# The UBC/SIRT5/DRP1 axis regulates mitochondrial dynamics to alleviate Staphylococcus aureus-induced oxidative stress and senescence in bovine mammary epithelial cells

## TL;DR

This study shows how S. aureus infection causes mitochondrial stress and cell aging in cow mammary cells, and how SIRT5 helps protect against this damage.

## Contribution

The study identifies the UBC–SIRT5–DRP1 pathway as a novel mechanism linking S. aureus infection to mitochondrial dysfunction and cellular senescence.

## Key findings

- S. aureus infection reduces SIRT5 levels, leading to mitochondrial fragmentation and oxidative stress.
- SIRT5 overexpression protects against mitochondrial stress and cellular senescence in infected cells.
- UBC promotes SIRT5 degradation, which increases DRP1 accumulation and mitochondrial fission.

## Abstract

Staphylococcus aureus (S. aureus)–driven senescence of bovine mammary epithelial cells is a key determinant of mammary gland health, yet its molecular basis remains poorly defined. Sirtuin 5 (SIRT5), a mitochondria-localized desuccinylase, may play an important regulatory role in this process. This study aimed to elucidate the mechanisms by which S. aureus drives cellular senescence and to define the contribution of the SIRT5–mitochondrial axis to delaying senescence. We found pronounced oxidative stress and cellular senescence in mammary tissues from cows with S. aureus mastitis, accompanied by marked downregulation of SIRT5. In an S. aureus-infected epithelial cell model, infection induced mitochondrial stress characterized by excessive mitochondrial fragmentation, loss of membrane potential, and increased mitochondrial superoxide, along with oxidative damage and cellular senescence. Mechanistically, S. aureus toxins and the toxin-induced inflammatory response cooperatively drove mitochondrial stress, which in turn increased intracellular bacterial burden and exacerbated cell death. During infection, SIRT5 protein abundance was significantly reduced. Mass spectrometry and co-immunoprecipitation analyses indicated that infection upregulated the ubiquitin-conjugating enzyme ubiquitin C (UBC), enhanced its interaction with SIRT5, and promoted ubiquitin-mediated degradation of SIRT5. Loss of SIRT5 increased succinylation of dynamin-related protein 1 (DRP1), inhibited its ubiquitin-mediated degradation, and led to its excessive accumulation on the outer mitochondrial membrane, thereby promoting excessive mitochondrial fission. Functionally, SIRT5 overexpression markedly alleviated mitochondrial stress, oxidative damage, and senescence phenotypes. When mitochondrial fission was forcibly enhanced, the cytoprotective effect of SIRT5 was substantially weakened, confirming that SIRT5 acts through a pathway dependent on mitochondrial integrity. Collectively, S. aureus infection releases toxins and induces inflammatory injury, during which UBC-mediated SIRT5 degradation activates DRP1-dependent mitochondrial hyper-fragmentation, aggravating mitochondrial stress, oxidative stress, and mammary epithelial cell senescence. These findings identify SIRT5 as a critical regulator of redox and mitochondrial homeostasis in mammary epithelial cells and a potential therapeutic target for mitigating oxidative damage associated with bovine mastitis.

Staphylococcus aureus (S. aureus) is a leading cause of bovine mastitis, an intramammary infection that reduces milk yield, increases antibiotic use, and results in substantial economic loss. One way this bacterium damages the udder is by driving bovine mammary epithelial cells into premature senescence, but how infection triggers this process has not been fully understood. Here, we show that S. aureus infection induces an inflammatory response via its secreted toxins, which in turn provokes mitochondrial stress. We identify the mitochondrial enzyme Sirtuin 5 (SIRT5) as a key defender in this context. During infection, the host ubiquitin-conjugating enzyme ubiquitin C (UBC) targets SIRT5 for degradation. Loss of SIRT5 leads to accumulation of the mitochondrial fission protein dynamin-related protein 1 (DRP1), causing excessive mitochondrial fragmentation, enhanced oxidative damage, increased intracellular bacterial burden, and aggravated cellular senescence. Restoring SIRT5 helps to preserve mitochondrial integrity and reduces oxidative injury and senescence; however, this protection is weakened when mitochondrial fission is forcibly maintained at a high level. Together, our findings reveal that the UBC–SIRT5–DRP1 pathway links S. aureus infection to mitochondrial damage and cellular senescence, and they highlight SIRT5 as a potential therapeutic target to protect the bovine mammary gland (S1 Fig).