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Biomedical subjects

A M Chakrabarty

Publications and source records attributed to A M Chakrabarty.

At least 19 recordsLinked to original sources
PubMed2003

Microbial genes and enzymes in the degradation of chlorinated compounds.

Microorganisms are well known for degrading numerous natural compounds. The synthesis of a multitude of chlorinated compounds by the chemical industry and their release into the natural environment have created major pollution problems. Part of the cause of such pollution is the inability of natural microorganisms to efficiently degrade synthetic chlorinated compounds. Microorganisms are, however, highly adaptable to changes in the environment and have consequently evolved the genes that specify the degradation of chlorinated compounds to varying degrees. Highly selective laboratory techniques have also enabled the isolation of microbial strains capable of utilizing normally recalcitrant highly chlorinated compounds as their sole source of carbon and energy. The evolution and role of microbial genes and enzymes, as well as their mode of regulation and genetic interrelationships, have therefore been the subjects of intense study. This review emphasizes the genetic organization and the regulation of gene expression, as well as evolutionary considerations, regarding the microbial degradation of chlorobenzoates, chlorocatechols, and chlorophenoxyacetic acids.

Bacteria↗
PubMed2003

Environmental biotechnology in the postgenomics era.

The sequencing of the human genome and many microbial genomes has provided new opportunities to study the environmental impact on life processes, leading to development of new technologies that can be protected by patenting. Development of such new technologies has, however, led in some cases to judicial intervention because of their controversial nature. This article illustrates some of the trends in postgenomics biotechnology development and the attendant legal and ethical considerations.

Biotechnology↗
PubMed2003

Induction of apoptosis in macrophages by Pseudomonas aeruginosa azurin: tumour-suppressor protein p53 and reactive oxygen species, but not redox activity, as critical elements in cytotoxicity.

Azurin is a copper-containing protein involved in electron transfer during denitrification. We reported recently that purified azurin demonstrates cytotoxicity to macrophages by forming a complex with the tumour-suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of proapoptotic activity (Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T. K., and Chakrabarty, A. M. 2002, Infect Immun70: 7054-7062). It is, however, not known whether the oxidoreductase (redox) activity of azurin or the involvement of copper is important for its cytotoxicity. We have isolated apo-azurin devoid of copper and site-directed mutants that are redox negative because of either replacement of a cysteine residue (Cys-112) involved in co-ordination with copper or mutational replacement of two methionine residues (Met-44 and Met-64) that are present in the hydrophobic patch of azurin and allow interaction of azurin with its redox partner cytochrome c551. We demonstrate that, although the wild type (wt) and the Cys-112 Asp mutant azurin can form complexes with the tumour-suppressor protein p53 and generate high levels of reactive oxygen species (ROS), the redox-negative Met-44LysMet-64Glu mutant azurin is defective in complex formation with p53, generates low levels of ROS and lacks appreciable cytotoxicity towards macrophages. Thus, complex formation with p53 and ROS generation, rather than azurin redox activity, are important in the cytotoxic action of azurin towards macrophages.

Amino Acid Sequence↗
PubMed2003

Redox proteins in mammalian cell death: an evolutionarily conserved function in mitochondria and prokaryotes.

Mammalian cell mitochondria are believed to have prokaryotic ancestry. Mitochondria are not only the powerhouse of energy generation within the eukaryotic cell but they also play a major role in inducing apoptotic cell death through release of redox proteins such as cytochrome c and the apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity. Recent evidence indicates that some present day prokaryotes release redox proteins that induce apoptosis in mammalian cells through stabilization of the tumour suppressor protein p53. p53 interacts with mitochondria either directly or through activation of the genes for pro-apoptotic proteins such as Bax or NOXA or genes that encode redox enzymes responsible for the production of reactive oxygen species (ROS). The analogy between the ancient ancestors of present day bacteria, the mitochondria, and the present day bacteria with regard to their ability to release redox proteins for triggering mammalian cell death is an interesting example of functional conservation during the hundreds of millions of years of evolution. It is possible that the ancestors of the present day prokaryotes released redox proteins to kill the ancestors of the eukaryotes. During evolution of the mitochondria from prokaryotes as obligate endosymbionts, the mitochondria maintained the same functions to programme their own host cell death.

Amino Acid Sequence↗
PubMed2003

Energy-generating enzymes of Burkholderia cepacia and their interactions with macrophages.

