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Bacterial pathogens employ a variety of tactics to persist in their host and promote infection. Pathogens often target host organelles in order to benefit their survival, either through manipulation or subversion of their function. Mitochondria are regularly targeted by bacterial pathogens owing to their diverse cellular roles, including energy production and regulation of programmed cell death. However, disruption of normal mitochondrial function during infection can be detrimental to cell viability because of their essential nature. In response, cells use multiple quality control programs to mitigate mitochondrial dysfunction and promote recovery. In this review, we will provide an overview of mitochondrial recovery programs including mitochondrial dynamics, the mitochondrial unfolded protein response (UPR(mt)), and mitophagy. We will then discuss the various approaches used by bacterial pathogens to target mitochondria, which result in mitochondrial dysfunction. Lastly, we will discuss how cells leverage mitochondrial recovery programs beyond their role in organelle repair, to promote host defense against pathogen infection.

Objectives Metallo-β-lactamases (MBLs) are an emerging class of antimicrobial resistance enzymes that degrade β-lactam antibiotics, including last-resort carbapenems. Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) are increasingly prevalent, but treatment options are limited. While several serine-dependent β-lactamase inhibitors are formulated with commonly prescribed β-lactams, no MBL inhibitors are currently approved for combinatorial therapies. New compounds that target MBLs to restore carbapenem activity against CPE are therefore urgently needed. Herein we identified and characterized novel synthetic peptide inhibitors that bound to and inhibited NDM-1, which is an emerging β-lactam resistance mechanism in CPE. Methods We leveraged Surface Localized Antimicrobial displaY (SLAY) to identify and characterize peptides that inhibit NDM-1, which is a primary carbapenem resistance mechanism in CPE. Lead inhibitor sequences were chemically synthesized and MBCs and MICs were calculated in the presence/absence of carbapenems. Kinetic analysis with recombinant NDM-1 and select peptides tested direct binding and supported NDM-1 inhibitor mechanisms of action. Inhibitors were also tested for cytotoxicity. Results We identified approximately 1700 sequences that potentiated carbapenem-dependent killing against NDM-1 Escherichia coli. Several also enhanced meropenem-dependent killing of other CPE. Biochemical characterization of a subset indicated the peptides penetrated the bacterial periplasm and directly bound NDM-1 to inhibit enzymatic activity. Additionally, each demonstrated minimal haemolysis and cytotoxicity against mammalian cell lines. Conclusions Our approach advances a molecular platform for antimicrobial discovery, which complements the growing need for alternative antimicrobials. We also discovered lead NDM-1 inhibitors, which serve as a starting point for further chemical optimization.

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

Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis-regulatory sequences (enhancers and promoters), trans-regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis-regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade.

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.

SUMMARY Metazoan cell death mechanisms are diverse and include numerous non-apoptotic programs. One program called entosis involves the invasion of live cells into their neighbors and is known to occur in cancers. Here, we identify a developmental function for entosis: to clear the male-specific linker cell in C. elegans. The linker cell leads migration to shape the gonad and is removed to facilitate fusion of the gonad to the cloaca. We find that the linker cell is cleared in a manner involving cell-cell adhesions and cell-autonomous control of uptake through linker cell actin. Linker cell entosis generates a lobe structure that is deposited at the site of gonad-to-cloaca fusion and is removed during mating. Inhibition of lobe scission inhibits linker cell death, demonstrating that the linker cell invades its host while alive. Our findings demonstrate a developmental function for entosis: to eliminate a migrating cell and facilitate gonad-to-cloaca fusion, which is required for fertility., Graphical Abstract, In Brief Entosis is a cell death mechanism, previously observed in cancer cell populations, that involves the invasion of live cells into their neighbors. Lee et al. now show that entosis has a developmental function in C. elegans, clearing the linker cell during gonad formation.

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.

Mitochondria are multifaceted and indispensable organelles required for cell performance. Accordingly, dysfunction to mitochondria can result in cellular decline and possibly the onset of disease. Cells use a variety of means to recover mitochondria and restore homeostasis, including the activation of retrograde pathways such as the mitochondrial unfolded protein response (UPRmt). In this Minireview, we will discuss how cells adapt to mitochondrial stress through UPRmt regulation. Furthermore, we will explore the current repertoire of biological functions that are associated with this essential stress-response pathway.

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.

