Community-acquired Respiratory Distress Syndrome Toxin Research Articles

Loop-Mediated Isothermal Amplification Coupled With Nanoparticle-Based Biosensor: A Rapid and Sensitive Method to Detect Mycoplasma pneumoniae.

Mycoplasma pneumoniae (MP), the causative agent of MP pneumonia (MPP), has posed a substantial burden to public health owing to a lack of rapid and effective diagnostic methods. Here, we designed a loop-mediated isothermal amplification (LAMP)-based assay, termed LAMP, combined with a nanoparticle-based lateral flow biosensor (LAMP-LFB) for rapid and sensitive diagnosis of MP.-LAMP-LFB included a set of six primers targeting the community-acquired respiratory distress syndrome (CARDS) toxin gene and was performed optimally at 63°C for only 30 min. The resulting LAMP products could be visually indicated by LFB within 2 min, thus the whole process could be accomplished within an hour. MP-LAMP-LFB’s sensitivity was 50 fg per reaction, which was in complete accordance with these results obtained from real-time turbidity and visual detection reagent (VDR). MP-LAMP-LFB had no cross-reactivity with other pathogens that had similar clinical presentations. Our assay was further validated using 100 nasopharyngeal swab samples collected from children suspected of MPP, and the result was compared with the real-time PCR method. With a positive rate of 50%, the data indicated that MP-LAMP-LFB is a sensitive test for MP detection in clinical settings. Collectively, the MP-LAMP-LFB assay targeting the CARDS toxin gene was a rapid, highly sensitive, and specific test that could be widely applied in point-of-care settings and basic medical facilities in rural areas.

High Expression of MUC5AC, MUC5B, and Layilin Plays an Essential Role in Prediction in the Development of Plastic Bronchitis Caused by MPP.

Plastic bronchitis (PB) is a rare respiratory condition which can result in severe respiratory complications such as respiratory failure and death. Mycoplasma pneumoniae infection is a main etiology cause of plastic bronchitis. However, the pathogenesis of plastic bronchitis complicated by Mycoplasma pneumoniae pneumonia (MPP) has not yet been fully elucidated. Our article aims to explore biomarkers for early prediction of MPP cases complicated with plastic bronchitis. We utilized a protein chip to screen for significantly different proteins among the groups of healthy, general Mycoplasma pneumoniae pneumonia (GMPP) and refractory Mycoplasma pneumoniae pneumonia (RMPP) patients, where layilin exhibited a potent change across biology information technology. Next, we demonstrated the high expression of MUC5AC, MUC5B, and layilin in bronchoalveolar lavage fluid (BALF) of MPP cases complicated with plastic bronchitis. Further study suggested that the level of layilin had a positive correlation with both MUC5AC and MUC5B. A receiver operating characteristic (ROC) analysis was performed to assess the diagnostic values of MUC5AC, MUC5B, and layilin in MPP cases with PB. Data show that the three indicators have similar diagnostic ability for MPP children with plastic bronchitis. Then, we used different concentrations of community-acquired respiratory distress syndrome (CARDS) toxin or lipid-associated membrane proteins (LAMPs) to simulate an in vitro experiment. The in vitro assay revealed that CARDS toxin or LAMPs induced A549 cells to secrete MUC5AC, MUC5B, layilin, and proinflammatory factors. These findings suggest that MUC5AC, MUC5B, and layilin are correlated with MPP. The high expression of MUC5AC, MUC5B, and layilin play an essential role in prediction in the development of plastic bronchitis caused by MPP. The high expression of MUC5AC, MUC5B, and layilin may be relevant to the severity of illness.

