The obligate intracellular pathogen, Anaplasma phagocytophilum, is the causative agent of life-threatening diseases in humans and animals. A. phagocytophilum is an emerging tick-borne pathogen in the United States, Europe, Africa and Asia, with increasing numbers of infected people and animals every year. It is increasingly recognized that intracellular pathogens modify host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. Recent reports have shown that amino acids are central to the host-pathogen metabolic interaction. In this study, a genome-wide search for components of amino acid metabolic pathways was performed in Ixodes scapularis, the main tick vector of A. phagocytophilum in the United States, for which the genome was recently published. The enzymes involved in the synthesis and degradation pathways of the twenty amino acids were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis amino acid metabolic pathway components in response to A. phagocytophilum infection of tick tissues and ISE6 tick cells. Our analysis was focused on the interplay between carbohydrate and amino acid metabolism during A. phagocytophilum infection in ISE6 cells. The results showed that tick cells increase the synthesis of phosphoenolpyruvate (PEP) from tyrosine to control A. phagocytophilum infection. Metabolic pathway analysis suggested that this is achieved by (i) increasing the transcript and protein levels of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), (ii) shunting tyrosine into the tricarboxylic acid (TCA) cycle to increase fumarate and oxaloacetate which will be converted into PEP by PEPCK-M, and (iii) blocking all the pathways that use PEP downstream gluconeogenesis (i.e., de novo serine synthesis pathway (SSP), glyceroneogenesis and gluconeogenesis). While sequestering host PEP may be critical for this bacterium because it cannot actively carry out glycolysis to produce PEP, excess of this metabolite may be toxic for A. phagocytophilum. The present work provides a more comprehensive view of the major amino acid metabolic pathways involved in the response to pathogen infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.

• Klíčová slova
• Anaplasma phagocytophilum, Ixodes scapularis, glycerol- 3-phosphate, phosphoenolpyruvate, proteomics, transcriptomics,
• MeSH
• aminokyseliny metabolismus   MeSH
• Anaplasma phagocytophilum účinky léků   genetika   metabolismus   patogenita   MeSH
• anaplasmóza MeSH
• apoptóza MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčné linie MeSH
• citrátový cyklus MeSH
• fosfoenolpyruvát metabolismus   farmakologie   MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) metabolismus   MeSH
• genom bakteriální MeSH
• glukoneogeneze MeSH
• glykolýza MeSH
• interakce hostitele a patogenu fyziologie   MeSH
• klíště mikrobiologie   MeSH
• kyselina oxaloctová metabolismus   MeSH
• messenger RNA genetika   MeSH
• metabolické sítě a dráhy genetika   MeSH
• metabolismus sacharidů MeSH
• mitochondrie metabolismus   MeSH
• proteomika metody   MeSH
• serin metabolismus   MeSH
• transkriptom MeSH
• tyrosin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• bakteriální proteiny MeSH
• fosfoenolpyruvát MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) MeSH
• kyselina oxaloctová MeSH
• messenger RNA MeSH
• serin MeSH
• tyrosin MeSH

The energetic landscape of the allosteric regulatory mechanism of rabbit muscle pyruvate kinase (RMPK) was characterized by isothermal titration calorimetry (ITC). Four novel insights were uncovered. (1) ADP exhibits a dual property. Depending on the temperature, ADP can regulate RMPK activity by switching the enzyme to either the R or T state. (2) The assumption that ligand binding to RMPK is state-dependent is only correct for PEP but not Phe and ADP. (3) The effect of pH on the regulatory behavior of RMPK is partly due to the complex pattern of proton release or absorption linked to the multiple linked equilibria which govern the activity of the enzyme. (4) The R <--> T equilibrium is accompanied by a significant DeltaC(p), rendering RMPK most sensitive to temperature under physiological conditions. To rigorously test the validity of conclusions derived from the ITC data, in this study a fluorescence approach, albeit indirect, that tracks continuous structural perturbations was employed. Intrinsic Trp fluorescence of RMPK in the absence and presence of substrates phosphoenolpyruvate (PEP) and ADP, and the allosteric inhibitor Phe, was measured in the temperature range between 4 and 45 degrees C. For data analysis, the fluorescence data were complemented by ITC experiments to yield an extended data set allowing more complete characterization of the RMPK regulatory mechanism. Twenty-one thermodynamic parameters were derived to define the network of linked interactions involved in regulating the allosteric behavior of RMPK through global analysis of the ITC and fluorescent data sets. In this study, 27 independent curves with more than 1600 experimental points were globally analyzed. Consequently, the consensus results substantiate not only the conclusions derived from the ITC data but also structural information characterizing the transition between the active and inactive states of RMPK and the antagonism between ADP and Phe binding. The latter observation reveals a novel role for ADP in the allosteric regulation of RMPK.

