Parallel ecological and evolutionary responses to selection in a natural bacterial community.

Evolution can occur over ecological timescales, suggesting a potentially important role for rapid evolution in shaping community trait distributions. However, evidence of concordant eco-evolutionary dynamics often comes from in vitro studies of highly simplified communities, and measures of ecological and evolutionary dynamics are rarely directly comparable. Here, we quantified how ecological species sorting and rapid evolution simultaneously shape community trait distributions by tracking within- and between-species changes in a key trait in a complex bacterial community. We focused on the production of siderophores; bacteria use these costly secreted metabolites to scavenge poorly soluble iron and to detoxify environments polluted with toxic nonferrous metals. We found that responses to copper-imposed selection within and between species were ultimately the same-intermediate siderophore levels were favored-and occurred over similar timescales. Despite being a social trait, this level of siderophore production was selected regardless of whether species evolved in isolation or in a community context. Our study suggests that evolutionary selection can play a pivotal role in shaping community trait distributions within natural, highly complex, bacterial communities. Furthermore, trait evolution may not always be qualitatively affected by interactions with other community members.

Ecological dependencies and the illusion of cooperation in microbial communities.

Ecological dependencies - where organisms rely on other organisms for survival - are a ubiquitous feature of life on earth. Multicellular hosts rely on symbionts to provide essential vitamins and amino acids. Legume plants similarly rely on nitrogen-fixing rhizobia to convert atmospheric nitrogen to ammonia. In some cases, dependencies can arise via loss-of-function mutations that allow one partner to benefit from the actions of another. It is common in microbiology to label ecological dependencies between species as cooperation - making it necessary to invoke cooperation-specific frameworks to explain the phenomenon. However, in many cases, such traits are not (at least initially) cooperative, because they are not selected for because of the benefits they confer on a partner species. In contrast, dependencies in microbial communities may originate from fitness benefits gained from genomic-streamlining (i.e. Black Queen Dynamics). Here, we outline how the Black Queen Hypothesis predicts the formation of metabolic dependencies via loss-of-function mutations in microbial communities, without needing to invoke any cooperation-specific explanations. Furthermore we outline how the Black Queen Hypothesis can act as a blueprint for true cooperation as well as discuss key outstanding questions in the field. The nature of interactions in microbial communities can predict the ability of natural communities to withstand and recover from disturbances. Hence, it is vital to gain a deeper understanding of the factors driving these dynamic interactions over evolutionary time.

SCFFBXW7 regulates G2-M progression through control of CCNL1 ubiquitination.

FBXW7, which encodes a substrate-specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various proteins involved in cellular proliferation. Here, using genome-wide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.

Cultivating antimicrobial resistance: how intensive agriculture ploughs the way for antibiotic resistance.

Antimicrobial resistance (AMR) is a growing threat to public health, global food security and animal welfare. Despite efforts in antibiotic stewardship, AMR continues to rise worldwide. Anthropogenic activities, particularly intensive agriculture, play an integral role in the dissemination of AMR genes within natural microbial communities - which current antibiotic stewardship typically overlooks. In this review, we examine the impact of anthropogenically induced temperature fluctuations, increased soil salinity, soil fertility loss, and contaminants such as metals and pesticides on the de novo evolution and dissemination of AMR in the environment. These stressors can select for AMR - even in the absence of antibiotics - via mechanisms such as cross-resistance, co-resistance and co-regulation. Moreover, anthropogenic stressors can prime bacterial physiology against stress, potentially widening the window of opportunity for the de novo evolution of AMR. However, research to date is typically limited to the study of single isolated bacterial species - we lack data on how intensive agricultural practices drive AMR over evolutionary timescales in more complex microbial communities. Furthermore, a multidisciplinary approach to fighting AMR is urgently needed, as it is clear that the drivers of AMR extend far beyond the clinical environment.

Stress causes interspecific facilitation within a compost community.

Ecological theory predicts interactions between species to become more positive under abiotic stress, while competition should prevail in more benign environments. However, experimental tests of this stress gradient hypothesis in natural microbial communities are lacking. We test this hypothesis by measuring interactions between 10 different members of a bacterial community inhabiting potting compost in the presence or absence of toxic copper stress. We found that copper stress caused significant net changes in species interaction signs, shifting the net balance towards more positive interactions. This pattern was at least in part driven by copper-sensitive isolates - that produced relatively small amounts of metal-detoxifying siderophores - benefitting from the presence of other species that produce extracellular detoxifying agents. As well as providing support for the stress gradient hypothesis, our results highlight the importance of community-wide public goods in shaping microbial community composition.

