Comprehensive lung microbial gene and genome catalogs assist the mechanism survey of Mesomycoplasma hyopneumoniae strains causing pig lung lesions

Understanding the community structure of the lower respiratory tract microbiome is crucial for elucidating its roles in respiratory tract diseases. However, there are few studies about this topic due to the difficulty in obtaining microbial samples from both healthy and disease individuals. Here, using 744 high-depth metagenomic sequencing data of lower respiratory tract microbial samples from 675 well-phenotyped pigs, we constructed a lung microbial gene catalog containing the largest scale of 10,031,593 nonredundant genes to date, 44.8% of which are novel. We obtained 356 metagenome-assembled genomes (MAGs) which were further clustered into 256 species-level genome bins with 41.8% being first reported in the current databases. Based on these data sets and through integrated analysis of the isolation of the related bacterial strains, in vitro infection, and RNA sequencing, we identified and confirmed that Mesomycoplasma hyopneumoniae (M. hyopneumoniae) MAG_47 and its adhesion-related virulence factors (VFs) were associated with lung lesions in pigs. Differential expression levels of adhesion- and immunomodulation-related VFs likely determined the heterogenicity of adhesion and pathogenicity among M. hyopneumoniae strains. M. hyopneumoniae adhesion activated several pathways, including nuclear factor kappa-light-chain-enhancer of activated B, mitogen-activated protein kinase, cell apoptosis, T helper 1 and T helper 2 cell differentiation, tumor necrosis factor signaling, interleukin-6/janus kinase 2/signal transducer and activator of transcription signaling, and response to reactive oxygen species, leading to cilium loss, epithelial cell‒cell barrier disruption, and lung tissue lesions. Finally, we observed the similar phylogenetic compositions of the lung microbiome between humans with Mycoplasma pneumoniae and pigs infected with M. hyopneumoniae. The results provided important insights into pig lower respiratory tract microbiome and its relationship with lung health.