Behrendt, C. | bioRxiv Science

bioRxiv2026

Ubp3 mediates dynamic deubiquitination of mitochondria upon induction of mitophagy

Ensuring quality and maintenance of mitochondria within eukaryotic cells is important for cellular fitness. One critical pathway that needs to be tightly controlled to achieve this is mitophagy, which is negatively regulated by the deubiquitinase Ubp3 in Saccharomyces cerevisiae. Here we show that beyond this established role of Ubp3, it is critical to mediate substantial temporal changes of the mitochondrial ubiquitin landscape during mitophagy. Isolated mitochondria displayed extensive ubiquitination under steady-state conditions, whereas rapamycin-mediated induction of mitophagy/autophagy led to a progressive elimination of mitochondrial ubiquitination. A systematic screening analysis revealed that Ubp3 and Ubp4 are crucial regulators of rapamycin-induced mitochondrial deubiquitination. Deletion of Bre5, a cofactor of Ubp3, or of the UBI4 gene, encoding Ubi4 required for mitophagy, did not impair deubiquitination of mitochondria during mitophagy, indicating that Ubp3 has additional functions. We further showed that Ubi4 acts epistatic to Ubp3 for regulating mitophagy, supporting that mitophagy does not simply depend on the extent of mitochondrial ubiquitination, but rather on specific ubiquitinated substrates. Consistent with the role of Ubp3 in vivo, purified Ubp3 or its catalytic domain alone were sufficient to efficiently deubiquitinate isolated mitochondria in vitro in a Bre5-independent manner. Together, these findings highlight Ubp3 as a prominent rheostat of mitochondrial ubiquitin remodelling, revealing mitochondrial ubiquitin storage and its dynamic release as another layer of stress regulation by ubiquitin-dependent quality control pathways.

cell biology↗

bioRxiv2023

Susceptible bacteria survive antibiotic treatment in the mammalian gastrointestinal tract without evolving resistance

In vitro systems have provided great insight into the mechanisms of antibiotic resistance. Yet, in vitro approaches cannot reflect the full complexity of what transpires within a host. As the mammalian gut is host to trillions of resident bacteria and thus a potential breeding ground for antibiotic resistance, we sought to better understand how gut bacteria respond to antibiotic treatment in vivo. Here, we colonized germ-free mice with a genetically barcoded antibiotic pan-susceptible Escherichia coli clinical isolate and then administered the antibiotic cefepime via programmable subcutaneous pumps which allowed for closer emulation of human parenteral antibiotic pharmacokinetics/dynamics. After seven days of antibiotics, we were unable to culture E. coli from feces. We were, however, able to recover barcoded E. coli from harvested gastrointestinal (GI) tissue, despite high GI tract and plasma cefepime concentrations. Strikingly, these E. coli isolates were not resistant to cefepime but had acquired mutations - most notably in the wbaP gene, which encodes an enzyme required for the initiation of the synthesis of the polysaccharide capsule and lipopolysaccharide O antigen - that increased their ability to invade and survive within intestinal cells, including cultured human colonocytes. Further, these E. coli mutants exhibited a persister phenotype when exposed to cefepime, allowing for greater survival to pulses of cefepime treatment when compared to the wildtype strain. Our findings highlight a mechanism by which bacteria in the gastrointestinal tract can adapt to antibiotic treatment by increasing their ability to persist during antibiotic treatment and invade intestinal epithelial cells where antibiotic concentrations are substantially reduced.

microbiology↗