CD46 in Meningococcal Disease Linda Johansson, et al. Science 301, 373 (2003); DOI: 10.1126/science.1086476

The following resources related to this article are available online at www.sciencemag.org (this information is current as of August 18, 2008 ):

This article cites 25 articles, 10 of which can be accessed for free: http://www.sciencemag.org/cgi/content/full/301/5631/373#otherarticles This article has been cited by 41 article(s) on the ISI Web of Science. This article has been cited by 17 articles hosted by HighWire Press; see: http://www.sciencemag.org/cgi/content/full/301/5631/373#otherarticles This article appears in the following subject collections: Medicine, Diseases http://www.sciencemag.org/cgi/collection/medicine Information about obtaining reprints of this article or about obtaining permission to reproduce this article in whole or in part can be found at: http://www.sciencemag.org/about/permissions.dtl

Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2003 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.

Downloaded from www.sciencemag.org on August 18, 2008

Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/cgi/content/full/301/5631/373

REPORTS

11. 12. 13. 14.

15. J. Grimsby et al., unpublished data. 16. T. Ferre, A. Pujol, E. Riu, F. Bosch, A. Valera, Proc. Natl. Acad. Sci. U.S.A. 93, 7225 (1996). 17. M. Shiota et al., Diabetes 50, 622 (2001). 18. U. J. Desai et al., Diabetes 50, 2287 (2001). 19. N. Morral et al., Hum. Gene Ther. 13, 1561 (2002). 20. F. M. Matschinsky, Diabetes 45, 223 (1996). 21. I. H. Segel, Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems (Wiley, New York, 1993), pp. 227–231. 22. I. R. Sweet, G. Li, H. Najafi, D. Berner, F. M. Matschinsky, Am. J. Physiol. 271, E606 (1996). 23. We thank L. Ping, S. Tannu, D. Lucas-McGady, and A.

CD46 in Meningococcal Disease Linda Johansson,1 Anne Rytko¨nen,1 Peter Bergman,2 Barbara Albiger,5 Helena Ka¨llstro¨m,3 Tomas Ho¨kfelt,4 Birgitta Agerberth,2 Roberto Cattaneo,6 Ann-Beth Jonsson1* The human-specific bacterial pathogen Neisseria meningitidis is a major cause of sepsis and/or meningitis. The pili of N. meningitidis interact with CD46, a human cell-surface protein involved in regulation of complement activation. Transgenic mice expressing human CD46 were susceptible to meningococcal disease, because bacteria crossed the blood-brain barrier in these mice. Development of disease was more efficient with piliated bacteria after intranasal, but not intraperitoneal, challenge of CD46 transgenic mice, suggesting that human CD46 facilitates pilusdependent interactions at the epithelial mucosa. Hence, the human CD46 transgenic mice model is a potentially useful tool for studying pathogenesis and for vaccine development against meningococcal disease. Understanding how bacteria cross the bloodbrain barrier is a key issue in the study of bacterial meningitis. Neisseria meningitidis

infection is characterized by bacterial dissemination from the nasopharyngeal mucosa to the blood and cerebrospinal fluid (CSF). How

Aglione for biological characterization; N. Doliba and M. Vatamaniuk for islet studies; and J. Sterner for editorial comments. Supporting Online Material www.sciencemag.org/cgi/content/full/301/5631/370/ DC1 Materials and Methods Figs. S1 to S6 Table S1 References 3 March 2003; accepted 13 June 2003

this bacterium crosses the epithelial barrier at the nasopharynx and the blood-brain barrier is poorly understood (1). Initial adherence to target epithelial cells is mediated by long filamentous surface pili that recognize the human cell-surface receptor CD46 (2). CD46 is a complement regulator and host-cell receptor for several pathogens, including measles virus, herpes virus 6, Streptococcus pyoMicrobiology and Tumor Biology Center, Nobels va¨g 16, Box 280, Karolinska Institutet, SE-171 77 Stockholm, Sweden. 2Department of Medical Biochemistry and Biophysics, 3Department of Cell and Molecular Biology, 4Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden. 5Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden. 6Molecular Medicine Program, Mayo Clinic, Guggenheim 1838, 200 First Street SW, Rochester, MN 55905, USA. 1