We previously demonstrated that several clinical and environmental isolates of Burkholderia cepacia secreted ATP-utilizing enzymes to the medium; the secretion of these enzymes by cystic fibrosis lung isolate strain 38 was shown to be greatly enhanced in the presence of alpha(2)-macroglobulin. Fractionation of the growth medium of cystic fibrosis isolate strain 71 belonging to genomovar I demonstrated the presence of two additional proteins, homologues of Pseudomonas aeruginosa azurin and cytochrome c(551), which are normally involved in electron transfer during denitrification. A Q-Sepharose column flowthrough fraction of the growth medium of B. cepacia strain 71 enriched with the azurin and cytochrome c(551) homologues triggered apoptosis in macrophages and mast cells, leading to their death. Incubation of the Q-Sepharose column flowthrough fraction with antiazurin and anti-cytochrome c(551) antibodies greatly reduced cell death. We cloned and hyperexpressed a gene from B. cepacia strain 71 that encodes the homologue of P. aeruginosa azurin. Such azurin homologues were detected in the growth medium of several strains belonging to genomovars I, III, and VI but not in the growth medium of strains belonging to other genomovars. The growth medium of the strains that elaborated the azurin homologue had high cytotoxicity towards macrophages. Purified azurin homologue was shown to induce apoptosis in macrophages in a caspase-dependent manner and was localized in both the cytosol and nucleus when incubated with or microinjected into macrophages. This is an interesting example of the interaction of a bacterial protein normally involved in cellular energetics with macrophages to effect their cell death.

Active Transport, Cell Nucleus↗
PubMed2002

The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53.

Two redox proteins, azurin and cytochrome c(551) elaborated by Pseudomonas aeruginosa, demonstrate significant cytotoxic activity towards macrophages. Azurin can enter macrophages, localize in the cytosol and nuclear fractions, and induce apoptosis. Two redox-negative mutants of azurin have less cytotoxicity than does wild-type (wt) azurin. Azurin has been shown to form a complex with the tumor suppressor protein p53, a known inducer of apoptosis, thereby stabilizing it and enhancing its intracellular level. A higher level of reactive oxygen species (ROS), generated during treatment of macrophages with wt azurin, correlates with its cytotoxicity. Treatment with some ROS-removing antioxidants greatly reduces azurin-mediated cytotoxicity, thus demonstrating a novel virulence property of this bacterial redox protein.

Animals↗
PubMed2001

Adenylate kinase as a virulence factor of Pseudomonas aeruginosa.

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.

Adenine Nucleotides↗
PubMed2000

Clinical and environmental isolates of Burkholderia cepacia exhibit differential cytotoxicity towards macrophages and mast cells.

Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Various environmental isolates of B. cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi. Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides. Various efforts to distinguish between clinical and environmental isolates of B. cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B. cepacia strains proposed for environmental release. We now report that several clinical strains of B. cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP. Several environmental strains had reduced activity in this regard. We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5'-nucleotidase activity, the level of secretion of the 5'-nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains. The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as alpha2-macroglobulin. As macrophage-or mast cell surface-associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP-using enzymes generate various phosphorylated or non-phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B. cepacia to evade host defence.

Adenosine Diphosphate↗
PubMed2000

Phagocytic cell killing mediated by secreted cytotoxic factors of Vibrio cholerae.

Vibrio cholerae strain VB1 secretes a number of enzymes into the outside medium that utilize ATP as a substrate. Such enzymes are found in the outside medium during the mid-log phase of growth, when the optical density at 650 nm is about 0.4, and they demonstrate nucleoside diphosphate kinase (Ndk), 5' nucleotidase, and adenylate kinase (Ak) activities. We report that the filtered growth medium of V. cholerae, as well as the flowthrough fraction of a green Sepharose column during fractionation of the growth medium, had very little cytotoxicity by itself towards macrophages and mast cells but exhibited significant cytotoxicity in the presence of exogenous ATP. Such fractions, harboring 5' nucleotidase, Ndk, and presumably other ATP-utilizing enzymes, demonstrated enhanced macrophage and mast cell death; periodate-oxidized-ATP (oATP)-treated macrophage and mast cells or such cells exposed to 0.1 mM Mg(2+), where surface-associated P2Z receptors could not be activated, were not susceptible to subsequent ATP addition. Microscopic visualization of mast cells clearly demonstrated cell morphological changes such as swelling, vacuolization, and nuclear fragmentation following treatment with ATP and the growth medium of V. cholerae; however, these effects were suppressed if the mast cells were pretreated with oATP. These results strongly imply that the secreted ATP-utilizing enzymes of V. cholerae modulate the external ATP levels of the macrophage and mast cells, leading to their accelerated death, presumably through activation of P2Z receptors. Thus, development of inhibitors for such enzymes may reduce the level of V. cholerae infection; alternatively, mutations in such genes may eliminate V. cholerae survival in the gut and contribute to a safer live vaccine.

