INFECTION AND IMMUNITY, Sept. 2009, p. 3578–3587 Vol. 77, No. 90019-9567/09/$08.00�0 doi:10.1128/IAI.01424-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Meningococcal ABC-Type L-Glutamate Transporter GltT IsNecessary for the Development of Experimental

Meningitis in Mice�†Roberta Colicchio,1 Susanna Ricci,2 Florentia Lamberti,3 Caterina Pagliarulo,4 Chiara Pagliuca,3

Velia Braione,2 Tiziana Braccini,2 Adelfia Tala,5 Donatella Montanaro,6 Sergio Tripodi,7Marcella Cintorino,7 Giancarlo Troncone,6,8 Cecilia Bucci,5 Gianni Pozzi,2

Carmelo B. Bruni,3,10 Pietro Alifano,5* and Paola Salvatore3,9*IRCCS Fondazione SDN, 80143 Naples,1 Dipartimento di Biologia Molecolare, LA.M.M.B., Universita di Siena, 53100 Siena,2

D.B.P.C.M. “L. Califano,” Universita di Napoli “Federico II,” 80131 Naples,3 D.S.B.A., Universita del Sannio,82100 Benevento,4 Di.S.Te.B.A., Universita del Salento, 73100 Lecce,5 Unita di Patologia Comparativa,Ceinge s.c.ar.l. Biotecnologie Avanzate, 80145 Naples,6 Dipartimento di Patologia Umana e Oncologia,

Universita di Siena, 53100 Siena,7 Dipartimento di Scienze Biomorfologiche e Funzionali, Universita diNapoli “Federico II,” 80131 Naples,8 Facolta di Scienze Biotecnologiche, Universita di

Napoli “Federico II,” 80131 Naples,9 and Ceinge s.c.ar.l. Biotecnologie Avanzate,80145 Naples,10 Italy

Received 20 November 2008/Returned for modification 21 January 2009/Accepted 5 June 2009

Experimental animal models of bacterial meningitis are useful to study the host-pathogen interactionsoccurring at the cerebral level and to analyze the pathogenetic mechanisms behind this life-threateningdisease. In this study, we have developed a mouse model of meningococcal meningitis based on the intracis-ternal inoculation of bacteria. Experiments were performed with mouse-passaged serogroup C Neisseriameningitidis. Survival and clinical parameters of infected mice and microbiological and histological analysis ofthe brain demonstrated the establishment of meningitis with features comparable to those of the disease inhumans. When using low bacterial inocula, meningococcal replication in the brain was very efficient, with a1,000-fold increase of viable counts in 18 h. Meningococci were also found in the blood, spleens, and livers ofinfected mice, and bacterial loads in different organs were dependent on the infectious dose. As glutamateuptake from the host has been implicated in meningococcal virulence, mice were infected intracisternally withan isogenic strain deficient in the ABC-type L-glutamate transporter GltT. Noticeably, the mutant was atten-uated in virulence in mixed infections, indicating that wild-type bacteria outcompeted the GltT-deficientmeningococci. The data show that the GltT transporter plays a role in meningitis and concomitant systemicinfection, suggesting that meningococci may use L-glutamate as a nutrient source and as a precursor tosynthesize the antioxidant glutathione.

Neisseria meningitidis is a narrow-host-range microorganismthat colonizes the nasopharyngeal mucosae of approximately10% of healthy subjects and only rarely causes life-threateningdiseases, such as meningitis and sepsis. In industrialized coun-tries, more than 60% of patients present with meningitis with-out septic shock (5). The pathogenesis of meningococcal dis-ease consists of several steps, including invasion of thebloodstream from the nasopharyngeal mucosa, survival in theblood, and entry into the central nervous system (CNS) bycrossing the blood-brain barrier (57). When N. meningitidismassively replicates in the blood with concomitant endotoxinshedding, high levels of proinflammatory mediators are sys-

temically released. In this case, fulminant meningococcal sepsismay occur even without induction of meningitis (5, 57). Incontrast, if bacteria multiply in the blood poorly, they mayreach the CNS, with subsequent development of meningitis(4, 57).

Progress in understanding the pathogenesis of meningococ-cal disease and developing effective drugs and vaccines hasbeen hampered by the lack of valuable animal models of dis-ease. Indeed, humans are the only natural hosts for this mi-croorganism due to high specificity of both meningococcalsurface structures (i.e., type IV pili and opacity proteins) andiron uptake systems for human receptors (36, 58) and transportproteins (43). Phase variation of meningococcal antigens dur-ing infection has further complicated the development of ex-perimental disease models (9, 54).

The long history of animal models of meningococcal diseasedates back to 1907, when meningitis was induced in monkeys(10). Presently, two rodent models (mouse and rat) and tworoutes of infection (intraperitoneal [i.p.] and intranasal [i.n.])are employed to induce meningococcal disease. The i.p. modeldoes not mimic the natural route of infection in humans butoffers the advantage of inducing severe sepsis. The i.n. route is

* Corresponding author. Mailing address for Paola Salvatore:D.B.P.C.M. “L. Califano,” Universita di Napoli “Federico II,”80131 Naples, Italy. Phone: (39) 081 7462058. Fax: (39) 0817703285. E-mail: psalvato@unina.it. Mailing address for PietroAlifano: Di.S.Te.B.A., Universita del Salento, 73100 Lecce, Italy.Phone: (39) 0832 298856. Fax: (39) 0832 298626. E-mail: alifano@ilenic.unile.it.

