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Objective To investigate the effects of Chlamydia trachomatis plasmid protein pORF5 on cellular mitophagy and mitochondrial fission and to elucidate whether its mechanism is related to Drp1 activation. Methods HeLa cells stably expressing pORF5 and control cells were constructed by lentiviral transfection. After serum starvation treatment, the expression levels of autophagy-related proteins—microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, and p62—were determined by Western blotting. Co-localization of LC3 and translocase of outer mitochondrial membrane 20 (TOMM20) was assessed by indirect immunofluorescence. Mitochondria were stained with MitoTracker Red CMXRos, and mitochondrial morphology was observed and analyzed through confocal laser scanning microscopy. Dynamin-related protein 1 (Drp1) phosphorylation and its translocation to mitochondria were examined by Western blotting and indirect immunofluorescence. To explore the role of Drp1 in autophagy and mitochondrial fission, we pretreated cells with the Drp1-specific mitochondrial division inhibitor 1 (Mdivi-1). Changes in mitochondrial morphology and Drp1 translocation were evaluated by confocal microscopy and indirect immunofluorescence. Then, Western blotting was employed to determine the expression levels of autophagy-related proteins, and indirect immunofluorescence assay to analyze LC3 fluorescence intensity and its co-localization with TOMM20. Results Compared with the control group, pORF5 significantly upregulated the expression of LC3-Ⅱ and Beclin-1, downregulated the expression of p62, and enhanced the co-localization of LC3 and TOMM20. pORF5 expression led to the fragmentation of the mitochondrial network structure. It promoted Drp1 phosphorylation at Ser616 and enhanced Drp1 translocation to mitochondria. Inhibition of Drp1 with Mdivi-1 attenuated Drp1 phosphorylation and translocation, resulting in elongated mitochondrial morphology. In addition, the Mdivi-1 inhibitor group showed downregulated expression of LC3-Ⅱ and Beclin-1, upregulated the expression of p62, and attenuated co-localization of LC3 and TOMM20. Conclusion The C. trachomatis plasmid protein pORF5 may induce mitophagy and mitochondrial fission by promoting Drp1 phosphorylation and its mitochondrial translocation.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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