidua basalis underlying the placental disc (32). This is the most profoundly infected site of the placental region. Maternal decidual stromal cells are a major constituent of this region (33), and we have demonstrated that these cells and their associated extracellular matrix are responsible for inhibiting macrophage functions both in vivo and in vitro (32, 34, 35). Our next experiments were designed to determine whether HLY- Listeria cells were virulent in decidual tissue. As discussed below, we employed the deciduoma-ofpseudopregnancy model because of its similarities to the decidua basalis of true pregnancy and because it allowed us to inject defined numbers of HLY+ and HLY- Listeria cells directly into the uterus, a maneuver which was technically not possible in true pregnancy. L. monocytogenes injected into the uterine horns of mice made pseudopregnant by hormone treatment causes the formation of deciduomas (35). Such deciduomas are similar to the decidua basalis of true pregnancy in that maternal decidual cells are a major component (1, 2, 28, 29, 37, 38). Macrophage antilisteria re-
LuJ -I
-
HLY+
HLY-
Loglo bacterial titers of neonatal mice receiving an intraperitoneal injection of either HLY+ or HLY- Listeria cells on day 4 after birth. HLY+, 104 L. monocytogenes CNL 85/163 cells per neonate; HLY-, 107 L. monocytogenes CNL 85/162 cells per neonate. Three days after inoculation, the mice were sacrificed and bacterial growth in an organ block containing heart, lungs, liver, and spleen was determined. The values shown are the means and standard errors from four animals. FIG. 2.
5
4
3.1
2
NOTES
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INFECT. IMMUN.
SPLE EN
RT UTERUS
LFT UTERUS
FIG. 3. Loglo bacterial titers of pseudopregnant mice receiving HLY+ Listeria cells in the left uterine horn and HLY- Listeria cells in the right uterine horn. HLY+, 5 x 10' CNL 85/163 cells (solid bars); HLY-, 5 x 104 CNL 85/162 cells (open bars). HLY+ Listeria colonies were distinguished from HLY- colonies by the presence of hemolysis around the former on Trypticase soy agar-blood plates. Mice were sacrificed 3 days after receiving Listeria inoculation. The values shown are the means and standard errors from five mice.
sponses are inhibited in the deciduoma of pseudopregnancy in a manner exactly analogous to that of the decidua basalis of true pregnancy (35). Deciduomas were induced by methods previously published by our laboratory (35). Briefly, 1 week before inducing deciduoma, female B10.A x BALB/c mice 2 months old, were primed with CNL 85/163 (HLY+) by injection of 104 bacteria into the lateral tail veins. One week later, the animals were anesthetized with tribromoethanol (Avertin) and bilateral oophorectomies were performed. Six days after the oophorectomies, subcutaneous hormone injections were begun. Each animal received 0.1 mg of estrogen daily for 3 days, rested for 2 days without hormone injections, and then was injected with a mixture of estrogen (10 ng) and progesterone (1 mg) for daily 3 more days. Six hours following the third estrogen-progesterone injection, the animals were anesthetized and each uterine horn was injected with 100 ,ul of the appropriate Listeria strain. The mice continued to receive progesterone injections for the next 3 days. The morning following the last injection, the mice were sacrificed and the tissues were analyzed for Listeria infection as described above. For Fig. 3, the left uterine horns of pseudopregnant mice were injected with HLY+ Listeria cells and the right uterine horns of the same mice were injected with 100 times more HLY- Listeria cells. After 3 days, Listeria growth in the individual uterine horns and spleens was determined. HLY+ and HLY- Listeria cells were distinguished by the presence or absence of hemolysis on Trypticase soy agar-blood plates. Note that there was a spread of HLY+ Listeria cells from the initial site of injection in the left uterine horn to the spleen and right uterine horn. There was no detectable growth of HLY- Listeria cells. For Fig. 4, pseudopregnant mice were injected in both uterine horns with the same Listeria strain. One group of mice received HLY+ Listeria cells; the other received
A) HLY+ 7
B) HLY7
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FIG. 4. Loglo bacterial titers in pseudopregnant mice receiving either HLY+ or HLY- Listeria cells into their uterine horns. Each mouse received only one Listeria strain. (A) HLY+, 5 x 102 CNL 85/163 cells injected into each uterine horn; (B) HLY-, 5 x 104 CNL 85/162 cells. Mice were sacrificed 3 days after receiving the Listeria injection. The values shown are the means and standard errors from three mice.