Transposable temperate phages promote the evolution of divergent social strategies in Pseudomonas aeruginosa populations.

Transposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Using experimental evolution of Pseudomonas aeruginosa in iron-limited and iron-rich environments, where the cost of producing cooperative iron-chelating siderophores is high and low, respectively, we show that transposable phages promote divergence into extreme siderophore production phenotypes. Iron-limited populations with transposable phages evolved siderophore overproducing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations in pvd genes, encoding pyoverdine biosynthesis, and pqs genes, encoding quinolone signalling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies.

Temperate phages both mediate and drive adaptive evolution in pathogen biofilms.

Temperate phages drive genomic diversification in bacterial pathogens. Phage-derived sequences are more common in pathogenic than nonpathogenic taxa and are associated with changes in pathogen virulence. High abundance and mobilization of temperate phages within hosts suggests that temperate phages could promote within-host evolution of bacterial pathogens. However, their role in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of three temperate phages active in cystic fibrosis (CF) lung infections, including the transposable phage, ɸ4, which is closely related to phage D3112. Populations grew as free-floating biofilms in artificial sputum medium, mimicking sputum of CF lungs where P. aeruginosa is an important pathogen and undergoes evolutionary adaptation and diversification during chronic infection. Although bacterial populations adapted to the biofilm environment in both treatments, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. Populations evolving with phages exhibited a greater degree of parallel evolution and faster selective sweeps than populations without phages. Phage ɸ4 integrated randomly into the bacterial chromosome, but integrations into motility-associated genes and regulators of quorum sensing systems essential for virulence were selected in parallel, strongly suggesting that these insertional inactivation mutations were adaptive. Temperate phages, and in particular transposable phages, are therefore likely to facilitate adaptive evolution of bacterial pathogens within hosts.

Ecological selection of siderophore-producing microbial taxa in response to heavy metal contamination.

Some microbial public goods can provide both individual and community-wide benefits, and are open to exploitation by non-producing species. One such example is the production of metal-detoxifying siderophores. Here, we investigate whether conflicting selection pressures on siderophore production by heavy metals - a detoxifying effect of siderophores, and exploitation of this detoxifying effect - result in a net increase or decrease. We show that the proportion of siderophore-producing taxa increases along a natural heavy metal gradient. A causal link between metal contamination and siderophore production was subsequently demonstrated in a microcosm experiment in compost, in which we observed changes in community composition towards taxa that produce relatively more siderophores following copper contamination. We confirmed the selective benefit of siderophores by showing that taxa producing large amounts of siderophore suffered less growth inhibition in toxic copper. Our results suggest that ecological selection will favour siderophore-mediated decontamination, with important consequences for potential remediation strategies.

Adaptation to public goods cheats in Pseudomonas aeruginosa.

Cooperation in nature is ubiquitous, but is susceptible to social cheats who pay little or no cost of cooperation yet reap the benefits. The effect such cheats have on reducing population productivity suggests that there is selection for cooperators to mitigate the adverse effects of cheats. While mechanisms have been elucidated for scenarios involving a direct association between producer and cooperative product, it is less clear how cooperators may suppress cheating in an anonymous public goods scenario, where cheats cannot be directly identified. Here, we investigate the real-time evolutionary response of cooperators to cheats when cooperation is mediated by a diffusible public good: the production of iron-scavenging siderophores by Pseudomonas aeruginosa We find that siderophore producers evolved in the presence of a high frequency of non-producing cheats were fitter in the presence of cheats, at no obvious cost to population productivity. A novel morphotype independently evolved and reached higher frequencies in cheat-adapted versus control populations, exhibiting reduced siderophore production but increased production of pyocyanin-an extracellular toxin that can also increase the availability of soluble iron. This suggests that cooperators may have mitigated the negative effects of cheats by downregulating siderophore production and upregulating an alternative iron-acquisition public good. More generally, the study emphasizes that cooperating organisms can rapidly adapt to the presence of anonymous cheats without necessarily incurring fitness costs in the environment they evolve in.

High virulence sub-populations in Pseudomonas aeruginosa long-term cystic fibrosis airway infections.