† Supplemental material for this article may be found at http://iai.asm.org/.

� Published ahead of print on 15 June 2009.

3578

useful to analyze disease pathogenesis, but animals may de-velop lung infection before sepsis, in contrast to humans, whodo not generally present with pneumonia. The model estab-lished in the rat is based on i.p. injection of neonatal animalswith rat-passaged meningococci to increase bacterial virulence(40), and it has largely been used in vaccine studies (14, 41, 59).Mouse models of meningococcal disease are generally basedon administration of an exogenous iron source to animals priorto infection in order to favor bacterial multiplication in thehost (17, 42). The i.p. mouse model was instrumental to assessprotection from meningococcal challenge (14, 33, 34). i.n.models have mainly used neonatal mice (28, 38) to analyzedisease pathogenesis and virulence of meningococcal isolates(29, 37). Interestingly, meningococcemia was also induced bythe i.n. route in adult mice superinfected with influenza A virus(1). To overcome some shortcomings of traditional models,alternative strategies have also been explored (8), including theuse of transgenic mice expressing human CD46 (18) or trans-ferrin (60). However, hardly any effort has been made to es-tablish models of meningitis (rather than sepsis), and to ourknowledge, no such a model has been developed in mice in-fected by the intracranic route.

N. meningitidis is able to obtain and synthesize nutrientsessential for its survival in the different environments withinthe human host during infection. A genome-wide analysis ofvirulence genes required for systemic meningococcal infectionin the rat showed that about half encode enzymes involved inmetabolism and transport of nutrients (51). There is evidencethat L-glutamate uptake from the host is critical for meningococ-cal infection in both cell and animal infection models (30, 51). Wehave demonstrated that GltT, an ABC-type transporter for L-glutamate, is essential for meningococcal growth/survival in in-fected cells (30) and that invasive isolates hyperexpress gdhA,encoding the NADP-specific L-glutamate dehydrogenase (35). In-deed, meningococci are naturally auxotrophic for L-glutamateand use it as a carbon (and nitrogen) source by supplying thetricarboxylic acid cycle with 2-oxoglutarate when glucose or lac-tate is limiting (30, 35). Due to this auxotrophy, host L-glutamateis essential for the biosynthesis of several amino acids and theantioxidant glutathione (http://www.genome.ad.jp/dbgetbin/get_pathway?org_name�nme&mapno�00251).

In this study, we have developed a novel model of menin-gococcal meningitis based on intracisternal (i.cist.) infection ofadult mice. The model was used to assess the virulence of amutant strain deficient in the L-glutamate transporter GltT.Here, evidence that the GltT mutant is attenuated comparedto the wild-type strain is provided, suggesting that uptake ofL-glutamate is crucial for the development of meningococcalmeningitis in mice.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The meningococcal strains used inthis study are the serogroup C 93/4286 isolate and its isogenic mutant 93/4286�gltT, which is devoid of the ABC-type L-glutamate transporter GltT. The93/4286 strain, belonging to the ET-37 hypervirulent lineage, was kindly providedby Novartis Vaccine and Diagnostics, Siena, Italy. Meningococci were culturedon blood agar medium (Oxoid) or gonococcus (GC) (Oxoid) agar/broth supple-mented with 1% (vol/vol) Polyvitox (Oxoid) at 37°C with 5% CO2. When needed,erythromycin (Sigma-Aldrich) was added to a final concentration of 7 �g ml�1.Escherichia coli strain DH5� was used in cloning procedures. This strain was

grown in Luria-Bertani (LB) (Oxoid) medium. To allow plasmid selection, LBmedium was supplemented with ampicillin (50 �g ml�1).

DNA procedures, plasmids, and transformation of meningococci. High-mo-lecular-weight genomic DNA from N. meningitidis strains was prepared as pre-viously reported (6). DNA fragments were isolated by using acrylamide slab gelsand recovered by electroelution as described before (39). Amplification reactionswere as follows: 45 s of denaturation at 94°C, 45 s of annealing at 65°C, and 60 sof extension at 72°C for a total of 30 cycles. Reactions were carried out in aPerkin-Elmer Cetus DNA Thermal Cycler 480 using oligonucleotidesNMB1965-1 and NMB1965-2 (30). The DNA of strain 93/4286 was used as atemplate. Southern blot hybridizations were carried out according to standardprotocols (39). 32P labeling of DNA fragments was performed by random prim-ing using the Klenow fragment of E. coli DNA polymerase I and [�-32P]dGTP(3,000 Ci mmol�1) (39).

The Neisseria-E. coli shuttle plasmids pDEX and pDE�NMB1965 were aspreviously described (30, 35). The NMC1937 open reading frame of strain93/4286, coding for the permease of the GltT transporter, was genetically inac-tivated by single crossover using plasmid pDE�NMB1965. The resulting mutantwas named 93/4286�gltT. Transformation experiments were performed by using0.1 to 1 �g of plasmid DNA as previously described (11). Transformants wereselected on GC agar medium supplemented with erythromycin. Gene inactiva-tion was demonstrated by Southern blot hybridization using a 482-bp-longNMB1965-specific 32P-labeled probe (30). Primer synthesis and DNA sequenc-ing were performed by Ceinge Biotecnologie Avanzate s.c.ar.l., Naples, Italy.DNA sequence analysis was carried out by using the GeneJockey SequenceProcessor software (Biosoft).