Investigation of the relationship between community-acquired respiratory distress syndrome toxin and the high-mobility group box protein 1-toll-like receptors-myeloid differentiation factor 88 signaling pathway in Mycoplasma pneumoniae pneumonia

BackgroundIn recent years, reports of refractory Mycoplasma pneumoniae pneumonia (RMPP) have gradually increased, including reports on how these conditions threaten the lives of children. However, the specific mechanism of Mycoplasma pneumoniae pneumonia (MPP) remains unclear. This study aimed to investigate the relationship between community-acquired respiratory distress syndrome toxin (CARDS TX) and High-mobility group box protein 1-Toll-like receptors-Myeloid differentiation factor 88 (HMGB1-TLRs-MyD88) in MPP and to examine the immune pathogenesis of Mycoplasma pneumoniae infection.MethodsChildren who were diagnosed with MPP and examined by bronchoscopy were included in the MPP group. Additionally, children who underwent bronchoscopy because of bronchial foreign bodies in the same period were included in the control group. Gene expression of CARDS TX, HMGB1, Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), MyD88, and cluster of differentiation 14 (CD14) in bronchoalveolar lavage fluid (BALF) were detected using real-time reverse transcription-polymerase chain reaction. Correlations between CARDS TX and HMGB1-TLRs-MyD88 were analyzed.ResultsCARDS TX, HMGB1, TLR2, MyD88, and CD14 mRNA expression in BALF in the MPP group was significantly higher than that in the control group (all P < 0.05). CARDS TX mRNA expression was positively correlated with HMGB1, TLR2, MyD88, and CD14 mRNA expression (all P < 0.05). Furthermore, HMGB1 mRNA expression was positively correlated with TLR2, MyD88, and CD14 mRNA expression (all P < 0.05).ConclusionsCARDS TX may participate in the immune pathogenesis of MPP through the HMGB1-TLRs/CD14-MyD88 pathway.

Role of Mycoplasma pneumoniae CARDS toxin in inducing mitochondrial damage and inflammation.

Abstract Known for its ability to cause damaging airway inflammation and decreased lung compliance, Mycoplasma pneumoniae does so through the production of the Community-Acquired Respiratory Distress Syndrome Toxin (CARDS toxin.) This toxin plays a key role in the inflammation seen during infection. Once M. pneumoniae is able to establish infection in the lungs and produce CARDS toxin, harmful inflammation followed by cell death and tissue injury follows. Although CARDS toxin is known to have vacuolating and ADP-ribosylating properties, the exact mechanism by which it triggers an inflammatory response is still being elucidated. Experimental evidence from our lab supports that CARDS toxin can localize to the mitochondria in vitro, and this localization likely triggers mitochondrial damage and an increase in cell death. In our current study, recombinant CARDS toxin was administered to lung epithelial cells and macrophages exogenously, as well as endogenously via the expression of the CARDS toxin gene on a mammalian expression plasmid. Endogenously-expressed CARDS toxin was analyzed by western blot. Additionally, markers of mitochondrial damage and cytokine production were measured. The ADP-ribosylation and vacuolation properties of CARDS toxin were also tested for their ability to trigger inflammatory cytokine production. Our results support our hypothesis that CARDS toxin localization to the mitochondria is required to trigger mitochondrial damage and thus promote inflammation. By better understanding the mechanism through which CARDS toxin affects the mitochondria, we will gain more insight into how M. pneumoniae infects the host, and which therapeutic strategies could be most effective for future outbreaks. Supported by grant from NIH T34GM008073

