T cell subsets including effector (T), regulatory (T), and memory (T) cells are characterized by distinct metabolic profiles that influence their differentiation and function. Previous research suggests that engagement of long-chain fatty acid oxidation (LC-FAO) supports Foxp3 T cell and T cell survival. However, evidence for this is mostly based on inhibition of Cpt1a, the rate-limiting enzyme for LC-FAO, with the drug etomoxir. Using genetic models to target Cpt1a specifically in T cells, we dissected the role of LC-FAO in primary, memory, and regulatory T cell responses. Here we show that the ACC2/Cpt1a axis is largely dispensable for T, T, or T cell formation, and that the effects of etomoxir on T cell differentiation and function are independent of Cpt1a expression. Together our data argue that metabolic pathways other than LC-FAO fuel T or T differentiation and suggest alternative mechanisms for the effects of etomoxir that involve mitochondrial respiration.
Sigma factors are essential global regulators of transcription initiation in bacteria which confer promoter recognition specificity to the RNA polymerase core enzyme. They provide effective mechanisms for simultaneously regulating expression of large numbers of genes in response to challenging conditions, and their presence has been linked to bacterial virulence and pathogenicity. In this study, we constructed nine his-tagged sigma factor expressing and/or deletion mutant strains in the opportunistic pathogen Pseudomonas aeruginosa. To uncover the direct and indirect sigma factor regulons, we performed mRNA profiling, as well as chromatin immunoprecipitation coupled to high-throughput sequencing. We furthermore elucidated the de novo binding motif of each sigma factor, and validated the RNA- and ChIP-seq results by global motif searches in the proximity of transcriptional start sites (TSS). Our integrated approach revealed a highly modular network architecture which is composed of insulated functional sigma factor modules. Analysis of the interconnectivity of the various sigma factor networks uncovered a limited, but highly function-specific, crosstalk which orchestrates complex cellular processes. Our data indicate that the modular structure of sigma factor networks enables P. aeruginosa to function adequately in its environment and at the same time is exploited to build up higher-level functions by specific interconnections that are dominated by a participation of RpoN.
Gold complexes with N-heterocyclic carbene (NHC) ligands represent a promising class of metallodrugs for the treatment of cancer or infectious diseases. In this report, the synthesis and the biological evaluation of halogen-containing NHC-Au -Cl complexes are described. The complexes 1 and 5 a-5 f displayed good cytotoxic activity against tumor cells, and cellular uptake studies suggested that an intact Au-NHC fragment is essential for the accumulation of high amounts of both the metal and the NHC ligand. However, the bioavailability was negatively affected by serum components of the cell culture media and was influenced by likely transformations of the complex. One example (5 d) efficiently induced apoptosis in vincristine- and daunorubicin-resistant P-glycoprotein overexpressing Nalm-6 leukemia cells. Cellular uptake studies with this compound showed that both the wild-type and resistant Nalm-6 cells accumulated comparable amounts of gold, indicating that the gold drug was not excreted by P-glycoprotein or other efflux transporters. The effective inhibition of mammalian and bacterial thioredoxin reductases (TrxR) was confirmed for all of the gold complexes. Antibacterial screening of the gold complexes showed a particularly high activity against Gram-positive strains, reflecting their high dependence on an intact Trx/TrxR system. This result is of particular interest as the inhibition of bacterial TrxR represents a relatively little explored mechanism of new anti-infectives.
e Pseudomonas aeruginosa is distinguished by its broad metabolic diversity and its remarkable capability for adaptation, which relies on a large collection of transcriptional regulators and alternative sigma () factors. The largest group of alternative factors is that of the extracytoplasmic function (ECF) factors, which control key transduction pathways for maintenance of envelope homeostasis in response to external stress and cell growth. In addition, there are specific roles of alternative factors in regulating the expression of virulence and virulence-associated genes. Here, we analyzed a deletion mutant of the ECF factor SigX and applied mRNA profiling to define the SigX-dependent regulon in P. aeruginosa in response to low-osmolarity-medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and cyclic di-GMP (c-di-GMP) signaling. In accordance, a sigX deletion mutant exhibited altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels, and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative factor SigX, revealing its role in complex virulence-associated phenotypes in P. aeruginosa. Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can be distinguished by its exceptional high capability to adapt and survive in various and challenging habitats and hosts, including animals, plants, and the human host. The necessary means for bacterial adaptation processes critically rely on sensing and quickly responding to the specific extracellular conditions encountered. One common way to achieve rapid activation of genes in response to fluctuating environmental conditions is the use of extracytoplasmic function (ECF) sigma () factors that are especially abundant in P. aeruginosa (1, 2). ECF factors serve as important regulators, and they are increasingly recognized as factors regulating expression of virulence genes and virulence-associated genes (3-5). The activity of most of the ECF factors are modulated by inner membrane sensor proteins that act as antisigma factors. An off-switch of the anti-sigma factor in response to specific environmental changes thereby presumably leads to the release of the cognate factor and thus allows recruitment of the RNA polymerase to initiate expression of the specific factordependent gene regulon (6). So far, cell envelope stress, iron limitation, and oxidative stress have been demonstrated to play a pivotal role during host infection and were described to activate ECF factors (7,8). In addition to th...
BackgroundThere continues to be a great need for better biomarkers and host-directed treatment targets for community-acquired pneumonia (CAP). Alterations in phospholipid metabolism may constitute a source of small molecule biomarkers for acute infections including CAP. Evidence from animal models of pulmonary infections and sepsis suggests that inhibiting acid sphingomyelinase (which releases ceramides from sphingomyelins) may reduce end-organ damage.MethodsWe measured concentrations of 105 phospholipids, 40 acylcarnitines, and 4 ceramides, as well as acid sphingomyelinase activity, in plasma from patients with CAP (n = 29, sampled on admission and 4 subsequent time points), chronic obstructive pulmonary disease exacerbation with infection (COPD, n = 13) as a clinically important disease control, and 33 age- and sex-matched controls.ResultsPhospholipid concentrations were greatly decreased in CAP and normalized along clinical improvement. Greatest changes were seen in phosphatidylcholines, followed by lysophosphatidylcholines, sphingomyelins and ceramides (three of which were upregulated), and were least in acylcarnitines. Changes in COPD were less pronounced, but also differed qualitatively, e.g. by increases in selected sphingomyelins. We identified highly accurate biomarkers for CAP (AUC ≤ 0.97) and COPD (AUC ≤ 0.93) vs. Controls, and moderately accurate biomarkers for CAP vs. COPD (AUC ≤ 0.83), all of which were phospholipids. Phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins were also markedly decreased in S. aureus-infected human A549 and differentiated THP1 cells. Correlations with C-reactive protein and procalcitonin were predominantly negative but only of mild-to-moderate extent, suggesting that these markers reflect more than merely inflammation. Consistent with the increased ceramide concentrations, increased acid sphingomyelinase activity accurately distinguished CAP (fold change = 2.8, AUC = 0.94) and COPD (1.75, 0.88) from Controls and normalized with clinical resolution.ConclusionsThe results underscore the high potential of plasma phospholipids as biomarkers for CAP, begin to reveal differences in lipid dysregulation between CAP and infection-associated COPD exacerbation, and suggest that the decreases in plasma concentrations are at least partially determined by changes in host target cells. Furthermore, they provide validation in clinical blood samples of acid sphingomyelinase as a potential treatment target to improve clinical outcome of CAP.
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