Mice. Eight-week-old female outbred CD1 mice weighing 24 to 30 g wereobtained from Charles River (Calco, Italy). Animals were allowed to settle in thenew environment for 1 week before the experiments were performed. All animalexperiments were approved by the local ethics committee (document no. 754/03,12.9.03) and were carried out according to institutional guidelines.

Mouse model of meningococcal meningitis. Mice were infected by the i.cist.route by a technique described by Koedel et al. to induce meningitis by Strepto-coccus pneumoniae (20). In vivo pilot experimentation was carried out usinglaboratory-grown N. meningitidis. Because the results were not compellingenough, subsequent experiments were all performed with mouse-passaged bac-teria to increase meningococcal virulence. Briefly, animals were lightly anesthe-tized (50 mg/kg ketamine and 3 mg/kg xylazine). Approximately 107 CFU ofbacteria in a total volume of 20 �l were inoculated by hand-puncturing thecisterna magna of mice using a 29-gauge needle (Artsana, Italy). Meningococciwere recovered 24 h later by homogenizing the brain with a screen mesh in 1 mlof GC medium. Passaged bacteria were grown to early exponential phase in GCbroth to an optical density of 0.7 at 600 nm, corresponding to approximately 7 �108 CFU ml�1. Bacteria were stored frozen at �80°C in GC broth supplementedwith 10% (vol/vol) glycerol (Carlo Erba) until use.

Prior to infection, passaged bacteria were thawed at room temperature, har-vested by centrifugation for 5 min at 1,800 � g, and resuspended in fresh GCbroth containing iron dextran (5 mg kg�1). Viable counts were performed onblood agar plates to determine the exact number of CFU. Approximately 5 hbefore infection, animals were injected i.p. with iron dextran (250 mg kg�1).Bacteria were injected by the i.cist. route as described above. Animals weremonitored for seizures due to inoculation.

Animal survival, clinical parameters, and CFU counts. Different bacterialdoses ranging from 105 to 107 CFU per mouse were used to inoculate animals(n � 4 to 12) by the i.cist. route with mouse-passaged wild-type or GltT-deficientmeningococci. Control mice were inoculated with GC broth. Every day through-out the whole experiment, animals were monitored for clinical symptoms (i.e.,ruffled fur, hunched appearance, hypothermia, weight loss, lethargy, or mori-bund). Body weight and temperature were measured by using a digital balance(Acculab) and a thermometer (Greisinger Elettronic), respectively. Rodentswere humanely killed before reaching the moribund state. Survival was recordedfor a week.

To determine the number of wild-type meningococci in organs, animals (n �16) were infected with 5 � 105 CFU/mouse and sacrificed at different time points(6, 12, 24, and 30 h) after infection. To compare virulence of wild-type versusGltT-deficient bacteria, two groups of mice (n � 30/group) were infected with107 CFU/mouse and sacrificed at 6, 24, and 48 h after challenge for organcollection. All moribund mice were humanely killed. Blood was withdrawn bycardiac puncture before sacrifice and added to a tube containing 3.8% sodiumcitrate. Brain, spleen, and liver were excised and homogenized in 1 ml of GCmedium. Viable counts were determined by plating 10-fold dilutions onto bloodagar plates.

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Mouse competition experiments. Frozen culture stocks of mouse-passaged93/4286 (erythromycin-sensitive) and 93/4286�gltT (erythromycin-resistant)strains were thawed, centrifuged, and resuspended in fresh GC broth with irondextran (5 mg kg�1) at the appropriate dilutions. Strains were mixed at a 1:1ratio, and mice (n � 9) were infected i.cist. with equivalent numbers of wild-typeand mutant bacteria. Each animal received 2 � 107 total bacteria in a volume of20 �l. Mice were killed at 6, 24, and 48 h after infection. Blood, brain, spleen, andliver were removed at each time point and treated as described above. Sampleswere plated onto blood agar plates with and without antibiotic selection todistinguish between strains. The competitive index (CI) was calculated as(CFUmutant/CFUwild type)output/(CFUmutant/CFUwild type)input, where the outputrepresents the viable bacteria recovered from a target organ at a certain timepoint after infection and the input is the bacterial mixture in the initial inoculum.A CI of 1 indicates decreased growth (and fitness) of the 93/4286�gltT mutantcompared to the wild type (2, 3). When no bacteria were recovered from one ormore mice, it was assumed that at least 1 CFU had been isolated. CI values arerepresented as means standard errors of the means (SEM).

