Confocal microscopic investigation of tubulin distribution and effect of paclitaxel on posttranslationally modified tubulins in sodium arsenite resistant Leishmania donovani

The affinity of arsenic towards the cytoskeleton leading to disturbance of tubulin polymerization is well known. Tubulin undergoes extensive posttranslational modifications which effect stability and dynamics of microtubules but little is known about the effect of antimicrotubule drugs on their distribution and function in kinetoplastid parasites such as Leishmania. The current study was undertaken to investigate the effect of continuous sodium arsenite exposure on the tubulin distribution profile in wild type and sodium arsenite resistant Leishmania donovani together with effect of paclitaxel, a tubulin-polymerizing agent, on that distribution using confocal microscopy. Immunofluorescence studies using specific monoclonal antibodies against alpha-tubulin and posttranslationally modified tubulins (acetylated and tyrosinated) have revealed distinct differences in the organization of microtubule arrays in wild type and sodium arsenite resistant L. donovani that is further affected by paclitaxel treatment. Microtubules are arranged in spiral arrays in wild type as compared to the longitudinal arrays in arsenite resistant L. donovani. The difference in microtubular structure organization may explain the parasite response to continuous drug pressure and illustrate the fundamental impact of arsenite on microtubules in arsenite resistant L. donovani.     

Altered expression, polymerisation and cellular distribution of alpha-/beta-tubulins and apoptosis-like cell death in arsenite resistant Leishmania donovani promastigotes

Studies in mammalian systems have shown specific affinity of arsenite for tubulin proteins. The sodium m-arsenite (NaAsO2) resistant Leishmania donovani used in this study is resistant to 20 microM NaAsO2, which is a 13-fold increase in resistance compared to the wild type. Data presented in this study shows decreased expression of alpha- and beta-tubulin in wild type L. donovani promastigotes on exposure to NaAsO2 from 0.0016 to 5.0 microM (IC50 in the wild type strain) in a dose-dependent manner. alpha- and beta-tubulins in the resistant strain show decreased expression levels only at 65.0 microM NaAsO2 (IC50 in the resistant strain). Treatment with respective IC50 concentrations of NaAsO2 caused alterations in tubulin polymerisation dynamics and deregulated the cellular distribution of the microtubules in wild type and resistant strains. The NaAsO2-induced cell death exhibited characteristics of apoptosis-like DNA laddering and fragmentation in both the affected wild type and resistant cells. However, poly(ADP-ribose)polymerase cleavage was evident in the wild type strain but not in the resistant strain.     

Structural insights on the small subunit of DNA topoisomerase I from the unicellular parasite Leishmania donovani

Leishmania donovani, the causative organism of visceral leishmaniasis, contains a unique heterodimeric DNA topoisomerase IB (LdTop1). The catalytically active enzyme consists of a large subunit (LdTop1L), which contains the non-conserved N-terminal end and a phylogenetically conserved core domain, and of a small subunit (LdTop1S) which harbours the C-terminal region with a characteristic tyrosine residue in the active site. Heterologous co-expression of LdTop1L and LdTop1S in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme which can be used for structural studies. The role played by the non-conserved N-terminal extension of LdTop1S in both relaxation activity and CPT sensitivity of LdTop1 has been examined co-expressing the full-length LdTop1L with several deletions of LdTop1S lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 174 amino acids of LdTop1S are dispensable in terms of relaxation activity and DNA cleavage. It is also described that the trapping of the covalent complex between LdTop1 and DNA by CPT requires a pentapeptide between amino acid residues 175 and 179 of LdTop1S. Our results suggest the crucial role played by the N-terminal extension of the small subunit of DNA topoisomerase I.     

Elevated levels of polyamines and trypanothione resulting from overexpression of the ornithine decarboxylase gene in arsenite-resistant Leishmania

The levels of trypanothione, a glutathione-spermidine conjugate, are increased in the protozoan parasite Leishmania selected for resistance to the heavy metal arsenite. The levels of putrescine and spermidine were increased in resistant mutants. This increase is mediated by overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Gene overexpression is generally mediated by gene amplification in Leishmania but, here, the mRNA and the enzymatic activity of ODC are increased without gene amplification. This RNA overexpression is stable when cells are grown in the absence of the drug and does not result from gene rearrangements or from an increased rate of RNA synthesis. Transient transfections suggest that mutations in the revertant cells contribute to these elevated levels of RNA. Stable transfection of the ODC gene increases the level of trypanothione, which can contribute to arsenite resistance. In addition to ODC overexpression, the gene for the ABC transporter PGPA is amplified in the mutants. The co-transfection of the ODC and PGPA genes confers resistance in a synergistic fashion in partial revertants, also suggesting that PGPA recognizes metals conjugated to trypanothione.     

