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

Abstract

Mycoplasma 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.

Disulfide bond ofMycoplasma pneumoniaecommunity‐acquired respiratory distress syndrome toxin is essential to maintain the ADP‐ribosylating and vacuolating activities

“…The reactions were stopped as indicated above, and the samples were resolved on 4-12% Nu-PAGE gel and Coomassie stained (i and ii), transferred to nitrocellulose membrane, and analysed by immunoblot using anti-CARDS toxin carboxy (iii) or amino terminal (iv) antibodies. We showed that CARDS toxin uses retrograde transport to reach the ER through Golgi complex by utilising a distinct KELED motif (Ramasamy et al, 2018). To induce cytotoxicity in host cells, bacterial toxins use multiple pathways to reach their intracellular targets.…”

“…Molecular weight markers are indicated on the right directly involved in vacuole formation (Figure 3), it is possible that the loss of the intrachain disulfide bond alters the subsequent trafficking of the toxin or proper processing and/or its stability. Altering the KELED motif interrupted CARDS toxin retrograde trafficking to ER, which in turn blocked ADP-ribosylating and vacuolating activities (Ramasamy et al, 2018). For example, diphtheria toxin reaches the cytosol by passing through early and late endosomes and by translocating across the endosomal membrane in a disulfide bond reduction-dependent cleavage event (Falnes et al, 1994).…”

“…The four additional Cys residues in the CARDS structure are shown as single yellow spheres CARDS toxin-mediated vacuolation (Ramasamy et al, 2018). (a) Examination of disulfide bond formation in wild-type (WT) and C → S mutant CARDS toxins.…”

“…To further explore CARDS toxin trafficking to the ER, we used ER-SNAP-cMyc tag as we recently described (Ramasamy et al, 2018). Incubation of benzylguanine (BG)-coupled WT toxin or …”

“…The C-terminal region of CARDS toxin (Domains 2 and 3) contains the cell receptor binding and vacuolating activities . In vulnerable target cells, CARDS toxin traffics along the retrograde pathway utilising a unique KELED amino acid sequence that mediates translocation of CARDS toxin to the endoplasmic reticulum (ER) followed by activation of ADPRT and vacuolating activities (Ramasamy et al, 2018). In vulnerable target cells, CARDS toxin traffics along the retrograde pathway utilising a unique KELED amino acid sequence that mediates translocation of CARDS toxin to the endoplasmic reticulum (ER) followed by activation of ADPRT and vacuolating activities (Ramasamy et al, 2018).…”