The AP-1-like transcription factor ChAP1 balances tolerance and cell death in the response of the maize pathogen Cochliobolus heterostrophus to a plant phenolic.

Fungal pathogens need to contend with stresses including oxidants and antimicrobial chemicals resulting from host defenses. ChAP1 of Cochliobolus heterostrophus, agent of Southern corn leaf blight, encodes an ortholog of yeast YAP1. ChAP1 is retained in the nucleus in response to plant-derived phenolic acids, in addition to its well-studied activation by oxidants. Here, we used transcriptome profiling to ask which genes are regulated in response to ChAP1 activation by ferulic acid (FA), a phenolic abundant in the maize host. Nuclearization of ChAP1 in response to phenolics is not followed by strong expression of genes needed for oxidative stress tolerance. We, therefore, compared the transcriptomes of the wild-type pathogen and a ChAP1 deletion mutant, to study the function of ChAP1 in response to FA. We hypothesized that if ChAP1 is retained in the nucleus under plant-related stress conditions yet in the absence of obvious oxidant stress, it should have additional regulatory functions. The transcriptional signature in response to FA in the wild type compared to the mutant sheds light on the signaling mechanisms and response pathways by which ChAP1 can mediate tolerance to ferulic acid, distinct from its previously known role in the antioxidant response. The ChAP1-dependent FA regulon consists mainly of two large clusters. The enrichment of transport and metabolism-related genes in cluster 1 indicates that C. heterostrophus degrades FA and removes it from the cell. When this fails at increasing stress levels, FA provides a signal for cell death, indicated by the enrichment of cell death-related genes in cluster 2. By quantitation of survival and by TUNEL assays, we show that ChAP1 promotes survival and mitigates cell death. Growth rate data show a time window in which the mutant colony expands faster than the wild type. The results delineate a transcriptional regulatory pattern in which ChAP1 helps balance a survival response for tolerance to FA, against a pathway promoting cell death in the pathogen. A general model for the transition from a phase where the return to homeostasis dominates to a phase leading to the onset of cell death provides a context for understanding these findings.

Delivery of the gene encoding the tumor suppressor Sef into prostate tumors by therapeutic-ultrasound inhibits both tumor angiogenesis and growth.

Carcinomas constitute over 80% of all human cancer types with no effective therapy for metastatic disease. Here, we demonstrate, for the first time, the efficacy of therapeutic-ultrasound (TUS) to deliver a human tumor suppressor gene, hSef-b, to prostate tumors in vivo. Sef is downregulated in various human carcinomas, in a manner correlating with tumor aggressiveness. In vitro, hSef-b inhibited proliferation of TRAMP C2 cells and attenuated activation of ERK/MAPK and the master transcription factor NF-κB in response to FGF and IL-1/TNF, respectively. In vivo, transfection efficiency of a plasmid co-expressing hSef-b/eGFP into TRAMP C2 tumors was 14.7 ± 2.5% following a single TUS application. Repeated TUS treatments with hSef-b plasmid, significantly suppressed prostate tumor growth (60%) through inhibition of cell proliferation (60%), and reduction in blood vessel density (56%). In accordance, repeated TUS-treatments with hSef-b significantly inhibited in vivo expression of FGF2 and MMP-9. FGF2 is a known mitogen, and both FGF2/MMP-9 are proangiogenic factors. Taken together our results strongly suggest that hSef-b acts in a cell autonomous as well as non-cell autonomous manner. Moreover, the study demonstrates the efficacy of non-viral TUS-based hSef-b gene delivery approach for the treatment of prostate cancer tumors, and possibly other carcinomas where Sef is downregulated.

The cytotoxic Staphylococcus aureus PSMα3 reveals a cross-α amyloid-like fibril.

Amyloids are ordered protein aggregates, found in all kingdoms of life, and are involved in aggregation diseases as well as in physiological activities. In microbes, functional amyloids are often key virulence determinants, yet the structural basis for their activity remains elusive. We determined the fibril structure and function of the highly toxic, 22-residue phenol-soluble modulin α3 (PSMα3) peptide secreted by Staphylococcus aureus PSMα3 formed elongated fibrils that shared the morphological and tinctorial characteristics of canonical cross-ß eukaryotic amyloids. However, the crystal structure of full-length PSMα3, solved de novo at 1.45 angstrom resolution, revealed a distinctive "cross-α" amyloid-like architecture, in which amphipathic α helices stacked perpendicular to the fibril axis into tight self-associating sheets. The cross-α fibrillation of PSMα3 facilitated cytotoxicity, suggesting that this assembly mode underlies function in S. aureus.

Plant phenolic acids induce programmed cell death of a fungal pathogen: MAPK signaling and survival of Cochliobolus heterostrophus.

Plant aromatic compounds provide signals and a nutrient source to pathogens, and also act as stressors. Structure-activity relationships suggest two pathways sensing these compounds in the maize pathogen Cochliobolus heterostrophus, one triggering a stress response, and one inducing enzymes for their degradation. Focusing on the stress pathway, we found that ferulic acid causes rapid appearance of TUNEL-positive nuclei, dispersion of histone H1:GFP, hyphal shrinkage, and eventually membrane damage. These hallmarks of programmed cell death (PCD) were not seen upon exposure to caffeic acid, a very similar compound. Exposure to ferulic acid dephosphorylated two MAP kinases: Hog1 (stress activated) and Chk1 (pathogenicity related), while increasing phosphorylation of Mps1 (cell integrity related). Mutants lacking Hog1 or Chk1 are hypersensitive to ferulic acid while Mps1 mutants are not. These results implicate three MAPK pathways in the stress response. Ferulic acid and the antifungal fludioxonil have opposite additive effects on survival and on dephosphorylation of Hog1, which is thus implicated in survival. The results may explain why some fungal pathogens of plants undergo cell death early in host invasion, when phenolics are released from plant tissue.

Sef is an inhibitor of proinflammatory cytokine signaling, acting by cytoplasmic sequestration of NF-κB.

The NF-κB transcription factor controls diverse biological processes. According to the classical model, NF-κB is retained in the cytoplasm of resting cells via binding to inhibitory, IκB proteins and translocates into the nucleus upon their ligand-induced degradation. Here we reveal that Sef, a known tumor suppressor and inhibitor of growth factor signaling, is a spatial regulator of NF-κB. Sef expression is regulated by the proinflammatory cytokines tumor necrosis factor and interleukin-1, and Sef specifically inhibits "classical" NF-κB (p50:p65) activation by these ligands. Like IκBs, Sef sequesters NF-κB in the cytoplasm of resting cells. However, contrary to IκBs, Sef continues to constrain NF-κB nuclear entry upon ligand stimulation. Accordingly, endogenous Sef knockdown markedly enhances stimulus-induced NF-κB nuclear translocation and consequent activity. This study establishes Sef as a feedback antagonist of proinflammatory cytokines and highlights its potential to regulate the crosstalk between proinflammatory cytokine receptors and receptor tyrosine kinases.