EseN is an
This work has global significance in the catfish industry, which provides food for increasing global populations.
Like many Gram‐negative pathogens,
- NSF-PAR ID:
- 10459863
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Proteins: Structure, Function, and Bioinformatics
- Volume:
- 87
- Issue:
- 11
- ISSN:
- 0887-3585
- Page Range / eLocation ID:
- p. 931-942
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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ABSTRACT Edwardsiella ictaluri type III secretion system effector with phosphothreonine lyase activity. In this work, we demonstrate that EseN inactivates p38 and c-Jun-N-terminal kinase (JNK) in infected head-kidney-derived macrophages (HKDMs). We have previously reported inactivation of extracellular-regulated kinase 1/2 (ERK1/2). Also, for the first time, we demonstrated that EseN is involved in the inactivation of 3-phosphoinositide-dependent kinase 1 (PDK1), which has not been previously demonstrated for any of the EseN homologs in other species. We also found that EseN significantly affected mRNA expression ofIL-10 , pro-apoptoticbaxa , andp53 , but had no significant effect on anti-apoptoticbcl2 or pro-apoptotic apoptotic peptidase activating factor 1. EseN is also involved in the inhibition of caspase-8 and caspase-3/7 but does not affect caspase-9 activity. Repression of apoptosis was further confirmed with flow cytometry using Alexa Fluor 647-labeled annexin V and propidium iodide. In addition, we found that theE. ictaluri T3SS is essential for the inhibition of IL-1β maturation, but EseN is not involved in this process. EseN did not affect cell pyroptosis, as indicated by the lack of EseN impact on the release of lactate dehydrogenase from infected HKDM. The transmission electron microscopy data also indicate that HKDM infected with WT or aneseN mutant died by apoptosis, while HKDM infected with the T3SS mutant more likely died by pyroptosis. Collectively, our results indicate thatE. ictaluri EseN is involved in inactivation of ERK1/2, p38, JNK, and PDK1 signaling pathways that lead to modulation of cell death among infected HKDMs.IMPORTANCE E. ictaluri is a leading cause of disease loss, and EseN is an important player inE. ictaluri virulence. TheE. ictaluri T3SS effector EseN plays an essential role in establishing infection, but the specific role EseN plays is not well characterized. EseN belongs to a family of phosphothreonine lyase effectors that specifically target host mitogen activated protein kinase (MAPK) pathways important in regulating host responses to infection. No phosphothreonine lyase equivalents are known in eukaryotes, making this family of effectors an attractive target for indirect narrow-spectrum antibiotics. Targeting of major vault protein and PDK1 kinase by EseN has not been reported in EseN homologs in other pathogens and may indicate unique functions ofE. ictaluri EseN. EseN targeting of PDK1 is particularly interesting in that it is linked to an extraordinarily diverse group of cellular functions. -
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Summary Pathogens secrete effector proteins into host cells to suppress host immunity and promote pathogen virulence, although many features at the molecular interface of host–pathogen interactions remain to be characterized. In a yeast two‐hybrid assay, we found that the
Pseudomonas syringae effector HopZ1a interacts with the Arabidopsis transcriptional regulator Abscisic Acid Repressor 1 (ABR1). Further analysis revealed that ABR1 interacts with multipleP. syringae effectors, suggesting that it may be targeted as a susceptibility hub. Indeed, loss‐of‐functionabr1 mutants exhibit reduced susceptibility to a number ofP. syringae strains. The ABR1 protein comprises a conserved APETALA2 (AP2) domain flanked by long regions of predicted structural disorder. We verified the DNA‐binding activity of the AP2 domain and demonstrated that the disordered domains act redundantly to enhance DNA binding and to facilitate transcriptional activation by ABR1. Finally, we compared gene expression profiles from wild‐type andabr1 plants following inoculation withP. syringae , which suggested that the reduced susceptibility ofabr1 mutants is due to the loss of a virulence target rather than an enhanced immune response. These data highlight ABR1 as a functionally important component at the host–pathogen interface. -
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Summary GacS/GacA is a conserved two‐component system that functions as a master regulator of virulence‐associated traits in many bacterial pathogens, including
Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/GacA‐regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogenPseudomonas aeruginosa , GacS/GacA negatively regulates the expression of type III secretion system (T3SS)‐encoding genes. However, in the plant pathogenic bacteriumPseudomonas syringae , strain‐to‐strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we re‐evaluated the function of GacA inP. syringae pv.tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence insideArabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface‐inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulatingP. syringae virulence. -