Effects of Mycoplasma pneumoniae CARDS Toxin on mitochondrial homeostasis

Abstract Mycoplasma pneumoniae is an atypical bacterial pathogen known to cause mild respiratory infections and pneumonia. M. pneumoniae produces Community-Acquired Respiratory Distress Syndrome toxin (CARDS toxin), a virulence factor that plays a primary role in pathogenesis. CARDS toxin is known to have dramatic vacuolating and inflammatory properties, however these properties have not been fully characterized. Recent evidence from our lab has supported that the toxin localizes on the mitochondria, affecting homeostasis and triggering inflammation. Our current study is focused on characterizing the effects of CARDS toxin on mitochondrial stress and production of mitochondrial reactive oxygen species (ROS). A549 lung epithelial cells and IMR-90 lung fibroblasts were treated with recombinant CARDS toxin exogenously, and mitochondrial ROS was measured. Additionally, we sought to determine whether ADP-ribosylation or vacuolization were required for CARDS toxin-induced mitochondrial stress by expressing CARDS toxin truncations on a mammalian expression plasmid. Endogenously expressed CARDS toxin truncations were fused to either EGFP or mCherry2. CARDS toxin was detected by western blot and IFA was used to track CARDS toxin localization in the cell. Our findings support our hypothesis that CARDS toxin disrupts mitochondrial homeostasis and triggers mitochondrial ROS production. Our results provide valuable insight into the effects of CARDS toxin on mitochondrial function, which will help us better characterize the role of CARDS toxin during M. pneumoniae pathogenesis. Supported by NIH T34GM008073

Vaccine using community-acquired respiratory distress syndrome toxin as an antigen against Mycoplasma pneumoniae in mice

Mycoplasma pneumoniae (Mp) is one of the most common causes of bacterial community-acquired pneumonia in humans. Because of the frequent epidemics and the emergence of antibiotic-resistant Mp, vaccines for Mp are urgently needed to ameliorate the pneumonia and secondary complications. The community-acquired respiratory distress syndrome (CARDS) toxin produced by Mp is a pathogenic factor that induces severe inflammatory responses in lung. Although blocking CARDS toxin is expected to mitigate the severity of Mp pneumonia, the potential of CARDS toxin as a vaccine antigen has not been assessed. Here, we examined the effectiveness of vaccine using recombinant CARDS toxin (rCARDS toxin) as an antigen in mice. Immunization with rCARDS toxin induced both rCARDS toxin- and Mp-specific antibody responses, indicating that CARDS toxin is located on the surface of Mp. In addition, immunization with rCARDS toxin decreased not only lung injury, neutrophil infiltration, and the production of inflammatory cytokines but also the persistence of Mp in lung after Mp challenge. Furthermore, we elucidated that the CARDS toxin on the surface of Mp facilitates the adherence of Mp to epithelial cells. In conclusion, we have demonstrated the potential of rCARDS toxin as a vaccine antigen to ameliorate Mp pneumonia by suppressing the inflammatory responses induced by Mp and the persistence of Mp in lung. These data support the development of novel vaccines for Mp pneumonia.

Community-Acquired Respiratory Distress Syndrome Toxin: Unique Exotoxin for M. pneumoniae.

Mycoplasma pneumoniae infection often causes respiratory diseases in humans, particularly in children and adults with atypical pneumonia and community-acquired pneumonia (CAP), and is often exacerbated by co-infection with other lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disorder. Community-acquired respiratory distress syndrome toxin (CARDS TX) is the only exotoxin produced by M. pneumoniae and has been extensively studied for its ADP-ribosyltransferase (ADPRT) activity and cellular vacuolization properties. Additionally, CARDS TX induces inflammatory responses, resulting in cell swelling, nuclear lysis, mucus proliferation, and cell vacuolization. CARDS TX enters host cells by binding to the host receptor and is then reverse transported to the endoplasmic reticulum to exert its pathogenic effects. In this review, we focus on the structural characteristics, functional activity, distribution and receptors, mechanism of cell entry, and inflammatory response of CARDS TX was examined. Overall, the findings of this review provide a theoretical basis for further investigation of the mechanism of M. pneumoniae infection and the development of clinical diagnosis and vaccines.

Evaluation of the CARDS toxin and its fragment for the serodiagnosis of Mycoplasma pneumoniae infections.