Histological and immunofluorescence analysis. For histological analysis, ani-mals were inoculated with 107 CFU of bacteria from either mouse-passagedwild-type or GltT-deficient strains and sacrificed when moribund. Mice injectedwith GC medium were used as controls. Brains were excised, fixed in formalin for24 h, and then embedded in paraffin according to standard procedures. Brainswere sectioned along a coronal plane (31). Tissue blocks were cut in 5-�msections, stained with hematoxylin-eosin according to standard techniques, andexamined by using routine light microscopy. In both mouse-passaged wild-typeand GltT-deficient strains, the extent and degree of inflammatory infiltrates inthe brain were evaluated in meningeal, intraparenchimal, and ventricular tissueareas (Table 1). Immunofluorescence was performed to distinguish betweenextracellular and intracellular meningococci in the brains of infected mice. Allincubations were carried out at room temperature for 60 min unless statedotherwise. After being washed in phosphate-buffered saline (PBS), samples wereincubated with polyclonal rabbit antibodies raised against whole meningococci(anti-N. meningitidis 6121; ViroStat) and then stained with anti-rabbit immuno-globulin G conjugated with tetramethylrhodamine isothiocyanate (TRITC)(Dako). To detect intracellular bacteria, TRITC-stained tissues were permeab-ilized for 10 min with 0.25% saponin (Sigma-Aldrich) in PBS. Samples wereincubated with anti-N. meningitidis antibodies (in PBS and 0.25% saponin) andthen treated with fluorescein isothiocyanate (FITC) (Dako)-conjugated anti-rabbit immunoglobulin G diluted in PBS and 0.25% saponin as previously de-scribed (48, 53). Primary and secondary antibodies were diluted 1:500 and 1:200,respectively. Successful permeabilization was verified using a control antibody(MAB318 [Chemicon], 1:300) specific for an intraneuronal cytoplasmic target(tyrosine hydroxylase) followed by Alexa Fluor-conjugated anti-mouse antibod-ies (Molecular Probes, 1:1,000) (see Fig. S1 in the supplemental material). DAPI(4�,6�-diamidino-2-phenylindole) (Sigma-Aldrich; 1:1,000) staining was per-formed to reveal nuclei. Extra- and intracellular bacteria were visualized using aZeiss Axioskop2 Plus fluorescence microscope (Zeiss).

Statistical analysis. Differences in survival of mice inoculated with the wildtype or the mutant were analyzed by using the Fisher exact test. The two-tailedStudent t test was employed to analyze differences in body weight and temper-ature between different animal groups (P 0.05). CFU counts in different organsand time points were represented as numbers of bacteria isolated from single

mice, and means SEM for each animal group were calculated. Differences inbacterial loads between mice infected with the wild type and the mutant weredetermined with the Mann-Whitney U test (P 0.05), (45).

RESULTS

Development of a murine model of meningococcal meningi-tis. The model was established by using a method previouslydescribed to induce pneumococcal meningitis (20). OutbredCD1 mice were infected directly into the cisterna magna(i.cist.) with the group C 93/4286 isolate. As treatment withexogenous iron enhances virulence of N. meningitidis (17, 38,42), animals were injected i.p. with iron dextran prior to i.cist.infection.

Preliminary experiments were performed with both the lab-oratory and the mouse-passaged 93/4286 isolates. While bothstrains induced meningitis (according to histopathology; seebelow), the laboratory strain was impaired in causing systemicinfection (data not shown). Therefore, mouse-passaged bacte-ria were chosen to develop the model. Three groups of micewere infected with 105, 106, and 107 CFU of strain 93/4286, andsurvival over time was recorded (Fig. 1A). No mice died due toinfection with 105 CFU, while 89% and 46% of rodents sur-vived meningococcal challenge with 106 and 107 CFU, respec-tively. Mouse death generally happened within the first 72 hafter meningococcal inoculation, and the median times todeath for animal groups inoculated with 106 CFU and 107 CFUwere 169 h and 144 h, respectively (data not shown). No animaldied or developed postinfection seizures after i.cist. injectionof fresh culture medium.

As rodents with bacterial meningitis generally become hy-pothermic and lose weight (20), the body weights and temper-atures of animals infected with 107 CFU of the N. meningitidis93/4286 strain were recorded once per day for the duration ofthe experiment. In accordance with the results on mouse mor-tality (Fig. 1A), larger variations occurred in the first 72 h afteri.cist. injection, when mice showed a weight loss of 23% (Fig.1B) and a temperature drop of 20% (Fig. 1C). The bodyweights and temperatures of uninfected mice remained stablethroughout the experiment. Differences between the infectedgroup and the control group were significant in the first 3 to 4days postchallenge (Fig. 1B and C). In animals surviving me-ningococcal challenge, both clinical parameters began to nor-malize within a week after infection (Fig. 1B and C).

Time course of meningococcal replication in mice afteri.cist. infection. To determine the numbers of meningococci inbrain, spleen, liver, and blood at different stages of disease,animals were injected i.cist. with a sublethal dose of the mouse-passaged 93/4286 strain and sacrificed at different time points(6, 12, 24, and 30 h) after challenge. In the brain, following aninitial reduction of viable cells (3 log 2.17 log CFU) com-pared to the inoculum (5 � 105 CFU), there was a steadyincrease of CFU counts over time. At 30 h after infection,bacteria reached large numbers (6.04 log 5.94 log CFU),demonstrating that meningococci can grow exponentially inthe CNS (Fig. 2A). Concurrently with brain infection, menin-gococci were also recovered from the blood, spleen, and liver(Fig. 2B to D). At 30 h postchallenge, infection was clearedfrom the bloodstream (Fig. 2B), whereas large bacterial loadswere still present in the spleen (Fig. 2C) and liver (Fig. 2D).

