Community-acquired Respiratory Distress Syndrome Research Articles

A one-pot, one-step CRISPR platform (CRISPR-one) for nucleic acid detection: Application for the detection of Mycoplasma pneumoniae

The conventional CRISPR-mediated detection technologies typically involve two separate steps: target pre-amplification and CRISPR-mediated signal detection. This multi-step procedure is not only cumbersome but also prone to cross-contamination. Here, we propose a new CRISPR-mediated nucleic acid detection methodology, termed CRISPR-one (CRISPR-mediated testing in one-tube, one-pot). At a constant temperature, CRISPR-one carries out both multiple cross displacement amplification (MCDA) for target pre-amplification and CRISPR/AapCas12b-based cleavage for signal detection in a single reaction pot. The novel CRISPR-one technique is employed for detecting Mycoplasma pneumoniae (M. pneumoniae), a causative agent for community-acquired pneumonia in children. M. pneumoniae CRISPR-one targets the CARDS (community-acquired respiratory distress syndrome toxin) gene and performs at 60 ºC for 50 min with only basic instrumentation required. The results can be easily visualized through a real-time fluorescence instrument, with the entire process taking less than an hour. The lowest detection limit of M. pneumoniae CRISPR-one was 50 fg (∼55 copies) of genomic DNA per reaction, and no cross-reactivity with non-M. pneumoniae templates was observed. In clinical applications, the CRISPR-one assay demonstrated a high level of agreement with both the microfluidic chip method (Kappa value: 0.95) and real-time PCR (Kappa value: 0.93) when analyzing 135 oropharynx swab samples. There is no statistical difference in detection efficiency between our assay and other methods. Together, the CRISPR-one developed here offers a rapid, convenient, highly sensitive, and specific approach for pathogen detection (e.g., M. pneumoniae), serving as an invaluable tool for clinical diagnosis and epidemiologic surveillance, particularly in resource-limited laboratories.

Yinqin Qingfei granules alleviate Mycoplasma pneumoniae pneumonia via inhibiting NLRP3 inflammasome-mediated macrophage pyroptosis.

Mycoplasma pneumoniae pneumonia (MPP) is a prevalent respiratory infectious disease in children. Given the increasing resistance of M. pneumoniae (MP) to macrolide antibiotics, the identification of new therapeutic agents is critical. Yinqin Qingfei granules (YQQFG), a Chinese patent medicine formulated specifically for pediatric MPP, lacks a clear explanation of its mechanism. The primary components of YQQFG were identified using LC-MS/MS. In vitro, RAW264.7 cells infected with MP underwent morphological examination via scanning electron microscopy. Drug-containing serum was prepared, and its intervention concentration was determined using the CCK-8 assay. The active components of YQQFG were molecularly docked with NLRP3 protein using Autodock Vina software. A RAW264.7 cell line overexpressing NLRP3 was created using lentivirus to pinpoint the target of YQQFG. In vivo, MPP model mice were established via nasal instillation of MP. Lung damage was assessed by lung index and H&E staining. Pyroptosis-associated protein levels in cells and lung tissue were measured by western blot, while interleukin (IL)-1β and IL-18 levels in cell supernatants and mouse serum were quantified using ELISA. Immunofluorescence double staining of lung tissue sections was conducted to assess the correlation between NLRP3 protein expression and macrophages. The expression of the community-acquired respiratory distress syndrome toxin (CARDS TX) was evaluated by qPCR. 25 effective components with favorable oral bioavailability were identified in YQQFG. Both in vitro and in vivo studies demonstrated that YQQFG substantially reduced the expression of the NLRP3/Caspase-1/GSDMD pathway, decreasing the release of IL-1β and IL-18, and inhibited MP exotoxin. Molecular docking indicated strong affinity between most YQQFG components and NLRP3 protein. Lentivirus transfection and immunofluorescence double staining confirmed that YQQFG significantly suppressed NLRP3 expression in macrophages, outperforming azithromycin (AZM). The combination of YQQFG and AZM yielded the optimal therapeutic effect for MPP. YQQFG mitigates inflammatory responses by suppressing NLRP3 inflammasome-mediated macrophage pyroptosis, thereby ameliorating MP-induced acute lung injury. YQQFG serves as an effective adjunct and alternative medication for pediatric MPP treatment.