*To whom correspondence should be addressed. Email: [email protected]

Fig. 1. CD46 expression pattern in mouse tissue and detection of bacteria in the brain. (A) CD46 expression in tissue homogenates immunoblotted with a serum containing rabbit polyclonal antibodies to CD46 (21). The upper band represents the BC isoform and the lower band represents the C isoform. Molecular masses are shown on the right in kilodaltons. (B and C) CD46 was detected in meninges (arrowhead) and blood vessel walls. No staining was seen in the nontransgenic brain (asterisk). Bars indicate 100 ␮m (B) and 20 ␮m (C). (D) CD46 (green) and the astrocyte-marker glial fibrillary acidic protein (GFAP) (red) show complementary distribution occasionally in close contact (arrowhead). Bar, 20 ␮m. (E) Strong CD46 expression in the choroid plexus of the lateral ventricle. Bar, 100 ␮m. (F and G) N. meningitidis was identified in the choroid plexus on day 1 postintraperitoneal challenge. (G) shows higher magnification of the box in (F). Bars, 20 ␮m (F) and 2.5 ␮m (G). (H to J) Colocalization of N. meningitidis (red) and CD46 (green) in a blood vessel wall. ( J) shows the merged image. Bar, 10 ␮m. (K and L) CD46 detected in trachea of CD46 transgenic mouse (K) and not detected in nontransgenic mouse (L). CD46 is mainly localized along the basolateral side of the epithelial cells (arrowheads). Asterisks indicate unspecific fluorescence in subepithelial tissue. Arrows indicate apical and basal border of the epithelial layer. Bars, 20 ␮m. Mice tissues were processed as described in (22–24). CD46 monoclonal antibody (1:10,000, NeoMarkers, Freemont, CA), goat antibody to GFAP (1:100, Santa Cruz, Santa Cruz, CA), and N. meningitidis antiserum (1: 200, U.S. Biological, Swampscott, MA) were detected with appropriate secondary antibodies. The sections were analyzed by confocal microscopy as previously described in (22).

www.sciencemag.org SCIENCE VOL 301 18 JULY 2003

Downloaded from www.sciencemag.org on August 18, 2008

5. 6. 7. 8. 9. 10.

H. H. Kazazian et al., Eds. ( Wiley, New York, 2002), pp. 1437–1442. G. Velho et al., J. Clin. Invest. 98, 1755 (1996). K. Clement et al., Diabetologia 39, 82 (1996). E. A. Davis et al., Diabetologia 42, 1175 (1999). Y. Liang et al., Biochem. J. 309, 167 (1995). J. Takeda et al., J. Biol. Chem. 268, 15200 (1993). M. Gidh-Jain et al., Proc. Natl. Acad. Sci. U.S.A. 90, 1932 (1993). S. P. Miller et al., Diabetes 48, 1645 (1999). B. Glaser et al., N. Engl. J. Med. 338, 226 (1998). H. B. Christesen et al., Diabetes 51, 1240 (2002). See supporting data on Science Online.

373

REPORTS

374

plexus (Fig. 1, F and G), and in the microvascular endothelia of CD46 transgenic mice (Fig. 1, H to J). An isogenic pair of meningococcal JB515 strains expressing or not expressing pili (18) were used to evaluate the role of pili in infection of CD46 transgenic mice by the intranasal route. If the mice were

pretreated with antibiotics to diminish the normal bacterial flora, intranasal challenge of CD46 transgenic mice with 109 piliated N. meningitidis caused 15% mortality (P ⬍ 0.05), whereas nonpiliated N. meningitidis caused no signs of disease (Fig. 3, A and B). Intranasal challenge did not affect mice that were not pretreated with antibiotics.