TABLE 1. Histological analysis of brain tissue from mice afterinfection with the mouse-passaged wild-type or

GltT-deficient strain

Strain Inoculuma

(CFU/mouse)

Inflammation (PMN/high power field)b

Meninges Intracerebralregions Ventricules

93/4286 107 ��� ��� ���93/4286 106 �� �� ��93/4286�gltT 107 � � �93/4286�gltT 106 � � �

a Mice were infected with 106 and 107 CFU of mouse-passaged wild-type ormutant strain. Control mice were inoculated with 20 �l GC broth medium.

b Levels of inflammation in the brain were evaluated by counting the numbersof PMN per field in different brain areas (meninges, intraparenchimal regions,and ventricles) in a high power (�400) field. �, 0 PMN; �, 10 PMN; ��, 10to 50 PMN; ���, �50 PMN.

3580 COLICCHIO ET AL. INFECT. IMMUN.

Interestingly, bacteria persisted in the liver (3.27 log 2.91 logCFU at 30 h), suggesting that this organ may represent a targetsite for meningococcal replication (26). When inoculation wasdone with the laboratory strain instead of the mouse-passagedisolate, it was no longer possible to isolate bacteria from theblood, spleen, and liver at 2 days after infection (data notshown). This observation underlines the importance of mousepassage for induction of systemic meningococcal disease.

Histopathological characterization of the meningitis model.In order to prove the establishment of meningitis and study thefeatures of disease, we performed both histological analysisand immunofluorescence staining of the brains from moribundmice inoculated with the mouse-passaged 93/4286 strain. Atlate disease stages, animals presenting with typical signs ofmeningitis (hunchbacked, photophobic, and lethargic) weresacrificed, and brains were processed for either hematoxylin-eosin or immunofluorescence staining. Histological features

for both mouse-passaged wild-type and GltT-deficient strainsare reported in Table 1.

Major histological changes were observed in the brains ofinfected mice (Fig. 3). Moderate inflammatory infiltrates ofpolymorphonuclear cells (PMN) were observed in the lepto-meningeal (Fig. 3B) and ventricular (Fig. 3D) spaces of in-fected rodents compared to control animals injected with GCbroth (Fig. 3A and C). In the regions with severe inflammation,PMN cellular exudates were also entrapped in a dense fibrinnet. Analysis of the hippocampi from animals infected with N.meningitidis revealed the presence of regions with neuronalshrinkage (Fig. 3F) in comparison with control subjects (Fig.3E). Inflammatory infiltrates and extravasation of red bloodcells were also visible (Fig. 3F). Histological alterations inmeningeal and ventricular spaces were also observed in thebrains of mice inoculated with the laboratory group C strain(data not shown), suggesting that mouse passage is not essen-tial for the establishment of meningitis.

To demonstrate the presence of bacteria at the infectionsite, immunofluorescence staining of the brain tissue was car-ried out by using an antimeningococcal serum followed byTRITC- and FITC-conjugated antibodies. Bacteria (mostlycocci and diplococci) were observed in both meningeal andparenchimal areas. While intracellular localization of bacteriawas rare (Fig. 4B), meningococci were mainly found extracel-lularly (Fig. 4A and C). Samples from uninfected animals re-vealed no fluorescence (Fig. 4D to F). A control sample

FIG. 1. Survival and clinical parameters of mice infected by thei.cist. route with group C N. meningitidis. (A) Three groups of CD1mice (n � 4 to 12) were infected i.cist. with three different doses (105,106, and 107 CFU/mouse) of the mouse-passaged 93/4286 strain. Micewere monitored for a week, and survival was recorded. Results areexpressed as percent survival over time. (B and C) Variation of bodyweight (B) and temperature (C) following i.cist. injection of CD1 micewith 107 CFU of the 93/4286 strain (squares). Results for controlanimals are also shown (circles). Clinical parameters were measuredonce per day for 7 days. Results are indicated as means SEM.Asterisks indicate statistical significance (�, P 0.05; ��, P 0.01; ���,P 0.001).

FIG. 2. Time course of bacterial loads in different organs followingi.cist. infection with N. meningitidis 93/4286. CD1 mice (n � 16) wereinfected by the i.cist. route with 5 � 105 CFU of the mouse-passaged93/4286 strain. Animals were sacrificed at 6, 12, 24, and 30 h afterinfection (four mice per time point). Brain (A), blood (B), spleen (C),and liver (D) specimens were collected, and viable counts were deter-mined. Results are expressed as mean (SEM) log of CFU numbersper organ or ml of blood at different time points after inoculation.

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stained for cytoplasmic tyrosine hydroxylase confirmed tissuepermeabilization (see Fig. S2 in the supplemental material).

These data demonstrate that the i.cist. murine model iseffective at inducing meningococcal meningitis with clinicaland histopathologic features that mimic the disease in humans.