The Anthropocene will be characterized by increased environmental disturbances, leading to the survival of stress-tolerant organisms, particularly in the oceans, where novel marine diseases and elevated temperatures are re-shaping ecosystems. These environmental changes underscore the importance of identifying mechanisms which promote stress tolerance in ecologically important non-model species such as reef-building corals. Mitochondria are central regulators of cellular stress and have dedicated recovery pathways including the mitochondrial unfolded protein response, which increases the transcription of protective genes promoting protein homeostasis, free radical detoxification and innate immunity. In this investigation, we identify a mitochondrial unfolded protein response in the endangered Caribbean coralOrbicella faveolata, by performingin vivofunctional replacement using a transcription factor (Of-ATF5) originating from a coral in the model organismCaenorhabditis elegans. In addition, we use RNA-seq network analysis and transcription factor-binding predictions to identify a transcriptional network of genes likely to be regulated by Of-ATF5 which is induced during the immune challenge and temperature stress. Overall, our findings uncover a conserved cellular pathway which may promote the ability of reef-building corals to survive increasing levels of environmental stress.

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.

During organogenesis, individual cells must commit to and execute specific cell fates. However, the molecular mechanisms linking cell fate specification to fate execution and morphogenesis remain a largely unexplored area in developmental biology. The Caenorhabditis elegans vulva is an excellent model to dissect the molecular pathways linking cell fate specification and execution during organogenesis. We have recently identified a conserved nuclear zinc finger transcription factor called VAB-23 that plays essential roles during vulval torid formation in the larva and ventral epidermal closure in the embryo. VAB-23 regulates the transcription of specific target genes including smp-1 Semaphorin. EGFR/RAS/MAPK signaling upregulates via the HOX protein LIN-39 the expression of VAB-23 in the 1° vulval cell lineage, indicating that cell fate specification and execution are temporally overlapping and tightly linked processes. Here, we discuss the roles of VAB-23 in morphogenesis and the implications of its regulation on the spatio-temporal control of organogenesis.

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.

Adenine nucleotide translocators (ANTs) belong to the mitochondrial carrier family (MCF) of proteins. ATP production and consumption are tightly linked to ANTs, the kinetics of which have been proposed to play a key regulatory role in mitochondrial oxidative phosphorylation. ANTs are also recognized as a central component of the mitochondrial permeability transition pore associated with apoptosis. Although ANTs have been investigated in a range of vertebrates, including human, mouse and cattle, and invertebrates, such as Drosophila melanogaster (vinegar fly), Saccharomyces cerevisiae (yeast) and Caenorhabditis elegans (free-living nematode), there has been a void of information on these molecules for parasitic nematodes of socio-economic importance. Exploring ANTs in nematodes has the potential lead to a better understanding of their fundamental roles in key biological pathways and might provide an avenue for the identification of targets for the rational design of nematocidal drugs. In the present article, we describe the discovery of an ANT from Haemonchus contortus (one of the most economically important parasitic nematodes of sheep and goats), conduct a comparative analysis of key ANTs and their genes (particularly ant-1.1) in nematodes and other organisms, predict the functional roles utilizing a combined genomic-bioinformatic approach and propose ANTs and associated molecules as possible drug targets, with the potential for biotechnological outcomes.

The sequence and expression profile of Tv-mab-23, a gene encoding a DM domain-containing protein from the parasitic nematode Trichostrongylus vitrinus, were investigated. The gene, containing an open reading frame (ORF) of 537 bp, encoded a predicted protein which had an overall amino acid identity of 45% to the MAB-23 molecule of the free-living nematode Caenorhabditis elegans. High levels of conservation were particularly predominant at the amino terminus of the proteins, with 86% identity over the first 58 amino acid residues in a region containing the highly conserved zinc module of the DM domain and conserved regions of the recognition helix of DM domain-containing transcription factors. Tv-mab-23 was expressed in a stage-specific manner in T. vitrinus. The highest levels of expression in fourth stage larvae coincide with the period in which profound gender-specific alterations in physiology occur in both the parasitic and free-living nematodes.

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.

Mitochondria are highly dynamic and structurally complex organelles that provide multiple essential metabolic functions. Mitochondrial dysfunction is associated with neurodegenerative conditions such as Parkinson's disease, as well as bacterial infection. Here, we explore the roles of mitochondrial autophagy (mitophagy) and the mitochondrial unfolded protein response (UPR(mt)) in the response to mitochondrial dysfunction, focusing in particular on recent evidence on the role of mitochondrial import efficiency in the regulation of these stress pathways and how they may interact to protect the mitochondrial pool while initiating an innate immune response to protect against bacterial pathogens.