• MeSH
• adenosindifosfát chemie   MeSH
• aktivace enzymů MeSH
• alosterická regulace MeSH
• chemické modely MeSH
• energetický metabolismus MeSH
• entropie MeSH
• fenylalanin chemie   MeSH
• fluorescenční spektrometrie * metody   MeSH
• fosfoenolpyruvát chemie   MeSH
• kosterní svaly enzymologie   MeSH
• králíci MeSH
• ligandy MeSH
• pyruvátkinasa antagonisté a inhibitory   chemie   metabolismus   fyziologie   MeSH
• terciární struktura proteinů MeSH
• tryptofan chemie   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenosindifosfát MeSH
• fenylalanin MeSH
• fosfoenolpyruvát MeSH
• ligandy MeSH
• pyruvátkinasa MeSH
• tryptofan MeSH

In this study we characterized aro mutants of Salmonella enterica serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the aroA, aroD, and aroC mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate aro gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the aroD mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of murA. To confirm this, we artificially overexpressed murA from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of aro mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response.

• MeSH
• albuminy farmakologie   MeSH
• alkyltransferasy a aryltransferasy genetika   MeSH
• aminokyseliny aromatické biosyntéza   genetika   MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální geny * MeSH
• bakteriální léková rezistence genetika   MeSH
• buněčná membrána účinky léků   genetika   MeSH
• buněčná stěna účinky léků   genetika   MeSH
• EDTA farmakologie   MeSH
• fosfoenolpyruvát metabolismus   MeSH
• klonování DNA MeSH
• komplement farmakologie   MeSH
• mutace MeSH
• ovotransferin farmakologie   MeSH
• plazmidy genetika   MeSH
• Salmonella enteritidis účinky léků   enzymologie   genetika   MeSH
• Salmonella typhimurium účinky léků   enzymologie   genetika   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• sérum MeSH
• testy genetické komplementace MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• albuminy MeSH
• alkyltransferasy a aryltransferasy MeSH
• aminokyseliny aromatické MeSH
• antibakteriální látky MeSH
• EDTA MeSH
• fosfoenolpyruvát MeSH
• komplement MeSH
• ovotransferin MeSH
• UDP-N-acetylglucosamine 1-carboxyvinyltransferase MeSH Prohlížeč

• MeSH
• adenosinmonofosfát metabolismus   MeSH
• difosfáty metabolismus   MeSH
• fosfoenolpyruvát metabolismus   MeSH
• Giardia lamblia enzymologie   MeSH
• glykolýza MeSH
• kinetika MeSH
• pyruvátfosfátdikinasa izolace a purifikace   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• adenosinmonofosfát MeSH
• difosfáty MeSH
• fosfoenolpyruvát MeSH
• pyruvátfosfátdikinasa MeSH

In a 14-day experiment, weaned and adult rats were given ad libitum isocaloric diets with a mounting casein content (5, 10, 15, 25 and 40% by weight) and growth parameters of protein biological value, PER and NPR, and the utilization parameters NPU (body protein) and LPU (liver protein) were determined together with phosphoenolpyruvate carboxykinase (gluconeogenetic enzyme) and pyruvate kinase (glycolytic enzyme) activity in the animals' liver. The decrease in all the biological value parameters in weaned rats on 25% and 40% casein diets and in adult rats on 15%, 25% and 40% casein diets shows that these concentrations are too high for the organism. The decrease in PER and diminished weight and body and liver nitrogen increments in both age groups in animals with a low protein intake is evidence that 5% casein is an inadequate concentration. The optimum diet for weaned rats is thus a 15% casein diet and for adult rats a 10% casein diet, as confirmed by the linear correlation between weight increments, body and liver nitrogen and protein intake and also by gluconeogenetic enzyme activity. Under the given experimental conditions the study is a contribution to the determination of optimum physiological doses of proteins.

• MeSH
• dietní proteiny aplikace a dávkování   metabolismus   MeSH
• dusík metabolismus   MeSH
• fosfoenolpyruvát metabolismus   MeSH
• fosfoenolpyruvátkarboxylasa metabolismus   MeSH
• glukoneogeneze MeSH
• játra enzymologie   metabolismus   MeSH
• kaseiny aplikace a dávkování   MeSH
• krysa rodu Rattus MeSH
• odstavení MeSH
• tělesná hmotnost MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dietní proteiny MeSH
• dusík MeSH
• fosfoenolpyruvát MeSH
• fosfoenolpyruvátkarboxylasa MeSH
• kaseiny MeSH