Host Cell Metabolism Research Articles

Monitoring of protein profiles for the optimization of recombinant fermentation processes using public domain databases.

The expression of human superoxide dismutase in fed-batch fermentation of E. coli HMS174(DE3)(pET3ahSOD) was studied as model system. Due to the frequently used strong T7 promoter system a high metabolic load is exerted, which triggers stress response mechanisms and finally leads to the differentiation of the host cell. As a consequence, host cell metabolism is partly shifted from growth to survival accompanied by significant alterations of the protein pattern. In terms of process optimization two-dimensional electrophoresis deserves as a powerful tool to monitor these changes on protein level. For the analysis of samples derived from different states of recombinant protein production wide-range Immobiline Dry Strips pH 3-10 were used. In order to establish an efficient procedure for accelerated process optimization and to avoid costly and time-consuming analysis like mass spectrometry (MS), a database approach for the identification of significant changes of the protein pattern was evaluated. On average, 935 spots per gel were detected, whereby 50 are presumably stress-relevant. Out of these, 24 proteins could be identified by using the SWISS-2DPAGE database (www.expasy.ch/ch2d/). The identified proteins are involved in regulatory networks, energy metabolism, purine and pyrimidine nucleotide synthesis and translation. By this database approach, significant fluctuations of individual proteins in relation to recombinant protein production could be identified. Seven proteins show strong alterations (>100%) directly after induction and can therefore be stated as reliable marker proteins for the assessment of stress response. For distinctive interpretation of this highly specific information, a bioinformatic and statistic tool would be essential in order to perceive the role and contribution of individual proteins in stress response.

Methanococcus jannaschii Uses a Pyruvoyl-dependent Arginine Decarboxylase in Polyamine Biosynthesis

The genome sequence of the hyperthermophilic methanogen Methanococcus jannaschii contains homologs of most genes required for spermidine polyamine biosynthesis. Yet genomes from neither this organism nor any other euryarchaeon have orthologs of the pyridoxal 5'-phosphate-dependent ornithine or arginine decarboxylase genes, required to produce putrescine. Instead, as shown here, these organisms have a new class of arginine decarboxylase (PvlArgDC) formed by the self-cleavage of a proenzyme into a 5-kDa subunit and a 12-kDa subunit that contains a reactive pyruvoyl group. Although this extremely thermostable enzyme has no significant sequence similarity to previously characterized proteins, conserved active site residues are similar to those of the pyruvoyl-dependent histidine decarboxylase enzyme, and its subunits form a similar (alphabeta)(3) complex. Homologs of PvlArgDC are found in several bacterial genomes, including those of Chlamydia spp., which have no agmatine ureohydrolase enzyme to convert agmatine (decarboxylated arginine) into putrescine. In these intracellular pathogens, PvlArgDC may function analogously to pyruvoyl-dependent histidine decarboxylase; the cells are proposed to import arginine and export agmatine, increasing the pH and affecting the host cell's metabolism. Phylogenetic analysis of Pvl- ArgDC proteins suggests that this gene has been recruited from the euryarchaeal polyamine biosynthetic pathway to function as a degradative enzyme in bacteria.

Bacterial secrets of secretion: EuroConference on the biology of type IV secretion processes.

Type IV secretion systems (TFSS) mediate secretion or direct cell-to-cell transfer of virulence factors (proteins or protein-DNA complexes) from many Gram-negative animal, human and plant pathogens, such as Agrobacterium tumefaciens, Bartonella tribocorum, Bordetella pertussis, Brucella suis, Helicobacter pylori, Legionella pneumophila and Rickettsia prowazekii, into eukaryotic cells. Bacterial conjugation is also classified as a TFSS-like process mediating the spread of broad-host-range plasmids between Gram-negative bacteria such as RP4 and R388, which carry antibiotic resistance genes. Genetic, biochemical, cell biological and structural biology experiments led to significant progress in the understanding of several aspects of TFSS processes. X-ray crystallography revealed that homologues of the A. tumefaciens inner membrane-associated proteins VirB11 and VirD4 from H. pylori and R388, respectively, may form channels for substrate translocation or assembly of the transmembrane TFSS machinery. Biochemical and cell biological experiments revealed interactions between components of the periplasmic core components VirB8, VirB9 and VirB10, which may form the translocation channel. Analysis of A. tumefaciens virulence proteins VirE2 and VirF suggested that the periplasmic translocation route of the pertussis toxin from B. pertussis may be more generally valid than previously anticipated. Secretion and modification of toxins from H. pylori and L. pneumophila profoundly affect host cell metabolism, thus entering the discipline of cellular microbiology. Finally, results from genome sequencing projects revealed the presence of up to three TFSS in a single organism, and the analysis of their interplay and adaptation to different functions will be a future challenge. TFSS-carrying plasmids were discovered in different ecosystems, suggesting that genetic exchange may speed up their evolution and adaptation to different cell-cell interactions.

