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Infection and Immunity, October 2006, p. 5506-5512, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00655-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Differential Role of Lipooligosaccharide of Neisseria meningitidis in Virulence and Inflammatory Response during Respiratory Infection in Mice

Maria Leticia Zarantonelli,1* Michel Huerre,2 Muhamed-Kheir Taha,1 and Jean-Michel Alonso1

Unité des Neisseria,1 Unité de Recherche et d'Expertise Histotechnologie et Pathologie, Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris Cedex 15, France2

Received 24 April 2006/ Returned for modification 7 June 2006/ Accepted 7 July 2006


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ABSTRACT
 
Meningococcal lipooligosaccharide (LOS) induces a strong proinflammatory response in humans during meningococcal infection. We analyzed the role of LOS in the inflammatory response and virulence during the early infectious process in a mouse model of meningococcal respiratory challenge. An lpxA mutant strain (serogroup B) devoid of LOS (strain Z0204) could not persist in the lungs and did not invade the blood. The persistence in the lungs and invasion of the bloodstream by a rfaD mutant expressing truncated LOS with only lipid A and 3-deoxy-D-manno-2-octulosonic acid molecules (strain Z0401) was intermediate between those of the wild-type and Z0204 strains. Both LOS mutants induced acute pneumonia with the presence of infiltrating polymorphonuclear leukocytes in lungs. Although tumor necrosis factor alpha production was reduced in mice infected with the mutant of devoid LOS, both LOS mutants induced production of other proinflammatory cytokines, such as interleukin-1ß (IL-1ß), IL-6, and the murine IL-8 homolog KC. Together, these results suggest that meningococcal LOS plays a role during the early infectious and invasive process, and they further confirm that other, nonlipopolysaccharide components of Neisseria meningitidis may significantly contribute to the inflammatory reaction of the host.


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INTRODUCTION
 
Neisseria meningitidis is a gram-negative bacterium that lives in the human nasopharynx, and it is commonly found among the commensal flora of about 10% of asymptomatic carriers. However, this bacterium can provoke severe systemic infections such as septicemia (with or without shock) and meningitis. Endotoxin is a major structural component of the outer membranes of gram-negative bacteria. Endotoxin, and particularly lipid A, potently induces the inflammatory response during meningococcal infection. The severity of meningococcal disease is thought to be linked to the degree of the inflammatory response induced during invasive infection (14, 25). Lipid A anchors the inner core, comprising two 3-deoxy-D-manno-2-octulosonic acid (KDO) and heptose (Hep) residues of meningococcal lipooligosaccharide (LOS) in the outer membrane (9). Meningococcal lipid A has a symmetrical structure. The lpxA gene adds the O-linked 3-OH (C12-3-OH) fatty acyl chains to positions 3 and 3' of the glucosamine disaccharide (19). Another gene, lpxD, adds the N-linked 3-OH (C14-3-OH) to positions 2 and 2'. Two others genes, lpxL1 and lpxL2, are involved in lipid A acyloxyacylation and may add a C12 chain to the N-linked C14-3-OH (24). The rfaD gene encodes the ADP-L-glycero-D-mannoheptose-6-epimerase, which is responsible for the biosynthesis of the lipooligosaccharide precursor ADP-L-glycero-D-mannoheptose. The rfaD mutant produces LOS with only lipid A and the KDO molecules (27). Meningococcal lpxA mutants are devoid of LOS but are viable (18, 27) despite the (KDO)2-lipid A structure being required for cell viability in other bacterial species (5). However, lpxA mutants seem to be heterodiploid for the lpxA gene, as they harbor both the wild-type and the insertionally inactivated lpxA alleles. An LOS– phenotype can result from the negative transdominance of the inactivated lpxA allele (27).

N. meningitidis LOS seems to induce a proinflammatory cytokine response either independently (17) or through the CD14/Toll-like receptor 4 (TLR4) pathway (28). This CD14/TLR4 activation requires the KDO moiety. Meningococcal lipid A expressed by KDO-deficient meningococci is much less biologically active than (KDO)2-containing meningococcal LOS (28). The impact on inflammatory responses of different meningococcal LOSs has been analyzed under ex vivo conditions using cell lines or peripheral blood mononuclear cells (3, 23). It was shown that lpxA mutants may be less toxic due to a reduced capacity to induce tumor necrosis factor alpha (TNF-{alpha}), although they were still able to induce a significant inflammatory response (23).

However, there has yet to be a global analysis of the role of LOS in the inflammatory response during infection. In particular, the induction of the local response in the respiratory tract, the route of entry of N. meningitidis, has not been addressed. This study aimed to analyze the impact of the absence of LOS on the inflammatory response and on virulence during the early infectious process, using a mouse model of meningococcal respiratory challenge (1).


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MATERIALS AND METHODS
 
Strains of N. meningitidis used and culture conditions. N. meningitidis LNP14912 is a serogroup B clinical isolate with a B:15:P1-7 phenotype and an L3,7,9 immunotype. It belongs to the ST32 (ET-5) clonal complex. Strain Z0204 is a viable isogenic mutant that is completely devoid of LOS, constructed by insertional inactivation of the lpxA gene. This mutant is heterodiploid for lpxA/lpxA::aph-3'. The absence of LOS was confirmed as previously described (27). Strain Z0401 is an isogenic mutant with a truncated LOS, constructed by transformation of the LNP14912 parent strain with the recombinant plasmid pLZ2 harboring the inactivated rfaD gene by insertion of the aph-3' cassette (27). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining were used to confirm the production of a truncated LOS by this rfaD mutant, as previously described (27). All N. meningitidis strains were grown on gonococcal broth containing Kellogg's supplement (10), and 100 µg/ml of kanamycin was added when needed. Plates were incubated at 37°C under a 5% CO2 atmosphere.

Electron microscopy. Bacteria were fixed with 2.5% glutaraldehyde (Sigma) in 0.1 M cacodylate buffer (pH 7.2), postfixed with a mixture of 1% osmium tetroxide, and then incubated at room temperature for 1 h with 2% uranyl acetate in Michaelis buffer (pH 6.0). Samples were dehydrated using an increasing series of ethanol concentrations and then embedded in epoxy resin. Ultrathin sections (70 to 80 nm) were cut with a diamond knife in a Leica Ultracut S microtome. Sections were placed on 200-mesh copper grids, stained with 2% uranyl acetate and lead citrate, and then examined using a JEOL 1200 transmission electron microscope operating at 80 kV with a charge-coupled device Megaview camera.

Gas chromatography analysis of cellular fatty acid composition. A bacterial suspension containing 2 x 1010 CFU was prepared from each strain, as described previously (27). Cells were lysed and saponified by incubation with a sodium hydroxide-methanol solution at 100°C for 30 min. The tubes containing the lysate were cooled, and the fatty acids were methylated by heating with a H2SO4-methanol solution at 70°C for 1 h. The tubes were rapidly cooled in an ice bath, and methylated compounds were extracted with chloroform. The aqueous phase was removed, and the fatty acid methyl esters in the organic phase were separated on a fused-silica capillary column (25 m by 0.25 mm [inner diameter]) coated with (methyl 5%) phenyl silicone (df = 0.25 µm [Quadrex]) and installed in a Shimadzu GC-14B apparatus. The compounds were identified by comparing retention times with those of standards processed in the same way.

Animal experiments for the evaluation of virulence in strain LNP14912 and its isogenic LOS mutants. The virulences of strain LNP14912 and its isogenic LOS mutants (Z0204 and Z0401) were compared in the model of dual influenza A virus (IAV)-N. meningitidis infection in BALB/c mice (1). The experimental design was approved by the Institut Pasteur Review Board. Five-week-old female mice (Janvier, France) were anesthetized with tribromoethanol (Sigma-Aldrich) at 250 mg/kg, and mild IAV infection was induced by intranasal administration of 250 PFU. The mice were then superinfected intranasally 7 days later with standardized bacterial suspensions of 5 x 107 CFU of each bacterial strain. CFU in samples of blood and lung homogenates were counted 3 h and 24 h after challenge. We tested three mice at each time point, and the experiments were repeated twice. CFU from mutant Z0204 were counted in the presence and absence of kanamycin.