Mycoplasma pneumoniae (M. pneumoniae) is an important pathogen in community-acquired pneumonia. The community-acquired respiratory distress syndrome (CARDS) toxin is the only known virulence factor of M. pneumoniae. It is worth exploring whether this toxin can be used as a candidate antigen for the serodiagnosis of M. pneumoniae. In this study, the full-length, N-terminal, and C-terminal regions of the CARDS toxin were expressed and purified, and serological reactions were evaluated using ELISA. A total of 184 serum samples were collected and tested using a commercialized test kit. Eighty-seven samples were positive, and 97 samples were negative for infection. The purified recombinant proteins were used as antigens to test the serum via indirect ELISA. The sensitivity of the CARDS toxin, the N-terminal region, and the C-terminal region were 90.8%, 90.8%, and 92.0%, respectively. The specificity of the CARDS toxin, the N-terminal region, and the C-terminal region were 85.6%, 73.2%, and 93.8%, respectively. All three CARDS toxin proteins exhibited good reactivity, of which the C-terminal region had a good discrimination ability in human sera. This may have a potential diagnostic value for M. pneumoniae infections.

Interleukin-23 derived from CD16+ monocytes drives IL-17 secretion by TLR4 pathway in children with mycoplasma pneumoniae pneumonia

AimsThe study aimed to investigate whether IL-23 is amplified in monocyte subsets of MP pneumonia and to determine its relevant pathway. Materials and methodsWe firstly analyze the IL-23p19 expression in monocyte subgroups in MP pneumonia patients and healthy controls subjects by using flow cytometry. Then, we also analyzed the percentage of IL-17+γδT cells and Th17 cells in patients with MP pneumonia and controls subjects. At the same time, the relation between IL-23 and IL-17 were also assessed. Furthermore, we constructed the recombinant community-acquired respiratory distress syndrome (CARDS) toxin and intend to stimulate peripheral blood mononuclear cells and RAW264.7 cells in vitro. IL-23p19 was detected by flow cytometry and the mRNA levels were measured by real-time PCR. Finally, TLR4 pathway was also investigated by TAK242 inhibitor. Key findingsIt turned out that the expression of IL-23p19 was increased in CD14brightCD16+ monocyte of MP pneumonia patients than controls subjects. The patients with MP pneumonia had significantly higher the percentage of IL-17+γδT cells and Th17 cells than controls subjects. Interestingly, the levels of IL-23 were positively related to IL-17 in MP pneumonia patients. CD16+ monocytes and RAW264.7 cells, respectively can be induced by CARDS toxin to secrete IL-23 by TLR4 pathway in vitro. SignificanceThese results indicated that IL-23-IL-17+γδT/Th17 axis may play a role in the pathogenesis of MP pneumonia, whereas IL-23 derived from CD16+ monocytes was expanded in MP pneumonia by TLR4 pathway.

Mycoplasma pneumoniae biofilms grown in vitro: traits associated with persistence and cytotoxicity.

The atypical bacterial pathogen Mycoplasma pneumoniae is a leading etiological agent of community-acquired pneumonia in humans; infections are often recalcitrant, recurrent and resistant to antibiotic treatment. These characteristics suggest a mechanism that facilitates long-term colonization in hosts. In an in vitro setting, M. pneumoniae forms biofilms that are unusual in that motility plays no more than a very limited role in their formation and development. Given the unusual nature of M. pneumoniae biofilms, open questions remain concerning phenotypes associated with persistence, such as what properties might favour the bacteria while minimizing host damage. M. pneumoniae also produces several cytotoxic molecules including community-acquired respiratory distress syndrome (CARDS) toxin, H2S and H2O2, but how it deploys these agents during growth is unknown. Whereas several biochemical techniques for biofilm disruption were ineffective, sonication was required for disruption of M. pneumoniae biofilms to generate individual cells for comparative studies, suggesting unusual physical properties likely related to the atypical cell envelope. Nonetheless, like for other bacteria, biofilms were less susceptible to antibiotic inhibition and complement killing than dispersed cells, with resistance increasing as the biofilms matured. CARDS toxin levels and enzymatic activities associated with H2S and H2O2 production were highest during early biofilm formation and decreased over time, suggesting attenuation of virulence in connection with chronic infection. Collectively, these findings result in a model of how M. pneumoniae biofilms contribute to both the establishment and propagation of M. pneumoniae infections, and how both biofilm towers and individual cells participate in persistence and chronic disease.

High co-expression of TNF-\u03b1 and CARDS toxin is a good predictor for refractory Mycoplasma pneumoniae pneumonia

BackgroundEarly distinction between refractory M. pneumoniae pneumonia (RMPP) and non-RMPP (NRMPP) is still difficult. The community-acquired respiratory distress syndrome (CARDS) toxin can induce inflammatory and histopathological phenotypes associated with M. pneumoniae infection. This study aimed to investigate the clinical significance of CARDS toxin and pro-inflammatory cytokines in children with RMPP and to explore whether CARDS toxin can induce TNF-α expression.MethodsLevels of CARDS toxin and cytokines in BALF from control and children with MPP were determined by real-time PCR and ELISA, respectively. A receiver-operating characteristic (ROC) analysis was performed to assess the diagnostic values of CARDS toxin, TNF-α, and IL-6 in RMPP. The recombinant CARDS toxin was constructed and prepared at different concentrations for stimulation of RAW264.7 cells. After co-culture with CARDS toxin, cytokines were detected by ELISA and the mRNA levels were measured by real-time PCR. Effects of CARDS toxin and TNF-α on inflammatory cell infiltration and mucus secretion in mouse lungs were also evaluated.ResultsLevels of CARDS toxin, TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were significantly higher in RMPP cases compared with NRMPP cases. Furthermore, TNF-α had better diagnostic ability for differentiation of RMPP with AUC of 0.824 and Youden index of 0.692 compared with CARDS toxin and IL-6. Moreover, CARDS toxin was positively correlated with TNF-α level in MPP cases. In vitro assay revealed that CARDS toxin induced RAW264.7 macrophages to secrete TNF-α. Further in vivo assay showed that TNF-α deletion partially abrogated the CARDS toxin-mediated induction of inflammatory cell infiltration and mucus secretion in mouse lungs.ConclusionsThe high co-expression of TNF-α and CARDS toxin in BALF is a good diagnostic biomarker for differentiating children with RMPP and NRMPP.

Disulfide bond of Mycoplasma pneumoniae community-acquired respiratory distress syndrome toxin is essential to maintain the ADP-ribosylating and vacuolating activities.

Mycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia among hospitalised children in United States and worldwide. Community-acquired respiratory distress syndrome (CARDS) toxin is a key virulence determinant of M. pneumoniae. The N-terminus of CARDS toxin exhibits ADP-ribosyltransferase (ADPRT) activity, and the C-terminus possesses binding and vacuolating activities. Thiol-trapping experiments of wild-type (WT) and cysteine-to-serine-mutated CARDS toxins with alkylating agents identified disulfide bond formation at the amino terminal cysteine residues C230 and C247. Compared with WT and other mutant toxins, C247S was unstable and unusable for comparative studies. Although there were no significant variations in binding, entry, and retrograde trafficking patterns of WT and mutated toxins, C230S did not elicit vacuole formation in intoxicated cells. In addition, the ADPRT domain of C230S was more sensitive to all tested proteases when compared with WT toxin. Despite its in vitro ADPRT activity, the reduction of C230S CARDS toxin-mediated ADPRT activity-associated IL-1β production in U937 cells and the recovery of vacuolating activity in the protease-released carboxy region of C230S indicated that the disulfide bond was essential not only to maintain the conformational stability of CARDS toxin but also to properly execute its cytopathic effects.

Clinical significance of the measurement of toxin level of community-acquired respiratory distress syndrome in children with Mycoplasma pneumoniae pneumonia