Fig. 2. Neisserial crossing of the blood-brain barrier requires expression of human CD46. (A to D) Intraperitoneal challenge of CD46 transgenic mice and nontransgenic C57BL/6 mice with piliated N. meningitidis JB515 (serogroup W-135) or FAM20 (serogroup C) demonstrated dose-dependent responses for both strains. As expected, the two strains required different challenge doses. Bacteria were suspended in G C base (GCB) (Gifco, Beckton Dickinson, Sparks, Maryland) liquid and injected intraperitoneally in 5- to 7-week-old animals. Bacterial doses were verified by viable count. Eight CD46 transgenic mice and eight nontransgenic mice were challenged. Black, dashed, and gray lines represent challenge doses of 4⫻108, 2⫻108, or 108, respectively [in (B)], and 108, 107, or 106, respectively [in (D)]. Nontransgenic mice survived all tested doses [(A) and (C)]. (E) The cisterna magna was punctured on day 1 or day 2 after intraperitoneal challenge with 108 N. meningitidis. CSF was checked for absence of red blood cells and spread on GCB plates for viable count. Bacteria were not detected in CSF of nontransgenic mice. Shown is the average CSF viable counts of five mice. CFU, colony-forming units.

Downloaded from www.sciencemag.org on August 18, 2008

genes, as well as pathogenic Neisseria spp. (3–7 ). In humans, CD46 is expressed as four major isoforms—BC1, BC2, C1, and C2— depending on the alternative splicing of a region encoding an extracellular domain and the choice between one of two cytoplasmic tails, Cyt-1 and Cyt-2 (8, 9). Receptor-binding activates host-cell signal transduction, leading to intimate contact between bacterial membrane components and host cell-surface receptors (10 –12). Bacterial uptake and invasion of the host cells may follow (13). A complete understanding of meningococcal disease requires an animal model that resembles the infection process in the human host. To mimic the human host, we used CD46 transgenic mice that express human CD46 with human-like tissue specificity (14). The CD46 expression pattern in tissue extracts from CD46 transgenic mice and nontransgenic C57BL/6 mice were similar to those of prior analyses (14, 15) (Fig. 1A). Immunohistochemical analysis detected CD46 at the blood-brain barrier level, that is, on epithelial cells of the choroid plexus, in the meninges ( pia mater), and in microvascular endothelia (Fig. 1, B to E). In the airways, CD46 expression was observed mainly along the basolateral surface of the epithelial cells of the trachea (Fig. 1, K and L). The CD46 transgenic mice were intraperitoneally challenged with wild-type N. meningitidis (serogroups W-135 and C). Mortality rates of CD46 transgenic mice were strain- and dose-dependent, whereas nontransgenic mice survived bacterial injection (Fig. 2, A to D). After intraperitoneal challenge, both CD46 transgenic and nontransgenic mice developed bacteremia ( Table 1), but lethal disease occurred only in CD46 transgenic mice. The bacterial counts were often lower in the nontransgenic mice than in CD46 transgenic mice, but the ranges overlapped. CD46 transgenic mice with 5 ⫻ 104 bacteria/ml in blood developed lethal disease, whereas nontransgenic mice with 1.5 ⫻ 106 bacteria/ml survived (16 ). CD46 transgenic mice challenged with Escherichia coli, N. lactamica, N. meningitidis preheated to 95°C, or a lipopolysaccharide (LPS)-deficient N. meningitidis mutant (17 ) were not visibly affected and did not develop bacteremia (Table 1). The cisterna magna was punctured at day 1 and day 2 postchallenge to check whether bacteria reached the CSF. High bacterial counts were found in the CSF of CD46 transgenic mice (Fig. 2E) but were not seen in the CSF of nontransgenic mice, although this group had high bacterial blood counts. Immunostaining of cryosections of tissues from infected mice revealed bacteria in the meninges, in the choroid

Table 1. Challenge of mice with bacterial strains and mutants. Blood samples were collected from the tail and spread on GCB plates for viable count. Bacteria were verified by gram-staining, microscopy, and an oxidase test. CFU from surviving animals are given as means ⫾SD. Bacteria/ml in blood was tested at day 1 postchallenge. Dashed entries indicate no detectable levels of bacteria in blood. wt, wild type.

