The recent observation that M. pneumoniae
possesses an ADP-ribosylating and vacuolating cytotoxin (designated CARDS toxin) provides a mechanism to explain the host cell injury observed with M. pneumoniae
). The respiratory tract pathogens Corynebacterium diphtheriae
and Bordetella pertussis
both produce ADP-ribosylating toxins critical in the pathogenesis of their respective diseases. The control of clinical diphtheria with the use of diphtheria toxoid (inactivated ADP-ribosylating diphtheria toxin) in immunizations attests to the essential role of this toxin in causing disease (31
). The role of CARDS toxin in M. pneumoniae
–associated disease remains to be dissected.
This study sought to compare the relationship of quantitative cultures, CARDS toxin production, cytokine response, and disease manifestations of mice infected with three different strains of M. pneumoniae. We demonstrated that all three strains produced CARDS toxin; however, pulmonary disease severity was associated with strain-dependent replication and persistence and concentration of CARDS toxin produced. The concentration of CARDS toxin in the BAL of mice inoculated with strain S1 was significantly greater than that of mice inoculated with the other two strains and appeared to be directly related to strain S1 colonization and survival properties. Interestingly, this is consistent with replication and survival properties of S1, especially during the early stages of mycoplasma colonization and establishment of infection. This virulence-related property of S1 has been repeatedly observed by us (unpublished data). At later days post infection, values for M. pneumoniae quantitative culture, CARDS toxin concentration, and HPS strongly correlated for all strains. In addition, mice inoculated with strain S1 displayed the greatest histologic lung inflammation. Although these results implicate CARDS toxin as contributing to the severity of M. pneumoniae disease, they do not clearly establish direct causality.
Along with the finding of significantly greater histologic pulmonary inflammation with strain S1, this strain also induced significantly greater concentrations of IL-12 and IFN-γ compared with the M129-B7 and M129-B9 strains, with no differences detected between strains for IL-4 concentrations. These results reconfirm the findings of recent investigations that have shown a significant role for IL-12 and IFN-γ in the immunopathogenesis of inflammation in Mycoplasma
respiratory infection (27
). It should be noted that the cytokine immunopathogenesis of M. pneumoniae
infection appears to be different between hosts with and without allergic sensitization of the airways, especially regarding pulmonary IL-4 (36
In support of a contributory role of CARDS toxin in M. pneumoniae
pathogenesis, we recently investigated the ability of recombinant CARDS toxin to elicit inflammation in the lungs of both mice and baboons. These animals responded to respiratory exposure of recombinant CARDS toxin in a dose-dependent manner with increased expression of the proinflammatory cytokines IL-1α, IL-1β, IL-6, IL-12, IL-17, TNF-α, and IFN-γ, as well as several growth factors and chemokines, including KC, IL-8, regulated upon activation normal T-cell expressed and secreted, and granulocyte colony–stimulating factor (G-CSF) G-CSF. Recombinant CARDS toxin exposure to the airways of these animals also resulted in cellular inflammatory responses characterized by a dose-dependent early vacuolization and cytotoxicity of the bronchiolar epithelium followed by a robust peribronchial and perivascular lymphocytic infiltration. Furthermore, recombinant CARDS toxin caused airway hyperreactivity in mice after toxin exposure as well as prolonged airway obstruction. The changes in airway function, cytokine expression, and cellular inflammation correlated temporally and were consistent with what has been reported in M. pneumoniae
infection. Therefore, these findings indicate that the response to M. pneumoniae
CARDS toxin parallels the pulmonary inflammatory responses and airway dysfunction observed with M. pneumoniae
Taken as a whole, the results of this investigation indicate that the severity of pulmonary disease caused by M. pneumoniae
can be strain- and toxin concentration–dependent. Consistent with this observation are the results attained from strains M129-B7 and M129-B9, which, given their similar background, were relatively comparable for all endpoints. This contrasts with a previous in vitro
investigation that found that proinflammatory cytokine production was not dependent on infecting M. pneumoniae
). Although in our investigation the S1 strain (subtype 2) was considerably more virulent than the two related M129 strains (subtype 1), it would be premature to state that all subtype 2 strains are more virulent than subtype 1 strains. However, it may be of value for future epidemiological investigations to determine the microbial characteristics of infecting M. pneumoniae
organisms regarding CARDS toxin production (high or low) and subtype colonization and persistence, as these microbial factors may be important in clinical manifestations of infection, such as disease severity, wheezing, or encephalitis.