Construction and virulence evaluation of an L-glutamatetransporter mutant in the meningitis model. Glutamate isinvolved in neuronal damage caused by pneumococcal andgroup B streptococcal meningitis (23, 44), and its levels arealso increased in the cerebrospinal fluid (CSF) of patients with

FIG. 3. Histological analysis of the brains of mice infected with N. meningitidis 93/4286. CD1 mice were infected by the i.cist. route with 107

CFU of the mouse-passaged 93/4286 strain and humanely killed after 24 h (B, D, and F). Control mice were injected i.cist. with GC broth (panelsA, C, and E). Brains were excised, fixed in formalin, embedded in paraffin, and stained with hematoxylin-eosin. (A) Meninges from control mice(magnification, �100). (B) Inflammation in the subarachnoid space of infected animals (magnification, �50); subarachnoidal accumulation ofPMN is shown in the inset (magnification, �100). (C) Ventricular space from an uninfected mouse (magnification, �50). (D) Acute inflammationin ventricular spaces of the brain from infected mice (magnification, �25); PMN can be seen at higher magnification (�50) in the inset. (E andF) Brain damage in the hippocampus (magnification, �200) with manifest neuronal shrinkage and extravasation of red blood cells (F) comparedto control animals (E). Scale bars in each panel indicate the magnification.

3582 COLICCHIO ET AL. INFECT. IMMUN.

bacterial meningitis (15, 49). To analyze the importance ofglutamate in the pathogenesis of meningococcal disease, anL-glutamate transporter mutant of the group C 93/4286 strainwas constructed and tested using the i.cist. meningitis mousemodel.

An isogenic mutant deficient in the L-glutamate uptake sys-tem GltT was obtained by insertional inactivation of theNMC1937 gene, coding for the permease component of theABC-type transporter. Southern blot analysis confirmed inser-tion of the erythromycin resistance cassette by single crossoverinto the NMC1937 gene. By using an NMC1937-specific probe,two HincII DNA fragments of the expected sizes (1,718 bp and951 bp) were detected in the 93/4286�gltT mutant, comparedto a single 1,975-bp HincII fragment observed in the parental93/4286 strain (see Fig. S2 in the supplemental material).

The virulence of the GltT-deficient strain was assessed in thei.cist. meningitis model by analyzing animal survival at differentdoses and mouse clinical parameters over time. Six groups ofanimals were infected with 105, 106, and 107 CFU of mouse-passaged wild-type or mutant meningococci. No animal dieddue to infection with 105 and 106 CFU of the mutant, while89% survival was recorded for mice inoculated with 106 CFUof the wild-type strain. At the largest dose of 107 CFU, therewas 64% survival in the group infected with the GltT-deficientstrain, compared to 46% survival of mice infected with the93/4286 strain (Fig. 5A). However, no statistically significantdifferences between the groups were found (Fisher exact test).Clinical parameters of mice infected with 107 CFU of theGltT-deficient strain were consistent with the increased sur-vival observed in rodents inoculated with the mutant comparedto animals injected with the wild type. Percent reductions ofboth body weight (Fig. 5B) and temperature (Fig. 5C) werelower in mice infected with the GltT mutant (weight loss �

17.5%; temperature drop � 7.4%) than in control animals thathad received the parental strain (weight loss � 24.4%; tem-perature drop � 17.6%). Differences in body weight and tem-perature between the two animal groups were significant (P 0.05) at days 3 and 1 after infection, respectively (Fig. 5Band C).

Reduction of virulence of the GltT-deficient strain was fur-ther confirmed by histological analysis of the brain tissue frommice inoculated with the mutant. Mild inflammatory infiltrateswere restricted to meningeal areas, with neither intracerebralnor intraventricular involvement, compared to brain samplesfrom mice infected with the mouse-passaged wild-type strain,which presented with severe inflammation in all brain areas(Table 1).

Mice infected with the GltT-deficient mutant clear systemicinfection compared to animals inoculated with the wild type.To evaluate clearance of wild-type and mutant bacteria frominfected mice, two groups of animals were inoculated with 107

CFU of either the 93/4286 or the 93/4286�gltT strain. Bothstrains were passaged in mice prior to infection. Mice weresacrificed at different disease stages (6, 24, and 48 h afterinfection), and viable counts on brain, blood, spleen, and liversamples were determined. At 24 and 48 h after infection,bacterial loads in the brains of mice challenged with the mu-tant were 385- and 154-fold lower than those in animals in-fected with the parental strain, respectively (Fig. 6A). Viablecounts of GltT-deficient meningococci in the brain decreasedover time down to 4.5 log 4.17 log CFU/brain at 48 hpostinoculation, and clearance from the infection site occurredin 20% of subjects. In contrast, infection was not yet eradicatedfrom the CNSs of mice that had received the wild-type strain(6.69 log 6.36 log CFU/brain at 48 h) (Fig. 6A). Systemically,meningococcal infection caused by the GltT-deficient mutant

FIG. 4. Immunofluorescence analysis of the brains of animals infected with the 93/4286 strain. CD1 mice were infected by the i.cist. route withthe mouse-passaged 93/4286 strain (107 CFU/mouse) and sacrificed 24 h later. Brains were sectioned and treated with an antimeningococcal serumfollowed by TRITC- or FITC-conjugated secondary antibodies. Intracellular bacteria were visualized by using FITC-conjugated antibodies aftertissue permeabilization with saponin. (A) Extracellular bacteria as revealed by TRITC-conjugated antibodies. (B) Intracellular meningococci aftercell permeabilization and treatment with FITC-conjugated antibodies. (C) TRITC/FITC overlay indicates that most meningococci are extracel-lular. (D to F) Brain sections from uninfected mice were used as controls. Bars, 10 �m.

