Study of Mycoplasma pneumoniae CARDS Toxin in a lung fibroblast transfection model 2310308

Introduction Mycoplasma pneumoniae is an atypical respiratory pathogen that causes bronchiolitis and bacterial pneumonia. During infection, M. pneumoniae generates a three domain protein called Community-Acquired Respiratory Distress Syndrome (CARDS) toxin. Intracellular trafficking and processing activate its ADP-ribosylation and vacuolization properties. While ADP-ribosylation activity originates from the first domain, it is unclear where vacuolization activity originates. Lung fibroblasts play a crucial role in maintaining lung tissue integrity throughout life by recruiting immune cells during infection, regulating inflammation, and shaping lung tissue repair. Chronic M. pneumoniae infection is associated with airway remodeling and fibrosis, however the effects of CARDS toxin on fibroblast function are unclear. We hypothesize that CARDS toxin vacuolization disrupts lung fibroblast ability to regulate inflammation and initiate lung repair. Methods An ADP-ribosylation-deficient mutant of CARDS toxin was cloned for mammalian and bacterial overexpression and validated by sequencing. Mammalian vectors were transfected into IMR-90 lung fibroblasts to visualize CARDS toxin localization and fibroblast function and viability. Results Transfected fibroblasts showed similar pathology as recombinant CARDS toxin-treated fibroblasts, as well as high counts of cell death and vacuolization. CARDS-GFP signal was localized around the nucleus, confirming previous immunofluorescence observations. Comparable vacuolization was also seen between transfected and recombinant CARDS toxin-treated fibroblasts. Conclusion IMR-90 fibroblast transfection serves as an effective model for studying effects of M. pneumoniae CARDS toxin. Visualization of GFP-CARDS toxin movement in real time also provides critical insight into CARDS toxin trafficking and fibroblast responses. Funding Source n/a Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)