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Infection and Immunity, April 2003, p. 1849-1855, Vol. 71, No. 4
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.4.1849-1855.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of an Improved Mouse Model of Meningococcal Colonization

Kyungcheol Yi,1* David S. Stephens,1,2,3 and Igor Stojiljkovic1

Department of Microbiology and Immunology,1 Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322,2 Laboratories of Microbial Pathogenesis, Veterans Affairs Medical Center, Atlanta, Georgia 303033

Received 30 October 2002/ Returned for modification 16 December 2002/ Accepted 14 January 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Studies of meningococcal pathogenesis have been severely restricted due to the absence of an adequate animal model. Given the significance of iron in meningococcal pathogenesis, we developed a model of Neisseria meningitidis colonization in outbred adult mice that included daily administration of iron dextran. While receiving iron, the animals were inoculated intranasally with the initial doses of bacterial suspension. Meningococci were recovered from the animals by nasopharyngeal washes. Approximately half of the animals inoculated with 107 CFU remained colonized 13 days after the initial bacterial inoculation. The model was further evaluated with genetically defined isogenic serogroup B mutant strains, and the colonization capabilities of the mutants were compared to that of the wild-type parent. A mutant that produces truncated lipooligosaccharide (KDO2-lipid A) and a mutant defective in capsule transport were dramatically impaired in colonization. A mutant defective in pilus transport (pilQ) showed moderately impaired colonization. The immunological aspect of the model was also evaluated by challenging mice after immunization with homologous whole-cell meningococci. The immunized mice were protected from colonization of the homologous strain. In this model, long-term meningococcal colonization was maintained, allowing us to study the effects of specific genetic mutation on colonization. In addition, this model allows investigation of the role of active immune response against meningococci.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neisseria meningitidis is now the leading cause of serious bacterial meningitis in the United States and is the leading cause in many countries worldwide, despite effective antibiotics and partially protective vaccines (3). Meningococci commonly colonize the nasopharynx of healthy individuals and can be isolated from 5 to 10% of the population in nonepidemic settings (34). Meningococcal acquisition in the nasopharynx may, in certain individuals, progress to an invasive disease resulting in fatal meningitis and/or sepsis.

Humans are the only natural host of meningococci. The bacterial virulence and colonization strategies are highly adapted to humans and have been difficult to study in animal models (41). Availability of iron is a major determinant of successful meningococcal infection, because iron is sequestered by the host, and an extremely low concentration of iron is maintained in the serum and at the mucosal surface. The inability of meningococci to cause disease in mice and other animals has been associated with the need of meningococci to use human transferrin or human lactoferrin as a source of iron (32). Meningococcal host specificity is due in part to the specificity of the iron-acquisition receptors on the surface of the bacteria, because the transferrin and lactoferrin receptors are specific for the corresponding human substrates (32, 33). This specificity for iron acquisition has restricted the development of the adequate animal model for studying the pathogenesis of meningococci. In this regard, enhancement of meningococcal virulence by the addition of iron in adult mice has been documented. Prior treatment of adult mice with human holo-transferrin causes bacteremia after intraperitoneal (i.p.) injection of the bacteria (14). Similarly, a parenteral dosage of iron dextran greatly enhances infection in animals challenged with meningococci (14).

Meningococcal disease has been modeled with infant mice, since infant rodents, for reasons not fully understood, are more susceptible to meningococci (29-31). Intranasal challenge with meningococci in infant mice produces invasive infection, mimicking the course of the meningococcal disease in humans (29). In the infant mouse model, administration of the iron to the animals further enhances the infection by the meningococci (20, 29-31). However, a major disadvantage of the infant mouse/rat model is the narrow window of infection, because mice and rats over 10 days old do not show signs of meningococcal disease (20, 31).

Recently, an animal model of Neisseria gonorrhoeae, a close relative of meningococcus, was developed in female BALB/c mice as a surrogate model for human infection (15). Prolonged genital tract infection in mice was achieved by treatment with estradiol and antibiotics. In this study, we have developed a convenient and economical animal model using adult outbred mice. Utilizing the importance of iron in the pathogenesis of meningococcus, we were able to establish colonization in the nasopharynx of the mouse by administering an iron source systemically and locally. Because meningococcal nasopharyngeal colonization is the first step of host-parasite interaction in the natural meningococcal infection, we first addressed these events by using this model. The model provides insight into understanding the early events of meningococcal disease.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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).


