Abstract Mycoplasma pneumoniae is the leading cause of bacterial pneumonia in children hospitalized with community acquired pneumonia (CAP) and the second most common cause of bacterial CAP in adults in the United States. Persistent and recurring M. pneumoniae infection leads to severe respiratory disorders, including asthma and COPD, and a range of extrapulmonary pathologies. Until recently, it was unknown how a respiratory pathogen, like M. pneumoniae, induces cytopathology and exaggerated inflammatory responses that cause airway injury, dysfunction and remodeling. We identified a novel M. pneumoniae ADP- ribosylating and vacuolating toxin designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. CARDS toxin alone elicits the characteristic airway inflammation, lung histopathology, cellular vacuolation, mucus metaplasia and pulmonary dysfunction in intoxicated rodents and primates that are observed during infection with M. pneumoniae. The amino terminal domain of full length (FL) CARDS toxin (i.e., N-CARDS) retains ADP-ribosyltransferase (ART) activity. N-CARDS selectively ADP-ribosylates NLRP3 of the NLRP3 inflammasome complex, resulting in inflammasome activation and subsequent release of IL- 1β, a potent pro-inflammatory cytokine. In preliminary results, we also show that CARDS toxin selectively ADP-ribosylates serine hydroxymethyltransferase (SHMT2), which is involved in one carbon metabolism, and EF1γ, which is involved in the transfer of aminoacyl-tRNAs to the ribosome. The unique carboxyl region of FL CARDS toxin (i.e., C-CARDS) selectively binds to receptors surfactant protein-A (SP-A), annexin A2 (AnxA2) and phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM). Internalization of FL CARDS toxin follows receptor-mediated binding, with subsequent ADP-ribosylation of host target proteins, vacuolation, hyperinflammation and cell/tissue histopathology and injury. Interestingly, C-CARDS alone causes both vacuole formation in human cells and eosinophilic inflammation in naïve mice, leading to an asthma-like phenotype. In this proposal, we intend to identify how ART and vacuolating properties of CARDS toxin trigger pro-inflammatory and pathologic responses in human WT, silenced or knockout cells and in WT and knockout mice. Based on our preliminary results, we hypothesize that both ADP-ribosylating and vacuolating activities contribute to the overall ability of CARDS toxin to initiate and sustain disease pathogenesis. We plan to test this hypothesis by – a) studying how CARDS toxin ART activities initiate inflammatory pathways and cytopathology, b) elucidating the role of receptor binding in CARDS toxin-mediated inflammation, c) characterizing how vacuolating activity promotes airway inflammation and injury, and d) analyzing the in vivo involvement of select ART and receptor targets in triggering CARDS toxin-mediated airway inflammation and lung pathology. Understanding the mechanisms by which ART and vacuolating act...