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.
Highlights
Mycoplasma pneumoniae is an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia [1,2]
Binding of recombinant CARDS (rCARDS) toxin to cell surfaces was determined by incubating HeLa cells with toxin (10 mg/ml) at 4uC for 30 min. rCARDS toxin was visualized as intense red fluorescence on the apical surfaces of cells using optical confocal planes; at this temperature cross sectional z series views did not detect toxin internalization (Fig. 1A)
Using fluorescent- and biotin-tagged rCARDS toxin, we showed that rCARDS toxin bound to mammalian cells in a concentration-dependent manner, followed by rapid internalization, which was temperature dependent and mediated by clathrinassociated endocytosis
Summary
Mycoplasma pneumoniae is an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia [1,2]. The interaction of M. pneumoniae with the airway epithelium results in significant cytopathology both in organ culture and in vivo [7,8,9]. The cytopathology induced by M. pneumoniae infection was linked in part to hydrogen peroxide and superoxide radicals generated by mycoplasma metabolism [7,10]. We identified a novel ADP-ribosylating and vacuolating cytotoxin of M. pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin capable of inducing cytopathology both in vitro and in vivo that reproduces the infectious process [11,12]
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