Search

Organisms use several strategies to mitigate mitochondrial stress, including the activation of the mitochondrial unfolded protein response (UPR

A dramatic rise of infections with antibiotic-resistant bacterial pathogens continues to challenge the healthcare field due to the lack of effective treatment regimes. As such, there is an urgent need to develop new antimicrobial agents that can combat these multidrug-resistant superbugs. Mitochondria are central regulators of metabolism and other cellular functions, including the regulation of innate immunity pathways involved in the defense against infection. The mitochondrial unfolded protein response (UPRmt) is a stress-activated pathway that mitigates mitochondrial dysfunction through the regulation of genes that promote recovery of the organelle. In the model organism Caenorhabditis elegans, the UPRmt also mediates an antibacterial defense program that combats pathogen infection, which promotes host survival. We sought to identify and characterize antimicrobial effectors that are regulated during the UPRmt. From our search, we discovered that the antimicrobial peptide CNC-4 is upregulated during this stress response. CNC-4 belongs to the caenacin family of antimicrobial peptides, which are predominantly found in nematodes and are known to have anti-fungal properties. Here, we find that CNC-4 also possesses potent antimicrobial activity against a spectrum of bacterial species and report on its characterization., Summary: The caenacin antimicrobial peptide CNC-4 is regulated by a mitochondrial recovery pathway and exhibits broad antibacterial activity.

The mitochondrial unfolded protein response (UPRmt) is a stress-activated pathway promoting mitochondrial recovery and defense against infection. In C. elegans, the UPRmt is activated during infection with the pathogen Pseudomonas aeruginosa—but only transiently. As this may reflect a pathogenic strategy to target a pathway required for host survival, we conducted a P. aeruginosa genetic screen to uncover mechanisms associated with this temporary activation. Here, we find that loss of the P. aeruginosa acyl-CoA dehydrogenase FadE2 prolongs UPRmt activity and extends host survival. FadE2 shows substrate preferences for the coenzyme A intermediates produced during the breakdown of the branched-chain amino acids valine and leucine. Our data suggests that during infection, FadE2 restricts the supply of these catabolites to the host hindering host energy metabolism in addition to the UPRmt. Thus, a metabolic pathway in P. aeruginosa contributes to pathogenesis during infection through manipulation of host energy status and mitochondrial stress signaling potential., Author summary The host engages multiple defense mechanisms in order to survive pathogen infection. Some pathogens have devised strategies to counteract these defense mechanisms to promote their success during infection. The mitochondrial unfolded protein response (UPRmt) is classically involved in resolving mitochondrial dysfunction but is also necessary to protect the host during infection with bacterial pathogens such as Pseudomonas aeruginosa. In C. elegans, while P. aeruginosa activates the UPRmt, chronic infection dampens this stress response pathway. Employing a genetic screen, we find that the novel P. aeruginosa acyl-CoA dehydrogenase FadE2, involved in the catabolism of the branched-chain amino acids valine and leucine, is associated with the repression of the UPRmt that limits host survival. Our findings suggest that competition for nutrients between the host and pathogen may dictate UPRmt activity and ultimately, host survival during infection.

Mitochondrial dysfunction is pervasive in human pathologies such as neurodegeneration, diabetes, cancer, and pathogen infections as well as during normal aging. Cells sense and respond to mitochondrial dysfunction by activating a protective transcriptional program known as the mitochondrial unfolded protein response (UPR(mt)), which includes genes that promote mitochondrial protein homeostasis and the recovery of defective organelles [1, 2]. Work in Caenorhabditis elegans has shown that the UPR(mt) is regulated by the transcription factor ATFS-1, which is regulated by organelle partitioning. Normally, ATFS-1 accumulates within mitochondria, but during respiratory chain dysfunction, high levels of reactive oxygen species (ROS), or mitochondrial protein folding stress, a percentage of ATFS-1 accumulates in the cytosol and traffics to the nucleus where it activates the UPR(mt) [2]. While similar transcriptional responses have been described in mammals [3, 4], how the UPR(mt) is regulated remains unclear. Here, we describe a mammalian transcription factor, ATF5, which is regulated similarly to ATFS-1 and induces a similar transcriptional response. ATF5 expression can rescue UPR(mt) signaling in atfs-1-deficient worms requiring the same UPR(mt) promoter element identified in C. elegans. Furthermore, mammalian cells require ATF5 to maintain mitochondrial activity during mitochondrial stress and promote organelle recovery. Combined, these data suggest that regulation of the UPR(mt) is conserved from worms to mammals.