BACKGROUND: Pseudomonas aeruginosa typically displays loss of virulence-associated secretions over the course of chronic cystic fibrosis infections. This has led to the suggestion that virulence is a costly attribute in chronic infections. However, previous reports suggest that overproducing (OP) virulent pathotypes can coexist with non-producing mutants in the CF lung for many years. The consequences of such within-patient phenotypic diversity for the success of this pathogen are not fully understood. Here, we provide in-depth quantification of within-host variation in the production of three virulence associated secretions in the Liverpool cystic fibrosis epidemic strain of P. aeruginosa, and investgate the effect of this phenotypic variation on virulence in acute infections of an insect host model. RESULTS: Within-patient variation was present for all three secretions (pyoverdine, pyocyanin and LasA protease). In two out of three patients sampled, OP isolates coexisted with under-producing mutants. In the third patient, all 39 isolates were under-producers of all three secretions relative to the transmissible ancestor LESB58. Finally, this phenotypic variation translated into variation in virulence in an insect host model. CONCLUSIONS: Within population variation in the production of P. aeruginosa virulence-associated secretions can lead to high virulence sub-populations persisting in patients with chronic CF infections.

Structural and pharmacological characterization of novel potent and selective monoclonal antibody antagonists of glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that, upon binding to its receptor (GIPr; a class B G-protein-coupled receptor), stimulates insulin secretion by beta cells in the pancreas. There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure to support studies on GIP biology. Here we describe the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog, and human GIP receptors with a Ki of 7 nm for the human GIPr. Gipg013 antagonizes the GIP receptor and inhibits GIP-induced insulin secretion in vitro and in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr extracellular domain (ECD) shows that the antibody binds through a series of hydrogen bonds from the complementarity-determining regions of Gipg013 Fab to the N-terminal α-helix of GIPr ECD as well as to residues around its highly conserved glucagon receptor subfamily recognition fold. The antibody epitope overlaps with the GIP binding site on the GIPr ECD, ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP.

Social evolution of toxic metal bioremediation in Pseudomonas aeruginosa.

Bacteria are often iron-limited, and hence produce extracellular iron-scavenging siderophores. A crucial feature of siderophore production is that it can be an altruistic behaviour (individually costly but benefitting neighbouring cells), thus siderophore producers can be invaded by non-producing social 'cheats'. Recent studies have shown that siderophores can also bind other heavy metals (such as Cu and Zn), but in this case siderophore chelation actually reduces metal uptake by bacteria. These complexes reduce heavy metal toxicity, hence siderophore production may contribute to toxic metal bioremediation. Here, we show that siderophore production in the context of bioremediation is also an altruistic trait and can be exploited by cheating phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. Specifically, we show that in toxic copper concentrations (i) siderophore non-producers evolve de novo and reach high frequencies, and (ii) producing strains are fitter than isogenic non-producing strains in monoculture, and vice versa in co-culture. Moreover, we show that the evolutionary effect copper has on reducing siderophore production is greater than the reduction observed under iron-limited conditions. We discuss the relevance of these results to the evolution of siderophore production in natural communities and heavy metal bioremediation.

Secondary messenger signalling influences Pseudomonas aeruginosa adaptation to sinus and lung environments.

Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Prolonged infection allows the accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonizing upper airway environments. Here, we model this process using an experimental evolution approach. Pseudomonas aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments-sinus and lungs, under CF and non-CF conditions-selected for loss of twitching motility, increased resistance to multiple antibiotic classes, and a hyper-biofilm phenotype. These traits conferred increased airway colonization potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching, and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included the loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection.

Proximity-driven site-specific cyclization of phage-displayed peptides.

Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvß3.

The evolution of bacterial mutation rates under simultaneous selection by interspecific and social parasitism.

Many bacterial populations harbour substantial numbers of hypermutable bacteria, in spite of hypermutation being associated with deleterious mutations. One reason for the persistence of hypermutators is the provision of novel mutations, enabling rapid adaptation to continually changing environments, for example coevolving virulent parasites. However, hypermutation also increases the rate at which intraspecific parasites (social cheats) are generated. Interspecific and intraspecific parasitism are therefore likely to impose conflicting selection pressure on mutation rate. Here, we combine theory and experiments to investigate how simultaneous selection from inter- and intraspecific parasitism affects the evolution of bacterial mutation rates in the plant-colonizing bacterium Pseudomonas fluorescens. Both our theoretical and experimental results suggest that phage presence increases and selection for public goods cooperation (the production of iron-scavenging siderophores) decreases selection for mutator bacteria. Moreover, phages imposed a much greater growth cost than social cheating, and when both selection pressures were imposed simultaneously, selection for cooperation did not affect mutation rate evolution. Given the ubiquity of infectious phages in the natural environment and clinical infections, our results suggest that phages are likely to be more important than social interactions in determining mutation rate evolution.

