[Effect of hsdS homologous gene duplication on pathogenicity-related phenotypes and gene expression in Mycoplasma pneumoniae].

Objective: To investigate the impact of the hsdS gene on the phenotypic characteristics and transcriptomic expression profiles of Mycoplasma pneumoniae (MP). Methods: This study is an experimental study. A total of 20 clinical MP strains were isolated from the clinical respiratory samples from the children with pneumonia in Shanghai Children's Hospital, between December 2023 and December 2024. MP strains were selected and divided into the multi-hsdS homologous group (MHG, n=10) and the oligo-hsdS homologous group (OHG, n=10) based on the number of hsdS homologous genes. The two groups were compared in terms of biofilm formation capacity, growth rate, morphological polarization of macrophages, and inflammatory cytokine secretion levels using independent-sample t-tests or Mann-Whitney U tests. Differentially expressed genes were analyzed by transcriptome sequencing and validated by RT-qPCR. Results: Compared with the OHG group, the MHG group exhibited significantly enhanced biofilm formation capacity (OD590: 0.33±0.06 vs. 0.28±0.05, t=3.364, P=0.001), accelerated growth rate (OD430/OD560: day 5: 4.03±1.51 vs. 2.81±1.80, t=2.852, P=0.042, day 6: 7.35±0.49 vs. 6.02±1.62, t=3.018, P=0.026), and an increased proportion of morphologically polarized macrophages (49.8%±15.7% vs. 29.6%±9.3%, t=2.657, P=0.026). Transcriptome sequencing revealed that the overall expression of hsdS homologous genes was upregulated in the MHG group (54.87±75.99 vs. 21.78±35.36, t=3.058, P=0.003), along with upregulation of topJ, MPN_RS00155, ftsZ, thyA, and multiple ATP synthase subunits (P<0.05). Except for MPN_RS03435, the expression levels of the remaining seven genes in the MHG group were significantly higher than those in the OHG group (topJ: t=3.144, P=0.014; MPN_RS00155: t=2.927, P=0.019; ftsZ: t=3.172, P=0.013; thyA: t=2.429, P=0.041; atpH: t=3.379, P=0.010; atpG: t=4.204, P=0.003; MPN_RS03405: t=2.368, P=0.045). Conclusion: The enhanced of hsdS homologous genes is closely associated with biofilm formation capacity, growth rate, and ability to induce morphologically macrophage polarization of MP strains, and upregulates the transcriptional expression of multiple phenotype-related genes.