Copper and iron are essential elements for cellular growth. Although bacteria have to overcome limitations of these metals by affine and selective uptake, excessive amounts of both metals are toxic for the cells. Here we investigated the influences of copper stress on iron homeostasis in Bacillus subtilis, and we present evidence that copper excess leads to imbalances of intracellular iron metabolism by disturbing assembly of iron-sulfur cofactors. Connections between copper and iron homeostasis were initially observed in microarray studies showing upregulation of Fur-dependent genes under conditions of copper excess. This effect was found to be relieved in a csoR mutant showing constitutive copper efflux. In contrast, stronger Fur-dependent gene induction was found in a copper efflux-deficient copA mutant. A significant induction of the PerR regulon was not observed under copper stress, indicating that oxidative stress did not play a major role under these conditions. Intracellular iron and copper quantification revealed that the total iron content was stable during different states of copper excess or efflux and hence that global iron limitation did not account for copperdependent Fur derepression. Strikingly, the microarray data for copper stress revealed a broad effect on the expression of genes coding for iron-sulfur cluster biogenesis (suf genes) and associated pathways such as cysteine biosynthesis and genes coding for iron-sulfur cluster proteins. Since these effects suggested an interaction of copper and iron-sulfur cluster maturation, a mutant with a conditional mutation of sufU, encoding the essential iron-sulfur scaffold protein in B. subtilis, was assayed for copper sensitivity, and its growth was found to be highly susceptible to copper stress. Further, different intracellular levels of SufU were found to influence the strength of Fur-dependent gene expression. By investigating the influence of copper on cluster-loaded SufU in vitro, Cu(I) was found to destabilize the scaffolded cluster at submicromolar concentrations. Thus, by interfering with iron-sulfur cluster formation, copper stress leads to enhanced expression of cluster scaffold and target proteins as well as iron and sulfur acquisition pathways, suggesting a possible feedback strategy to reestablish cluster biogenesis.

Copper is an essential cofactor for many enzymes, and at over a threshold level, it is toxic for all organisms. To understand the mechanisms underlying copper homeostasis of the gram-positive bacterium Bacillus subtilis, we have performed microarray studies under copper-limiting conditions. These studies revealed that the ycnJ gene encodes a protein that plays an important role in copper metabolism, as it shows a significant, eightfold upregulation under copper-limiting conditions and its disruption causes a growth-defective phenotype under copper deprivation as well as a reduced intracellular content of copper. Native gel shift experiments with the periplasmic N-terminal domain of the YcnJ membrane protein (135 residues) disclosed its strong affinity to Cu(II) ions in vitro. Inspection of the upstream sequence of ycnJ revealed that the ycnK gene encodes a putative transcriptional regulator, whose deletion caused an elevated expression of ycnJ, especially under conditions of copper excess. Further studies demonstrated that the recently identified copper efflux regulator CsoR also is involved in the regulation of ycnJ expression, leading to a new model for copper homeostasis in B. subtilis.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a poor prognosis. Pirfenidone is the first antifibrotic agent to be approved for IPF-treatment as it is able to slow down disease progression. However, there is no curative treatment other than lung transplantation. Because epigenetic alterations are associated with IPF, histone deacetylase (HDAC)-inhibitors have recently been proven to attenuate fibrotic remodeling in vitro and in vivo. This study compared the effects of pirfenidone with the pan-HDAC-inhibitor panobinostat/LBH589, a FDA-approved drug for the treatment of multiple myeloma, head-to-head on survival, fibrotic activity and proliferation of primary IPF-fibroblasts in vitro.MethodsPrimary fibroblasts from six IPF-patients were incubated for 24h with vehicle (0.25% DMSO), panobinostat (LBH589, 85 nM) or pirfenidone (2.7 mM), followed by assessment of proliferation and expression analyses for profibrotic and anti-apoptosis genes, as well as for ER stress and apoptosis-markers. In addition, the expression status of all HDAC enzymes was examined.ResultsTreatment of IPF-fibroblasts with panobinostat or pirfenidone resulted in a downregulated expression of various extracellular matrix (ECM)-associated genes, as compared to vehicle-treated cells. In agreement, both drugs decreased protein level of phosphorylated (p)-STAT3, a transcription factor mediating profibrotic responses, in treated IPF-fibroblasts. Further, an increase in histone acetylation was observed in response to both treatments, but was much more pronounced and excessive in panobinostat-treated IPF-fibroblasts. Panobinostat, but not pirfenidone, led to a significant suppression of proliferation in IPF-fibroblasts, as indicated by WST1- and BrdU assay and markedly diminished levels of cyclin-D1 and p-histone H3. Furthermore, panobinostat-treatment enhanced α-tubulin-acetylation, decreased the expression of survival-related genes Bcl-XL and BIRC5/survivin, and was associated with induction of ER stress and apoptosis in IPF-fibroblasts. In contrast, pirfenidone-treatment maintained Bcl-XL expression, and was neither associated with ER stress-induction nor any apoptotic signaling. Pirfenidone also led to increased expression of HDAC6 and sirtuin-2, and enhanced α-tubulin-deacetylation. But in line with its ability to increase histone acetylation, pirfenidone reduced the expression of HDAC enzymes HDAC1, -2 and -9.ConclusionsWe conclude that, beside other antifibrotic mechanisms, pirfenidone reduces profibrotic signaling also through STAT3 inactivation and weak epigenetic alterations in IPF-fibroblasts, and permits survival of (altered) fibroblasts. The pan-HDAC-inhibitor panobinostat reduces profibrotic phenotypes while inducing cell cycle arrest and apoptosis in IPF-fibroblasts, thus indicating more efficiency than pirfenidone in inactivating IPF-fibroblasts. We therefore believe that HDAC-inhibitors such as panobinostat can present a novel therapeutic strategy for IPF.