A link between an intracellular stress response, bacterial infection and triggering of the innate immune response is shown in Caenorhabditis elegans; exposure to the pathogen Pseudomonas aeruginosa caused activation of the transcription factor ATFS-1 and innate immunity that is regulated by the mitochondrial unfolded protein response. The mitochondrial unfolded protein response (UPRmt) is a stress response that activates transcription of nuclear-encoded mitochondrial chaperone genes to promote protein homeostasis within the mitochondrion. Here Mark Pellegrino et al. provide evidence that mitochondrial dysfunction, and activation of the UPRmt, leads to upregulation of innate immunity and promotes pathogen resistance in Caenorhabditis elegans exposed to Pseudomonas aeruginosa. Metazoans identify and eliminate bacterial pathogens in microbe-rich environments such as the intestinal lumen; however, the mechanisms are unclear. Host cells could potentially use intracellular surveillance or stress response programs to detect pathogens that target monitored cellular activities and then initiate innate immune responses1,2,3. Mitochondrial function is evaluated by monitoring mitochondrial protein import efficiency of the transcription factor ATFS-1, which mediates the mitochondrial unfolded protein response (UPRmt). During mitochondrial stress, mitochondrial import is impaired4, allowing ATFS-1 to traffic to the nucleus where it mediates a transcriptional response to re-establish mitochondrial homeostasis5. Here we examined the role of ATFS-1 in Caenorhabditis elegans during pathogen exposure, because during mitochondrial stress ATFS-1 induced not only mitochondrial protective genes but also innate immune genes that included a secreted lysozyme and anti-microbial peptides. Exposure to the pathogen Pseudomonas aeruginosa caused mitochondrial dysfunction and activation of the UPRmt. C. elegans lacking atfs-1 were susceptible to P. aeruginosa, whereas hyper-activation of ATFS-1 and the UPRmt improved clearance of P. aeruginosa from the intestine and prolonged C. elegans survival in a manner mainly independent of known innate immune pathways6,7. We propose that ATFS-1 import efficiency and the UPRmt is a means to detect pathogens that target mitochondria and initiate a protective innate immune response.

SummaryMorphogenesis is a developmental phase during which cell fates are executed. Mechanical forces shaping individual cells play a key role during tissue morphogenesis. By investigating morphogenesis of the Caenorhabditis elegans hermaphrodite vulva, we show that the force-generating actomyosin network is differentially regulated by NOTCH and EGFR/RAS/MAPK signaling to shape the vulval tube. NOTCH signaling activates expression of the RHO kinase LET-502 in the secondary cell lineage through the ETS-family transcription factor LIN-1. LET-502 induces actomyosin-mediated contraction of the apical lumen in the secondary toroids, thereby generating a dorsal pushing force. In contrast, MAPK signaling in the primary lineage downregulates LET-502 RHO kinase expression to prevent toroid contraction and allow the gonadal anchor cell to expand the dorsal lumen of the primary toroids. The antagonistic action of the MAPK and NOTCH pathways thus controls vulval tube morphogenesis linking cell fate specification to morphogenesis.

Initiating Mitochondrial Repair The mitochondrial unfolded protein response (UPRmt) mediates the up-regulation of nuclear encoded mitochondrial chaperone genes in response to mitochondrial dysfunction. How mitochondrial dysfunction is communicated to the nucleus is unclear, but requires the transcription factor, ATFS-1. Nargund et al. (p. 587 , published online 14 June) found that the key point of regulation in UPRmt signaling is mitochondrial protein import efficiency of ATFS-1. In addition to a nuclear localization sequence (NLS), ATFS-1 has a mitochondrial targeting sequence (MTS) that is necessary for UPRmt repression. ATFS-1 is normally imported efficiently into mitochondria and degraded by the Lon protease. However, in the presence of stress, some ATFS-1 fails to be imported into mitochondria and is trafficked to the nucleus. The juxtaposition of a C-terminal NLS to an N-terminal MTS in a transcriptional activator thus couples unfolded protein load in the mitochondrial matrix to a rectifying transcriptional response in the nucleus.

