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Abstract
Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.
Author Summary
Invasive pulmonary aspergillosis (IPA) is a life-threatening disease that affects primarily immunosuppressed patients. During the last decades the incidence of this disease that is accompanied by high mortality rates has increased. Since opportunistic pathogenic fungi, unlike other pathogens, do not express specific virulence factors, it is becoming more and more clear that the elucidation of fungal metabolism is an essential task to understand fungal pathogenicity and to identify novel antifungal targets. In this work we report genetic inactivation of the sulphur transcription regulator MetR in Aspergillus fumigatus and subsequent study of the resulting phenotypes and transcriptional deregulation of the mutant. Here we show that regulation of sulphur assimilation is an essential process for the manifestation of IPA. Moreover, a regulatory connection between sulphur metabolism and iron homeostasis, a further essential virulence determinant of A. fumigatus, is demonstrated in this study for the first time. A deeper knowledge of sulphur metabolism holds the promise of increasing our understanding of fungal virulence and might lead to improved antifungal therapy.
Abstract
In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5′-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA \((tRNA_i^{fMet})\)while binding of elongator \(tRNA^{Met}\) proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5′ met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence \((tRNA_i^{fMet})\) becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development.
Author Summary
Prokaryote metabolism is key for our understanding of bacterial virulence and pathogenesis and it is also an area with huge opportunity to identify novel targets for antibiotic drugs. Here, we have addressed the so far poorly characterized regulation of methionine biosynthesis in S. aureus. We demonstrate that methionine biosynthesis control in staphylococci significantly differs from that predicted for other Bacillales. Notably, involvement of a T-box instead of an S-box riboswitch separates staphylococci from other bacteria in the order. We provide, for the first time, direct experimental proof for an interaction of a methionyl-tRNA-specific T-box with its cognate tRNA, and the identification of initiator \((tRNA_i^{fMet})\) as the specific binding partner is an unexpected finding whose exact function in Staphylococcus metabolism remains to be established. The data further suggest that in staphylococci a range of regulatory elements are integrated to form a hierarchical network that elegantly limits costly (excess) methionine biosynthesis and, at the same time, reliably ensures production of the amino acid in a highly selective manner. Our findings open a perspective to exploit methionine biosynthesis and especially its T-box-mediated control as putative target(s) for the development of future anti-staphylococcal therapeutics.
In recent years, various forms of caloric restriction (CR) and amino acid or protein restriction (AAR or PR) have shown not only success in preventing age-associated diseases, such as type II diabetes and cardiovascular diseases, but also potential for cancer therapy. These strategies not only reprogram metabolism to low-energy metabolism (LEM), which is disadvantageous for neoplastic cells, but also significantly inhibit proliferation. Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumour types, with over 600,000 new cases diagnosed annually worldwide. With a 5-year survival rate of approximately 55%, the poor prognosis has not improved despite extensive research and new adjuvant therapies. Therefore, for the first time, we analysed the potential of methionine restriction (MetR) in selected HNSCC cell lines. We investigated the influence of MetR on cell proliferation and vitality, the compensation for MetR by homocysteine, the gene regulation of different amino acid transporters, and the influence of cisplatin on cell proliferation in different HNSCC cell lines.
All forms of restriction, from caloric to amino acid to glucose restriction, have been established in recent years as therapeutic options for various diseases, including cancer. However, usually there is no direct comparison between the different restriction forms. Additionally, many cell culture experiments take place under static conditions. In this work, we used a closed perfusion culture in murine L929 cells over a period of 7 days to compare methionine restriction (MetR) and glucose restriction (LowCarb) in the same system and analysed the metabolome by liquid chromatography mass spectrometry (LC-MS). In addition, we analysed the inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) over a period of 72 h. 2-DG induced very fast a low-energy situation by a reduced glycolysis metabolite flow rate resulting in pyruvate, lactate, and ATP depletion. Under perfusion culture, both MetR and LowCarb were established on the metabolic level. Interestingly, over the period of 7 days, the metabolome of MetR and LowCarb showed more similarities than differences. This leads to the conclusion that the conditioned medium, in addition to the different restriction forms, substantially reprogramm the cells on the metabolic level.