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  		TABLE 1. Characteristics of the strains used in this study

 
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-H2d H2-T18c 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).



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  		FIG. 2. Levels of IgA in nasopharyngeal washes of adult mice (Swiss Webster) colonized with meningococci over 13 days. The levels of IgA to the homologous strain are presented as ELISA readings at OD630 (x axis). The nasopharyngeal washes from colonized mice and a control mouse are indicated on the left (y axis). The ELISA readings from colonized mice (n = 6, mean = 0.065, standard deviation = 0.035, P = 0.009) were compared to the negative control value by using a one-sample test.

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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).



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  		FIG. 1. Course of adult outbred mice (Swiss Webster) colonization with the wild-type meningococcus. Number of mice colonized in percent (y axis) was monitored at day 1 and every 2 days for 13 days (x axis). The reference strain, IR4127, was instilled at 107(10-7; {blacktriangleup}), 106 (10-6; {triangleup}), or 105 (10-5; {circ}) CFU. The graph represents combined data from two experiments performed separately. The total number of mice used is indicated in the parenthesis on the right. The cumulative clearance was statistically different between the inoculum sizes (P = 0.001) by the log-rank test.

 
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.



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  		FIG. 3. Colonization of adult mice with LOS-, capsule-, or pilus-defective mutants. The number (percentage) of mice colonized (y axis) was monitored for 13 days (x axis). The rfaC ({triangleup}), lgtF (•), lst ({circ}), ctrA ({square}), and pilQ ({blacksquare}) mutants were inoculated into the corresponding groups of mice. The data from the wild-type strain ({blacktriangleup}) were from Fig. 1 and are included as a reference. Each inoculum was 107 CFU. The graph represents combined data from two experiments performed separately. The total number of mice used is indicated in parentheses on the right. The cumulative clearance between the wild-type and mutant strains was statistically different (P < 0.001) by the log-rank test. The growth rates of the rfaC ({triangleup}), ctrA ({square}), pilQ ({blacksquare}) mutants are presented, with that of wild type ({blacktriangleup}) given in the insert. The log numbers of CFU per milliliter (y axis) are plotted over 7 h (x axis).

 
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).



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  		FIG. 4. Colonization of CDM with LOS-defective (rfaC) and capsule-defective (ctrA) strains. CDM were challenged with rfaC ({blacktriangleup}) and ctrA (•) mutants. As references, normal mice were challenged with rfaC ({triangleup}) and ctrA ({circ}) mutants. The number (percentage) of mice colonized (y axis) was monitored every 2 days for 7 days (x axis). Each inoculum was 107 CFU. The graph represents the combined data from two experiments performed separately. The total number of mice used is indicated in parentheses on the right. 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).



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  		FIG. 5. Effect of immunization on mouse colonization by MC. The number (percentage) of mice colonized (y axis) was monitored every 2 days for 5 days (x axis). The immunized mice ({triangleup}) and the naive mice ({blacktriangleup}) were challenged with the homologous wild-type strain. The total number of mice used is indicated in parentheses on the right. The cumulative clearance between immunized mice and naïve mice was statistically different (P < 0.001) by the log-rank test.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The in vivo study of meningococcal pathogenesis has been severely restricted by the pathogen's strict host specificity and the morbidity and mortality of human disease. An animal model of meningococcal colonization to study the initial course of the disease and to facilitate the development of better vaccines is critically needed. Studies with infant rodents had limited success in producing bacteremia by intranasal inoculation (20). The animals were 4 to ~5 days old at the beginning of the experiment and maintained susceptibility for the next 5 days. This study describes the establishment of a model of long-term nasopharyngeal meningococcus colonization of adult mice by supplying the animals' daily iron. Both daily systemic iron dextran and intranasal priming with iron dextran were required to initiate colonization. The importance of iron related to meningococcal virulence in mice has been studied extensively (13, 14). We tested both human holo-transferrin and iron dextran to facilitate meningococcal colonization. Both iron sources supported the bacterial colonization in mice when given i.p. (human transferrin data not shown). However, iron dextran was chosen for the challenges because (i) iron dextran is relatively economical and showed very low toxicity (14), and (ii) the iron moiety was complexed with dextran, a low-molecular-weight polyglucose (4). Dextran is cleared rapidly by either metabolism or excretion. Parenteral administration of human transferrin may lead to immune responses, which should influence the model.

