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

Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
Molecular Microbiology (Impact Factor: 4.42). 02/2010; 76(5):1127-41. DOI: 10.1111/j.1365-2958.2010.07092.x
Source: PubMed


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.

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    • "The inflammation associated with the OVA model of asthma is characterized by the influx of eosinophils and Th2 T-cells and epithelial cell hyperplasia, and is similar in many respects to human allergic asthma. Our findings suggest that CARDS toxin is a significant virulence factor in the exacerbations of OVA-induced inflammation in mice infected with live M. pneumoniae, as mycoplasmas synthesize augmented levels of CARDS toxin in the airways [38] and rCARDS toxin alone recapitulates the exaggerated pathologies observed during M. pneumoniae infection of OVA-treated mice [17], [39]. These data strongly suggest that the exacerbation of allergic inflammation observed with M. pneumoniae infection of OVA-treated mice was due in part to the actions of the CARDS toxin. "
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    ABSTRACT: Mycoplasma pneumoniae causes a range of airway and extrapulmonary pathologies in humans. Clinically, M. pneumoniae is associated with acute exacerbations of human asthma and a worsening of experimentally induced asthma in mice. Recently, we demonstrated that Community Acquired Respiratory Distress Syndrome (CARDS) toxin, an ADP-ribosylating and vacuolating toxin synthesized by M. pneumoniae, is sufficient to induce an asthma-like disease in BALB/cJ mice. To test the potential of CARDS toxin to exacerbate preexisting asthma, we examined inflammatory responses to recombinant CARDS toxin in an ovalbumin (OVA) murine model of asthma. Differences in pulmonary inflammatory responses between treatment groups were analyzed by histology, cell differentials and changes in cytokine and chemokine concentrations. Additionally, assessments of airway hyperreactivity were evaluated through direct pulmonary function measurements. Analysis of histology revealed exaggerated cellular inflammation with a strong eosinophilic component in the CARDS toxin-treated group. Heightened T-helper type-2 inflammatory responses were evidenced by increased expression of IL-4, IL-13, CCL17 and CCL22 corresponding with increased airway hyperreactivity in the CARDS toxin-treated mice. These data demonstrate that CARDS toxin can be a causal factor in the worsening of experimental allergic asthma, highlighting the potential importance of CARDS toxin in the etiology and exacerbation of human asthma.
    PLoS ONE 07/2014; 9(7):e102613. DOI:10.1371/journal.pone.0102613 · 3.23 Impact Factor
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    • "experimental animals and is readily detectable in airway fluids (Kannan et al., 2010; 2011; 2012; Techasaensiri et al., 2010; Muir et al., 2011; Peters et al., 2011). Furthermore , purified recombinant CARDS toxin elicits multiple inflammatory and temporally changing histopathological patterns in rodents and primates in a manner similar to those observed during M. pneumoniae infection (Hardy et al., 2009). "
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    ABSTRACT: Community-acquired respiratory distress syndrome (CARDS) toxin from Mycoplasma pneumoniae is a 591 amino acid virulence factor with ADP-ribosyltransferase (ADPRT) and vacuolating activities. It is expressed at low levels during in vitro growth and at high levels during colonization of the lung. Exposure of experimental animals to purified recombinant CARDS toxin alone is sufficient to recapitulate the cytopathology and inflammatory responses associated with M. pneumoniae infection in humans and animals. Here, by molecular modeling, serial truncations and site-directed mutagenesis, we show that the N-terminal region is essential for ADP-ribosylating activity. Also, by systematic truncation and limited proteolysis experiments we identified a portion of the C-terminal region that mediates toxin binding to mammalian cell surfaces and subsequent internalization. In addition, the C-terminal region alone induces vacuolization in a manner similar to full-length toxin. Together, these data suggest that CARDS toxin has a unique architecture with functionally separable N-terminal and C-terminal domains.
    Molecular Microbiology 06/2014; 93(3). DOI:10.1111/mmi.12680 · 4.42 Impact Factor
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    • "Interestingly, CARDS toxin initiates proinflammatory responses that also mimic M. pneumoniae-induced pathologies in animal models [8], [12]. Furthermore, the high levels of CARDS toxin expression observed during M. pneumoniae infection in animal models and, importantly, the detection of CARDS toxin in the lungs and bronchoalveolar fluid of infected humans establish this protein as a bona fide virulence factor [12], [15], [16], [17], [18]. "
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    ABSTRACT: Bacterial toxins possess specific mechanisms of binding and uptake by mammalian cells. Mycoplasma pneumoniae CARDS (Community Acquired Respiratory Distress Syndrome) toxin is a 68 kDa protein, which demonstrates high binding affinity to human surfactant protein-A and exhibits specific biological activities including mono-ADP ribosylation and vacuolization. These properties lead to inflammatory processes in the airway and a range of cytopathologies including ciliostasis, loss of tissue integrity and injury, and cell death. However, the process by which CARDS toxin enters target cells is unknown. In this study, we show that CARDS toxin binds to mammalian cell surfaces and is internalized rapidly in a dose and time-dependent manner using a clathrin-mediated pathway, as indicated by inhibition of toxin internalization by monodansylcadaverine but not by methyl-β-cyclodextrin or filipin. Furthermore, the internalization of CARDS toxin was markedly inhibited in clathrin-depleted cells.
    PLoS ONE 05/2013; 8(5):e62706. DOI:10.1371/journal.pone.0062706 · 3.23 Impact Factor
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