Grundsätzlich sollte in dieser Arbeit der Einfluss der Glutamin-Restriktion als eine Form der Aminosäure-Restriktion auf das Proliferationsverhalten, den Stoffwechsel sowie die Morphologie der murinen Fibroblastenzelllinie L929 untersucht werden. Es sollte dabei auch gezeigt werden, ob sich Indizien für die Induktion eines LEM finden lassen. Weiterhin sollten die Polyamine Spermidin, Spermin, Putrescin und N-Acetylputrescin als CRMs diskutiert und ihr Einfluss auf das Proliferationsverhalten an sieben Zelllinien herausgestellt werden. Der antiproliferative Effekt der Polyamine Spermidin und Spermin auf das Wachstum der untersuchten Zelllinien konnte in dieser Arbeit bestätigt werden, wobei sich Spermin als potenter erwies als Spermidin. Eine durch Spermidin/Spermin induzierte Autophagie konnte in den Western Blots nicht signifikant nachgewiesen werden. Putrescin und N-Acetylputrescin zeigten nur leichte Wirkungen bei hoher Dosis (10 mM). Während Putrescin einen leicht antiproliferativen Effekt hatte, zeigte N-Acetylputrescin eine leicht proliferationssteigernde Wirkung. Die Autophagie-Induktion durch Methionin-Restriktion konnte durch den Nachweis Autophagie-assoziierter Proteine in den durchgeführten Western Blots bestätigt werden. Um regelmäßig von den positiven Effekten der Autophagie zu profitieren, wäre eine pflanzenbasierte Ernährungsweise sinnvoll und gut durchführbar, da Methionin hauptsächlich in tierischen Lebensmitteln und nur geringfügig in pflanzlichen Nahrungsmitteln vorkommt. Ein völliger Verzicht auf Methionin ist nicht empfehlenswert, da die Aminosäure essentiell ist und somit über die Nahrung aufgenommen werden muss. Um das Methionin-Level konstant, aber niedrig zu halten, erscheint eine Bedarfsdeckung über pflanzliche Nahrung als ideal. Die an den Zelllinien L929 und HeLa durchgeführten Proliferationsstudien zeigten, dass die Zellen selbst nach fünftägiger Glutamin- Restriktion geringfügig weiter proliferierten und keinerlei Merkmale des Zelltodes aufwiesen. Die massenspektrometrische Analyse der Modellzelllinie L929, welche über einen Zeitraum von fünf Tagen einer Glutamin-Restriktion ausgesetzt war, zeigte deutliche metabolische Veränderungen bei den über 150 untersuchten Metaboliten, die auf die Induktion eines LEM schließen lassen. Es konnte ein metabolischer Fingerabdruck nach 48 h für die Zelllinie L929 unter Glutamin-Restriktion definiert werden, der zukünftig als Referenz bei der Testung potentieller CRMs herangezogen werden kann.
Die Krebstherapie und Behandlung von Tumoren stellt für die moderne Medizin auch in Zukunft eine enorme Herausforderung dar. Trotz intensiver Forschung konnten in den letzten Jahrzehnten zwar zunehmend Fortschritte erzielt werden, allerdings muss das Spektrum an neuen Therapieformen und Möglichkeiten kontinuierlich erweitert werden. In den letzten Jahren haben die Kalorienrestriktion sowie die Aminosäuren- und Proteinrestriktion zunehmend an Bedeutung gewonnen, da sie einen erheblichen positiven Einfluss auf die Entstehung von altersassoziierten Erkrankungen wie z.B. Krebs haben. Allen Formen gemeinsam ist die Induktion eines Low-Energy-Metabolismus, der die Zellen in einen antiproliferativen und selbst-regenerierenden Zustand versetzt. In dieser Arbeit sollte untersucht werden, ob die Methionin-Restriktion als eine Form der Aminosäurerestriktion sich grundsätzlich als Therapieform im Plattenepithelkarzinom (HNSCC) eignet. Zusätzlich sollte ein einfaches zelluläres Modellsystem etabliert werden, das auf metaboler Ebene die Charakterisierung und Analyse des Low-Energy-Metabolismus ermöglicht. Es konnte aufgezeigt werden, dass die Methionin-Restriktion eine effektive Methode ist, um die Proliferation ausgesuchter Zelllinien des HNSCC zu inhibieren. Des Weiteren konnte aufgezeigt werden, dass der Einsatz von Aminosäure-Analoga eine weitere Möglichkeit darstellt, auf die Proliferation von Tumorzellen Einfluss zu nehmen. Die massenspektrometrische Analyse der murinen Zelllinie L929 mittels LC/MS lieferte über einen Zeitraum von 5 Tagen ein detailliertes Bild des Stoffwechsels von mehr als 150 Metaboliten unter Methionin-Restriktion. Durch die Definition eines charakteristischen Fingerabdrucks nach 48 h und eines nur wenige Metabolite umfassenden Fußabdrucks konnte ein murines Modellsystem etabliert werden, dass die Analyse von potentiellen Wirkstoffen, u.a. sogenannten caloric restriction mimetics, ermöglicht.
\(^{11}\)C-methionine (\(^{11}\)C-MET) is a new positron emission tomography (PET) tracer for the assessment of disease activity in multiple myeloma (MM) patients, with preliminary data suggesting higher sensitivity and specificity than \(^{18}\)F-fluorodeoxyglucose (\(^{18}\)F-FDG). However, the value of tumor burden biomarkers has yet to be investigated. Our goals were to corroborate the superiority of \(^{11}\)C-MET for MM staging and to compare its suitability for the assessment of metabolic tumor burden biomarkers in comparison to \(^{18}\)F-FDG. Twenty-two patients with newly diagnosed, treatment-naïve symptomatic MM who had undergone \(^{11}\)C-MET and \(^{18}\)F-FDG PET/CT were evaluated. Standardized uptake values (SUV) were determined and compared with total metabolic tumor volume (TMTV) for both tracers: total lesion glycolysis (TLG) and total lesion \(^{11}\)C-MET uptake (TLMU). PET-derived values were compared to Revised International Staging System (R-ISS), cytogenetic, and serologic MM markers such as M component, beta 2 microglobulin (B2M), serum free light chains (FLC), albumin, and lactate dehydrogenase (LDH). In 11 patients (50%), \(^{11}\)C-MET detected more focal lesions (FL) than FDG (p < 0.01). SUVmax, SUVmean, SUVpeak, TMTV, and TLMU were also significantly higher in \(^{11}\)C-MET than in \(^{18}\)F-FDG (p < 0.05, respectively). \(^{11}\)C-MET PET biomarkers had a better correlation with tumor burden (bone marrow plasma cell infiltration, M component; p < 0.05 versus p = n.s. respectively). This pilot study suggests that \(^{11}\)C-MET PET/CT is a more sensitive marker for the assessment of myeloma tumor burden than \(^{18}\)F-FDG. Its implications for prognosis evaluation need further investigation.