Objective To assess the level and trend of community acquired respiratory distress syndrome (CARDS) toxin after the infection of Mycoplasma pneumonia(MP), and evaluate the clinical characteristics, the level of immunoglobulin E (IgE) and interleukin-4 (IL-4) so as to find the association among these factors. Methods According to whether the child had wheezing symptoms, all the 63 children were divided into wheezing group (26 cases) and non-wheezing group (3 cases). The levels of CARDS toxin were respectively detected in the acute stage of MP infection, 3 and 6 months later after MP infection in different groups, moreover, IgE and IL-4 levels were monitored at the same time. Results (1)The mean level of IgE were (384.96±316.62)×103 IU/L and (87.32±66.32)×103 IU/L in wheezing group and non-wheezing group, respectively, and there was statistically significant difference (P<0.05). (2)The load of CARDS toxin in wheezing group and non-wheezing group were (1.87±0.62) Delta Rn and (1.15±0.48) Delta Rn in the stage of acute infection, respectively, and there was statistically significant difference (P<0.05). Nevertheless, differences between 2 groups after 3 months and 6 months were not significant.(3)In the acute stage, the level of CARDS toxin in the severe cases were higher than the mild cases[(2.37±0.37) Delta Rn vs (1.21±0.45) Delta Rn], and there was statistically significant difference (P<0.05). (4)IL-4 showed significant difference in the acute stage and 3 months later after acute infection between 2 groups, however, there were no difference between 2 groups after 6 months later.(5)The load of CARDS toxin showed no significant difference between 2 groups at 3 months [(0.96±0.35) vs. (0.99±0.40) Delta Rn, P=0.757] and 6 months [(0.67±0.20) vs. (0.69±0.32) Delta Rn, P=0.641] later after MP infection.(6)The children in wheezing group coughed for (24.89±7.04) days after acute infection and the last time for non-wheezing group was (16.46±4.79) days, and there was statistically significant difference(P=0.000). Conclusions The load of CARDS toxin decreased after acute MP infection and it was still detectable six months after onset in the blood.The level of CARDS toxin was associated with the cough and wheezing symptom and the severity of disease. Key words: Mycoplasma pneumoniae pneumonia; Community acquired respiratory distress syndrome; Toxin; Cough; Child

CARDS toxin is critical for mediating the early inflammatory response during Mycoplasma pneumoniae infection

Abstract Mycoplasma pneumoniae is a leading cause of bacterial community-acquired pneumonia and is implicated in initiation and exacerbation of asthma. One of the characteristic signs of M. pneumoniae infection in humans is an imbalanced proinflammatory immune response. A recently identified M. pneumoniae virulence factor is the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin, an ADP-ribosylating and vacuolating protein produced abundantly during infection that is capable of displaying inflammation and pathology similar to infection with live M. pneumoniae. Our prior studies have focused heavily on the potent inflammatory effects of CARDS toxin in vitro, however its exact role during infection is unclear. We hypothesized that an M. pneumoniae strain lacking CARDS toxin expression would be unable to trigger the robust inflammatory response in vivo that is necessary for M. pneumoniae pathogenesis. We genetically modified the clinical isolate M. pneumoniae S1 strain to generate a CARDS-deficient mutant we designated M7. M7 and S1 were used in vivo to identify changes in the proinflammatory response. Interestingly, we observed a drastic difference in the dynamics of M. pneumoniae infection during the early course of infection. Secretion of the cytokines TNF-α and CXCL1 were both significantly altered early during infection in M7-infected mice. The effects of this response led to a significant impact on Mycoplasma survival. By utilizing a novel CARDS toxin-deficient mutant of M. pneumoniae, our findings provide valuable insight into our understanding of the innate immune response against M. pneumoniae and implicate CARDS toxin as a potentially important target for therapeutic intervention during M. pneumoniae infection.

Mycoplasma pneumoniae Community-Acquired Respiratory Distress Syndrome Toxin Uses a Novel KELED Sequence for Retrograde Transport and Subsequent Cytotoxicity.