Bacterial strain N. meningitidis N. meningitidis N. meningitidis N. meningitidis N. lactamica E. coli BL21

Bacteria/ml in blood CD46

C57BL/6

JB515 (wt) 1 ⫻ 105 ⫾ 1 ⫻ 105 1 ⫻ 102 ⫾ 3 ⫻ 102 FAM20 (wt) 3 ⫻ 106 ⫾ 4 ⫻ 106 3 ⫻ 105 ⫾ 8 ⫻ 105 FAM20 heated 95°C – – FAM20 lpxA mutant – – – – – –

18 JULY 2003 VOL 301 SCIENCE www.sciencemag.org

Surviving mice CD46 C57BL/6 0% 0% 100% 100% 100% 100%

100% 100% 100% 100% 100% 100%

Ref no. (18) (18, 25) (18, 25) (17) (26) (27)

REPORTS the CD46 transgenic mice were more sensitive to intraperitoneal infection with nonpiliated bacteria, suggesting that an additional, as-yet-unknown bacterial factor (or factors) must access and exploit the CD46 receptor during the intraperitoneal route of infection. Interaction with human CD46 is a critical step for the onset of bacterial meningitis. By promoting passage of the bloodbrain barrier and translocation from blood to CSF, CD46 mediates access to the meninges. CD46 is an enriched protein at the blood-brain barrier (19); hence, its importance supports the role of CD46 in meningococcal meningitis. CD46 regulates T cell–mediated inflammatory responses and the contact hypersensitivity reaction, with different effects depending on the nature of its cytoplasmic tail: Cyt-1 engagement suppresses inflammation, whereas Cyt-2 in-

Fig. 3. Intranasal but not intraperitoneal challenge of CD46 transgenic mice requires piliated bacteria for development of disease. (A and B) Mice were treated with 400 ␮g/ml of trimethoprim in drinking water and injected twice a day intraperitoneally with 600 ␮g of streptomycin sulfate and 600 ␮g of vancomycin for 2 days. Antibiotics were not given 1 day before intranasal challenge with 40 ␮l containing 109 piliated JB515 (serogroup W135) (P⫹) or its isogenic nonpiliated mutant JB515.P (with an insertionally inactivated pilE gene) (P–). Challenge with piliated bacteria was lethal in 4 of 27 (15%) of CD46 transgenic mice, whereas all of 27 nontransgenic mice challenged survived (P ⬍ 0.05, Fischer’s exact test). Intranasal challenge with nonpiliated bacteria left all 27 mice unaffected. Only mice with lethal disease developed bacteremia. (C to F) Fifteen CD46 transgenic mice and 15 nontransgenic mice were challenged intraperitoneally with 2 ⫻ 108 piliated JB515 or nonpiliated JB515.P. Blood samples were collected from the tail and spread on GCB plates for viable count. Black and dashed lines represent mice infected with P⫹ or P⫺ bacteria, respectively. Mean CFU from surviving animals are shown. The difference in mortality between piliated and nonpiliated bacteria was significant at days 2, 3, and 4 postchallenge (P ⬍ 0.05, Fischer’s exact test). The JB515 isogenic strains used carried only one pilC gene. The JB515 isogenic pair, as well as several JB515 clones reisolated at day 1 from intraperitoneally challenged mice, expressed identical Opa PilC, and LPS repertoires (16), as determined by either immunoblotting (Opa and PilC) or tris-Tricine gel electrophoresis of purified LPS.

creases it (20). Because Cyt-2 is preferentially expressed in brain tissue and is tyrosine-phosphorylated by Neisseria (12), it is possible that Cyt-2 abundance in the brain facilitates bacterial meningitis, enhances inflammatory responses, and explains the lethal effect of neisserial infection in CD46 transgenic mice. This transgenic mouse model reproduces many of the manifestations of meningococcal infection that could prove useful for more rapid testing and evaluation of new potential vaccine candidates against group B meningococci, as well as elucidating other molecular aspects of neisserial pathogenesis and offering a model for other forms of meningitis. References and Notes

