Vaccination of BALB/c Mice with an Avirulent Mycoplasma pneumoniae P30 Mutant Results in Disease Exacerbation upon Challenge with a Virulent Strain

All M. pneumoniae strains, including PI1428 (passage 12) (5) and M129 (passage 13), were cultured at 37°C in fortified commercial (FC) or Hayflick's medium (10% horse serum, 5% yeast extract). The generation and characterization of P30 mutants II-3 and II-3R have been previously described (24, 32). Isogenic P30 mutant P-130 was generated from an M. pneumoniae transposon (Tn) mutant library (min-Tn pMT85, an aliquot of the same mouse challenge lot of strain PI1428, was used to generate the library) using the haystack mutagenesis approach, as previously described (40). Gentamicin sulfate (80 μg/ml) was added to the medium for the propagation of the transposon mutant. For mouse studies, frozen 50-μl aliquots of each strain were thawed, and 10 ml of FC medium was added to the tube. Cultures were incubated at 37°C with gentle shaking at 120 RPM on an orbital shaker. After 5 h of incubation, optical density at 620 nm (OD₆₂₀) measurements were aseptically conducted on a spectrophotometer to estimate CFU counts per ml of culture, and color changing unit (CCU) measurements were conducted using 10-fold serial dilutions to ensure that spectrophotometric measurements represented live bacteria. Samples were then centrifuged at 2,000 × g for 10 min at 4°C, the supernatant was decanted, and the pellet was suspended in a volume of fresh medium that was calculated to give the desired concentration, given the total CFU measured in the tube (calculated as follows: CFU/ml × volume [in ml] before centrifugation = CFU [pellet]; CFU [pellet]/X volume = CFU [desired]/volume [desired], where “X” is the variable volume added to the pellet).

Protein expression was analyzed by Western blotting as previously described (4) but using antibodies and concentrations described elsewhere (10). Transposon mutant P-130 was assessed qualitatively for hemadsorption as previously described (16). Satellite colony formation was examined after 60 h of growth in borosilicate glass chamber slides (Nunc Nalgene, Naperville, IL) as a qualitative assessment of gliding motility or at 37°C for 18 h for quantitative calculation of gliding motility for individual cells (>1,000 cells observed for gliding frequency and at least 38 cells per strain for mean and corrected gliding velocities), as previously described (18).

All animal experiments were conducted in accordance with our approved Institutional Animal Care and Use Committee protocol (protocol A09-012). Mycoplasma and murine virus-free BALB/c mice (2 months old, female) were acquired from Charles River Laboratories (Worcester, MA). Mice were allowed to acclimate in filter-top cages (5 mice per cage) for 1 week in a biosafety hood prior to use. Mice were sedated using vaporized isoflurane before being handled. Four animal experiments were conducted: one to assess the virulence of mutant P-130 (study 1), another to assess the virulence of mutant II-3R (study 2), a vaccination and challenge study to determine the efficacy of the P30 mutant P-130 as a vaccine candidate (study 3), and a vaccination and challenge study of the immune response after challenge (study 4). Groups of mice were inoculated by the intranasal route with 50 μl of 7 × 10⁷ CFU (studies 1 and 2) or 1 × 10⁷ CFU (studies 3 and 4) of the respective M. pneumoniae strain (n = 5 for each group except study 4, for which n = 3). For studies 1 and 2, negative-control mice were inoculated with sterile FC medium and positive-control mice were inoculated with wild-type PI1428 (study 1) or M129 (study 2). For studies 3 and 4, negative-control mice were given sterile FC medium on day 0 (vaccination) and day 21 (challenge), positive-control mice were sham vaccinated with sterile FC medium (day 0) and challenged with virulent strain PI1428 (day 21), and test groups were vaccinated with P30 mutant strain P-130 (day 0) and challenged with strain PI1428 (day 21). Mice were humanely sacrificed 4 days postchallenge via cervical dislocation after all experiments, and the lungs were immediately harvested for histopathology and mycoplasma culture (studies 1, 2, and 3) or subjected to bronchoalveolar lavage (study 4). For mycoplasma recovery, the lower right lung was removed and placed into 3 ml of FC medium, briefly vortexed, and incubated for 3 h at 37°C. The remaining tissue was inflated with 10% neutral buffered formalin and allowed to fix for histopathologic evaluation. After 3 h of incubation, mycoplasma recovery samples were passed through a 0.45-μm filter and transferred to new tubes. Acidic cultures (as indicated by a color shift from red to orange or yellow) were adjusted to an approximate pH of 7.4 by adding 10 N NaOH until a red color was restored. Quantification of recovery cultures was performed by assessing CCU in neat and 10-fold serial dilutions (50 μl into 450 μl FC medium, out to 10⁻⁵ dilution). Samples were incubated for 28 days or until a color shift was observed.

