Metabolism the Difficile Way: The Key to the Success of the Pathogen Clostridioides difficile

Strains of Clostridioides difficile cause detrimental diarrheas with thousands of deaths worldwide. The infection process by the Gram-positive, strictly anaerobic gut bacterium is directly related to its unique metabolism, using multiple Stickland-type amino acid fermentation reactions coupled to Rnf complex-mediated sodium/proton gradient formation for ATP generation. Major pathways utilize phenylalanine, leucine, glycine and proline with the formation of 3-phenylproprionate, isocaproate, butyrate, 5-methylcaproate, valerate and 5-aminovalerate. In parallel a versatile sugar catabolism including pyruvate formate-lyase as a central enzyme and an incomplete tricarboxylic acid cycle to prevent unnecessary NADH formation completes the picture. However, a complex gene regulatory network that carefully mediates the continuous adaptation of this metabolism to changing environmental conditions is only partially elucidated. It involves the pleiotropic regulators CodY and SigH, the known carbon metabolism regulator CcpA, the proline regulator PrdR, the iron regulator Fur, the small regulatory RNA CsrA and potentially the NADH-responsive regulator Rex. Here, we describe the current knowledge of the metabolic principles of energy generation by C. difficile and the underlying gene regulatory scenarios

Metabolism the Difficile Way: The Key to the Success of the Pathogen Clostridioides difficile

Strains of Clostridioides difficile cause detrimental diarrheas with thousands of deaths worldwide. The infection process by the Gram-positive, strictly anaerobic gut bacterium is directly related to its unique metabolism, using multiple Stickland-type amino acid fermentation reactions coupled to Rnf complex-mediated sodium/proton gradient formation for ATP generation. Major pathways utilize phenylalanine, leucine, glycine and proline with the formation of 3-phenylproprionate, isocaproate, butyrate, 5-methylcaproate, valerate and 5-aminovalerate. In parallel a versatile sugar catabolism including pyruvate formate-lyase as a central enzyme and an incomplete tricarboxylic acid cycle to prevent unnecessary NADH formation completes the picture. However, a complex gene regulatory network that carefully mediates the continuous adaptation of this metabolism to changing environmental conditions is only partially elucidated. It involves the pleiotropic regulators CodY and SigH, the known carbon metabolism regulator CcpA, the proline regulator PrdR, the iron regulator Fur, the small regulatory RNA CsrA and potentially the NADH-responsive regulator Rex. Here, we describe the current knowledge of the metabolic principles of energy generation by C. difficile and the underlying gene regulatory scenarios

Metabolic Reprogramming of Clostridioides difficile During the Stationary Phase With the Induction of Toxin Production

The obligate anaerobe, spore forming bacterium Clostridioides difficile (formerly Clostridium difficile) causes nosocomial and community acquired diarrhea often associated with antibiotic therapy. Major virulence factors of the bacterium are the two large clostridial toxins TcdA and TcdB. The production of both toxins was found strongly connected to the metabolism and the nutritional status of the growth environment. Here, we systematically investigated the changes of the gene regulatory, proteomic and metabolic networks of C. difficile 630Δerm underlying the adaptation to the non-growing state in the stationary phase. Integrated data from time-resolved transcriptome, proteome and metabolome investigations performed under defined growth conditions uncovered multiple adaptation strategies. Overall changes in the cellular processes included the downregulation of ribosome production, lipid metabolism, cold shock proteins, spermine biosynthesis, and glycolysis and in the later stages of riboflavin and coenzyme A (CoA) biosynthesis. In contrast, different chaperones, several fermentation pathways, and cysteine, serine, and pantothenate biosynthesis were found upregulated. Focusing on the Stickland amino acid fermentation and the central carbon metabolism, we discovered the ability of C. difficile to replenish its favored amino acid cysteine by a pathway starting from the glycolytic 3-phosphoglycerate via L-serine as intermediate. Following the growth course, the reductive equivalent pathways used were sequentially shifted from proline via leucine/phenylalanine to the central carbon metabolism first to butanoate fermentation and then further to lactate fermentation. The toxin production was found correlated mainly to fluxes of the central carbon metabolism. Toxin formation in the supernatant was detected when the flux changed from butanoate to lactate synthesis in the late stationary phase. The holistic view derived from the combination of transcriptome, proteome and metabolome data allowed us to uncover the major metabolic strategies that are used by the clostridial cells to maintain its cellular homeostasis and ensure survival under starvation conditions

The Impact of Pyroglutamate: Sulfolobus acidocaldarius Has a Growth Advantage over Saccharolobus solfataricus in Glutamate-Containing Media

Microorganisms are well adapted to their habitat but are partially sensitive to toxic metabolites or abiotic compounds secreted by other organisms or chemically formed under the respective environmental conditions. Thermoacidophiles are challenged by pyroglutamate, a lactam that is spontaneously formed by cyclization of glutamate under aerobic thermoacidophilic conditions. It is known that growth of the thermoacidophilic crenarchaeon Saccharolobus solfataricus (formerly Sulfolobus solfataricus) is completely inhibited by pyroglutamate. In the present study, we investigated the effect of pyroglutamate on the growth of S. solfataricus and the closely related crenarchaeon Sulfolobus acidocaldarius. In contrast to S. solfataricus, S. acidocaldarius was successfully cultivated with pyroglutamate as a sole carbon source. Bioinformatical analyses showed that both members of the Sulfolobaceae have at least one candidate for a 5-oxoprolinase, which catalyses the ATP-dependent conversion of pyroglutamate to glutamate. In S. solfataricus, we observed the intracellular accumulation of pyroglutamate and crude cell extract assays showed a less effective degradation of pyroglutamate. Apparently, S. acidocaldarius seems to be less versatile regarding carbohydrates and prefers peptidolytic growth compared to S. solfataricus. Concludingly, S. acidocaldarius exhibits a more efficient utilization of pyroglutamate and is not inhibited by this compound, making it a better candidate for applications with glutamate-containing media at high temperatures