Mycobacterium bovis BCG but not Mycobacterium leprae induces TNF-alpha secretion in human monocytic THP-1 cells.

In this study, we compared the level of TNF-alpha secretion induced in monocytic THP-1 cells after phagocytosis of Mycobacterium leprae, the causative agent of leprosy, and M. bovis BCG, an attenuated strain used as a vaccine against leprosy and tuberculosis. The presence of M. leprae and BCG was observed in more than 80% of the cells after 24 h of exposure. However, BCG but not M. leprae was able to induce TNF-alpha secretion in these cells. Moreover, THP-1 cells treated simultaneously with BCG and M. leprae secreted lower levels of TNF-alpha compared to cells incubated with BCG alone. M. leprae was able, however, to induce TNF-alpha secretion both in blood-derived monocytes as well as in THP-1 cells pretreated with phorbol myristate acetate. The inclusion of streptomycin in our cultures, together with the fact that the use of both gamma-irradiated M. leprae and heat-killed BCG gave similar results, indicate that the differences observed were not due to differences in viability but in intrinsic properties between M. leprae and BCG. These data suggest that the capacity of M. leprae to induce TNF-alpha is dependent on the stage of cell maturation and emphasize the potential of this model to explore differences in the effects triggered by vaccine strain versus pathogenic species of mycobacteria on the host cell physiology and metabolism.

Metabolic Control Through L Calcium Channel, PKC and Opioid Receptors Modulation by an Association of Naloxone and Calcium Salts

beta-endorphins (beta-ends) are released from the anterior pituitary and from lymphocytes directly into inflamed tissue in response to stress and pain. At the site of inflammation and trauma, the link of beta- ends to opioid receptors hyperpolarizes nerve terminal, by blocking L-calcium gated channels, induces modifications of receptor stereoisomerism and alters the bond-energy. Opioids increase potassium and decrease calcium and sodium currents through interactions with G-protein. In some pathologies, it has been found a loss of desensitization and down regulation of opioid receptors by means of Ca++ blocking that, in turn, inhibits PKC-activation. The physiopathological mechanism dependent on the high concentration of linked opioids affects cellular level of Ca++, ATP and NADH. This biochemical reaction exerts deep influence on energetic cell status and metabolism. In gram negative bacteria, expression of mu-receptors on cell surface has been observed, with a possibility to interfere with host cell metabolism. There are many human and veterinary pathologies in which the reported mechanisms are well known: polycystic ovary syndrome, gross cystic breast disease, milk fever, ruminal tympanites, pyometra, equine colic syndrome, ovarian follicular cyst in dairy cows, calcium deficit in post-partum cows, uterine involution in cows. Also incoming pathologies such as Electro-Magnetic-Field exposure may induce alteration of calcium channel activity through the same mechanism. On clinical bases, it has been pointed out that the therapeutic administration of an association of calcium salts and naloxone controls calcium turnover, pain and functional activity of endocrine glands, via down regulation/desensitization of opioid receptors, PKC stimulation and energy restoration.

Persistence of the influenza A/WSN/33 virus RNA at midbrain levels of immunodefective mice.

Strains of influenza A virus are known to infect specific subpopulations of neurons in the mouse brain. Here we report that all segments of the genome of the neurotropic influenza A virus, strain WSN/33, can persist in the brains of immunodefective transporter associated with Antigen Processing 1 (TAP1) mutant mice. Ten to 17 months after injection of virus into the olfactory bulbs, viral RNA encoding the nonstructural NS1 protein was detected in sections from the brain at midbrain levels by RT-PCR in almost all animals. Both negative-strand genomic RNA (vRNA) and positive-strand RNA, including mRNA, were found. RNA encoding nucleoprotein and polymerases, which form the replicative complex of the virus, were detected in fewer brains. RNA encoding envelope proteins were found only in occasional brains. No viral cDNA could be identified. This observation shows that certain regions of the brain in immunodefective mice may harbor the genome of influenza A virus including the NS1 gene, the products of which may play a regulatory role in host-cell metabolism.

Target site selection by Tn7: attTn7 transcription and target activity.

The bacterial transposon Tn7 inserts at high frequency into a specific site called attTn7, which is present in the chromosomes of many bacteria. We show here that transcription of a nearby gene, glmS, decreases the frequency of Tn7 insertion into attTn7, thus providing a link between Tn7 transposition and host cell metabolism.

Molecular pathology and structural features of enteroviral replication. Toward understanding the pathogenesis of viral heart disease.

Enteroviruses of the Picornaviridae and primarily coxsackieviruses of group B (CVB) can be detected in humans and various experimental murine models of acute myocarditis and chronic heart muscle diseases indicating enterovirus persistence in the myocardium. Persistent myocardial infection is characterized by restricted viral replication and gene expression in myocytes capable of sustaining chronic inflammation. Viral cytotoxicity was found to be crucial for organ pathology both during acute and persistent infection. In-situ hybridization experiments at the cellular and subcellular level have demonstrated that virus replication is associated with severe structural changes of the cardiomyocyte cytoarchitecture at any stage of the disease. In tissue culture experiments and transgenic mice, it was shown that restricted replication and gene expression of the virus are capable of inducing myocytopathic effects. Investigations at the molecular level revealed that interference of coxsackievirus replication with the cellular metabolism is mediated by cleavage of host cell proteins by virus-encoded proteinases. Notably, there is also evidence that enteroviruses are able to activate specific cellular signal transduction pathways in the course of infection, thus promoting enteroviral replication. In summary, these data indicate that mutual influences of virus replication and subsequent modifications of the host cell metabolism are crucial for cardiac injury and dysfunction during acute and chronic disease.

A temperature-regulated replicon-based DNA expression system.

We present a temperature-regulated, alphavirus replicon-based DNA expression system. The system is regulated by a viral temperature-sensitive RNA-dependent RNA replicase, creating a temperature-dependent RNA amplification loop. Because of this positive feedback, the system exhibits both low background and high inducibility. We observed 700-fold induction in transiently transfected cells, and over 104-fold induction in stably transfected cells. The high stringency of inducibility allowed the generation of stable cell lines expressing a highly toxic protein upon temperature shift. These data suggest that the present expression system could simplify bioprocess engineering strategies, especially in situations where the cloned protein has detrimental effects on host cell metabolism.

Spindle Bodies of Heliothis armigera Entomopoxvirus Develop in Structures Associated with Host Cell Endoplasmic Reticulum

Immunoelectron microscopy has shown that morphogenesis of spindle bodies (SB) of Heliothis armigera entomopoxvirus involves an iterative process of condensation, aggregation, and crystallization of the major constituent protein (fusolin) within the perinuclear space and endoplasmic reticulum (ER) of infected cells and in vesicles derived from ER constituents. The ER-specific chaperone BiP has been observed to be associated with developing SBs at all stages of this process, and it is postulated that its sequestration within these bodies may have consequences for host cell metabolism.

Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: a novel mechanism for activation of p38.

Recent evidence indicates activated mitogen-activated protein kinase (MAPK) p38 has a critical function in human cytomegalovirus (HCMV) viral DNA replication in infected human fibroblasts. To elucidate the mechanism of HCMV-mediated p38 activation, we have performed a detailed analysis of p38 activation and the kinases associated with this activation at different times postinfection. We demonstrate that p38 kinase activity is strongly increased following viral infection. Inhibition of this activity significantly inhibited HCMV-induced hyperphosphorylation of pRb and phosphorylation of heat shock protein 27, suggesting that p38 activation is involved in virus-mediated changes in host cell metabolism throughout the course of infection. We then provide evidence that p38 activation is mediated by different mechanisms at early times versus later times of infection. At early times of infection (8 to 14 h postinfection [hpi]), when p38 activation is first observed, no significant activation of the three kinases which can directly phosphorylate p38 (namely, MKK3, MKK6, and MKK4) is detected. Using vectors which express dominant negative proteins, we demonstrate that basal MKK6 kinase activity is necessary for HCMV-mediated p38 activation at these early times of infection (12 hpi). Then, we use ATP depletion to show that at 12 hpi, HCMV inhibits dephosphorylation of activated p38. These two experiments suggest that HCMV activates p38 by inhibition of dephosphorylation of p38. In contrast to early times of infection, at later times of infection (48 to 72 hpi), increased MKK3/6, but not MKK4, activity is observed. These results indicate that at early times of HCMV infection, increased steady-state levels of activated p38 is mediated at least in part by inhibition of dephosphorylation of p38, while at later times of infection p38 activation is due to increased activity of the upstream kinases MKK3 and MKK6. These findings indicate that HCMV has developed multiple mechanisms to ensure activation of the MAPK p38, a kinase critical to viral infection.

Structured modeling of recombinant protein production in batch and fed-batch culture of baculovirus-infected insect cells.

The infection of insect cells with baculovirus was described in a mathematical model as a part of the structured dynamic model describing whole animal cell metabolism. The model presented here is capable of simulating cell population dynamics, the concentrations of extracellular and intracellularviral components, and the heterologous product titers. The model describes the whole processes of viral infection and theeffect of the infection on the host cell metabolism. Dynamic simulation of the model in batch and fed-batch mode gave goodagreement between model predictions and experimental data. Optimum conditions for insect cell culture and viral infectionin batch and fed-batch culture were studied using the model.

Metabolic approaches for the optimisation of recombinant fermentation processes.

The aim of this work was the establishment of a novel method to determine the metabolic load on host-cell metabolism resulting from recombinant protein production in Escherichia coli. This tool can be used to develop strategies to optimise recombinant fermentation processes through adjustment of recombinant-protein expression to the biosynthetic capacity of the host-cell. The signal molecule of the stringent-response network, guanosine tetraphosphate (ppGpp), and its precursor nucleotides were selected for the estimation of the metabolic load relating to recombinant-protein production. An improved analytical method for the quantification of nucleotides by ion-pair, high-performance liquid chromatography was established. The host-cell response upon overexpression of recombinant protein in fed-batch fermentations was investigated using the production of human superoxide dismutase (rhSOD) as a model system. E. coli strains with different recombinant systems (the T7 and pKK promoter system) exerting different loads on host-cell metabolism were analysed with regard to intracellular nucleotide concentration, rate of product formation and plasmid copy number.

Deletion of the fiber gene induces the storage of hexon and penton base proteins in PML/Sp100‐containing inclusions during adenovirus infection

The present study has documented changes in the in situ distribution of viral DNA and capsid proteins in 293 cells infected with fiber gene-deleted adenoviruses. It shows that infection results in the intense production of progeny viruses which appear morphologically intact although they are devoid of fiber-coding sequence in their genome and hence of fiber protein in their capsid. The data confirm, therefore, that fiber protein is not essential for the assembly of progeny viruses. The main contribution of our observations concerns specific intranuclear structures induced by infection with either wild-type or fiber gene-deleted viruses. These clear amorphous inclusions contain two cellular proteins, PML and Sp100, which in non-infected cells co-localize to a special type of nuclear bodies. PML and Sp100 nuclear bodies appear to directly modulate or to be altered in a wide variety of situations including viral infections, cell death and transformation. In cells infected with fiber gene-deleted viruses, the clear amorphous inclusions now accumulate non-used hexon and penton base proteins, whereas the absence of fiber protein prevents the assembly of capsid proteins in crystallin arrays. Taken together, the data suggest that the clear amorphous inclusions may correspond to storage sites of structural and regulatory proteins. Consequently, these virus-induced structures may promote the productive cycle of adenoviruses by regulating the amount of over-produced viral proteins and the shutoff of the host cell metabolism.

Deletion of the fiber gene induces the storage of hexon and penton base proteins in [formula omitted] inclusions during adenovirus infection

The present study has documented changes in the in situ distribution of viral DNA and capsid proteins in 293 cells infected with fiber gene-deleted adenoviruses. It shows that infection results in the intense production of progeny viruses which appear morphologically intact although they are devoid of fiber-coding sequence in their genome and hence of fiber protein in their capsid. The data confirm, therefore, that fiber protein is not essential for the assembly of progeny viruses. The main contribution of our observations concerns specific intranuclear structures induced by infection with either wild-type or fiber gene-deleted viruses. These clear amorphous inclusions contain two cellular proteins, PML and Sp100, which in non-infected cells co-localize to a special type of nuclear bodies. PML and Sp100 nuclear bodies appear to directly modulate or to be altered in a wide variety of situations including viral infections, cell death and transformation. In cells infected with fiber gene-deleted viruses, the clear amorphous inclusions now accumulate non-used hexon and penton base proteins, whereas the absence of fiber protein prevents the assembly of capsid proteins in crystallin arrays. Taken together, the data suggest that the clear amorphous inclusions may correspond to storage sites of structural and regulatory proteins. Consequently, these virus-induced structures may promote the productive cycle of adenoviruses by regulating the amount of over-produced viral proteins and the shutoff of the host cell metabolism.

Virulence of Mycoplasma synoviae in poultry: a review

Mycoplasma synoviae belongs to the class Mollicutes which includes the mycoplasmas, the ureaplasmas, the acholeplasmas and the spiroplasmas. These organisms, most of which are parasites or commensals of plants, insects and animals, are among the smallest prokaryotes capable of independent life. Numerous factors have been associated with the pathogenicity of Mycoplasma infection, including cytadhesion to host target cells, evasion of host immune defences, stimulation of host immune-related self injury, toxin production and harmful effects on host cell metabolism. In animals, mycoplasmas usually colonise mucosal epithelial surfaces of the upper respiratory and urogenital tracts and the mammary gland; however, some mycoplasmas have the potential to invade systemically. Strains of M. synoviae differ in the severity with which they cause disease in poultry, from subclinical infection to upper respiratory disease and, occasionally, septicaemia and synovitis. Current findings indicate that the pathogenicity of M. synoviae in poultry involves attachment and colonisation of the organisms in target tissues; however, one or more as yet unidentified factors seem also to be necessary.

Preclinical aspects of lamivudine and famciclovir against hepatitis B virus.

Chemotherapy for chronic hepatitis B virus (HBV) infection is inherently difficult for a variety of reasons that are related to unusual features of both HBV replication strategy and host cell metabolism. Previous attempts to treat chronic HBV infection using nucleoside analogues have been almost universally disappointing, but several recently developed nucleoside analogues have been identified as potent, non-toxic inhibitors of HBV replication. These fall into two broad categories: nucleosides having the 'unnatural' L-configuration, and deoxyguanosine analogues with modified-sugar configurations, represented by lamivudine and penciclovir respectively. Both lamivudine and penciclovir (in its orally available form, famciclovir) have progressed to phase III clinical trials against chronic HBV infection, with promising preliminary results. However, chemotherapy for chronic HBV is necessarily long term, which increases the risks for development of viral resistance and cumulative toxicity. Such risks might be minimized by the use of appropriate drug combinations, rational selection of which requires knowledge of the pharmacokinetics and mechanisms of action of the individual agents. An appreciation of cellular deoxynucleoside metabolism and its regulation, the complexities of which are still emerging, is an indispensable aid to understanding the biological activities of deoxynucleoside analogues. The modes of action of lamividine and penciclovir, and how these two deoxynucleoside analogues may interact in vitro and in vivo as inhibitors of HBV replication, are examined here in the context of cellular deoxynucleoside metabolism.

A new aspect to the origin and evolution of eukaryotes.

One of the most important omissions in recent evolutionary theory concerns how eukaryotes could emerge and evolve. According to the currently accepted views, the first eukaryotic cell possessed a nucleus, an endomembrane system, and a cytoskeleton but had an inefficient prokaryotic-like metabolism. In contrast, one of the most ancient eukaryotes, the metamonada Giardia lamblia, was found to have formerly possessed mitochondria. In sharp contrast with the traditional views, this paper suggests, based on the energetic aspect of genome organization, that the emergence of eukaryotes was promoted by the establishment of an efficient energy-converting organelle, such as the mitochondrion. Mitochondria were acquired by the endosymbiosis of ancient alpha-purple photosynthetic Gram-negative eubacteria that reorganized the prokaryotic metabolism of the archaebacterial-like ancestral host cells. The presence of an ATP pool in the cytoplasm provided by this cell organelle allowed a major increase in genome size. This evolutionary change, the remarkable increase both in genome size and complexity, explains the origin of the eukaryotic cell itself. The loss of cell wall and the appearance of multicellularity can also be explained by the acquisition of mitochondria. All bacteria use chemiosmotic mechanisms to harness energy; therefore the periplasm bounded by the cell wall is an essential part of prokaryotic cells. Following the establishment of mitochondria, the original plasma membrane-bound metabolism of prokaryotes, as well as the funcion of the periplasm providing a compartment for the formation of different ion gradients, has been transferred into the inner mitochondrial membrane and intermembrane space. After the loss of the essential function of periplasm, the bacterial cell wall could also be lost, which enabled the naked cells to establish direct connections among themselves. The relatively late emergence of mitochondria may be the reason why multicellularity evolved so slowly.

Persistence and expression of the herpes simplex virus genome in the absence of immediate-early proteins.

The immediate-early (IE) proteins of herpes simplex virus (HSV) function on input genomes and affect many aspects of host cell metabolism to ensure the efficient expression and regulation of the remainder of the genome and, subsequently, the production of progeny virions. Due to the many and varied effects of IE proteins on host cell metabolism, their expression is not conducive to normal cell function and viability. This presents a major impediment to the use of HSV as a vector system. In this study, we describe a series of ICP4 mutants that are defective in different subsets of the remaining IE genes. One mutant, d109, does not express any of the IE proteins and carries a green fluorescent protein (GFP) transgene under the control of the human cytomegalovirus IE promoter (HCMVIEp). d109 was nontoxic to Vero and human embryonic lung (HEL) cells at all multiplicities of infection tested and was capable of establishing persistent infections in both of these cell types. Paradoxically, the genetic manipulations that were required to eliminate toxicity and allow the genome to persist in cells for long periods of time also dramatically lowered the level of transgene expression. Efficient expression of the HCMVIEp-GFP transgene in the absence of ICP4 was dependent on the ICP0 protein. In d109-infected cells, the level of transgene expression was very low in most cells but abundant in a small subpopulation of cells. However, expression of the transgene could be induced in cells containing quiescent d109 genomes weeks after the initial infection, demonstrating the functionality of the persisting genomes.

Interaction of the Second Coding Exon of Tat with Human EF-1δ Delineates a Mechanism for HIV-1-Mediated Shut-Off of Host mRNA Translation

HIV-1 Tat has pleiotropic functions. While its most studied role is to activate transcription from the retroviral long terminal repeat (LTR)-promoter, Tat also has functions as a secretable growth factor, a T-cell activator, and an inducer of cellular apoptosis, amongst others. For its transcriptional function, the first coding exon of Tat appears wholly sufficient; however, lentiviruses (HIVs and SIVs) maintain and conserve a second coding exon for Tat. While the function(s) of the second exon of Tat has remained largely unknown, its integrity in lentiviral genomes suggests biological importance, possibly a role in non-transcriptional activities. To understand better the biology of the second exon of Tat in HIV-1 infection of cells, we have searched for cellular proteins that bind specifically to this protein domain. Here, we report that the human translation elongation factor 1-delta (EF-1δ) binds to the second exon of HIV-1 Tat. Interaction between Tat and EF-1δ dramatically reduces the efficiency of the translation of cellular, but not viral, mRNAs. These findings suggest that a non-transcriptional activity of Tat modulates cellular protein synthesis, thereby affecting the metabolism of host cells.