After fixation for 48 h in 10% buffered formalin, tissues were routinely processed and stained as previously described (40). Perivascular and peribronchial lymphocytic infiltrates were graded in increasing severity with scores ranging from 0 (no visible lesions) to 4 (marked lesions), in increments of 1. Lesion score evaluations were performed in a blinded fashion by a board-certified veterinary pathologist who was unaware of the experimental groups.

Processing of bronchoalveolar lavage fluid (BALF) and subsequent differential leukocyte counts were conducted as described by Siddiqui et al. (36). Data are represented as the percentage of each leukocyte cell type per 500 total white blood cells. BALF supernatants were concentrated 10-fold using Amicon Ultra-4 3K centrifugal filter devices (Millipore Corp., Billerica, MA), per the manufacturer's instructions, before cytokine analysis. Cytokine concentrations in 10× BALF (interleukin 1β [IL-1β], IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17, gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) were measured using a custom-made Bioplex array system (Bio-Rad, Hercules, CA) on a Bio-Rad Bioplex-200 system, with recombinant murine cytokines as standards. The postassay sample concentration and limits of detection were reported, taking into consideration the 10× sample concentration prior to analysis. Luminex bead array software was used to analyze the data.

Lesion scores for all experiments were analyzed between the test group and the positive- and negative-control groups by the nonparametric Kruskal-Wallis analysis of variance (ANOVA) on ranks test with a Student-Newman-Keuls (SNK) post hoc test for multiple pairwise comparisons between groups (α = 0.05). Quantitative gliding motility, differential leukocyte counts, and cytokine concentrations for all experiments were analyzed between the test group and the positive- and negative-control groups by the parametric one-way ANOVA test with a Student-Newman-Keuls post hoc test for multiple pairwise comparisons between groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001). A surrogate value which fell below the limit of detection (lod) was used for statistical analysis of BALF cytokine concentrations in animals by subtracting 0.1 from the lod. All data were analyzed using the software program SigmaPlot for Windows, version 11.0 (Systat Software Inc., San Jose, CA).

An isogenic M. pneumoniae P30 mutant was isolated from a library of more than 1,900 Tn mutants generated in strain PI1428 using the haystack mutagenesis approach. This mutant was designated P-130, as the 130th clone picked from the PI1428 mutant library. The Tn insertion mapped to nucleotide 156 (of 825) of the P30 gene, resulting in disruption of 81% of the coding sequence.

P-130 protein expression of cytadherence and cytoskeletal proteins (P200, HMW1, HMW2, HMW3, P1, B, C, P65, P41, P30, P28, and P24) was assessed by Western blotting and compared to the previously characterized P30 mutant II-3 and wild-type M129 (Fig. 1A). No P30 protein was detected in P-130 (as expected), and P65 was also noticeably reduced, as previously described for other P30 mutants (21). Of note, mutant P-130 also had reduced steady-state levels of the HMW3 protein, a phenotype that has not been previously described for P30 mutants. The gene for HMW3 is immediately downstream of the gene for P30, and they appear to be cotranscribed (43). Disruption of P30 in transposon mutant P-130 may have a polar effect on HMW3 transcription, whereas the frameshift in mutant II-3 may not, accounting for the difference in HMW3 expression in these mutants. To ensure that the observed reduction in protein levels for P30, P65, and HMW3 was not due to nucleotide (and hence amino acid/antigenic) differences between strains M129 (for which antibody binding has been verified for each protein) and PI1428 (the parental strain of P-130), we generated a draft sequence of the complete genome of strain PI1428 (data not shown) and compared these genes with the published M129 genome (12). No amino acid differences were observed in these predicted proteins, consistent with the conclusion that levels of proteins P30, P65, and HMW3 in mutant P-130 were reduced as a result of disruption of the P30 gene.

The previously characterized P30 mutant II-3 has been shown to have marked reductions in hemadsorption and gliding motility (18, 32). Erythrocyte binding, satellite growth, and individual cell gliding for mutant P-130 were also negligible compared to those for M129 (Fig. 1B and C). Thus, the P30 mutant P-130 exhibits deficient attachment and motility phenotypes, similar to those observed in previously characterized P30 mutants.