Histological studies. Three hours after N. meningitidis challenge, the mice were killed by intraperitoneal injection of 300 mg/kg sodium pentobarbital. Blood was removed by retro-orbital puncture. The lungs were removed and immersed in 10% formaldehyde for 8 days. These were then further processed and embedded in paraffin. Lung sections were stained with hematoxylin-eosin and Giemsa stain. The presence of N. meningitidis in the lungs was detected by incubation with a specific anti-serogroup B antibody. Briefly, deparaffinized lung sections were immersed in citrate buffer and treated with proteinase K. Endogenous peroxidase activity was blocked with H2O2 and a rabbit anti-N. meningitidis serogroup B antibody (specific for the capsular polysaccharide antigen) was added at a dilution of 1:50. The tissue sections were incubated for 1 h at room temperature and washed with phosphate-buffered saline. A biotinylated goat anti-rabbit immunoglobulin G antibody (Dako EO432), diluted 1:500, was added and the section incubated for 45 min. Sections were then treated with horseradish peroxidase-conjugated streptavidin (Dako PO397), and the reaction products were revealed by aminoethylcarbazole/H2O2. The preparations were then counterstained in hematoxylin.

Cytokine and chemokine profile. Lung homogenates were prepared in 1 ml of RPMI 1640 medium (Gibco) either 7 days after IAV infection (control mice) or 3 h after N. meningitidis superinfection and centrifuged at 1,800 x g. The supernatants were collected and stored at –80°C. Cytokines (TNF-{alpha}, interleukin-ß [IL-1ß], and IL-6) and chemokines (murine IL-8 homolog [KC], macrophage inflammatory protein-1{alpha} [MIP-1{alpha}], JE/monocyte chemoattractant protein-1 [MCP-1], and RANTES) were quantified by enzyme-linked immunosorbent assay (Quantikine; R&D Systems Europe, Abingdon, Oxon, United Kingdom). We used a two-tailed Student t test for statistical analysis, with a P value of ≤0.05 being considered statistically significant.


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RESULTS
 
Cell morphology of LOS mutants. We compared the morphologies of the two LOS mutants, Z0204 (lpxA/lpxA::aph-3' heterodiploid, completely devoid of LOS) and Z0401 (rfaD::aph-3', expressing truncated LOS), by transmission electron microscopy to evaluate the influence of the rfaD and lpxA mutations on the cell structure of N. meningitidis. The rfaD mutant exhibited a morphology similar to that of the wild-type strain, with a typical diplococcal shape and a normal septation. By contrast, the lpxA mutant devoid of LOS showed segments of the outer membrane detached from the bacterial bodies, with large and empty detached fragments being observed. In several cases, two separated bacteria remained linked, sharing a detached membrane. However, it was possible to distinguish the inner and outer membranes (Fig. 1A).


Figure 1
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  		FIG. 1. (A) Electron microscopy images of the wild-type LNP14912 strain and both isogenic LOS mutant strains, Z0401 (rfaD::aph-3', expressing truncated LOS) and Z0204 (lpxA/lpxA::aph3' heterodiploid, completely devoid of LOS). (B and D) Bacterial counts in the lungs and blood of IAV-infected mice challenged intranasally with the wild-type strain and the isogenic LOS mutants Z0401, Z0204, and Z0204 revertant. Data are means ± standard deviations for groups of three mice per time point in two independent experiments. (C) Gas chromatograms of cellular fatty acid methyl esters from the LNP14912, Z0204, and Z0204 revertant strains. Relevant peaks are given in each chromatogram. The arrow shows the absence of the methyl ester derived from 3-OH-C12:0, which is a specific fatty acid from lipid A. C12:0, dodecanoate; 3-OH-C12:0, 3-hydroxydodecanoate; C14:0, tetradecanoate, 3-OH-C14:0, 3-hydroxytetradecanoate; C16:1, hexadecenoate; C16:0, hexadecanoate.

