Development and Evaluation of an Improved Mouse Model of Meningococcal Colonization

Bacterial strains.

The meningococcal strains used in this study are listed in Table 1. A spontaneous streptomycin-resistant strain was selected as a reference strain from a clinical isolate, with a serogroup B N. meningitidis strain, IR2781 (27). Meningococci were grown on GC medium base (Difco, Becton Dickinson, Sparks, Md.) plates (GCB plates) containing Kellog's supplements and incubated at 37°C under 5% CO₂ tension or in liquid cultures in GC broth containing Kellog's supplements at 37°C with 5% CO₂ with agitation_. When necessary, appropriate antibiotics were added to the medium as follows: streptomycin, 750 μg/ml; kanamycin, 100 μg/ml; and spectinomycin, 70 μg/ml. Transformation was performed as previously described (27).

Mouse model.

Female outbred mice [Swiss Webster, Crl:CFW(SW) BR, Charles River Laboratories] were used for most of the experiments. In some experiments, complement-deficient mice (CDM) (B10.D2-H2^d H2-T18^c Hc⁰/oSnJ; Jackson Laboratories) were used. These CDM lack the C5 component of the complement pathway. Mice received 200 mg of iron dextran (Dexferrum; American Regent Laboratories) per kg of body weight i.p. in 100 μl 2 to 3 h before intranasal inoculation with meningococci. Designated numbers of CFU of meningococci in 20 μl of GC broth were atraumatically inoculated into the right nare of the animal. After 3 h of the meningococcal inoculation, 15 μl of iron dextran (50 μg/μl) was given intranasally. The i.p. iron dextran was administered once a day during the duration of the colonization experiment. In some instances, human holo-transferrin (Sigma) in 200 μl of phosphate-buffered saline (PBS) (pH 7.4) was injected into the mice (800 mg per body weight) in the place of iron dextran. Human holo-transferrin was used for the subsequent intranasal inoculation (1.5 mg per mouse in a volume of 15 μl) and for the daily dosage. Nasopharyngeal washes were carried out by nasally instilling 35 μl of GC broth. Liquid was recovered from the mouth and plated on the modified Thayer-Martin medium to monitor for meningococcus growth. The number (percentage) of mice colonized with meningococci over a period of 13 days was monitored.

ELISA.

The enzyme-linked immunosorbent assay (ELISA) was performed as follows. Levels of immunoglobulin A (IgA) to the homologous strain were assayed in the nasopharyngeal washes of the mice colonized with the wild-type strain for 13 days or longer. The wells of microtiter plates (Maxisorp; Nunc) were coated overnight at 4°C with the whole-cell bacteria. Prior to coating, bacteria were grown overnight on GCB plates and resuspended in coating buffer (0.1 M sodium carbonate, 0.1 M sodium bicarbonate, pH 9.6) to an optical density at 600 nm (OD₆₀₀) of 0.2. The wells were blocked with 3% bovine serum albumin (Sigma) in PBS for 1 h. Nasopharyngeal washes were collected as described above, and the volumes were adjusted to 100 μl. The washes were placed in the coated microtitier wells and probed with horseradish peroxidase-labeled antimouse IgA (Sigma). Reactions were visualized by applying the wells with tetramethylbenzidine with hydrogen peroxide (TMB Soluble; Calbiochem) and read at 630 nm after a 30-min incubation.

Immunization.

Mice were immunized with viable whole bacteria of N. meningitidis strain IR2781 suspended in PBS. Bacteria were first grown on GCB plates overnight as described above. The bacterial cells were resuspended in PBS (pH 7.4) to an OD₆₀₀ of 0.4. The mice received 100 μl of resuspended bacterial preparation i.p. Additional i.p. immunizations were performed twice at 9-day intervals.

Statistical analysis.

All of the mouse colonization data were subjected to statistical analysis to determine significance. The Kaplan-Meier method was used to estimate cumulative clearance. Cumulative clearance between groups (strains, challenges, and inoculum sizes) was compared by log-rank tests. Statistical tests were two sided. A P value of ≤0.05 was considered statistically significant. The data from the ELISA reading were analyzed by using a one-sample test (see Fig. 2).