In previous work, mutant II-3 was inoculated into hamsters to assess its virulence in vivo and was shown to be avirulent 14 days postinoculation (24). Given that II-3 was a naturally arising hemadsorption-negative mutant, it is not known if other mutations that contribute to the attenuated phenotype potentially exist in this strain. To confirm that P30 is indeed essential for virulence, we intranasally inoculated BALB/c mice with a high dose of 7 × 10⁷ CFU of isogenic mutant P-130, PI1428 (positive control), or sterile FC medium (negative control) and performed pathological assessments (Fig. 2A) and mycoplasma recovery (Fig. 2B) from the lungs 4 days postinoculation (the time point which corresponds with peak lesions in these mice [17]). Consistent with the attenuation of mutant II-3 in hamsters, P-130 was avirulent in the lungs of mice based on pathological assessments and the absence of mycoplasma recovery. These data indicate that cytadhesin P30 is essential for the virulence of M. pneumoniae in BALB/c mice.

Given the similar avirulent phenotype shared by P30 mutants II-3 and P-130, as well as the lack of hemadsorption and gliding motility of both strains, interest was raised in the virulence phenotype of II-3 revertant II-3R, which exhibits near-wild-type hemadsorption but severely reduced gliding motility (18). Again, mice were intranasally inoculated and lesions/recovery was assessed 4 days later. II-3R was not recoverable from the lungs of mice (Fig. 2D), and lesions in this group were significantly less severe than those observed in the M129-inoculated animals (Fig. 2C), indicating that this mutant is also attenuated. Thus, it appears that gliding motility, independent of hemadsorption, is essential for the virulence and in vivo survival of M. pneumoniae in BALB/c mice.

Given that a high dose of the gliding-motility and hemadsorption P30 mutant P-130 resulted in no lung lesions after intranasal inoculation in BALB/c mice, we initiated a vaccination/challenge study to explore whether this mutant might serve as a potential vaccine candidate. A standard dose of 1 × 10⁷ CFU of P130 was intranasally administered to groups of mice, and 3 weeks later the animals were challenged with a standard dose of strain PI1428. Negative controls included animals inoculated with sterile FC medium at the vaccination and challenge time points, while sham-vaccinated (FC medium) and challenged (PI1428) mice served as positive controls. Four days postchallenge, pathological assessments and mycoplasma recovery were assessed as described above (Fig. 3A and B). Given the lack of recovery of this mutant in the virulence experiment, any recovered mycoplasmas were considered to be wild-type PI1428, so additional genotyping was not conducted. Lesion scores were compared between the vaccinated group and controls, and vaccinated animals were statistically different from both sham-vaccinated and negative controls (which were also significantly different from each other). The lesion scores in the vaccinated group were actually higher than those observed in the sham-vaccinated group, indicating that disease exacerbation had occurred upon challenge with a virulent strain. There was also more frequent mycoplasma recovery in the vaccinated group, indicating that the increased severity of the lesions may be beneficial for the in vivo survival of M. pneumoniae or that persistence of the bacterium resulted in more severe disease, perhaps as a result of immune dysregulation.

An understanding of the mechanism(s) of disease exacerbation is essential for rational design of an M. pneumoniae vaccine. Eosinophilic and neutrophilic infiltration was noted in the lesions of many animals in the vaccination study; hence, a second vaccination/challenge experiment was conducted and BALF was collected to determine the proportions of different leukocytes (Fig. 4) and cytokines (Fig. 5) in lavage samples. Differential counts (based on cell morphology and staining characteristics) of macrophages, neutrophils, eosinophils, and lymphocytes were performed with BALF samples from each animal after cytocentrifugation and cell staining. A statistically significant reduction in the percentage of macrophages was observed in the vaccinated group in part due to the significant increase in the proportion of eosinophils. Conversely, the sham-vaccinated group had a significantly higher proportion of neutrophils than all other groups. Thus, vaccination with mutant P-130 appears to dysregulate the postchallenge infiltration of neutrophils observed in sham-vaccinated controls to eosinophilic infiltration.

The concentration of various cytokines (IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17, IFN-γ, and TNF-α) in BALF was determined using a Luminex bead assay. These cytokines were chosen to evaluate the potential Th1/Th2/Th17 responses that may influence the infiltration of different populations of white blood cells into the lung mucosa. There was a statistically significant increase in the presence of IL-1β and IL-17 in the BALF of vaccinated animals compared to controls. IL-4, IL-10, IL-12p70, and IFN-γ were not detected in any samples above the limit of detection. Of note, IL-6 and TNF-α were detected at higher levels in two out of three mice in the vaccinated group than in controls. The expression of IL-1β, IL-6, and TNF-α (in the absence of IL-4, IFN-γ, or IL-10) has been shown to augment IL-17 responses (reviewed in reference 29) and may contribute to the exacerbated disease observed in the vaccinated mice.