Chromosome-level genome editing uncovers aneuploidy addiction in cancer.

Publishing in Science, Girish and colleagues achieve chromosome-level genome editing to reveal a requirement for aneuploidy in breast and melanoma cancers. Authors developed and leveraged ReDACT (restoring disomy in aneuploid cells using CRISPR targeting) to generate isogenic models of aneuploidy and demonstrate that some cancers are addicted to increased copy number of specific chromosome arms.

Modulation of Wnt-ß-catenin signaling with antibodies: therapeutic opportunities and challenges.

Since the recognition that mutations in components of the Wnt-ß-catenin pathway underlie some human cancers, considerable attention has been dedicated to developing therapeutic modalities to block its activity. Despite numerous efforts, no drug directly inhibiting Wnt signaling is currently clinically available. Conversely, activating the Wnt pathway in a specific manner has recently been made possible with new molecules mimicking the activity of Wnt proteins, thus offering new possibilities for controlling tissue stem cell activity and for the rational treatment of various degenerative conditions. We describe the landscape of antibody modalities that modulate the Wnt-ß-catenin pathway, and detail the advances and challenges in both cancer and regenerative medicine drug development.

Community composition drives siderophore dynamics in multispecies bacterial communities.

BACKGROUND: Intraspecific public goods are commonly shared within microbial populations, where the benefits of public goods are largely limited to closely related conspecifics. One example is the production of iron-scavenging siderophores that deliver iron to cells via specific cell envelope receptor and transport systems. Intraspecific social exploitation of siderophore producers is common, since non-producers avoid the costs of production but retain the cell envelope machinery for siderophore uptake. However, little is known about how interactions between species (i.e., interspecific interactions) can shape intraspecific public goods exploitation. Here, we predicted that strong competition for iron between species in diverse communities will increase costs of siderophore cooperation, and hence drive intraspecific exploitation. We examined how increasing microbial community species diversity shapes intraspecific social dynamics by monitoring the growth of siderophore producers and non-producers of the plant-growth promoting bacterium Pseudomonas fluorescens, embedded within tree-hole microbial communities ranging from 2 to 15 species. RESULTS: We find, contrary to our prediction, that siderophore production is favoured at higher levels of community species richness, driven by increased likelihood of encountering key species that reduce the growth of siderophore non-producing (but not producing) strains of P. fluorescens. CONCLUSIONS: Our results suggest that maintaining a diverse soil microbiota could partly contribute to the maintenance of siderophore production in natural communities.

Relative Biological Effectiveness (RBE) of [64Cu]Cu and [177Lu]Lu-NOTA-panitumumab F (ab')2 radioimmunotherapeutic agents vs. γ-radiation for decreasing the clonogenic survival in vitro of human pancreatic ductal adenocarcinoma (PDAC) cells.