Caenorhabditis elegans is an excellent model to observe cell movements and shape changes during the morphogenesis of the egg-shaped embryo into an elongated tube-like larva. Although much is known about the structural determinants involved in epidermal morphogenesis, relatively little is known about the transcriptional and post-transcriptional regulatory networks involved.Here, we describe the identification and functional characterization of the novel nuclear protein VAB-23, which belongs to a conserved protein family found in all metazoans. C. elegans VAB-23 is essential for ventral closure and elongation of the embryo. Time-lapse analysis indicates that VAB-23 is required for the formation of proper cell contacts between contralateral pairs of ventral epidermal cells. Tissue-specific rescue experiments reveal a function of VAB-23 in ventral neuroblasts that control the enclosure of the embryo by the overlaying epidermal cells. Finally, we provide evidence suggesting a role of VAB-23 in post-transcriptional gene regulation. We thus propose that VAB-23 regulates the expression of multiple secreted guidance cues in ventral neuroblasts that direct the migration of the overlaying epidermal cells. Members of the VAB-23 family may perform similar functions during morphogenesis in other metazoans.

A full-length cDNA (Tv-ant-1) encoding an adenine nucleotide translocator (ANT or ADP/ATP translocase) (Tv-ANT-1) was isolated from Trichostrongylus vitrinus (order Strongylida), an economically important parasitic nematode of small ruminants. The uninterrupted open reading frame (ORF) of 894 nucleotides encoded a predicted protein of 297 amino acids, containing characteristic motifs [RRRMMM] and PX(D,E)XX(K,R). Comparison with selected sequences from the free-living nematode Caenorhabditis elegans, cattle and human showed that Tv-ANT-1 is relatively conserved. Sequence identity was the greatest in and near the consensus sequence RRRMMM, and in the six hydrophobic regions predicted to be associated with alpha-helices and to traverse the cell membrane. Phylogenetic analyses of selected amino acid sequence data, using the neighbor-joining and maximum parsimony methods, revealed Tv-ANT-1 to be most closely related to the molecule (Ce-ANT-3) inferred from the tag-61 gene of C. elegans. Comparison of the genomic organization of the full-length Tv-ant-1 gene was similar to that of tag-61. Analysis of the region (5'-UTR) upstream of Tv-ant-1 identified some promoter components, including GATA transcription factor, CAAT and E-box elements. Transcriptional analysis by reverse transcription polymerase chain reaction (RT-PCR) showed that Tv-ant-1 was transcribed in all developmental stages of T. vitrinus, including the first- to fourth- stage larvae (L(1)-L(4)) as well as female and male adults. RNA interference, conducted by feeding C. elegans with double-stranded RNA (dsRNA) from Tv-ant-1 cDNA (using the homologous gene from C. elegans as a positive control), revealed no gene silencing. In spite of nucleotide identities of 100% in 23-30 bp stretches of sequence between the genes Tv-ant-1 and tag-61, these identities seem to be insufficient to achieve effective silencing in C. elegans using the parasite homologue/orthologue Tv-ant-1. This first insight into an ANT of T. vitrinus provides a foundation for exploring its role in developmental and/or survival processes of trichostrongylid nematodes.

