Variation in colonization, ADP-ribosylating and vacuolating cytotoxin, and pulmonary disease severity among mycoplasma pneumoniae strains

Abstract

Rationale: Mycoplasma pneumoniae was recently discovered to produce an ADP-ribosylating and vacuolating cytotoxin, designated CARDS toxin, which is hypothesized to be a primary pathogenic mechanism responsible for M. pneumoniae-induced pulmonary inflammation. It is unknown if cytotoxin production varies with M. pneumoniae strain or if variation in cytotoxin production affects pulmonary disease severity.

Objectives: To examine the production of CARDS toxin by various strains of M. pneumoniae and compare the disease manifestations elicited by these strains in an experimental model of M. pneumoniae respiratory infection.

Methods: BALB/c mice were inoculated once intranasally with SP4 broth (negative control) or three different M. pneumoniae strains: M129-B7, M129-B9, or S1. Mice were assessed at 1, 2, 4, 7, 10, and 14 days after inoculation. Outcome variables included comparisons among M. pneumoniae strains relative to bronchoalveolar lavage (BAL) M. pneumoniae quantitative culture, CARDS toxin-based PCR, and CARDS toxin protein determinations, as well as cytokine and chemokine concentrations. Graded lung histopathologic score (HPS) was also assessed.

Measurements and main results: CARDS toxin concentrations were significantly increased in mice inoculated with strain S1 compared with mice inoculated with M129-B7 or M129-B9 strains. Quantitative M. pneumoniae culture and polymerase chain reaction were also significantly greater in mice infected with S1 strain compared with the other two strains, as were lung HPS and concentrations of IFN-γ, IL-12, IL-1α, macrophage inflammatory protein-1α, and keratinocyte-derived chemokine. In addition, a significant positive correlation was found between CARDS toxin concentration and lung HPS.

Conclusions: CARDS toxin concentrations in BAL are directly linked to the ability of specific M. pneumoniae strains to colonize, replicate, and persist, and elicit lung histopathology. This variation among strains may predict the range in severity of pulmonary disease observed among patients.

Figure 1.

Quantitative Mycoplasma pneumoniae (Mp) cultures of bronchoalveolar lavage (BAL) fluid samples from mice inoculated with three different strains of Mp. Lines represent results from 5 to 10 mice per group at each time point from repeated experiments. Values shown are the means ± SD (error bars). * P < 0.05 between the M129-B7 and S1 groups at the time point by one-way analysis of variance (ANOVA) followed by pairwise multiple comparisons. ^δ P < 0.05 between the M129-B9 and S1 groups at the time point by one-way ANOVA followed by pairwise multiple comparisons. CFU = colony-forming units.

Figure 2.

ADP-ribosylating and vacuolating cytotoxin (CARDS) real-time polymerase chain reaction (PCR) of bronchoalveolar lavage (BAL) fluid samples from mice inoculated with SP4, M129-B7, M129-B9, and S1 strains of Mycoplasma pneumoniae. Lines represent results from 6 to 10 mice per group at each time point from repeated experiments. Values shown are the medians and the 25th to 75th percentile (error bars). * P < 0.05 between M129-B7 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^ P < 0.05 between M129-B9 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ⁺ P < 0.05 between S1 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. * P < 0.05 between M129-B7 and S1 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^δ P < 0.05 between M129-B9 and S1 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^γ P < 0.05 between M129-B7 and M129-B9 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons.

Figure 3.

ADP-ribosylating and vacuolating cytotoxin (CARDS) concentration in bronchoalveolar lavage (BAL) fluid sample from mice inoculated with SP4, M129-B7, M129-B9, and S1 strains of Mycoplasma pneumoniae. Lines represent results from 6 to 10 mice per group at each time point from repeated experiments. Values shown are the medians and the 25th to 75th percentile (error bars). * P < 0.05 between M129-B7 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^ P < 0.05 between M129-B9 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ⁺ P < 0.05 between S1 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. * P < 0.05 between M129-B7 and S1 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^δ P < 0.05 between M129-B9 and S1 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^γ P < 0.05 between M129-B7 and M129-B9 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons.

Figure 4.

Lung histopathology score (HPS) from mice inoculated with SP4, M129-B7, M129-B9, and S1 strains of Mycoplasma pneumoniae. Lines represent results from 7 to 10 mice per group at each time point from repeated experiments. Values shown are the medians and the 25th to 75th percentile (error bars). * P < 0.05 between M129-B7 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^ P < 0.05 between M129-B9 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ⁺ P < 0.05 between S1 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. * P < 0.05 between M129-B7 and S1 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^δ P < 0.05 between M129-B9 and S1 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^γ P < 0.05 between M129-B7 and M129-B9 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons.

Figures 5.

Representative lung histopathology from mice 4 days after inoculation with M129-B7, M129-B9, and S1 strains of Mycoplasma pneumoniae. a = arteriole; b = bronchiole; v = vein; Hematoxylin and eosin; ×10 and inset ×40.

Figure 6.

Representative lung histopathology from mice 7 days after inoculation with M129-B7, M129-B9, and S1 strains of Mycoplasma pneumoniae. Hematoxylin and eosin, X2.

Figure 7.

Representative lung histopathology from mice 14 days after inoculation with M129-B7, M129-B9, and S1 strains of Mycoplasma pneumoniae. b = bronchiole; v = vein. Hematoxylin and eosin, ×40.

Figure 8.

(a–e) Cytokine and chemokine concentrations in bronchoalveolar lavage (BAL) fluid specimens in mice inoculated with SP4, M129-B7, M129-B9, and S1 strains of Mycoplasma pneumoniae. Lines represent results from 8 to 10 mice per group at each time point from repeated experiments. Values shown are the medians and the 25th to 75th percentile (error bars). * P < 0.05 between M129-B7 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^ P < 0.05 between M129-B9 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ⁺ P < 0.05 between S1 and SP4 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. * P < 0.05 between M129-B7 and S1 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^δ P < 0.05 between M129-B9 and S1 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons. ^γ P < 0.05 between M129-B7 and M129-B9 groups at the time point by Kruskal-Wallis test followed by pairwise multiple comparisons.