Effect of LOS alteration on meningococcal virulence. We investigated the role of LOS mutations in meningococcal virulence by comparing the kinetics of in vivo growth/survival of the wild-type strain LNP14912 with those of both LOS mutants in the mouse model of dual IAV-N. meningitidis infection (1). The bacterial loads in the lungs and blood were scored 3 h and 24 h after intranasal bacterial challenge with 5 x 107 CFU. As Z0204 is a heterodiploid lpxA/lpxA::aph3' mutant, CFU were counted on GCB medium supplemented or not with 100 µg/ml of kanamycin. We observed a considerable decrease in CFU counts, of at least three orders of magnitude, in the lungs at 3 h for the mutant devoid of LOS. At 24 h, this mutant was completely cleared from the lungs (Fig. 1B). CFU counts in lungs infected with strain Z0401 (expressing truncated LOS) were found to be intermediate between those in lungs infected with the wild-type and Z0204 strains. We detected no bacteremia for either LOS mutant (Fig. 1B). Viable bacteria (2 x 105 CFU) from the lungs of one mouse infected with mutant Z0204 (heterodiploid lpxA/lpxA::aph-3') were recovered only on a GCB plate, whereas no colonies grew in kanamycin-supplemented GCB plates. When passaged on kanamycin-supplemented GCB plates, these colonies were sensitive to kanamycin, suggesting that the bacteria had lost the aph-3' gene. Such bacteria may correspond to lpxA::lpxA revertants. PCR analysis showed these bacteria to be lpxA::lpxA revertants (named Z0204 revertant). We detected no PCR product when DNA from this revertant was amplified using oligonucleotides binding to the aph-3' gene (data not shown). We analyzed the cellular fatty acid composition of this revertant strain by gas chromatography. Hydroxylated fatty acids 3-OH-C12:0 and 3-OH-C14:0 derived exclusively from lipid A were observed (Fig. 1C) indicating that the Z0204 revertant strain had recovered the LOS+ phenotype. We analyzed the virulence of this revertant by intranasal challenge of IAV-infected mice and found that the Z0204 revertant had recovered the virulent phenotype (Fig. 1D). It showed meningococcal infection kinetics in lungs and blood similar to those of the parent LNP14912 strain, suggesting a recovery of virulence after loss of the inactivated lpxA allele.

Histological characterization of acute meningococcal infection in mice. The alveolar architecture of lungs from IAV-infected mice at day 7 of viral infection (prior to the meningococcal challenge) was normal. No recruitment of inflammatory cells, especially macrophages and lymphocytes, was observed within the alveoli, indicating that the mice had recovered from viral pneumonia (Fig. 2A). The microscopic examinations of lung sections 3 h after bacterial challenge with strain LNP14912 revealed an intense inflammation of the alveolar septa (Fig. 2B), including focal recruitment of polymorphonuclear cells within the alveoli. We also observed a similar and intense inflammatory reaction when mice were infected with LOS mutant strains. Both isogenic LOS mutants were able to induce acute pneumonia with the presence of infiltrating polymorphonuclear leukocytes (Fig. 2, compare panels C and D with panel B). Moreover, immunohistochemistry using a rabbit polyclonal serum directed against the serogroup B meningococcal capsule revealed bacteria in the lungs.


Figure 2
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  		FIG. 2. Histological examination of lung sections 3 h after bacterial challenge. Compared with controls (A), IAV-infected mice superinfected with strain LNP14912 (B) showed an intense inflammation of the alveolar septa. A similar and intense inflammatory reaction was also observed when mice were infected with isogenic LOS mutants expressing a truncated LOS (Z0401) (C) or devoid of LOS (Z0204) (D).

Cytokine and chemokine production in the lungs during acute experimental meningococcal infection. We have previously reported that meningococcal IAV-N. meningitidis-infected mice showed a local cytokine response in the lungs. Therefore, we quantitatively studied the local production of the proinflammatory cytokines TNF-{alpha}, IL-1ß, and IL-6 in lung homogenates 3 h after bacterial challenge to asses the role of meningococcal LOS in inducing the inflammatory response during acute experimental infection. As a control, we evaluated cytokine production in the lungs of mice 7 days after IAV infection. After infection, all three strains, including the mutant devoid of LOS, increased the production of IL-1ß and IL-6 versus that by control mice. Moreover, the levels of induction of these two cytokines by the two mutants were not significantly different from that by the wild-type strain (P = 0.164 and P = 0.140 for IL-1ß and IL-6, respectively, for the mutant Z0204) (Fig. 3). As expected, the mutant devoid of LOS (Z0204) induced a lower production of TNF-{alpha} than the wild-type strain (P = 0.023). The TNF-{alpha} levels induced by the mutant devoid of LOS were similar to those observed for the control mice. The deep-rough mutant (Z0401) induced lower production of TNF-{alpha} than the wild-type strain, although this difference was not significant (P = 0.073) (Fig. 3).


