FMRP, an RNA-binding Protein Induced by the Mycoplasma Pneumoniae CARDS Toxin, Regulates Multiciliogenesis and Inflammation
Authors: Yuling Xu1, Tingyu Yang2, Shuai Shao2, Fengjiao Liu1, Nan Song1, Jieqiong Li1
Affiliations:
1Medical Research Center, Beijing Institute of Respiratory Medicine and Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, China;
2Department of Respiratory and Critical Care Medicine, Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, China.
Introduction
Mycoplasma pneumoniae (M. pneumoniae) is an atypical pathogen lacking a cell wall and is a leading cause of community-acquired pneumonia, particularly in school-age children. The community-acquired respiratory distress syndrome (CARDS) toxin, a unique exotoxin produced by M. pneumoniae, is known to induce cell swelling and vacuolization. However, the precise functional characteristics of the CARDS toxin remain incompletely understood.
Motile cilia, which extend from specialized epithelial surfaces, play essential roles in development and disease by facilitating the movement or clearance of fluids and particles through coordinated beating. Fragile X mental retardation protein (FMRP), encoded by the Fmr1 gene, is widely expressed in various organs and is enriched in the axonemal central lumen of cilia. FMRP has critical roles in multiciliated cell differentiation and multiciliogenesis. Recent studies have also shown that FMRP is expressed in the lungs, where it protects the airways from xenobiotic stress.
This study investigates the role of ciliary FMRP in the pulmonary immune response during M. pneumoniae infection, focusing on the effects of the CARDS toxin on ciliary structure and function, as well as the potential therapeutic implications of targeting FMRP.
Materials and Methods
All animal experiments were approved by the Capital Medical University Animal Care and Use Committee (No. AEEI-2024-117). BALB/c mice were intratracheally instilled with recombinant CARDS toxin (700 pmol/L) to simulate M. pneumoniae infection, with heat-inactivated CARDS toxin used as a control. Seven days post-procedure, tracheal tissue, lung tissue, and bronchoalveolar lavage fluid (BALF) samples were collected and analyzed.
RNA sequencing (RNA-seq) was performed to compare transcriptome signatures between control and CARDS-treated mice. Principal component analysis (PCA) and gene set enrichment analysis were conducted to identify differentially expressed genes and enriched pathways. Whole-mount immunofluorescence and hematoxylin-eosin staining were used to examine ciliary distribution and structure in mouse tracheal tissue.
Proteomic analysis of BALF samples from patients with M. pneumoniae pneumonia (MPP) and non-MPP diseases was performed using liquid chromatography–tandem mass spectrometry. Clinical samples were approved by the Ethics Committee of Beijing Chao-Yang Hospital (2021-Ke-501), and written informed consent was obtained.
Results
CARDS-treated mice exhibited minor body weight loss compared to controls. Hematoxylin-eosin staining revealed bronchial epithelial damage, thickened alveolar septa, and inflammatory cell infiltration in the lungs of CARDS-treated mice. BALF protein levels, proinflammatory cytokine expression, and immune cell infiltration were also increased in CARDS-treated mice, indicating an inflammatory response.
RNA-seq analysis showed clear separation in transcriptome profiles between control and CARDS-treated groups. Gene set enrichment analysis and Gene Ontology annotation indicated that differentially expressed genes were highly enriched in pathways related to motile cilia, cilium organization, and cilium movement. These genes are essential for normal ciliary structure and function, including processes such as axial elongation, microtubule assembly, ciliary trafficking, and formation of the transition zone and basal body.
Whole-mount immunofluorescence staining of tracheal tissue revealed defects in motile cilia in CARDS-treated mice. The number of basal cells (krt5-positive cells) increased in tracheal tissue from CARDS-treated mice, indicating damage to the epithelial barrier and priming for proliferation and differentiation. Expression levels of cilia-related genes were decreased in the lungs of CARDS-treated mice.
FMRP expression was downregulated in CARDS-treated mice, accompanied by ciliary injury. Proteomic analysis of BALF samples from patients with MPP showed downregulation of proteins related to cilium morphogenesis, movement, and organization, consistent with the transcriptomic data from experimental mice. FMRP expression was also downregulated in BALF samples from patients with MPP.
Dihydroxyphenylglycine (DHPG), an agonist of group I metabotropic glutamate receptors, was found to upregulate FMRP expression in mouse tracheobronchial epithelial cells. In mice treated with DHPG five days after CARDS toxin injection, the toxin-mediated changes in FMRP expression and ciliary morphology were reversed. Lung tissue damage, cytokine gene expression levels, and the percentages of CD68+ alveolar macrophages and Ly6G+ neutrophils were significantly reduced after DHPG treatment.
Discussion
This study demonstrates that the CARDS toxin damages motile cilia in the respiratory tract and decreases FMRP expression in ciliated cells. These changes are consistent with observations in patients with MPP, suggesting that FMRP plays a key role in CARDS-mediated ciliary injury and the maintenance of pulmonary immune homeostasis.
The findings indicate that pharmacological activation of FMRP can protect the lungs from CARDS toxin-induced damage. Approaches targeting the cilia–FMRP axis may help address drug resistance in the treatment of M. pneumoniae infection.
Conclusion
In conclusion, the CARDS toxin disrupts normal ciliary gene expression and damages motile cilia in the respiratory tract, leading to decreased FMRP expression in ciliated cells. These changes are consistent with observations in patients with MPP, highlighting the importance of FMRP in ciliary function and pulmonary immune homeostasis. Pharmacological activation of FMRP, such as through DHPG treatment, may offer a therapeutic strategy to mitigate CARDS toxin-induced lung damage and inflammation.
Funding
This work was supported by Beijing Chao-Yang Hospital Golden Seeds Foundation (No. CYJZ202312), the Capital’s Funds for Health Improvement and Research (No. 2022-1-1061), and Beijing Research Ward Demonstration Construction Project (No. BCRW202110).
Conflicts of Interest
None.
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DOI: doi.org/10.1097/CM9.0000000000003474
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