Model for meningococcal colonization of outbred mice.

Outbred adult mice (Swiss Webster) were selected in an effort to closely reflect colonization of human populations. A daily dose of systemic iron was required to establish meningococcal colonization of outbred adult mice. In addition, intranasal local priming with iron dextran on day 0 was necessary to have all of the mice colonized on day 1. In contrast to the work of Jerse with gonococcus (15), prior antibiotic treatment of mice was not necessary for meningococcal colonization in our experiments (data not shown). The number (percentage) of mice colonized with meningococci over 13 days was plotted (Fig. 1). Three groups of mice intranasally received the serogroup B wild-type strain (IR4127) at 10⁷, 10⁶, and 10⁵ CFU, respectively. Forty-two percent of the mice inoculated with 10⁷ CFU remained colonized with meningococci after 13 days (Fig. 1). The cumulative clearance between the inoculum sizes was statistically different (P = 0.001) by the log-rank test. Nasopharyngeal washes from mice that had been colonized for more than 13 days contained mouse IgA to the homologous meningococcus strain, indicating the presence of active mucosal immune responses against colonizing meningococci (Fig. 2).

Colonization of adult mice with mutants.

Adult mice were intranasally challenged with meningococcus mutants to evaluate the roles of specific genes in meningococcal colonization. First, three mutants that did not express wild-type lipooligosaccharide (LOS) due to inactivation of the LOS biosynthesis genes (rfaC, lgtF, and lst) were studied. These mutants produce truncated LOS molecules: the rfaC mutant synthesizes only KDO₂-lipid A (18), while the lgtF mutant expresses the Hep₂(GlcNAc)KDO₂-lipid A (17, 18). The LOS from the lst mutant is not sialylated due to a defect in LOS sialyltransferase (17, 18). Figure 3 represents the number of mice colonized with corresponding mutant strains in three groups. The rfaC mutant (IR5389) was severely impaired in the colonization capability, while the lgtF (IR5475) and lst (IR5476) mutants showed moderately attenuated colonization. Likewise, the capsule-deficient ctrA strain (IR5390) showed severely impaired colonization (Fig. 3). The ctrA mutant is capsule deficient due to inability to transport the capsule to the cell surface. As another control, a pilQ mutant that does not allow pilus biogenesis was examined (8). The PilQ protein functions in the terminal process of pilus expression, and inactivation of the gene affects the competence for the natural transformation (8). The pilQ mutant was moderately impaired in colonization (Fig. 3). The cumulative clearance was statistically different between the wild-type and mutant strains (P < 0.001) by the log-rank test.

The in vitro growth rates of the mutants were compared with that of wild-type parent to determine whether gene inactivation affected the growth of the mutants, which could reduce the degree of the nasopharyngeal colonization. The mutants grew at rates similar to that of the wild type, indicating that the gene inactivation of the mutants did not affect the in vitro growth of the bacteria (Fig. 3, insert).

Role of complement in the colonization of rfaC and ctrA mutant strains.

Complement-mediated bactericidal activity is the major defense mechanism against meningococcal infection (7, 19, 25). Capsule and LOS appeared to play critical roles in meningococcal colonization in normal adult mice, because mutants devoid of capsule or expressing a truncated LOS (rfaC) failed to effectively colonize in this model. This led us to further investigate the role of complement in the colonization of the rfaC and ctrA mutants by using CDM. Complement was partially responsible for the failure of colonization for both mutants, since colonization by the mutants was partially restored in the CDM (Fig. 4). The cumulative clearance between four test groups was compared by the log-rank test (P < 0.001).

Effect of immunization on colonization of the mouse nasopharynx by meningococci.

The effect of parenteral immunization on colonization of adult mice by nasopharyngeal administration of the homologous meningococcus strain was evaluated. The animals were intranasally challenged after a course of three immunizations with the whole bacterial cell (see Materials and Methods). None of the immunized mice became colonized with the homologous strain, indicating that the parenteral immunization effectively protects mice from nasopharyngeal colonization by homologous meningococci (Fig. 5). ELISA detected a high level of serum IgG to the homologous strain in sera from all immunized mice, while IgA was not detected (data not shown).