INTRODUCTION Eukaryotes contain a highly conserved signaling pathway that becomes rapidly activated when adenosine triphosphate (ATP) levels decrease, as happens during conditions of nutrient shortage or mitochondrial dysfunction. The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is activated within minutes of energetic stress and phosphorylates a limited number of substrates to biochemically rewire metabolism from an anabolic state to a catabolic state to restore metabolic homeostasis. AMPK also promotes prolonged metabolic adaptation through transcriptional changes, decreasing biosynthetic genes while increasing expression of genes promoting lysosomal and mitochondrial biogenesis. The transcription factor EB (TFEB) is a well-appreciated effector of AMPK-dependent signals, but many of the molecular details of how AMPK controls these processes remain unknown. RATIONALE The requirement of AMPK and its specific downstream targets that control aspects of the transcriptional adaptation of metabolism remain largely undefined. We performed time courses examining gene expression changes after various mitochondrial stresses in wild-type (WT) or AMPK knockout cells. We hypothesized that a previously described interacting protein of AMPK, folliculin-interacting protein 1 (FNIP1), may be involved in how AMPK promotes increases in gene expression after metabolic stress. FNIP1 forms a complex with the protein folliculin (FLCN), together acting as a guanosine triphosphate (GTP)–activating protein (GAP) for RagC. The FNIP1-FLCN complex has emerged as an amino acid sensor to the mechanistic target of rapamycin complex 1 (mTORC1), involved in how amino acids control TFEB activation. We therefore examined whether AMPK may regulate FNIP1 to dominantly control TFEB independently of amino acids. RESULTS AMPK was found to govern expression of a core set of genes after various mitochondrial stresses. Hallmark features of this response were activation of TFEB and increases in the transcription of genes specifying lysosomal and mitochondrial biogenesis. AMPK directly phosphorylated five conserved serine residues in FNIP1, suppressing the function of the FLCN-FNIP1 GAP complex, which resulted in dissociation of RagC and mTOR from the lysosome, promoting nuclear translocation of TFEB even in the presence of amino acids. FNIP1 phosphorylation was required for AMPK to activate TFEB and for subsequent increases in peroxisome proliferation–activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) mRNAs. Cells in which the five serines in FNIP1 were mutated to alanine were unable to increase lysosomal and mitochondrial gene expression programs after treatment with mitochondrial poisons or AMPK activators despite the presence and normal regulation of all other substrates of AMPK. By contrast, neither AMPK nor its control of FNIP1 were needed for activation of TFEB after amino acid withdrawal, illustrating the specificity to energy-limited conditions. CONCLUSION Our data establish FNIP1 as the long-sought substrate of AMPK that controls TFEB translocation to the nucleus, defining AMPK phosphorylation of FNIP1 as a singular event required for increased lysosomal and mitochondrial gene expression programs after metabolic stresses. This study also illuminates the larger biological question of how mitochondrial damage triggers a temporal response of repair and replacement of damaged mitochondria: Within early hours, AMPK-FNIP1–activated TFEB induces a wave of lysosome and autophagy genes to promote degradation of damaged mitochondria, and a few hours later, TFEB–up-regulated PGC1⍺ and ERR⍺ promote expression of a second wave of genes specifying mitochondrial biogenesis. These insights open therapeutic avenues for several common diseases associated with mitochondrial dysfunction, ranging from neurodegeneration to type 2 diabetes to cancer. Mitochondrial damage activates AMPK to phosphorylate FNIP1, stimulating TFEB translocation to the nucleus and sequential waves of lysosomal and mitochondrial biogenesis. After mitochondrial damage, activated AMPK phosphorylates FNIP1 (1), causing inhibition of FLCN-FNIP1 GAP activity (2). This leads to accumulation of RagC in its GTP-bound form, causing dissociation of RagC, mTORC1, and TFEB from the lysosome (3). TFEB is therefore not phosphorylated and translocates to the nucleus, inducing transcription of lysosomal or autophagy genes, with parallel increases in NT-PGC1α mRNA (4), which, in concert with ERRα (5), subsequently induces mitochondrial biogenesis (6). CCCP, carbonyl cyanide m-chlorophenylhydrazone; CLEAR, coordinated lysosomal expression and regulation; GDP, guanosine diphosphate; P, phosphorylation. [Figure created using BioRender]more » « less
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null (Ed.)Pseudomonas syringae are Gram-negative, plant pathogenic bacteria that use a type III secretion system (T3SS) to disarm host immune responses and promote bacterial growth within plant tissues. Despite the critical role for type III secretion in promoting virulence, T3SS-encoding genes are not constitutively expressed by P. syringae and must instead be induced during infection. While it has been known for many years that culturing P. syringae in synthetic minimal media can induce the T3SS, relatively little is known about host signals that regulate the deployment of the T3SS during infection. The recent identification of specific plant-derived amino acids and organic acids that induce T3SS-inducing genes in P. syringae has provided new insights into host sensing mechanisms. This review summarizes current knowledge of the regulatory machinery governing T3SS deployment in P. syringae, including master regulators HrpRS and HrpL encoded within the T3SS pathogenicity island, and the environmental factors that modulate the abundance and/or activity of these key regulators. We highlight putative receptors and regulatory networks involved in linking the perception of host signals to the regulation of the core HrpRS–HrpL pathway. Positive and negative regulation of T3SS deployment is also discussed within the context of P. syringae infection, where contributions from distinct host signals and regulatory networks likely enable the fine-tuning of T3SS deployment within host tissues. Last, we propose future research directions necessary to construct a comprehensive model that (a) links the perception of host metabolite signals to T3SS deployment and (b) places these host–pathogen signaling events in the overall context of P. syringae infection.more » « less
