The Immunopathologic Effects of Mycoplasma pneumoniae and Community-acquired Respiratory Distress Syndrome Toxin. A Primate Model.

Mycoplasma pneumoniae infection has been linked to poor asthma outcomes. M. pneumoniae produces an ADP-ribosylating and vacuolating toxin called community-acquired respiratory distress syndrome (CARDS) toxin that has a major role in inflammation and airway dysfunction. The objective was to evaluate the immunopathological effects in primates exposed to M. pneumoniae or CARDS toxin. A total of 13 baboons were exposed to M. pneumoniae or CARDS toxin. At Days 7 and 14, BAL fluid was collected and analyzed for cell count, percent of each type of cell, CARDS toxin by PCR, CARDS toxin by antigen capture, eosinophilic cationic protein, and cytokine profiles. Serum IgM, IgG, and IgE responses to CARDS toxin were measured. All animals had a necropsy for analysis of the histopathological changes on lungs. No animal developed signs of infection. The serological responses to CARDS toxin were variable. At Day 14, four of seven animals exposed to M. pneumoniae and all four animals exposed to CARDS toxin developed histological "asthma-like" changes. T cell intracellular cytokine analysis revealed an increasing ratio of IL-4/IFN-γ over time. Both M. pneumoniae and CARDS toxin exposure resulted in similar histopathological pulmonary changes, suggesting that CARDS toxin plays a major role in the inflammatory response.

NLRP3 Is a Critical Regulator of Inflammation and Innate Immune Cell Response during Mycoplasma pneumoniae Infection.

Mycoplasma pneumoniae is an atypical bacterial respiratory pathogen known to cause a range of airway inflammation and lung and extrapulmonary pathologies. We recently reported that an M. pneumoniae-derived ADP-ribosylating and vacuolating toxin called community-acquired respiratory distress syndrome (CARDS) toxin is capable of triggering NLRP3 (NLR-family, leucine-rich repeat protein 3) inflammasome activation and interleukin-1ß (IL-1ß) secretion in macrophages. However, it is unclear whether the NLRP3 inflammasome is important for the immune response during M. pneumoniae acute infection. In the current study, we utilized in vitro and in vivo models of M. pneumoniae infection to characterize the role of the NLRP3 inflammasome during acute infection. M. pneumoniae-infected macrophages deficient for inflammasome components NLRP3, ASC (apoptosis speck-like protein containing a caspase activation and recruitment domain), or caspase-1 failed to process and secrete IL-1ß. The MyD88/NF-κB signaling pathway was found to be critical for proinflammatory gene expression in macrophages infected with M. pneumoniae C57BL/6 mice deficient for NLRP3 expression were unable to produce IL-1ß in the airways during acute infection, and lack of this inflammatory response led to deficient immune cell activation and delayed bacterial clearance. These findings are the first to report the importance of the NLRP3 inflammasome in regulating the inflammatory response and influencing the progression of M. pneumoniae during acute infection.

Mycoplasma pneumoniae and health outcomes in children with asthma.

BACKGROUND: Acute infections with Mycoplasma pneumoniae (Mp) have been associated with worsening asthma in children. Mp can be present in the respiratory tract for extended periods; it is unknown whether the long-term persistence of Mp in the respiratory tract affects long-term asthma control. OBJECTIVE: To determine the effect of Mp on asthma control. METHODS: We enrolled 31 pediatric subjects 3 to 10 years of age with persistent asthma who completed up to 8 visits over a 24-month period. We detected Mp by antigen capture and polymerase chain reaction. Primary outcome measurements included symptom scores, quality of life, medication scores, oral corticosteroid use, health care usage, school absences, and exhaled breath condensate pH. RESULTS: Low levels of Mp community-acquired respiratory distress syndrome toxin were detected in 20 subjects (64.5%) at enrollment. Subjects with Mp positivity at a given visit had a .579 probability of remaining Mp positive at the subsequent visit, whereas those with Mp negativity had a .348 probability of becoming Mp positive at the following visit. The incidence of Mp overall was higher in the spring and summer months. Overall, we found no significant relation between the detection of Mp and worse outcome measurements at the same visit or at subsequent visits. CONCLUSION: The long-term persistence of Mp in the respiratory tract is common in children with asthma. However, the detection of Mp was not associated significantly with worse asthma symptoms, quality of life, health care usage, school absences, or exhaled breath condensate pH in this pediatric asthma cohort.

Mycoplasma pneumoniae in children with acute and refractory asthma.