5'-Nucleotidase↗
PubMed2000

Secretion of nucleoside diphosphate kinase by mucoid Pseudomonas aeruginosa 8821: involvement of a carboxy-terminal motif in secretion.

Nucleoside diphosphate kinase (Ndk) is a ubiquitous enzyme which functions in balancing the nucleotide pool of the cell. We have recently reported that in addition to being intracellular in both mucoid and nonmucoid Pseudomonas aeruginosa, Ndk is also secreted into the extracellular environment by mucoid P. aeruginosa cells. This secreted Ndk has biochemical activity similar to the intracellular Ndk and is 16 kDa in size. To demonstrate that Ndk is indeed secreted and to localize the secretion motif, we constructed an ndk knockout mutant, which lacks both intracellular and extracellular forms of Ndk. In this study, we report the construction of deletion derivatives made from the carboxy-terminal region of Ndk. These deletion derivatives were introduced into the ndk::Cm knockout mutant and were examined for the intracellular and extracellular presence of Ndk. It was observed that the carboxy-terminal 8-amino-acid region is required for the secretion of Ndk into the extracellular region. This region has the sequence DXXX, where X is a predominantly hydrophobic residue. Such sequences represent a conserved motif in proteins secreted by the type I secretory pathway in gram-negative microorganisms. To investigate the significance of this motif in the secretion of Ndk, we constructed a fusion protein of Ndk and the blue fluorescent protein (BFP) as well as a fusion protein of mutated Ndk (whose DTEV motif has been changed to AAAA) and the BFP. The presence of extracellular Ndk was detected only in the ndk::Cm knockout mutant harboring the wild-type BFP-Ndk protein fusion. We could not detect the presence of extracellular Ndk in the ndk::Cm knockout mutant containing the mutated BFP-Ndk protein fusion. In addition, we have also used immunofluorescence microscopy to localize the wild-type and mutated BFP-Ndk proteins in the cell. The significance of these observations is discussed.

Amino Acid Motifs↗
PubMed2000

Succinyl coenzyme A synthetase of Pseudomonas aeruginosa with a broad specificity for nucleoside triphosphate (NTP) synthesis modulates specificity for NTP synthesis by the 12-kilodalton form of nucleoside diphosphate kinase.

Pseudomonas aeruginosa secretes copious amounts of an exopolysaccharide called alginate during infection in the lungs of cystic fibrosis patients. A mutation in the algR2 gene of mucoid P. aeruginosa is known to exhibit a nonmucoid (nonalginate-producing) phenotype and showed reduced activities of succinyl-coenzyme A (CoA) synthetase (Scs) and nucleoside diphosphate kinase (Ndk), implying coregulation of Ndk and Scs in alginate synthesis. We have cloned and characterized the sucCD operon encoding the alpha and beta subunits of Scs from P. aeruginosa and have studied the role of Scs in generating GTP, an important precursor in alginate synthesis. We demonstrate that, in the presence of GDP, Scs synthesizes GTP using ATP as the phosphodonor and, in the presence of ADP, Scs synthesizes ATP using GTP as a phosphodonor. In the presence of inorganic orthophosphate, succinyl-CoA, and an equimolar amount of ADP and GDP, Scs synthesizes essentially an equimolar amount of ATP and GTP. Such a mechanism of GTP synthesis can be an alternate source for the synthesis of alginate as well as for the synthesis of other macromolecules requiring GTP such as RNA and protein. Scs from P. aeruginosa is also shown to exhibit a broad NDP kinase activity. In the presence of inorganic orthophosphate (P(i)), succinyl-CoA, and either GDP, ADP, UDP or CDP, it synthesizes GTP, ATP, UTP, or CTP. Scs was previously shown to copurify with Ndk, presumably as a complex. In mucoid cells of P. aeruginosa, Ndk is also known to exist in two forms, a 16-kDa cytoplasmic form predominant in the log phase and a 12-kDa membrane-associated form predominant in the stationary phase. We have observed that the 16-kDa Ndk-Scs complex present in nonmucoid cells, synthesizes all three of the nucleoside triphosphates from a mixture of GDP, UDP, and CDP, whereas the 12-kDa Ndk-Scs complex specifically present in mucoid cell predominantly synthesizes GTP and UTP but not CTP. Such regulation may promote GTP synthesis in the stationary phase when the bulk of alginate is synthesized by mucoid P. aeruginosa.