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was entirely cleared within 24 h, whereas none of the animalsinoculated with the wild type had eliminated bacteria from thebloodstream and internal organs. Two days after inoculation,average counts of the parental strain in the blood, spleen, andliver were still 3.55 log, 2.96 log, and 3.32 log CFU/ml blood ororgan, respectively (Fig. 6B to D). At 24 and 48 h after diseaseinduction, differences in bacterial loads between the two ani-mal groups were statistically significant for each organ tested(P 0.01 for the brain at 48 h; P 0.001 for all other samples).

L-Glutamate uptake contributes to virulence in mutant/wild-type mixed infections. To unambiguously define a role forthe GltT transporter in meningococcal disease, mice were in-fected i.cist. with both strains at a 1:1 ratio. Bacterial loads inbrain, blood, spleen, and liver were determined at 6, 24, and48 h after infection, and mean CIs were calculated (Fig. 7). Forevery time point and organ examined, the GltT-deficient strainwas less fit when competing with the wild type (CI 1). Early

(6 h) in infection, the wild type outgrew the mutant both in theCNS (CI � 0.14) and systemically (CI � 0.43 to 0.02). At laterstages of meningitis, the CI progressively decreased in thebrain to 0.014 0.08 (24 h) and 0.007 0.002 (48 h) (Fig. 7A).Differences in fitness between wild-type and mutant bacteriawere also pronounced in the spleens and livers of infectedanimals, where the mean CI values at 48 h postchallenge de-clined to 0.004 in both organs (Fig. 7C and D). Notably, thelargest defect in the growth of mutant meningococci was ob-served in the bloodstream at 24 h after i.cist. infection (CI �0.0002 0.00004) (Fig. 7B).

Altogether, these results demonstrate that the GltT-defi-cient strain is impaired in surviving in the murine host, stronglysuggesting that availability of intracellular L-glutamate is cru-cial for the development of experimental meningococcal dis-ease.

DISCUSSION

In the present study, we have established a model of menin-gococcal meningitis in outbred adult mice based on i.cist. in-oculation of bacteria. To our knowledge, no other model ofmeningococcal meningitis has been developed in mice infectedi.cist., although the i.cist. route of infection has been exploredto establish models of meningococcal meningitis in both the rat(19, 55) and the rabbit (56). As the highest rates of meningo-coccal disease are in young children, adolescents, and youngadults (12, 13), immunocompetent 8-week-old animals wereemployed rather than neonatal or infant subjects. Outbred(CD1) mice were preferred to inbred strains due to their cost-effectiveness and because they better mimic the natural varia-tion in infection occurring in human population. We chose thei.cist. (20) rather than the intracranic (7) route of infection,because the former ensures complete delivery of the inoculumdirectly into the cisterna magna, thereby facilitating bacterialreplication in the CSF. As shown by mouse survival, clinicalparameters, and histology, i.cist. inoculation was functional atinducing the disease. Large doses ( 107 CFU) of group C N.meningitidis were necessary to cause systemic infection anddeath of 50% of mice in comparison with S. pneumoniae,where the 50% lethal dose was approximately 102 CFU (7). Incontrast, the use of lower i.cist. inocula ( 105 CFU) supportedactive bacterial replication in the CSF but resulted in clearancefrom the bloodstream. Overall, the bacterial doses used in thisstudy were smaller with respect to the inocula (108 to 109 CFU)required to induce meningococcal disease in neonatal miceinjected via the i.n. route (29). Mouse passage was a key stepto develop the model. Indeed, microorganisms recovered fromthe brains of infected mice were capable of replicating in theCSF and blood of naïve rodents. In contrast, systemic survivalof the laboratory strain was severely hampered. Interestingly,both laboratory and mouse-passaged strains induced meningi-tis with histopathologic features mimicking those of the diseasein humans, suggesting that replication of meningococcal strains(even those that are poorly virulent) may be facilitated in theCSF due to CNS immunodeficiency (46, 47). Not only did themouse-passaged isolate grow efficiently in the CSF, but it alsopersisted in the spleen and liver. During the hypoferremicphase of neisserial infection, most of heme-derived iron re-mains associated within liver ferritin (26). As meningococci

FIG. 5. Survival and clinical parameters of mice after infection withwild-type or GltT-deficient N. meningitidis. (A) Three groups of CD1mice (n � 4 to 12) were infected i.cist. with 105, 106, and 107 CFU permouse of either the 93/4286 wild-type strain or the GltT mutant. Micewere monitored for a week, and survival was recorded. Results areexpressed as percent survival at different doses. (B and C) Variation ofbody weight (B) and temperature (C) following i.cist. injection of CD1mice with 107 CFU of the wild-type or GltT-deficient 93/4286 strain.Clinical parameters were measured once per day for 7 days. Resultsare indicated as means SEM. Asterisks indicate statistical signifi-cance (�, P 0.05).