Neural Learning of Vector Fields for Encoding Stable Dynamical Systems

Lemme A, Reinhart F, Neumann K, Steil JJ. Neural Learning of Vector Fields for Encoding Stable Dynamical Systems. Neurocomputing. 2014;141:3-14

Complex and flexible catabolism in Aromatoleum aromaticum pCyN1

Large quantities of organic matter are continuously deposited, and (a)biotic gradients intersect in the soil-rhizosphere, where biodegradation contributes to the global cycles of elements. The betaproteobacterial genus Aromatoleum comprises cosmopolitan, facultative denitrifying degradation specialists. Aromatoleum aromaticum. pCyN1 stands out for anaerobically decomposing plant-derived monoterpenes in addition to monoaromatic hydrocarbons, polar aromatics and aliphatics. The catabolic network's structure and flexibility in A. aromaticum pCyN1 were studied across 34 growth conditions by superimposing proteome profiles onto the manually annotated 4.37 Mbp genome. Strain pCyN1 employs three fundamentally different enzymes for C-H-bond cleavage at the methyl groups of p-cymene/4-ethyltoluene, toluene and p-cresol respectively. Regulation of degradation modules displayed substrate specificities ranging from narrow (toluene and cyclohexane carboxylate) via medium-wide (one module shared by p-cymene, 4-ethyltoluene, alpha-phellandrene, alpha-terpinene, gamma-terpinene and limonene) to broad (central benzoyl-CoA pathway serving 16 aromatic substrates). Remarkably, three variants of ATP-dependent (class I) benzoyl-CoA reductase and four different beta-oxidation routes establish a degradation hub that accommodates the substrate diversity. The respiratory system displayed several conspicuous profiles, e.g. the presence of nitrous oxide reductase under oxic and of low-affinity oxidase under anoxic conditions. Overall, nutritional versatility in conjunction with network regulation endow A. aromaticum pCyN1 with broad adaptability

Clostridioides difficile Activates Human Mucosal-Associated Invariant T Cells

Clostridioides difficile infection (CDI) causes severe inflammatory responses at the intestinal mucosa but the immunological mechanisms underlying CDI-related immunopathology are still incompletely characterized. Here we identified for the first time that both, non-toxigenic strains as well as the hypervirulent ribotypes RT027 and RT023 of Clostridioides difficile (formerly Clostridium difficile), induced an effector phenotype in mucosal-associated invariant T (MAIT) cells. MAIT cells can directly respond to bacterial infections by recognizing MR1-presented metabolites derived from the riboflavin synthesis pathway constituting a novel class of antigens. We confirmed functional riboflavin synthesis of C. difficile and found fixed bacteria capable of activating primary human MAIT cells in a dose-dependent manner. C. difficile-activated MAIT cells showed an increased and MR1-dependent expression of CD69, proinflammatory IFNγ, and the lytic granule components granzyme B and perforin. Effector protein expression was accompanied by the release of lytic granules, which, in contrast to other effector functions, was mainly induced by IL-12 and IL-18. Notably, this study revealed hypervirulent C. difficile strains to be most competent in provoking MAIT cell responses suggesting MAIT cell activation to be instrumental for the immunopathology observed in C. difficile-associated colitis. In conclusion, we provide first evidence for a link between C. difficile metabolism and innate T cell-mediated immunity in humans

Giant persistent photoconductivity in monolayer MoS2 field-effect transistors

Monolayer transition metal dichalcogenides (TMD) have numerous potential applications in ultrathin electronics and photonics. The exposure of TMD-based devices to light generates photo-carriers resulting in an enhanced conductivity, which can be effectively used, e.g., in photodetectors. If the photo-enhanced conductivity persists after removal of the irradiation, the effect is known as persistent photoconductivity (PPC). Here we show that ultraviolet light (λ = 365 nm) exposure induces an extremely long-living giant PPC (GPPC) in monolayer MoS2 (ML-MoS2) field-effect transistors (FET) with a time constant of ~30 days. Furthermore, this effect leads to a large enhancement of the conductivity up to a factor of 107. In contrast to previous studies in which the origin of the PPC was attributed to extrinsic reasons such as trapped charges in the substrate or adsorbates, we show that the GPPC arises mainly from the intrinsic properties of ML-MoS2 such as lattice defects that induce a large number of localized states in the forbidden gap. This finding is supported by a detailed experimental and theoretical study of the electric transport in TMD based FETs as well as by characterization of ML-MoS2 with scanning tunneling spectroscopy, high-resolution transmission electron microscopy, and photoluminescence measurements. The obtained results provide a basis for the defect-based engineering of the electronic and optical properties of TMDs for device applications

Learning modular policies for robotics

A promising idea for scaling robot learning to more complex tasks is to use elemental behaviors as building blocks to compose more complex behavior. Ideally, such building blocks are used in combination with a learning algorithm that is able to learn to select, adapt, sequence and co-activate the building blocks. While there has been a lot of work on approaches that support one of these requirements, no learning algorithm exists that unifies all these properties in one framework. In this paper we present our work on a unified approach for learning such a modular control architecture. We introduce new policy search algorithms that are based on information-theoretic principles and are able to learn to select, adapt and sequence the building blocks. Furthermore, we developed a new representation for the individual building block that supports co-activation and principled ways for adapting the movement. Finally, we summarize our experiments for learning modular control architectures in simulation and with real robots