Adenosine Triphosphate↗
PubMed1999

Secretion of ATP-utilizing enzymes, nucleoside diphosphate kinase and ATPase, by Mycobacterium bovis BCG: sequestration of ATP from macrophage P2Z receptors?

Mycobacterium bovis BCG secretes two ATP-scavenging enzymes, nucleoside diphosphate kinase (Ndk) and ATPase, during growth in Middlebrook 7H9 medium. In synthetic Sauton medium without any protein supplements, there is less secretion of these two enzymes unless proteins such as bovine serum albumin (BSA), ovalbumin or extracts of macrophages are added to the medium. There is a gradient of activity among various proteins in triggering the induction of secretion of these two enzymes. Other mycobacteria, such as M. smegmatis, primarily secrete Ndk, while M. chelonae does not appear to secrete either of these two enzymes. Purification of the enzymes from the culture filtrate of 7H9-grown M. bovis BCG cells and determination of the N-terminal amino-acid sequence have demonstrated a high level of sequence identity of one of the ATPases with DnaK, a heat shock chaperone, of M. tuberculosis and M. leprae, while that of Ndk shows significant identity with the Ndk of Myxococcus xanthus. As both Ndk and ATPase use ATP as a substrate, the physiological significance of the secretion of these two ATP-utilizing enzymes was explored. External ATP is important in the activation of macrophage surface-associated P2Z receptors, whose activation has been postulated to allow phagosome-lysosome fusion and macrophage cell death. We demonstrate that the presence of the filtrate containing these enzymes prevents ATP-induced macrophage cell death, as measured by the release of an intracellular enzyme, lactate dehydrogenase. In vitro complexation studies with purified Ndk/ATPase and hyperproduced P2Z receptor protein will demonstrate whether these enzymes may be used by mycobacteria to sequester ATP from the macrophage P2Z receptors, thereby preventing phagosome-lysosome fusion or macrophage apoptotic death.

Adenosine Triphosphatases↗
PubMed1999

Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae.

Alginate, a co-polymer of O-acetylated beta-1,4-linked D-mannuronic acid and L-guluronic acid, has been reported to function in the virulence of Pseudomonas syringae, although genetic studies to test this hypothesis have not been undertaken previously. In the present study, we used a genetic approach to evaluate the role of alginate in the pathogenicity of P. syringae pv. syringae 3525, which causes bacterial brown spot on beans. Alginate biosynthesis in strain 3525 was disrupted by recombining Tn5 into algL, which encodes alginate lyase, resulting in 3525.L. Alginate production in 3525.L was restored by the introduction of pSK2 or pAD4033, which contain the alginate biosynthetic gene cluster from P. syringae pv. syringae FF5 or the algA gene from P. aeruginosa respectively. The role of alginate in the epiphytic fitness of strain 3525 was assessed by monitoring the populations of 3525 and 3525.L on tomato, which is not a host for this pathogen. The mutant 3525.L was significantly impaired in its ability to colonize tomato leaves compared with 3525, indicating that alginate functions in the survival of strain 3525 on leaf surfaces. The contribution of alginate to the virulence of strain 3525 was evaluated by comparing the population dynamics and symptom development of 3525 and 3525.L in bean leaves. Although 3525. L retained the ability to form lesions on bean leaves, symptoms were less severe, and the population was significantly reduced in comparison with 3525. These results indicate that alginate contributes to the virulence of P. syringae pv. syringae 3525, perhaps by facilitating colonization or dissemination of the bacterium in planta.

Adaptation, Biological↗
PubMed1999

Cloning and characterization of polyphosphate kinase and exopolyphosphatase genes from Pseudomonas aeruginosa 8830.

Pseudomonas aeruginosa accumulates polyphosphates in response to nutrient limitations. To elucidate the function of polyphosphate in this microorganism, we have investigated polyphosphate metabolism by isolating from P. aeruginosa 8830 the genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which are involved in polyphosphate synthesis and degradation, respectively. The 690- and 506-amino-acid polypeptides encoded by the two genes have been expressed in Escherichia coli and purified, and their activities have been tested in vitro. Gene replacement was used to construct a PPK-negative strain of P. aeruginosa 8830. Low residual PPK activity in the ppk mutant suggests a possible alternative pathway of polyphosphate synthesis in this microorganism. Primer extension analysis indicated that ppk is transcribed from a sigmaE-dependent promoter, which could be responsive to environmental stresses. However, no coregulation between ppk and ppx promoters has been demonstrated in response to osmotic shock or oxidative stress.

Acid Anhydride Hydrolases↗