ABSTRACTMycoplasma pneumoniae is an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia (CAP). It has also been directly linked to reactive airway disease, asthma, and extrapulmonary pathologies. During its colonization, M. pneumoniae expresses a unique ADP-ribosylating and vacuolating cytotoxin designated community-acquired respiratory distress syndrome (CARDS) toxin. CARDS toxin persists and localizes in the airway in CAP patients, asthmatics, and trauma patients with ventilator-associated pneumonia. Although CARDS toxin binds to specific cellular receptors, is internalized, and induces hyperinflammation, histopathology, mucus hyperplasia, and other airway injury, the intracellular trafficking of CARDS toxin remains unclear. Here, we show that CARDS toxin translocates through early and late endosomes and the Golgi complex and concentrates at the perinuclear region to reach the endoplasmic reticulum (ER). Using ER-targeted SNAP-tag, we confirmed the association of CARDS toxin with the ER and determined that CARDS toxin follows the retrograde pathway. In addition, we identified a novel CARDS toxin amino acid fingerprint, KELED, that is required for toxin transport to the ER and subsequent toxin-mediated cytotoxicity.

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.

Mycoplasma pneumoniae and health outcomes in children with asthma

BackgroundAcute 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. ObjectiveTo determine the effect of Mp on asthma control. MethodsWe 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. ResultsLow 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. ConclusionThe 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 CARDS toxin is a unique virulence factor targeting the Th1/Th2 response for persistent infection

Abstract Mycoplasma pneumoniae is a leading cause of bacterial community-acquired pneumonia and is implicated in the initiation and exacerbation of asthma. Over 2 million cases of M. pneumoniae infection occur in the United States each year. However, with no surveillance system in place for reporting infections, the true scope of the problem may be vastly underestimated. One of the hallmark immunological effects of M. pneumoniae infection is an imbalanced Th1/Th2 response. A recently identified M. pneumoniae virulence factor is the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin, an ADP-ribosylating and vacuolating protein produced abundantly during infection that is capable of provoking a Th2 cytokine phenotype in mice. We hypothesize that CARDS toxin is utilized by M. pneumoniae in vivo to dampen the Th1 response and influence the Th2 response in order to establish a strong, persistent infection. The clinical isolate strain M. pneumoniae S1 abundantly produces CARDS toxin during infection and causes severe pathology. We utilized S1 as a template to generate a CARDS-deficient mutant we designated M7. M7 and S1 were used in vitro and in vivo to identify Th1- and Th2-specific cytokine changes during active infection. We observed significant differences in pathology between M7- and S1-infected mice, indicating that CARDS toxin directly impacted M. pneumoniae disease and persistence. Our findings provide insights to understand the elusive pathological mechanisms behind M. pneumoniae infection and implicate CARDS toxin as a potentially important target for therapeutic intervention during M. pneumoniae infection.

Inflammation-inducing Factors of Mycoplasma pneumoniae.

Mycoplasma pneumoniae, which causes mycoplasmal pneumonia in human, mainly causes pneumonia in children, although it occasionally causes disease in infants and geriatrics. Some pathogenic factors produced by M. pneumoniae, such as hydrogen peroxide and Community-Acquired Respiratory Distress Syndrome (CARDS) toxin have been well studied. However, these factors alone cannot explain this predilection. The low incidence rate of mycoplasmal pneumonia in infants and geriatrics implies that the strong inflammatory responses induced by M. pneumoniae coordinate with the pathogenic factors to induce pneumonia. However, M. pneumoniae lacks a cell wall and does not possess an inflammation-inducing endotoxin, such as lipopolysaccharide (LPS). In M. pneumoniae, lipoproteins were identified as an inflammation-inducing factor. Lipoproteins induce inflammatory responses through Toll-like receptors (TLR) 2. Because Mycoplasma species lack a cell wall and lipoproteins anchored in the membrane are exposed, lipoproteins and TLR2 have been thought to be important for the pathogenesis of M. pneumoniae. However, recent reports suggest that M. pneumoniae also induces inflammatory responses also in a TLR2-independent manner. TLR4 and autophagy are involved in this TLR2-independent inflammation. In addition, the CARDS toxin or M. pneumoniae cytadherence induces inflammatory responses through an intracellular receptor protein complex called the inflammasome. In this review, the inflammation-inducing factors of M. pneumoniae are summarized.

Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and "walking" pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.