Development of a high-brightness, genetically stable fluorescent reporter system for Mycoplasma pneumoniae and its application in host-pathogen interaction studies.

This study aimed to develop a genetically stable, high-brightness fluorescent reporter system for Mycoplasma pneumoniae (MP) and to demonstrate its utility in investigating host-pathogen interactions. The system is based on the mNeonGreen fluorescent protein. Its expression level was enhanced by utilizing the promoter of the MP tuf gene, while transposition efficiency was improved by codon optimization of the transposase gene. To facilitate the selection of recombinant mycoplasmas containing the transposon, strategies involving antibiotic resistance gene and promoter optimization were employed. Using random transposition via a miniTn4001 transposon vector, we achieved the efficient and stable integration of both mNeonGreen and an antibiotic resistance gene into the MP genome. Experimental results indicated that MP colonies electrotransformed with the chloramphenicol-resistant transposon exhibited normal morphology, whereas those with the tetracycline-resistant transposon showed abnormal morphology and growth defects. To evaluate the practicality of the resulting mNeonGreen-expressing recombinant MP strain (M129-mNeonGreen) for monitoring MP infection, particularly cellular adherence/invasion, we conducted infection assays using human bronchial epithelial cells (BEAS-2B) and human cervical adenocarcinoma cells (HeLa). Specific green fluorescence was observed by confocal microscopy when M129-mNeonGreen adhered to both BEAS-2B and HeLa cells, which was absent in cells infected with the wild-type M129 strain, confirming the utility of the recombinant strain for detecting MP infection. Furthermore, to validate the in vivo tracking capability of M129-mNeonGreen, BALB/c mice were infected with the recombinant strain. Frozen section analysis of lung tissues revealed specific green fluorescence signals three days post-infection, demonstrating the successful application of this reporter strain for in vivo tracing in a mouse model. In conclusion, this study successfully established a bright and genetically stable fluorescent reporter system for MP, providing a valuable tool for elucidating the in vivo and in vitro functionalities of MP.