VOL. 59, 1991
HLY- Listeria cells. As a precaution, the latter mice also received kanamycin sulfate to prevent growth of spontaneous revertants which had lost the transposon and its antibiotic resistance gene (3). In contrast to the Fig. 3 experiment, each individual mouse received only one Listeria strain. There were 100 to 1,000 times more Listeria cells in the spleens and uteri of mice receiving intrauterine HLY+ bacteria (Fig. 4A) than in those of mice receiving HLYbacteria (Fig. 4B), even though the latter mice received 100 times more Listeria cells. Thus, there was 1,000-fold growth of the HLY+ Listeria cells from the 1,000 bacteria initially injected into the uteri. We have previously performed detailed immunohistochemical studies of HLY+ Listeria growth in decidual tissues (32, 35). Listeria cells were found in both extra- and intracellular sites. At early stages of infection, inflammatory cells were not present and any intracellular bacteria were in decidual cells. In contrast, the number of HLY- Listeria cells in the uteri decreased to 1/100 of the initial 10,000 bacteria injected. Altogether, the data in Fig. 3 and 4 indicate that HLYListeria cells were not virulent even when injected directly into decidual tissue, a site at which macrophage functions are profoundly inhibited. In conclusion, macrophages play a major role in the host defense against Listeria infection. Recent data from our laboratory indicate that macrophage functions are inhibited in the murine neonate and placental region by two entirely different mechanisms. In the neonate, in contrast to the adult, concentrations of a-fetoprotein (8) and nonesterified docosahexaenoic acid (9) in serum are high. These molecules inhibit the ability of macrophages to be activated by gamma interferon (7, 11, 22) and thus inhibit an important step in the macrophage response against L. monocytogenes (17). Despite these inhibitors, we now find that listeriolysin is still required for virulence in neonatal infections. Listeriolysin is also required in the placental region, where macrophage functions are inhibited by a different mechanism. The decidua basalis is where the placenta is anchored in the maternal uterus. Infection at this site is a complex event because it involves not only the pathogen and the host defenses but also the maternal-fetal immunologic relationship. The latter may be considered an allograft-host relationship because of the paternal antigens of the fetus and placenta (see reviews in references 16 and 18). Macrophage functions are profoundly inhibited in the decidua basalis, perhaps to prevent maternal antifetal immunologic responses. We have previously shown that solid-phase molecules embedded in these decidual tissues inhibit macrophage functions (31, 32, 34, 35). Similar decidual tissue is present in the deciduoma of pseudopregnancy. We now report that despite the profoundly inhibited macrophage functions in the decidua and the neonate, listeriolysin is still required for virulence. One explanation of this is that listeriolysin is required for virulence because it allows the bacteria to survive within macrophages and other cells (4, 12, 14, 30). Since listeriolysin functions best at pH 5, which is found within phagolysosomes, but minimally at pH 7, which is found in the extracellular environment, it is unlikely to contribute to bacterial growth outside of cells. Listeriosis may be transmitted by contaminated food (25, 36). The development of regulatory guidelines to prevent such infections will require identification of virulence factors which differentiate pathogenic from nonpathogenic strains of this common bacterium. To our knowledge, this report is the first to demonstrate that production of listeriolysin is a virulence factor in infection of the most susceptible popula-
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tions, the neonate and decidual tissue of the fetoplacental unit. This work was supported by grants from the March of Dimes and NIH RO1-HD24797 and RO1 DK43634. Dianne B. McKay is supported by an institutional postdoctoral fellowship grant from the NIH (2T32-DK-07527). Christopher Y. Lu is the recipient of an NIH Research Career Development Award (K04-HD00862). We thank Miguel A. Vazquez for critically reviewing the manuscript. REFERENCES 1. Bell, S. C. 1983. Decidualization: regional differentiation and
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