Yersinia entomophaga Tc toxin is released by T10SS-dependent lysis of specialized cell subpopulations.

Disease-causing bacteria secrete numerous toxins to invade and subjugate their hosts. Unlike many smaller toxins, the secretion machinery of most large toxins remains enigmatic. By combining genomic editing, proteomic profiling and cryo-electron tomography of the insect pathogen Yersinia entomophaga, we demonstrate that a specialized subset of these cells produces a complex toxin cocktail, including the nearly ribosome-sized Tc toxin YenTc, which is subsequently exported by controlled cell lysis using a transcriptionally coupled, pH-dependent type 10 secretion system (T10SS). Our results dissect the Tc toxin export process by a T10SS, identifying that T10SSs operate via a previously unknown lytic mode of action and establishing them as crucial players in the size-insensitive release of cytoplasmically folded toxins. With T10SSs directly embedded in Tc toxin operons of major pathogens, we anticipate that our findings may model an important aspect of pathogenesis in bacteria with substantial impact on agriculture and healthcare.

Time-resolved proteomic analyses of senescence highlight metabolic rewiring of mitochondria.

Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early responding pathways, the degradation of branched-chain amino acid was increased, whereas the one carbon folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic flux analyses, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide a comprehensive view on the changes in mitochondrial proteome in senescent cells and reveal how the mitochondrial metabolism is rewired in senescent cells.

The mitochondrial protease OMA1 acts as a metabolic safeguard upon nuclear DNA damage.

The metabolic plasticity of mitochondria ensures cell development, differentiation, and survival. The peptidase OMA1 regulates mitochondrial morphology via OPA1 and stress signaling via DELE1 and orchestrates tumorigenesis and cell survival in a cell- and tissue-specific manner. Here, we use unbiased systems-based approaches to show that OMA1-dependent cell survival depends on metabolic cues. A metabolism-focused CRISPR screen combined with an integrated analysis of human gene expression data found that OMA1 protects against DNA damage. Nucleotide deficiencies induced by chemotherapeutic agents promote p53-dependent apoptosis of cells lacking OMA1. The protective effect of OMA1 does not depend on OMA1 activation or OMA1-mediated OPA1 and DELE1 processing. OMA1-deficient cells show reduced glycolysis and accumulate oxidative phosphorylation (OXPHOS) proteins upon DNA damage. OXPHOS inhibition restores glycolysis and confers resistance against DNA damage. Thus, OMA1 dictates the balance between cell death and survival through the control of glucose metabolism, shedding light on its role in cancerogenesis.

Regulation of mitochondrial proteostasis by the proton gradient.

Mitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multiproteomic approach to demonstrate the regulation of the m-AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca2+ /H+ exchanger in the mitochondrial inner membrane, which binds to and inhibits the m-AAA protease. TMBIM5 ensures cell survival and respiration, allowing Ca2+ efflux from mitochondria and limiting mitochondrial hyperpolarization. Persistent hyperpolarization, however, triggers degradation of TMBIM5 and activation of the m-AAA protease. The m-AAA protease broadly remodels the mitochondrial proteome and mediates the proteolytic breakdown of respiratory complex I to confine ROS production and oxidative damage in hyperpolarized mitochondria. TMBIM5 thus integrates mitochondrial Ca2+ signaling and the energetic status of mitochondria with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism.