A c-di-GMP Effector System Controls Cell Adhesion by Inside-Out Signaling and Surface Protein Cleavage

In Pseudomonas fluorescens Pf0-1 the availability of inorganic phosphate (Pi) is an environmental signal that controls biofilm formation through a cyclic dimeric GMP (c-di-GMP) signaling pathway. In low Pi conditions, a c-di-GMP phosphodiesterase (PDE) RapA is expressed, depleting cellular c-di-GMP and causing the loss of a critical outer-membrane adhesin LapA from the cell surface. This response involves an inner membrane protein LapD, which binds c-di-GMP in the cytoplasm and exerts a periplasmic output promoting LapA maintenance on the cell surface. Here we report how LapD differentially controls maintenance and release of LapA: c-di-GMP binding to LapD promotes interaction with and inhibition of the periplasmic protease LapG, which targets the N-terminus of LapA. We identify conserved amino acids in LapA required for cleavage by LapG. Mutating these residues in chromosomal lapA inhibits LapG activity in vivo, leading to retention of the adhesin on the cell surface. Mutations with defined effects on LapD's ability to control LapA localization in vivo show concomitant effects on c-di-GMP-dependent LapG inhibition in vitro. To establish the physiological importance of the LapD-LapG effector system, we track cell attachment and LapA protein localization during Pi starvation. Under this condition, the LapA adhesin is released from the surface of cells and biofilms detach from the substratum. This response requires c-di-GMP depletion by RapA, signaling through LapD, and proteolytic cleavage of LapA by LapG. These data, in combination with the companion study by Navarro et al. presenting a structural analysis of LapD's signaling mechanism, give a detailed description of a complete c-di-GMP control circuit—from environmental signal to molecular output. They describe a novel paradigm in bacterial signal transduction: regulation of a periplasmic enzyme by an inner membrane signaling protein that binds a cytoplasmic second messenger

GLUT1 gene is a potential hypoxic marker in colorectal cancer patients

<p>Abstract</p> <p>Background</p> <p>Tumor hypoxia is an important factor related to tumor resistance to radiotherapy and chemotherapy. This study investigated molecules synthesized in colorectal cancer cells during hypoxia to explore the possibility of developing molecular probes capable of detecting cell death and/or the efficiency of radiotherapy and chemotherapy.</p> <p>Methods</p> <p>At first, we incubated two human colorectal adenocarcinoma cell lines SW480 (UICC stage II) and SW620 (UICC stage III) cells in hypoxic (≤2% O₂, 93% N₂, and 5% CO₂) and normoxic conditions (20% O₂, 75% N₂, and 5% CO₂) for 24 h and 48 h. The relative expression ratio of GLUT1 mRNA in hypoxic conditions was analyzed by RT-PCR. Ten cancerous tissues collected from human colorectal cancer patients were examined. HIF-1α and HIF-2α levels were measured to indicate the degree of hypoxia, and gene expression under hypoxic conditions was determined. As a comparison, HIF-1α, HIF-2α, and GLUT1 levels were measured in the peripheral blood of 100 CRC patients.</p> <p>Results</p> <p>Hypoxia-induced lactate was found to be elevated 3.24- to 3.36-fold in SW480 cells, and 3.06- to 3.17-fold in SW620 cells. The increased relative expression ratio of GLUT1 mRNA, under hypoxic conditions was higher in SW620 cells (1.39- to 1.72-fold elevation) than in SW480 cells (1.24- to 1.66-fold elevation). HIF-1α and HIF-2α levels were elevated and GLUT1 genes were significantly overexpressed in CRC tissue specimens. The elevated ratio of GLUT1 was higher in stage III and IV CRC tissue specimens than in the stage I and II (2.97–4.73 versus 1.44–2.11). GLUT1 mRNA was also increased in the peripheral blood of stage II and III CRC patients as compared to stage I patients, suggesting that GLUT1 may serve as a hypoxic indicator in CRC patients.</p> <p>Conclusion</p> <p>In conclusion, this study demonstrated that GLUT1 has the potential to be employed as a molecular marker to indicate the degree of hypoxia experienced by tumors circulating in the blood of cancer patients.</p

Can metabolic plasticity be a cause for cancer? Warburg–Waddington legacy revisited

Fermentation of glucose to lactate in the presence of sufficient oxygen, known as aerobic glycolysis or Warburg effect, is a universal phenotype of cancer cells. Understanding its origin and role in cellular immortalization and transformation has attracted considerable attention in the recent past. Intriguingly, while we now know that Warburg effect is essential for tumor growth and development, it is thought to arise because of genetic and/or epigenetic changes. In contrast to the above, we propose that Warburg effect can also arise due to normal biochemical fluctuations, independent of genetic and epigenetic changes. Cells that have acquired Warburg effect proliferate rapidly to give rise to a population of heterogeneous progenitors of cancer cells. Such cells also generate more lactate and alter the fitness landscape. This dynamic fitness landscape facilitates evolution of cancer cells from its progenitors, in a fashion analogous to Darwinian evolution. Thus, sporadic cancer can also occur first by the acquisition of Warburg effect, then followed by mutation and selection. The idea proposed here circumvents the inherent difficulties associated with the current understanding of tumorigenesis, and is also consistent with many experimental and epidemiological observations. We discuss this model in the context of epigenetics as originally enunciated by Waddington

Metabolic alterations during the growth of tumour spheroids

Solid tumours undergo considerable alterations in their metabolism of nutrients in order to generate sufficient energy and biomass for sustained growth and proliferation. During growth, the tumour microenvironment exerts a number of influences (e.g. hypoxia and acidity) that affect cellular biology and the flux or utilisation of fuels including glucose. The tumour spheroid model was used to characterise the utilisation of glucose and describe alterations to the activity and expression of key glycolytic enzymes during the tissue growth curve. Glucose was avidly consumed and associated with the production of lactate and an acidified medium, confirming the reliance on glycolytic pathways and a diminution of oxidative phosphorylation. The expression levels and activities of hexokinase, phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase in the glycolytic pathway were measured to assess glycolytic capacity. Similar measurements were made for glucose-6-phosphate dehydrogenase, the entry point and regulatory step of the pentose-phosphate pathway (PPP) and for cytosolic malate dehydrogenase, a key link to TCA cycle intermediates. The parameters for these key enzymes were shown to undergo considerable variation during the growth curve of tumour spheroids. In addition, they revealed that the dynamic alterations were influenced by both transcriptional and posttranslational mechanisms

Metabolic alterations during the growth of tumour spheroids

Solid tumours undergo considerable alterations in their metabolism of nutrients in order to generate sufficient energy and biomass for sustained growth and proliferation. During growth, the tumour microenvironment exerts a number of influences (e.g. hypoxia and acidity) that affect cellular biology and the flux or utilisation of fuels including glucose. The tumour spheroid model was used to characterise the utilisation of glucose and describe alterations to the activity and expression of key glycolytic enzymes during the tissue growth curve. Glucose was avidly consumed and associated with the production of lactate and an acidified medium, confirming the reliance on glycolytic pathways and a diminution of oxidative phosphorylation. The expression levels and activities of hexokinase, phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase in the glycolytic pathway were measured to assess glycolytic capacity. Similar measurements were made for glucose-6-phosphate dehydrogenase, the entry point and regulatory step of the pentose-phosphate pathway (PPP) and for cytosolic malate dehydrogenase, a key link to TCA cycle intermediates. The parameters for these key enzymes were shown to undergo considerable variation during the growth curve of tumour spheroids. In addition, they revealed that the dynamic alterations were influenced by both transcriptional and posttranslational mechanisms

Structural Basis for c-di-GMP-Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis

X-ray crystallographic structural analyses of the bacterial transmembrane receptor LapD identify conserved molecular mechanisms that control biofilm formation in response to changes in the intracellular levels of the second messenger c-di-GMP

Defining the Molecular Basis of Tumor Metabolism: a Continuing Challenge Since Warburg's Discovery

Cancer cells are the product of genetic disorders that alter crucial intracellular signaling pathways associated with the regulation of cell survival, proliferation, differentiation and death mechanisms. the role of oncogene activation and tumor suppressor inhibition in the onset of cancer is well established. Traditional antitumor therapies target specific molecules, the action/expression of which is altered in cancer cells. However, since the physiology of normal cells involves the same signaling pathways that are disturbed in cancer cells, targeted therapies have to deal with side effects and multidrug resistance, the main causes of therapy failure. Since the pioneering work of Otto Warburg, over 80 years ago, the subversion of normal metabolism displayed by cancer cells has been highlighted by many studies. Recently, the study of tumor metabolism has received much attention because metabolic transformation is a crucial cancer hallmark and a direct consequence of disturbances in the activities of oncogenes and tumor suppressors. in this review we discuss tumor metabolism from the molecular perspective of oncogenes, tumor suppressors and protein signaling pathways relevant to metabolic transformation and tumorigenesis. We also identify the principal unanswered questions surrounding this issue and the attempts to relate these to their potential for future cancer treatment. As will be made clear, tumor metabolism is still only partly understood and the metabolic aspects of transformation constitute a major challenge for science. Nevertheless, cancer metabolism can be exploited to devise novel avenues for the rational treatment of this disease. Copyright (C) 2011 S. Karger AG, BaselFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed ABC UFABC, CCNH, Santo Andre, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Ciencias Biol, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Bioquim, São Paulo, BrazilUniv Fed Sao Carlos UFSCar, DFQM, Sorocaba, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Ciencias Biol, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Bioquim, São Paulo, BrazilFAPESP: 10/16050-9FAPESP: 10/11475-1FAPESP: 08/51116-0Web of Scienc

MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices

Clinical chronobiology: a timely consideration in critical care medicine

A fundamental aspect of human physiology is its cyclical nature over a 24-h period, a feature conserved across most life on Earth. Organisms compartmentalise processes with respect to time in order to promote survival, in a manner that mirrors the rotation of the planet and accompanying diurnal cycles of light and darkness. The influence of circadian rhythms can no longer be overlooked in clinical settings; this review provides intensivists with an up-to-date understanding of the burgeoning field of chronobiology, and suggests ways to incorporate these concepts into daily practice to improve patient outcomes. We outline the function of molecular clocks in remote tissues, which adjust cellular and global physiological function according to the time of day, and the potential clinical advantages to keeping in time with them. We highlight the consequences of "chronopathology", when this harmony is lost, and the risk factors for this condition in critically ill patients. We introduce the concept of "chronofitness" as a new target in the treatment of critical illness: preserving the internal synchronisation of clocks in different tissues, as well as external synchronisation with the environment. We describe methods for monitoring circadian rhythms in a clinical setting, and how this technology may be used for identifying optimal time windows for interventions, or to alert the physician to a critical deterioration of circadian rhythmicity. We suggest a chronobiological approach to critical illness, involving multicomponent strategies to promote chronofitness (chronobundles), and further investment in the development of personalised, time-based treatment for critically ill patients