3584 COLICCHIO ET AL. INFECT. IMMUN.

can obtain iron from ferritin (22), the liver may represent atarget organ for meningococcal replication. Altogether, thei.cist. model is effective at inducing both primary meningitisand disseminated meningococcal disease compared to i.p. ori.n. models, which are generally less suitable to study menin-gitis, as animals may die of sepsis before meningitis is estab-lished.

By using our murine model of meningitis, we tested thevirulence of a strain deficient in the L-glutamate transporterGltT. Data on the survival and clinical parameters of rodentsinfected with the mutant suggested virulence reduction, butstatistical significance was not compelling.

Analysis of meningococcal loads over time clearly provedthat the GltT mutant was attenuated in the model. In contrastto wild-type bacteria, the GltT-deficient strain was clearedsystemically the day after infection. The mutant did not exhibitactive replication into CSF but could still be isolated from thebrains of infected animals at 48 h after challenge, yet againsuggesting CNS immunodeficiency (46, 47). Despite persis-tence of the mutant in the CNS, meningeal inflammation waslimited or absent in comparison to that caused by the wild-typestrain, indicating that L-glutamate transport plays a role inestablishing the disease in the CNS. As assessment of virulenceby standard methods (i.e., 50% lethal dose calculation) is arelatively crude and insensitive approach that may fail at evi-dencing the contribution of a given factor to pathogenicity (3),we decided to use mixed infections to determine the degree ofvirulence attenuation of the GltT-deficient strain compared to

the wild type. Indeed, reduction of virulence was proven inmixed meningococcal infections, where the mutant was clearlyhampered compared to the parental strain. During diseaseprogression, the GltT-deficient strain showed a decreasing ca-pability to adapt to different body sites in the host. At 48 h afterinfection, CI values were comparable in each mouse compart-ment analyzed, indicating that the wild type outcompeted themutant strain in a time-dependent and organ-independentmanner. Attenuation of the GltT-deficient strain in the periph-eral compartments was consistent with recent work demon-strating that a knockout GltT mutant had reduced survival inhuman blood and was attenuated in a systemic mouse model ofinfection (27).

N. meningitidis is able to efficiently utilize only a few com-pounds as energy sources, including lactate, pyruvate, glucose,and maltose. Glucose and lactate are the predominant carbonsources in blood and at mucosal surfaces, respectively (24, 25).In the intracellular milieu, where levels of glucose are low, themain energy sources for meningococci are pyruvate, lactate,and certain amino acids, such as L-glutamate (16, 52). Theavailability of L-glutamate is considered critical for meningo-coccal infection in both cell (27, 30) and animal infection (27,51) models and is also instrumental in preventing oxidativeinjury, as L-glutamate is the precursor of glutathione (52). Inthis study, we demonstrated that L-glutamate uptake from themurine host is also required for bacterial replication and sur-vival in the CSF, suggesting that the L-glutamate may be im-portant in meningitis both for supporting meningococcal

FIG. 6. Bacterial loads over time in mice inoculated with wild-type or GltT-deficient N. meningitidis. Two groups of CD1 mice (n � 30/group)were infected i.cist. with 107 CFU of either the 93/4286 wild-type strain or the GltT mutant. Animals were sacrificed at 6, 24, and 48 h afterinfection. Brain (A), blood (B), spleen (C), and liver (D) specimens were collected, and viable counts were determined. Results are expressed aslog CFU numbers per organ or ml of blood at different time points after inoculation. Horizontal bars indicate mean logs of bacterial titers. Eachsymbol represents a single animal. Asterisks indicate statistical significance (��, P 0.01; ���, P 0.001).

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growth in the CNS and in preventing bacterial damage due tooxidative stress. Interestingly, the levels of L-glutamate in thebrain and CSF are strongly increased in animals and humanssuffering from bacterial meningitis (15, 49) and correlate withdisease severity (50). It has been hypothesized that duringmeningitis L-glutamate and other excitatory amino acids arereleased by both macrophages derived from blood monocytesand microglial cells during meningitis (50) and that release ofthese amino acids may result in membrane depolarization andcalcium influx, leading to energy failure and neuronal celldeath (32). These effects are mediated by the activation of theN-methyl-D-aspartate receptor complex. Therefore, it is be-lieved that L-glutamate release contributes to neuronal damageduring bacterial meningitis and that N-methyl-D-aspartate re-ceptor antagonists may be of therapeutic use (21, 44). In thisperspective, the use of L-glutamate analogues to both haltmeningococcal replication in the CSF and impede L-glutamateneurotoxic effects may represent an attractive therapeuticstrategy against bacterial meningitis.

ACKNOWLEDGMENTS

This work was supported by grants from Progetto MIUR Cofin 2006“Basi genetiche e molecolari della patogenicita batterica” (to PietroAlifano, Paola Salvatore, Gianni Pozzi, and Carmelo Bruno Bruni),Piano di Ateneo per la Ricerca (PAR) 2006 and 2007 (to Gianni Pozziand Susanna Ricci), and the European Commission EuroPatho-Genomics project, contract LSHB-CT-2005-512061 (to Gianni Pozzi).

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Editor: J. N. Weiser

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