Mitochondrial genomes (mitochondrial DNA, mtDNA) encode essential oxidative phosphorylation (OXPHOS) components. Because hundreds of mtDNAs exist per cell, a deletion in a single mtDNA has little impact. However, if the deletion genome is enriched, OXPHOS declines, resulting in cellular dysfunction. For example, Kearns-Sayre syndrome is caused by a single heteroplasmic mtDNA deletion. More broadly, mtDNA deletion accumulation has been observed in individual muscle cells and dopaminergic neurons during ageing. It is unclear how mtDNA deletions are tolerated or how they are propagated in somatic cells. One mechanism by which cells respond to OXPHOS dysfunction is by activating the mitochondrial unfolded protein response (UPR(mt)), a transcriptional response mediated by the transcription factor ATFS-1 that promotes the recovery and regeneration of defective mitochondria. Here we investigate the role of ATFS-1 in the maintenance and propagation of a deleterious mtDNA in a heteroplasmic Caenorhabditis elegans strain that stably expresses wild-type mtDNA and mtDNA with a 3.1-kilobase deletion (∆mtDNA) lacking four essential genes. The heteroplasmic strain, which has 60% ∆mtDNA, displays modest mitochondrial dysfunction and constitutive UPR(mt) activation. ATFS-1 impairment reduced the ∆mtDNA nearly tenfold, decreasing the total percentage to 7%. We propose that in the context of mtDNA heteroplasmy, UPR(mt) activation caused by OXPHOS defects propagates or maintains the deleterious mtDNA in an attempt to recover OXPHOS activity by promoting mitochondrial biogenesis and dynamics.

Mitochondrial diseases and aging are associated with defects in the oxidative phosphorylation machinery (OXPHOS), which are the only complexes composed of proteins encoded by separate genomes. To better understand genome coordination and OXPHOS recovery during mitochondrial dysfunction, we examined ATFS-1, a transcription factor that regulates mitochondria-to-nuclear communication during the mitochondrial UPR, via ChIP-sequencing. Surprisingly, in addition to regulating mitochondrial chaperone, OXPHOS complex assembly factor, and glycolysis genes, ATFS-1 bound directly to OXPHOS gene promoters in both the nuclear and mitochondrial genomes. Interestingly, atfs-1 was required to limit the accumulation of OXPHOS transcripts during mitochondrial stress, which required accumulation of ATFS-1 in the nucleus and mitochondria. Because balanced ATFS-1 accumulation promoted OXPHOS complex assembly and function, our data suggest that ATFS-1 stimulates respiratory recovery by fine-tuning OXPHOS expression to match the capacity of the suboptimal protein-folding environment in stressed mitochondria, while simultaneously increasing proteostasis capacity.

Morphogenesis represents a phase of development during which cell fates are executed. The conserved hox genes are key cell fate determinants during metazoan development, but their role in controlling organ morphogenesis is less understood. Here, we show that the C. elegans hox gene lin-39 regulates epidermal morphogenesis via its novel target, the essential zinc finger protein VAB-23. During the development of the vulva, the egg-laying organ of the hermaphrodite, the EGFR/RAS/MAPK signaling pathway activates, together with LIN-39 HOX, the expression of VAB-23 in the primary cell lineage to control the formation of the seven vulval toroids. VAB-23 regulates the formation of homotypic contacts between contralateral pairs of cells with the same sub-fates at the vulval midline by inducing smp-1 (semaphorin) transcription. In addition, VAB-23 prevents ectopic vulval cell fusions by negatively regulating expression of the fusogen eff-1. Thus, LIN-39 and the EGFR/RAS/MAPK signaling pathway, which specify cell fates earlier during vulval induction, continue to act during the subsequent phase of cell fate execution by regulating various aspects of epidermal morphogenesis. Vulval cell fate specification and execution are, therefore, tightly coupled processes.

Mitochondria are compartmentalized organelles essential for numerous cellular functions including ATP generation, iron-sulfur cluster biogenesis, nucleotide and amino acid metabolism as well as apoptosis. To promote biogenesis and proper function, mitochondria have a dedicated repertoire of molecular chaperones to facilitate protein folding and quality control proteases to degrade those proteins that fail to fold correctly. Mitochondrial protein folding is challenged by the complex organelle architecture, the deleterious effects of electron transport chain-generated reactive oxygen species and the mitochondrial genome's susceptibility to acquiring mutations. In response to the accumulation of unfolded or misfolded proteins beyond the organelle's chaperone capacity, cells mount a mitochondrial unfolded protein response (UPR mt ). The UPR mt is a mitochondria-to-nuclear signal transduction pathway resulting in the induction of mitochondrial protective genes including mitochondrial molecular chaperones and proteases to re-establish protein homeostasis within the mitochondrial protein-folding environment. Here, we review the current understanding of UPR mt signal transduction and the impact of the UPR mt on diseased cells. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.