INTRODUCTION: Our objective was to compare [64Cu]Cu-NOTA-panitumumab F(ab')2 and [177Lu]Lu-NOTA-panitumumab F(ab')2 radioimmunotherapy (RIT) agents for decreasing the clonogenic survival fraction (SF) in vitro of EGFR-positive human pancreatic ductal adenocarcinoma (PDAC) cell lines and estimate the relative biological effectiveness (RBE) vs. γ-radiation (XRT). METHODS: EGFR-positive PDAC cell lines (AsPC-1, PANC-1, MIAPaCa-2, Capan-1) and EGFR-knockout PANC-1 EGFR KO cells were treated in vitro for 18 h with (0-19.65 MBq; 72 nmols/L) of [64Cu]Cu-NOTA-panitumumab F(ab')2 or [177Lu]Lu-NOTA-panitumumab F(ab')2 or XRT (0-8 Gy) followed by clonogenic assay. The SF was determined after culturing single treated cells for 14 d. Cell fractionation studies were performed for cells incubated with 1 MBq (72 nmols/L) of [64Cu]Cu-NOTA-panitumumab F(ab')2 or [177Lu]Lu-NOTA-panitumumab F(ab')2 for 1, 4, or 24 h to estimate the time-integrated activity (Ã) on the cell surface, cytoplasm, nucleus and medium. Radiation absorbed doses in the nucleus were calculated by multiplying à by S-factors calculated by Monte Carlo N Particle (MCNP) modeling using monolayer cell culture geometry. The SF of PDAC cells was plotted vs. dose and fitted to a linear quadratic model to estimate the dose required to decrease the SF to 0.1 (D10). The D10 for RIT agents were compared to XRT to estimate the RBE. DNA double-strand breaks (DSBs) caused by [64Cu]Cu-NOTA-panitumumab F(ab')2 or [177Lu]Lu-NOTA-panitumumab F(ab')2 continuous exposure for 5 h or 20 h were probed by immunofluorescence for γ-H2AX. Relative EGFR expression of PDAC cells was assessed by flow cytometry (scored + to +++) and cell doubling times for untreated cells were determined. RESULTS: The D10 for [64Cu]Cu-NOTA-panitumumab F(ab')2 ranged from 9.1 Gy (PANC-1) to 39.9 Gy (Capan-1). The D10 for [177Lu]Lu-NOTA-panitumumab F(ab')2 ranged from 11.7 Gy (AsPC-1) to 170.8 Gy (Capan-1). The D10 for XRT ranged from 2.5 Gy (Capan-1) to 6.7 Gy (PANC-1 EGFR KO). D10 values were not correlated with EGFR expression over a relatively narrow range (++ to +++) or with cell doubling times. Based on D10 values, PANC-1 EGFR KO cells were 1.6-fold less sensitive than PANC-1 cells to [64Cu]Cu-NOTA-panitumumab F(ab')2 and 1.9-fold less sensitive to [177Lu]Lu-NOTA-panitumumab F(ab')2. The RBE for [64Cu]Cu-NOTA-panitumumab F(ab')2 ranged from 0.06 for Capan-1 cells to 0.45 for PANC-1 cells. The RBE for [177Lu]Lu-NOTA-panitumumab F(ab')2 ranged from 0.015 for Capan-1 cells to 0.28 for AsPC-1 cells. DNA DSBs were detected in PDAC cells exposed to [64Cu]Cu-NOTA-panitumumab F(ab')2 or [177Lu]Lu-NOTA-panitumumab F(ab')2 but were not correlated with the SF of the cells. CONCLUSIONS: We conclude that at the same dose delivered to the cell nucleus [64Cu]Cu-NOTA-panitumumab F(ab')2 and [177Lu]Lu-NOTA-panitumumab F(ab')2 were less radiobiologically effective than XRT for decreasing the SF of human PDAC cells, but [64Cu]Cu-NOTA-panitumumab F(ab')2 was more cytotoxic than [177Lu]Lu-NOTA-panitumumab F(ab')2 except for AsPC-1 cells which were more sensitive to [177Lu]Lu-NOTA-panitumumab F(ab')2. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: This study demonstrates that higher radiation doses may be required for RIT than XRT to achieve radiobiologically equivalent effects when used to treat PDAC.

CRISPR Screening in Tandem with Targeted mtDNA Damage Reveals WRNIP1 Essentiality.

A major impediment to the characterization of mtDNA repair mechanisms, in comparison to nuclear DNA repair mechanisms, is the difficulty of specifically addressing mitochondrial damage. Using a mitochondria-penetrating peptide, we can deliver DNA-damaging agents directly to mitochondria, bypassing the nuclear compartment. Here, we describe the use of a mtDNA-damaging agent in tandem with CRISPR/Cas9 screening for the genome-wide discovery of factors essential for mtDNA damage response. Using mitochondria-targeted doxorubicin (mtDox) we generate mtDNA double-strand breaks (mtDSBs) specifically in this organelle. Combined with an untargeted Dox screen, we identify genes with significantly greater essentiality during mitochondrial versus nuclear DNA damage. We characterize the essentially of our top hit - WRNIP1 - observed here for the first time to respond to mtDNA damage. We further investigate the mitochondrial role of WRNIP1 in innate immune signaling and nuclear genome maintenance, outlining a model that experimentally supports mitochondrial turnover in response to mtDSBs.