BACKGROUND: The presence of Mycoplasma pneumoniae has been associated with worsening asthma in children. Sensitive assays have been developed to detect M pneumoniae-derived community-acquired respiratory distress syndrome (CARDS) toxin. OBJECTIVES: To identify the frequency and persistence of M pneumoniae detection in respiratory secretions of children with and without asthma and to evaluate antibody responses to M pneumoniae and the impact of M pneumoniae on biological markers, asthma control, and quality of life. METHODS: We enrolled 143 pediatric patients (53 patients with acute asthma, 26 patients with refractory asthma, and 64 healthy controls; age range, 5-17 years) during a 20-month period with 2 to 5 follow-up visits. We detected M pneumoniae using CARDS toxin antigen capture and polymerase chain reaction and P1 adhesin polymerase chain reaction. Immune responses to M pneumoniae were determined by IgG and IgM levels directed against CARDS toxin and P1 adhesin. pH was measured in exhaled breath condensates, and asthma control and quality of life were assessed using the Asthma Control Test and Pediatric Asthma Quality of Life Questionnaire. RESULTS: M pneumoniae was detected in 64% of patients with acute asthma, 65% with refractory asthma, and 56% of healthy controls. Children with asthma had lower antibody levels to M pneumoniae compared with healthy controls. Exhaled breath condensate pHs and asthma control and quality of life scores were lower in M pneumoniae-positive patients with asthma. CONCLUSION: The results suggest that M pneumoniae detection is common in children, M pneumoniae detection is associated with worsening asthma, and children with asthma may have poor humoral immune responses to M pneumoniae.

Synthesis and distribution of CARDS toxin during Mycoplasma pneumoniae infection in a murine model.

Mice were infected with Mycoplasma pneumoniae and monitored for the synthesis and distribution of the unique adenosine diphosphate-ribosylating and vacuolating Community Acquired Respiratory Distress Syndrome (CARDS) toxin in bronchiolar lavage fluid (BALF) and lung. We noted direct relationships between the concentration of CARDS toxin and numbers of mycoplasma genomes in BALF and the degree of histologic pulmonary inflammation. Immunostaining of lungs revealed extensive colonization by mycoplasmas, including the detection of CARDS toxin in the corresponding inflamed airways. Lung lesion scores were higher during the early stages of infection, decreased gradually by day 14 postinfection, and reached substantially lower values at day 35. Infected mouse immunoglobulin (Ig) M and IgG titers were positive for CARDS toxin as well as for the major adhesin P1 of M. pneumoniae. These data reinforce the proposed pathogenic role of CARDS toxin in M. pneumoniae-mediated pathologies.

Mycoplasma pneumoniae Community Acquired Respiratory Distress Syndrome toxin expression reveals growth phase and infection-dependent regulation.

Mycoplasma pneumoniae causes acute and chronic respiratory infections, including tracheobronchitis and community acquired pneumonia, and is linked to asthma and an array of extra-pulmonary disorders. Recently, we identified an ADP-ribosylating and vacuolating toxin of M. pneumoniae, designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we analysed CARDS toxin gene (annotated mpn372) transcription and identified its promoter. We also compared CARDS toxin mRNA and protein profiles in M. pneumoniae during distinct in vitro growth phases. CARDS toxin mRNA expression was maximal, but at low levels, during early exponential growth and declined sharply during mid-to-late log growth phases, which was in direct contrast to other mycoplasma genes examined. Between 7% and 10% of CARDS toxin was localized to the mycoplasma membrane at mid-exponential growth, which was reinforced by immunogold electron microscopy. No CARDS toxin was released into the medium. Upon M. pneumoniae infection of mammalian cells, increased expression of CARDS toxin mRNA was observed when compared with SP-4 broth-grown cultures. Further, confocal immunofluorescence microscopy revealed that M. pneumoniae readily expressed CARDS toxin during infection of differentiated normal human bronchial epithelial cells. Analysis of M. pneumoniae-infected mouse lung tissue revealed high expression of CARDS toxin per mycoplasma cell when compared with M. pneumoniae cells grown in SP-4 medium alone. Taken together, these studies indicate that CARDS toxin expression is carefully controlled by environmental cues that influence its transcription and translation. Further, the acceleration of CARDS toxin synthesis and accumulation in vivo is consistent with its role as a bona fide virulence determinant.

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

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.

Persistence of community-acquired respiratory distress syndrome toxin-producing Mycoplasma pneumoniae in refractory asthma.

BACKGROUND: The role of Mycoplasma pneumoniae (Mp) in the initiation and persistence of asthma remains elusive. Mp community-acquired respiratory distress syndrome toxin (CARDS Tx) is a unique virulence factor that induces an intense lymphocytic response and exacerbates asthma in animal models. We sought to determine the incidence of Mp infection and the presence of CARDS Tx in subjects with refractory asthma (RA). METHODS: We conducted a prospective observational study in 64 subjects with RA. Respiratory secretions (sputum, nasal lavage, and throat swab) and blood were analyzed for the presence of CARDS Tx and P1 adhesin (P1) DNA by polymerase chain reaction (PCR), and CARDS Tx by antigen capture. Serum IgM and IgG antibodies to CARDS Tx were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Thirty-three of 64 subjects (52%) tested positive for Mp: 29 of 33 by CARDS Tx vs 10 of 33 by P1 assays. Ten subjects followed longitudinally for up to 633 days tested persistently positive for Mp. There were no significant differences in Mp-specific IgG responses between Mp-positive and Mp-negative groups. Eight of 10 subjects who tested persistently positive failed to mount a substantial IgG response to CARDS Tx, and up to 8 weeks of clarithromycin failed to eradicate Mp in five subjects. CONCLUSIONS: Subjects with RA may be chronically infected with Mp. PCR for CARDS Tx appears to be the most sensitive method of identifying Mp infection. Despite the persistence of Mp in subjects with RA, some subjects failed to mount an IgG response, and macrolide therapy was insufficient to eradicate Mp.

Interaction of Mycoplasma genitalium with host cells: evidence for nuclear localization.

Mycoplasma genitalium (Mg) is a mollicute that causes a range of human urogenital infections. A hallmark of these bacteria is their ability to establish chronic infections that can persist despite completion of appropriate antibiotic therapies and intact and functional immune systems. Intimate adherence and surface colonization of mycoplasmas to host cells are important pathogenic features. However, their facultative intracellular nature is poorly understood, partly due to difficulties in developing and standardizing cellular interaction model systems. Here, we characterize growth and invasion properties of two Mg strains (G37 and 1019V). Mg G37 is a high-passage laboratory strain, while Mg 1019V is a low-passage isolate recovered from the cervix. The two strains diverge partially in gene sequences for adherence-related proteins and exhibit subtle variations in their axenic growth. However, with both strains and consistent with our previous studies, a subset of adherent Mg organisms invade host cells and exhibit perinuclear targeting. Remarkably, intranuclear localization of Mg proteins is observed, which occurred as early as 30 min after infection. Mg strains deficient in adherence were markedly reduced in their ability to invade and associate with perinuclear and nuclear sites.

Cervicitis and genitourinary symptoms in women culture positive for Mycoplasma genitalium.

PROBLEM: Mycoplasma genitalium has been associated with male urethritis. We sought to relate M. genitalium to genitourinary signs and symptoms in women. METHOD OF STUDY: We compared 26 culture-positive women (group 1), 257 additional polymerase chain reaction-positive women (group 2), and 107 negative control women. We used logistic regression to evaluate signs and symptoms, controlling for co-infections, pregnancy, age, and intervention group assignment. RESULTS: Comparing group 1 with controls, we found significantly elevated odds ratios (ORs) for intermediate vaginal discharge (OR = 5.4; 95% confidence interval 1.01, 29.2) and action in response to discharge [3.9 (1.1, 13.5)]. Non-significant increases were observed for pathologic vaginal discharge [3.8 (0.78, 18.2)], pathologic dyspareunia [1.5 (0.25, 9.0)], vaginal odor [2.1 (0.75, 5.7)], and cervical mucopus [4.1 (0.74, 22.4)]. Group 2 results were similar, but showed no increase in cervical mucopus relative to controls. CONCLUSION: Infection with M. genitalium in women is independently related to increased genitourinary symptomatology.

Diagnostic assessment of Mycoplasma genitalium in culture-positive women.

Detection of Mycoplasma genitalium-mediated, chlamydia-negative nongonococcal urethritis and other M. genitalium-linked infectious etiologies has been very challenging. Although M. genitalium is considered a leading cause of genitourinary symptoms in men and women, extreme difficulties in its cultivation due to its highly fastidious nature and the lack of routine and effective diagnostic tests have slowed the generation of clinical data which directly implicate the presence of M. genitalium in disease pathogenesis. In this study, we compared enzyme-linked immunosorbent assays (ELISAs) and immunoblot and PCR assays in M. genitalium culture-positive women over 1 to 3 years of clinical visits to determine the usefulness of independent diagnostic strategies. Furthermore, the value of combinatorial diagnostic assessments is described, which provides insights into the dynamics of M. genitalium-host interactions. Overall, we show that neither ELISA nor PCR, alone or in combination, provides the sensitivity required to confidently predict the existence of viable M. genitalium organisms in cervical and vaginal samples. Additionally, culture-positive women exhibited a range of antibody responsiveness to M. genitalium based upon ELISA and immunoblot assessments, indicating immune diversity among this high-risk population.