Sergio Pichuantes

Catalases are NAD(P)H-dependent tellurite reductases

Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO3 2−) to the less toxic, insoluble metal, tellurium (Te°), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical.

Biochemical characterization of tellurite-reducing activities of Bacillus stearothermophilus V

Bacillus stearothermophilus V is a naturally occurring Gram-positive rod which exhibits resistance to potassium tellurite. Crude extracts of this bacterium catalyse the NADH-dependent, protease-sensitive reduction of K2TeO3 in vitro. Two fractions which showed the ability to reduce potassium tellurite (H1 and H2) were obtained. Fraction H1 behaved as a macroaggregate exhibiting a very high molecular mass that could not be estimated accurately. Upon electrophoresis in polyacrylamide gels in the presence of SDS, however, it was resolved into three distinct bands of 60, 41 and 37.5 kDa. On the other hand, an M(r) of 121 was determined for fraction H2 by means of gel filtration and high-pressure liquid chromatography. In SDS-PAGE a unique protein band of 60 kDa was observed, suggesting that it is actually a dimer. Both fractions showed pH and temperature optima of 7.5 and 57 degrees C, respectively. Concentrations of 2.5 M NaCl or 0.35 mM SDS inhibited fraction H2 almost completely, while fraction H1 retained 20% of its activity under the same conditions. Concentrations of 5 mM EDTA caused the activity of both fractions to increase 2-fold. In addition to reducing tellurite, they were also able to reduce Na2SeO3 and Na2SO3 in vitro.

The dihydrolipoamide dehydrogenase of Aeromonas caviae ST exhibits NADH-dependent tellurite reductase activity.

Potassium tellurite (K(2)TeO(3)) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of TeO32- in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms.

Cysteine Metabolism-Related Genes and Bacterial Resistance to Potassium Tellurite

Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of Geobacillus stearothermophilus V cysteine-related genes cobA, cysK, and iscS and Escherichia coli cysteine regulon genes under conditions that included the addition of K2TeO3 to the culture medium. Results showed that cell tolerance to tellurite correlates with the expression level of the cysteine metabolic genes and that these genes are up-regulated when tellurite is present in the growth medium.

The Product of the cysK Gene of Bacillus stearothermophilus V Mediates Potassium Tellurite Resistance in Escherichia coli

The nucleotide sequence of a 4,539 bp fragment of Bacillus stearothermophilus V mediating tellurite resistance in Escherichia coli was determined. Four ORFs of more than 150 amino acids encoding polypeptides of 244, 258, 308, and 421 residues were found in the restriction fragment. E. coli cells harboring a recombinant plasmid containing the Ter determinant express, when challenged with tellurite, a 32 kDa protein with an amino terminal sequence identical to the ten first residues of the 308 ORF. This ORF shows great similarity with the cysteine synthase gene (cysK) of a number of organisms. Recombinant clones carrying the active cysK gene have minimal inhibitory concentrations to K2TeO3 that were tenfold higher than those determined for the host strain or that of clones carrying ORFs 244, 258, and 421. Introduction of the B. stearothermophilus V cysK gene into a cysK strain of Salmonella typhimurium LT2 resulted in complementation of the mutation as well as transfer of tellurite resistance.

Expression of functional HIV-1 integrase in the yeastSaccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening

The integrase of the human immunodeficiency virus type 1 (HIV-1) has been expressed in yeast in order to investigate its potential lethal effect mediated by DNA damage. To this end, we have constructed an expression plasmid containing the retroviral integrase gene under the control of the inducible promotor ADH2/GAPDH which is regulated by the glucose concentration of the medium. Haploid yeast strain W303-1A did not appear to be clearly sensitive to HIV-1 integrase expression. However, disruption of theRAD 52 gene, which is involved in the repair of double-strand DNA breaks, strongly increased the deleterious effects of the retroviral enzyme in this yeast strain. The diploid strain constructed with W303-1A and an isogenic strain of the opposite mating type also showed a strong sensitivity to the HIV-1 integrase. Under yeast culture conditions allowing moderate integrase synthesis, the deleterious effect was totally abolished by missense integrase mutations, which are known to abolish HIV-1 integrase activities in vitro. We conclude that the lethal phenotype due to HIV-1 integrase expression in yeast may be closely related to the HIV-1 integration reaction in infected human cells, and that yeast may be a useful tool to study the HIV-1 integration process and to screen drugs capable of inhibiting HIV-1 integration in vivo.

Inactivation of the SNF5 transcription factor gene abolishes the lethal phenotype induced by the expression of HIV-1 integrase in yeast.

The ubiquitous human transcription factor Ini1 has been shown to interact with HIV-1 integrase (IN) and to stimulate in vitro the reactions catalyzed by this enzyme. We have previously used a yeast model to study the effect of HIV-1 IN expression (Caumont, A.B., Jamieson, G.A., Pichuantes, S., Nguyen, A.T., Litvak, S., Dupont, C. -H., 1996. Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening. Curr. Genet. 29, 503-510). Here, we describe the effect of the inactivation of the gene encoding for SNF5, a yeast transcription factor homologous to Ini1, on the lethality induced by the expression of HIV-1 IN in yeast. We observed that the retroviral IN was unable to perform its lethal activity in cells where the SNF5 gene has been disrupted, suggesting that SNF5 may play a role in the lethal effect induced by IN in yeast. SNF5 inactivation affects neither yeast viability nor expression of HIV-1 IN. Given the homology between SNF5 and its human counterpart Ini1, our results suggest that this factor may be important for IN activity in infected cells. Moreover, given the important role proposed for this transcription factor in the integration step and the fact that it is dispensable for cell viability, the interaction between Ini1/ySNF5 and HIV-1 IN should become a potential target in the search for new antiretroviral agents.

Differential distribution of Trypanosoma cruzi clones in human chronic chagasic cardiopathic and non-cardiopathic individuals.

PCR and Southern blot hybridization were used to determine the distribution of Trypanosoma cruzi clones in 37 chronic chagasic cardiopathic and non-cardiopathic patients. Parasite DNA amplified from peripheral blood or dejections of Triatoma infestans fed on patient blood was hybridized with probes containing hypervariable minicircle nucleotide sequences capable of detecting three sublineages of T. cruzi. Probes Z-I and Z-IIb detect unique sequences in lineages TcI and TcIIb, respectively. Probe Z-hybrid detects sequences of lineages TcIId and TcIIe. T. cruzi clones of the Z-I sublineage were detected in 62.2% of T. infestans dejections and 5.4% of peripheral blood samples. Clones of Z-IIb and Z-hybrid sublineages had similar distribution in blood and dejection samples. Interestingly, clones of the Z-IIb sublineage were significantly lower in cardiopathic than in non-cardiopathic patients (23.5% versus 75%; P=0.0006). Clones of the Z-hybrid sublineage were found in 29.4% of cardiopathic and 75% of non-cardiopathic patients, respectively (P=0.0051). By contrast, clones of sublineage Z-I were similarly distributed in both groups of patients. The low frequency of Z-IIb and Z-hybrid sublineage clones detected in cardiopathic patients suggests that the immunological mechanisms involved in controlling and eliminating these T. cruzi parasites may be detrimental to the host, leading to the development of chagasic cardiomyopathy.

High prevalence of cagA-positive strains in Helicobacter pylori-infected, healthy, young Chinese adults

Background: Cytotoxin‐associated gene A (cagA) has been implicated as a potential pathogenic marker for Helicobacter pylori‐induced severe gastroduodenal diseases. Although the prevalence of cagA‐positive strains has been reported in patient populations from developed countries, only limited information from developing countries is available.

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium.

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host .zntA gene product.