Figure 3
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  		FIG. 3. Lung cytokine levels in mice after challenges with the meningococcal wild-type strain and the LOS mutants. Cytokine and chemokine levels were measured 3 h after intranasal bacterial infection. IAV-infected mice on day 7 after viral infection were used as controls. Significant decreases in cytokine and/or chemokine production induced by the two mutants versus the wild-type strain are indicated with an asterisk (calculated using a two-tailed Student t test [P < 0.05]). Error bars indicate standard deviations.

Chemokines play a central role in recruiting and regulating the leukocyte traffic during the acute inflammatory response. Studies of patients with meningococcal disease have shown that plasma levels of MCP-1, MIP-1{alpha}, and IL-8 directly correlate with lipopolysaccharide (LPS) levels, whereas levels of RANTES are negatively correlated (11). Therefore, we quantified in vivo the levels of KC (the functional murine homologous of human IL-8), MIP-1{alpha}, RANTES, and MCP-1 production in the lungs 3 h after infection of mice with the wild-type strain and the LOS mutants to determine the importance of meningococcal LOS in inducing chemokines. All three strains induced production of significantly higher levels of all the tested chemokines in the lungs of infected mice than in the lungs of control mice (IAV infected). However, the levels of MIP-1{alpha}, RANTES, and MCP-1 induced by the wild-type strain and the rfaD mutant (Z0401) were significantly higher than those induced by the mutant devoid of LOS (Z0204) and by control mice (P < 0.05 in all cases). We observed no significant differences between the production of KC in lungs of mice infected with the wild-type or the LOS mutant strains.

We observed a similar pattern of cytokine and chemokine production when experiments were carried out with the same standardized inoculum of heat-inactivated bacteria instead (data not shown).


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DISCUSSION
 
LOS is a major virulence factor during meningococcal sepsis and meningitis. The severity of meningococcal disease is directly correlated with the levels of endotoxin in the plasma and cerebrospinal fluid (2). N. meningitidis is the first viable gram-negative bacterium harboring an inactivated lpxA gene, and thus it completely lacks LOS. We infected mice with a serogroup B N. meningitidis mutant devoid of LOS. Our dual IAV-meningococcal mouse model mimics the different steps of infectious meningococcal disease, with a local infection of the respiratory tract that progresses to meningococcemia (1). We have shown that the mutant devoid of LOS (Z0204) and the deep-rough LOS mutant (Z0401) were affected mostly in their ability to invade and persist in the bloodstream. In particular, the Z0204 mutant was very affected in its capacity to persist in the lungs and was unable to invade the blood. This shows that meningococcal LOS plays a major role during the early infectious and invasive process. The presence of the capsule in N. meningitidis is the major element in mediating serum resistance (26). However, LOS sialylation may be important when capsule is downregulated. This occurs during meningococcal infection and the crossing of the epithelium (4, 7). Our results suggest that the lack of LOS renders N. meningitidis more susceptible to in vivo complement-mediated bacteriolysis and opsonophagocytosis, both of which are major mechanisms of the early innate defense against meningococcal infections. This was further confirmed by the observation of the Z0204 revertant strain, which recovered its ability to synthesize LOS and recovered the same virulent phenotype as the parent LNP14912 strain. All these results are consistent with the expression of a complete LOS molecule being crucial for meningococcal survival in situations in which the principal mechanism allowing meningococci to escape the innate immune response, the neisserial capsule, is not present. It is possible that this takes place during meningococcal infection and the crossing of the epithelium (4, 7). Alternatively, the lack of LOS may have an indirect effect on meningococcal virulence through the alteration of bacterial integrity. Indeed, electron microscopy examination of the lpxA mutant showed alteration in the outer membrane (Fig. 1A). In summary, all these data show that the lack of LOS will affect multiple aspects of the bacterial behavior and host-pathogen interaction.

Electron microscopy examination (Fig. 1A) and data obtained by whole-cell ELISA (using monoclonal antibodies against two major outer membrane proteins, PorA and PorB) and by gas chromatography analysis of cellular fatty acid composition (data not shown) showed that the rfaD mutant has no detectable alteration in the integrity of the outer membrane. However, this mutant showed reduced virulence and cytokine production. These observations are in accord with a direct role of LOS in meningococcal virulence. Plant et al. recently showed that the meningococcal gmhB mutant, which expresses a truncated LOS molecule similar to that of the rfaD mutant, was avirulent in mice (12).