The Therapeutic Potential of Neutrophil Extracellular Traps and NLRP3 Inflammasomes in Mycoplasma pneumoniae Pneumonia

ABSTRACT Introduction Mycoplasma pneumoniae (MP) is the most common pathogen of community-acquired pneumonia in children. However, the role of neutrophil extracellular traps (NETs) in the pathogenesis of MP is unclear. Methods Both the level of NETs were detected between the 60 MP pneumonia patients and 20 healthy controls, whose the clinical characteristics were compared. Additionally, NETs formation induced by community-acquired respiratory distress syndrome (CARDS) toxin was also analyzed through transcriptome sequencing. Results The levels of cell-free DNA, Cit-H3, and MPO-DNA complexes were significantly increased in the patients with MP pneumonia. Importantly, both cell-free DNA and LDH were higher in hospitalized patients with severity than those without severity. In addition, CARDS toxin induced the NETs formation in vitro and in vivo. Transcriptomics GO and KEGG pathway analysis indicate that NOD like receptor signaling pathway and Toll-like receptor signaling pathway are significantly enriched. Finally, we found that DNase I significantly attenuated the higher levels of Cit-H3, and up-regulation of interleukin-1β (IL-1β) and interleukin-18 (IL-18) by down-regulating the expression of NLRP3 and Caspase1(p20) in the lung tissues. Discussion These results indicate that inhibiting excessive activation of NLRP3 inflammasomes, and NETs formation may alleviate MP pneumonia.

Immunomodulatory Effects of Fluoroquinolones in Community-Acquired Pneumonia-Associated Acute Respiratory Distress Syndrome.

Community-acquired pneumonia is reported as one of the infectious diseases that leads to the development of acute respiratory distress syndrome. The innate immune system is the first line of defence against microbial invasion; however, its dysregulation during infection, resulting in an increased pathogen load, stimulates the over-secretion of chemokines and pro-inflammatory cytokines. This phenomenon causes damage to the epithelial-endothelial barrier of the pulmonary alveoli and the leakage of the intravascular protein into the alveolar lumen. Fluoroquinolones are synthetic antimicrobial agents with immunomodulatory properties that can inhibit bacterial proliferation as well as exhibit anti-inflammatory activities. It has been demonstrated that the structure of fluoroquinolones, particularly those with a cyclopropyl group, exerts immunomodulatory effects. Its capability to inhibit phosphodiesterase activity leads to the accumulation of intracellular cAMP, which subsequently enhances PKA activity, resulting in the inhibition of transcriptional factor NF-κB and the activation of CREB. Another mechanism reported is the inhibition of TLR and ERK signalling pathways. Although the sequence of events has not been completely understood, significant progress has been made in comprehending the specific mechanisms underlying the immunomodulatory effects of fluoroquinolones. Here, we review the indirect immunomodulatory effects of FQs as an alternative to empirical therapy in patients diagnosed with community-acquired pneumonia.

Observational retrospective clinical study on clinical features of macrolide-resistant Mycoplasma pneumoniae pneumonia in Chinese pediatric cases

This study aimed to investigate differences in clinical characteristics and laboratory findings between children infected with Macrolide-Sensitive Mycoplasma pneumoniae (MSMP) and Macrolide-Resistant Mycoplasma pneumoniae (MRMP). Additionally, the research sought to identify laboratory markers for rapidly distinguishing refractory Mycoplasma pneumoniae pneumonia (RMPP) from ordinary Mycoplasma pneumoniae pneumonia (OMPP). In total, 265 Mycoplasma pneumoniae (MP) patients were included, with MRMP identified by specific point mutations in domain V of the 23S rRNA gene. A retrospective analysis compared the clinical courses and laboratory data, revealing that MRMP patients experienced prolonged febrile days (P = 0.004), elevated CRP levels (P < 0.001), and higher MP DNA loads than MSMP patients (P = 0.037). Based on clinical symptoms, MRMP was divided into RMPP (n = 56) and OMPP (n = 70), with RMPP demonstrating significantly increased IL-18, community-acquired respiratory distress syndrome (CARDS) toxins in nasopharyngeal aspirate, and serum CRP levels (P < 0.001; P = 0.006; P < 0.001). In conclusion, timely recognition of RMPP is crucial for enhancing prognosis. The identification of MRMP, coupled with proinflammatory cytokines such as IL-18, CARDS toxins, and CRP, emerges as promising markers with the potential to contribute significantly to diagnostic accuracy and prognosis assessment.

Rapid, ultrasensitive and highly specific diagnosis of Mycoplasma pneumoniae by a CRISPR-based detection platform.