1. X. Nassif, C. Pujol, P. Morand, E. Euge `ne, Mol. Microbiol. 32, 1124 (1999). 2. H. Ka¨llstro¨m, M. K. Liszewski, J. P. Atkinson, A.-B. Jonsson, Mol. Microbiol. 25, 639 (1997). 3. M. Manchester, M. K. Liszewski, J. P. Atkinson, M. B. Oldstone, Proc. Natl. Acad. Sci. U.S.A. 91, 2161 (1994). 4. R. E. Do¨rig, A. Marcil, A. Chopra, C. D. Richardson, Cell 75, 295 (1993). 5. N. Okada, M. K. Liszewski, J. P. Atkinson, M. Caparon, Proc. Natl. Acad. Sci. U.S.A. 92, 2489 (1995). 6. D. Naniche et al., J. Virol. 67, 6025 (1993). 7. F. Santoro et al., Cell 99, 817 (1999). 8. T. W. Post et al., J. Exp. Med. 174, 93 (1991). 9. D. F. Purcell et al., Immunogenetics 33, 335 (1991). 10. H. Ka¨llstro¨m, M. S. Islam, P.-O. Berggren, A.-B. Jonsson, J. Biol. Chem. 273, 21777 (1998). 11. B. P. Ayala et al., Cell. Microbiol. 3, 265 (2001). 12. S. W. Lee et al., J. Cell. Biol. 156, 951 (2002). 13. A. J. Merz, M. So, Annu. Rev. Cell. Dev. Biol. 16, 423 (2000). 14. B. Mrkic et al., J. Virol. 72, 7420 (1998). 15. C. Kemper et al., Clin. Exp. Immunol. 124, 180 (2001). 16. L. Johansson et al., unpublished data. 17. B. Albiger, L. Johansson, A.-B. Jonsson. Infect. Immun. 71, 155 (2003). 18. M. Rahman, H. Ka¨llstro¨m, S. Normark, A.-B. Jonsson, Mol. Microbiol. 25, 11 (1997). 19. E. V. Shusta, C. Zhu, R. J. Boado, W. M. Pardridge, J. Neuropathol. Exp. Neurol. 61, 597 (2002). 20. J. C. Marie et al., Nature Immunol. 3, 659 (2002). 21. M. K. Liszewski, J. P. Atkinson, J. Immunol. 156, 4415 (1996). 22. P. R. Brumovsky et al., Exp. Neurol. 174, 1 (2002). 23. A. H. Coons, in General Cytochemical Methods, J. F. Danielli, Ed. (Academic Press, New York, 1958), pp. 399 – 422. 24. J. C. Adams, J. Histochem. Cytochem. 40, 1457 (1992). 25. D. W. Dyer, W. McKenna, J. P. Woods, P. F. Sparling, Microb. Pathog. 3, 351 (1987). 26. A.-B. Jonsson, G. Nyberg, S. Normark, EMBO J. 10, 477 (1991). 27. F. W. Studier, B. A. Moffatt, J. Mol. Biol. 189, 113 (1986). 28. We thank M. Hagelin for excellent technical assistance. This work was supported by grants from the Swedish Research Council (Dnr 10846, Dnr 1121708A, and Dnr 04X-2887), Swedish Cancer Society, Magnus Bergvalls Stiftelse, Strategic Foundation (I & V program), Aventis, Karolinska Institutet, the Marianne and Marcus Wallenberg Foundation, the Knut and Alice Wallenberg Foundation, and BristolMyers Squibb (unrestricted neuroscience grant). B. Albiger was supported by a postdoctoral fellowship from Stiftelsen Wenner Grenska Samfundet.

Downloaded from www.sciencemag.org on August 18, 2008

Neisseria were only detected in blood of CD46 transgenic mice that developed lethal disease and not in nontransgenic mice (16 ). On the contrary, intraperitoneal challenge with piliated or nonpiliated N. meningitidis revealed that both variants cause bacteremia and mortality in transgenic mice but not in control mice (Fig. 3, C to F). These data demonstrate the importance of the normal bacterial flora at the nasopharynx of CD46 transgenic mice in reducing the chances of meningococcal disease, because mice not pretreated with antibiotics that were challenged intranasally did not develop bacteremia or disease symptoms. A possible explanation for the relatively low mortality rate (15%) could be the largely basolateral CD46 expression in epithelial cells of the throat. Only limited CD46 expression was detected on the apical surface of these cells (Fig. 1, K and L). Notably,

6 May 2003; accepted 11 June 2003

www.sciencemag.org SCIENCE VOL 301 18 JULY 2003

375