In the model of dual IAV-N. meningitidis infection of adult mice, N. meningitidis colonizes the lungs and induces inflammatory pneumonia followed by bacteremia. During the virus-induced susceptibility to meningococcal superinfection, a normal polymorphonuclear cell response to bacterial infection is observed, with an intense influx of polymorphonuclear leukocytes and monocytes (1). However, the local inflammatory response in the lungs seems to be LOS independent. Histological examination revealed no differences in lungs from mice infected with a meningococcal strain completely devoid of LOS and the wild-type strain. Both isogenic LOS mutants Z0204 and Z0401 induced an acute alveolitis, with equivalent intense influxes of polymorphonuclear cells.

However, the mutant devoid of LOS, Z0204, induced significantly lower levels of TNF-{alpha} than the wild-type strain. This is consistent with many studies of an LOS-free lpxA mutant of the N. meningitidis serogroup B strain H44/76 conducted in a cell model system (8, 16, 21, 22). The mutant devoid of LOS showed an enhanced release of membrane fragments. Membrane-associated proteins and peptidoglycan could be responsible for the proinflammatory response observed for this mutant. The mutant devoid of LOS induced significant levels of IL-1ß and IL-6 versus those in control mice. These levels were not significantly different from those induced by the wild-type strain. Moreover, we have recently shown that other meningococcal structures, such as peptidoglycan and pili, are inducers of an inflammatory response through the NF-{kappa}B-dependent signaling pathway (6, 20). Peptidoglycan is a major pathogen-associated molecular pattern that is sensed by the cytosolic Nod1/Nod2 proteins (6).

Cytokine production was quantified 3 hours after bacterial challenge. As a difference in bacterial counts in lungs was detected, the experiment was repeated by injecting mice with heat-killed bacteria (the same concentration of 5 x 107 bacteria). Indeed, live and heat-inactivated bacteria (wild type, rfaD mutant, and mutant devoid of LOS) were able to induce similar patterns of cytokine production (data not shown). These results suggest that the induction of cytokine production in lungs does not seem to be dependent on the bacterial growth/survival rate.

We report, for the first time, a differential induction of TNF-{alpha} and IL-1ß/IL-6 mediated by a meningococcal mutant devoid of LOS. This strongly suggests that meningococcal LOS is a central mediator of TNF-{alpha} induction in vivo. However, other non-LPS constituents of N. meningitidis contribute to the in vivo induction of IL-1ß and IL-6 (8, 16, 21, 22). Ramphal et al. (15) have recently shown a similar differential induction of TNF-{alpha} and IL-6 in a mouse model of Pseudomonas aeruginosa infection. TNF-{alpha} production in TLR2/4-deficient mice was severely impaired, whereas IL-6 production was observed, with no change in the neutrophil count in the lungs.

We observed a significant decrease in the production of the chemokines MIP-1{alpha}, RANTES, and MCP-1 after challenge with the mutant devoid of LOS. For both LOS mutants, the KC response in vivo was associated with an intense influx of polymorphonuclear leukocytes in lungs (Fig. 2C and D). Together, these findings strongly suggest that non-LPS components of N. meningitidis may contribute significantly to the inflammatory reaction of the host.

Although the proinflammatory action of meningococcal LOS is thought to be mediated by the CD14/TLR4 pathway (28), the cell-activating property of the H44/76 lpxA mutant is thought to be mediated by the CD14/TLR2 receptor pathway (8, 13). However, the differential pattern of cytokine and chemokine production observed in the absence of LOS suggests other unknown signaling pathways for the different proinflammatory cytokines and chemokines.


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ACKNOWLEDGMENTS
 
This work was supported by the Institut Pasteur.

We gratefully acknowledge Jean-Philippe Carlier for excellent assistance with gas chromatography, Huot Khun for histological studies, and Isabelle Bonne for the electron microscopy.


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FOOTNOTES
 
* Corresponding author. Mailing address: Unité des Neisseria, Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: 33 1 45 68 89 58. Fax: 33 1 41 60 30 34. E-mail: lzaranto{at}pasteur.fr. Back

Editor: J. N. Weiser


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Infection and Immunity, October 2006, p. 5506-5512, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00655-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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