A previous study by Mackinnon et al. (21) showed that the nasal colonization usually accompanied lung infection in the infant mice. In this study, animals colonized in their nasopharynxes did not have any viable bacteria in the lung.

Curiously, reducing the amount of normal flora in the nasopharyngeal cavity by antibiotic treatment did not affect the outcome of the colonization (data not shown). In the mouse model of gonococcal genital tract infection, antibiotic treatment was required to suppress inhibitory normal flora.

Binding of pilus to the host mucosal surface is an initial step in colonization (26, 35, 36, 39). Primary adherence by pili to the epithelial cells is followed by intimate contacts with secondary surface molecules, such as Opa/Opc proteins (6). The mouse colonization model with a pilQ mutant revealed that the mutant colonized less efficiently than the parent strain. Bacterial surface components other than pili contribute to bacterial adhesion to epithelial and endothelial cells (24, 28, 46). In the absence of proper pilus assembly function, Opc and Opa may facilitate adhesion of meningococci to the eukaryotic cells, especially if capsule expression is turned off or downregulated (44, 45)

Previous studies with capsule-defective mutants in an infant rat model demonstrated the importance of capsule for bacteremia (47). We found that capsule is required for effective colonization in our model and that complement is partially responsible for the defect in the ability of the capsule-deficient mutant to colonize.

The role of LOS in the pathogenesis of meningococci and gonococci has been studied extensively. Gonococcal LOS plays a role in the adherence of the organism to host cells in vitro (5, 23, 43). In an infant rat model, the meningococcus mutant defective in LOS sialylation did not cause bacteremia, even with a high dose of CFU (47). In our mouse model, an rfaC mutant with a truncated LOS structure (KDO2-lipid A) did not colonize. An earlier study with the same mutant showed its inability to cause bacteremia (37). Interestingly, an lgtF mutant, which had three additional inner core glycosyl residues on the LOS molecule compared with the rfaC mutant, and an lst mutant (sialylation defective) showed only moderately attenuated colonization (Fig. 3). The lgtF and lst mutants were similar in our assay despite the differences in the length of the truncated LOS, suggesting a role for LOS core structure in colonization.

The incidence of meningococcal disease inversely correlates to the level of bactericidal antibodies in human serum (9, 10). The role of complement-mediated bactericidal activity has been evidenced by the susceptibility to meningococcus of individuals deficient in the terminal component of complement. The role of antibodies to meningococci in preventing mucosal colonization has been documented. The rate of carriage of serogroup C was reduced in a group of military recruits vaccinated with the polysaccharide capsule from serogroup C or a combination of capsule polysaccharides from serogroups A and C (11, 38). These studies indirectly suggest a protective role of humoral antibodies in mucosal colonization. In other vaccine trials, however, vaccination with serogroup A capsule did not grant a reduction in carriage rate (2, 12). More recently, a significant mucosal immune response has been observed in young adults after immunization with meningococcal A+C conjugate and polysaccharide vaccines (1). Vaccination with meningococcal C conjugate polysaccharide reduced the rate of carriage of serogroup C meningococci by 66% among an adolescent population (22). In this study, hyperimmune mice with whole meningococci were protected from mucosal colonization by the homologous strain. Our results suggest that parenteral immunization could potentially prevent colonization and thereby prevent transmission.

In summary, a model for meningococcal colonization was developed with adult outbred mice. The colonization capabilities of several mutants and the prevention of colonization by immunization were demonstrated. While this model does not accurately mimic all aspects of interactions between the human nasopharyngeal tissue and meningococci, because the organism is highly adapted to humans and its receptors (CD46, human transferrin, lactoferrin, etc.), the model will allow us to study early events of meningococcal pathogenesis.


   ACKNOWLEDGMENTS
 
We thank Anthony Richardson for helpful suggestions.

This study was supported by an Emory University Research Committee award and Public Health Service grant AI472870-01A1.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Emory University School of Medicine, 1510 Clifton Rd. NE, Atlanta, GA 30322. Phone: (404) 727-5968. Fax: (404) 727-3659. E-mail: kyi{at}emory.edu. Back

Editor: F. C. Fang


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Infection and Immunity, April 2003, p. 1849-1855, Vol. 71, No. 4
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.4.1849-1855.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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