Open Access

Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 106 bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.

Regulated ATP-dependent proteolysis is a common feature of developmental processes and plays also a crucial role during environmental perturbations such as stress and starvation. The Bacillus subtilis MgsR regulator controls a subregulon within the stress- and stationary phase σB regulon. After ethanol exposition and a short time-window of activity, MgsR is ClpXP-dependently degraded with a half-life of approximately 6 min. Surprisingly, a protein interaction analysis with MgsR revealed an association with the McsB arginine kinase and an in vivo degradation assay confirmed a strong impact of McsB on MgsR degradation. In vitro phosphorylation experiments with arginine (R) by lysine (K) substitutions in McsB and its activator McsA unraveled all R residues, which are essentially needed for the arginine kinase reaction. Subsequently, site directed mutagenesis of the MgsR substrate was used to substitute all arginine residues with glutamate (R-E) to mimic arginine phosphorylation and to test their influence on MgsR degradation in vivo. It turned out, that especially the R33E and R94/95E residues (RRPI motif), the latter are adjacently located to the two redox-sensitive cysteines in a 3D model, have the potential to accelerate MgsR degradation. These results imply that selective arginine phosphorylation may have favorable effects for Clp dependent degradation of short-living regulatory proteins. We speculate that in addition to its kinase activity and adaptor function for the ClpC ATPase, McsB might also serve as a proteolytic adaptor for the ClpX ATPase in the degradation mechanism of MgsR.

Context: 3,5-Diiodo-<smlcap>L</smlcap>-thyronine (3,5-T₂) is a thyroid hormone metabolite which exhibited versatile effects in rodent models, including the prevention of insulin resistance or hepatic steatosis typically forced by a high-fat diet. With respect to euthyroid humans, we recently observed a putative link between serum 3,5-T₂ and glucose but not lipid metabolism. Objective: The aim of the present study was to widely screen the urine metabolome for associations with serum 3,5-T₂ concentrations in healthy individuals. Study Design and Methods: Urine metabolites of 715 euthyroid participants of the population-based Study of Health in Pomerania (SHIP-TREND) were analyzed by ¹H-NMR spectroscopy. Multinomial logistic and multivariate linear regression models were used to detect associations between urine metabolites and serum 3,5-T₂ concentrations. Results: Serum 3,5-T₂ concentrations were positively associated with urinary levels of trigonelline, pyroglutamate, acetone and hippurate. In detail, the odds for intermediate or suppressed serum 3,5-T₂ concentrations doubled owing to a 1-standard deviation (SD) decrease in urine trigonelline levels, or increased by 29-50% in relation to a 1-SD decrease in urine pyroglutamate, acetone and hippurate levels. Conclusion: Our findings in humans confirmed the metabolic effects of circulating 3,5-T₂ on glucose and lipid metabolism, oxidative stress and enhanced drug metabolism as postulated before based on interventional pharmacological studies in rodents. Of note, 3,5-T₂ exhibited a unique urinary metabolic profile distinct from previously published results for the classical thyroid hormones.

Microglia are the resident immune cells of the central nervous system (CNS) and play a major role in the regulation of brain homeostasis. To maintain their cellular protein homeostasis, microglia express standard proteasomes and immunoproteasomes (IP), a proteasome isoform that preserves protein homeostasis also in non-immune cells under challenging conditions. The impact of IP on microglia function in innate immunity of the CNS is however not well described. Here, we establish that IP impairment leads to proteotoxic stress and triggers the unfolded and integrated stress responses in mouse and human microglia models. Using proteomic analysis, we demonstrate that IP deficiency in microglia results in profound alterations of the ubiquitin-modified proteome among which proteins involved in the regulation of stress and immune responses. In line with this, molecular analysis revealed chronic activation of NF-κB signaling in IP-deficient microglia without further stimulus. In addition, we show that IP impairment alters microglial function based on markers for phagocytosis and motility. At the molecular level IP impairment activates interferon signaling promoted by the activation of the cytosolic stress response protein kinase R. The presented data highlight the importance of IP function for the proteostatic potential as well as for precision proteolysis to control stress and immune signaling in microglia function.

With more than 25 million people affected, heart failure (HF) is a global threat. As energy production pathways are known to play a pivotal role in HF, we sought here to identify key metabolic changes in ischemic- and non-ischemic HF by using a multi-OMICS approach. Serum metabolites and mRNAseq and epigenetic DNA methylation profiles were analyzed from blood and left ventricular heart biopsy specimens of the same individuals. In total we collected serum from n = 82 patients with Dilated Cardiomyopathy (DCM) and n = 51 controls in the screening stage. We identified several metabolites involved in glycolysis and citric acid cycle to be elevated up to 5.7-fold in DCM (p = 1.7 × 10−6 ). Interestingly, cardiac mRNA and epigenetic changes of genes encoding rate-limiting enzymes of these pathways could also be found and validated in our second stage of metabolite assessment in n = 52 DCM, n = 39 ischemic HF and n = 57 controls. In conclusion, we identified a new set of metabolomic biomarkers for HF. We were able to identify underlying biological cascades that potentially represent suitable intervention targets.