Mycoplasma pneumoniae (MP) is an important causative agent of morbidity and mortality among all age groups, especially among patients of extreme ages. Improved and readily available tests for accurate, sensitive and rapid diagnosis of MP infection is sorely needed. Here, we developed a CRISPR-Cas12b-based detection platform on the basis of recombinase polymerase amplification (RPA) for rapid, simple, and accurate diagnosis of MP infection, named MP-RPA-CRISPR. The RPA was employed for amplifying the community-acquired respiratory distress syndrome (CARDS) toxin gene of MP strains at the optimal reaction temperature 37°C. The resulting amplicons were decoded by the CRISPR-Cas12b-based detection platform, which was interpreted by real-time PCR system and by naked eye under blue light. The MP-RPA-CRISPR can detected down to 5 fg of genomic DNA templates of MP strains and accurately distinguish MP strains from non-MP strains without any cross-reactivity. A total of 96 bronchoalveolar lavage fluid (BALF)samples collected from patients suspected of respiratory infection were used to evaluate the clinical performance of the MP-RPA-CRISPR assay. As a result, our assay accurately diagnosed 45 MP-infected samples and 51 non-MP infected sample, and the results obtained from MP-RPA-CRISPR were consistent with microfluidic chip technology. In conclusion, our MP-RPA-CRISPR assay is a simple, rapid, portable and highly sensitive method to diagnose MP infection, which can be used as a promising tool in a variety of settings including clinical, field, and resource-limited aeras.

Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display.

Pathogenic species of Leptospira are etiologic agents of leptospirosis, an emerging zoonotic disease of worldwide extent and endemic in tropical regions. The growing number of identified leptospiral species sheds light to their genetic diversity and unique virulence mechanisms, many of them still remain unknown. Toxins and adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines with cross-protection and long-lasting effect against leptospirosis. For this aim, we used the shotgun phage display technique to unravel new proteins with adhesive properties. A shotgun library was constructed using fragmented genomic DNA from Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and pG8SAET phagemid vector. Selection of phages bearing new possible cell-binding antigens was performed against VERO cells, using BRASIL biopanning methodology. Analysis of selected clones revealed the hypothetical protein LIC10778, a potentially exposed virulence factor that belongs to the virulence-modifying (VM) protein family (PF07598), composed of 13 members in the leptospiral strain Fiocruz L1-130. Prediction of LIC10778 tertiary structure indicates that the protein contains a cellular-binding domain (N-terminal portion) and an unknown domain of no assigned activity (C-terminal portion). The predicted N-terminal domain shared structural similarities with the cell-binding and internalization domain of toxins like Ricin and Abrin, as well as to the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin in Mycoplasma pneumoniae. Interestingly, recombinant portions of the N-terminal region of LIC10778 protein showed binding to laminin, collagens I and IV, vitronectin, and plasma and cell fibronectins using overlay blotting technique, especially regarding the binding site identified by phage display. These data validate our preliminary phage display biopanning and support the predicted three-dimensional models of LIC10778 protein and other members of PF07598 protein family, confirming the identification of the N-terminal cell-binding domains that are similar to ricin-like toxins. Moreover, fluorescent fused proteins also confirmed that N-terminal region of LIC10778 is capable of binding to VERO and A549 cell lines, further highlighting its virulence role during host-pathogen interaction in leptospirosis probably mediated by its C-terminal domain. Indeed, recent results in the literature confirmed this assumption by demonstrating the cytotoxicity of a closely related PF07598 member.

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.

Additional effect of azithromycin over \u03b2-lactam alone for severe community-acquired pneumonia-associated acute respiratory distress syndrome: a retrospective cohort study

BackgroundCommunity-acquired pneumonia (CAP) is the most common cause of acute respiratory distress syndrome (ARDS). Although previous studies have suggested that macrolide therapy is beneficial for ARDS, its benefit for severe CAP-associated ARDS remains uncertain. Previous studies were limited in that they had a small sample size and included patients with non-pulmonary ARDS and those with pulmonary ARDS. This study aimed to investigate the additional effect of azithromycin when used with β-lactam compared with the effect of β-lactam alone in mechanically ventilated patients with CAP-associated ARDS.MethodsWe identified mechanically ventilated patients with CAP-associated ARDS between July 2010 and March 2015 using data in the Diagnosis Procedure Combination database, a Japanese nationwide inpatient database. We performed propensity score matching analysis to assess 28-day mortality and in-hospital mortality in mechanically ventilated patients with CAP-associated ARDS who received β-lactam with and without azithromycin within hospital 2 days after admission. The inverse probability of treatment weighting analysis was also conducted.ResultsEligible patients (n = 1257) were divided into the azithromycin group (n = 226) and the control group (n = 1031). The one-to-four propensity score matching analysis included 139 azithromycin users and 556 non-users. No significant difference was observed between the groups with respect to 28-day mortality (34.5% vs. 37.6%, p = 0.556) or in-hospital mortality (46.0% vs. 49.1%, p = 0.569). The inverse probability of treatment weighting analysis showed similar results.ConclusionsCompared with treatment with β-lactam alone, treatment with azithromycin plus β-lactam had no significant additional effect on 28-day mortality or in-hospital mortality in mechanically ventilated patients with CAP-associated ARDS. To the best of our knowledge, this study is the first to determine the effect of azithromycin in mechanically ventilated patients with CAP-associated ARDS.

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.