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes

Bacillus flexus strain SSAI1 isolated from agro-industry waste, Tuem, Goa, India displayed high arsenite resistance as minimal inhibitory concentration was 25 mM in mineral salts medium. This bacterial strain exposed to 10 mM arsenite demonstrated rapid arsenite oxidation and internalization of 7 mM arsenate within 24 h. The Fourier transformed infrared (FTIR) spectroscopy of cells exposed to arsenite revealed important functional groups on the cell surface interacting with arsenite. Furthermore, scanning electron microscopy combined with electron dispersive X-ray spectroscopy (SEM-EDAX) of cells exposed to arsenite revealed clumping of cells with no surface adsorption of arsenite. Transmission electron microscopy coupled with electron dispersive X-ray spectroscopic (TEM-EDAX) analysis of arsenite exposed cells clearly demonstrated ultra-structural changes and intracellular accumulation of arsenic. Whole-genome sequence analysis of this bacterial strain interestingly revealed the presence of large number of metal(loid) resistance genes, including aioAB genes encoding arsenite oxidase responsible for the oxidation of highly toxic arsenite to less toxic arsenate. Enzyme assay further confirmed that arsenite oxidase is a periplasmic enzyme. The genome of strain SSAI1 also carried glpF, aioS and aioE genes conferring resistance to arsenite. Therefore, multi-metal(loid) resistant arsenite oxidizing Bacillus flexus strain SSAI1 has potential to bioremediate arsenite contaminated environmental sites and is the first report of its kind.

     

Leishmania donovani bisubunit topoisomerase I gene fusion leads to an active enzyme with conserved type IB enzyme function

All eukaryotic topoisomerase I enzymes are monomeric enzymes, whereas the kinetoplastid family (Trypanosoma and Leishmania) possess an unusual bisubunit topoisomerase I. To determine what happens to the enzyme architecture and catalytic property if the two subunits are fused, and to explore the functional relationship between the two subunits, we describe here in vitro gene fusion of Leishmania bisubunit topoisomerase I into a single ORF encoding a new monomeric topoisomerase I (LdTOPIL-fus-S). It was found that LdTOPIL-fus-S is active. Gene fusion leads to a significant modulation of in vitro topoisomerase I activity compared to the wild-type heterodimeric enzyme (LdTOPILS). Interestingly, an N-terminal truncation mutant (1-210 amino acids) of the small subunit, when fused to the intact large subunit [LdTOPIL-fus-Delta(1-210)S], showed reduced topoisomerase I activity and camptothecin sensitivity in comparison to LdTOPIL-fus-S. Investigation of the reduction in enzyme activity indicated that the nonconserved 1-210 residues of LdTOPIS probably act as a 'pseudolinker' domain between the core and catalytic domain of the fused Leishmania enzyme, whereas mutational analysis of conserved His453 in the core DNA-binding domain (LdTOPIL) strongly suggested that its role is to stabilize the enzyme-DNA transition state through hydrogen bonding to one of the nonbridging oxygens. Taken together, our findings provide an insight into the details of the unusual structure of bisubunit topoisomerase I of Leishmania donovani.     

Assessment of the antibacterial activity of galangin against 4-quinolone resistant strains of Staphylococcus aureus

The flavonol galangin is present in numerous plants and is a major constituent of Helichrysum aureonitens, a perennial herb used by South African indigenes to treat infection. In the present study, the antibacterial activity of galangin was assessed against 17 strains of 4-quinolone resistant S. aureus using an agar dilution assay. It was determined that the flavonol had a minimum inhibitory concentration (MIC) of approximately 50 microg/ml against 16 of these strains, including those which exhibited 250- and 500-fold increases in norfloxacin resistance. The remaining one strain, which possessed an amino acid alteration in the GrlB subunit of topoisomerase IV, had increased susceptibility to galangin. Control strains of 4-quinolone sensitive S. aureus were also found to have MICs of 50 microg/ml. The topoisomerase IV enzyme may therefore be implicated in the antibacterial mechanism of action of galangin. Clearly however, there is no cross-resistance between galangin and the 4-quinolones, and the flavonol therefore warrants further investigation as an antibacterial agent.     

Proteomic analysis of wild type and arsenite-resistant Leishmania donovani

Leishmania donovani, causative organism for visceral leishmaniasis, is responsible for considerable mortality and morbidity worldwide. Generation of drug-resistant variants continue to challenge the chemotherapy, the mainstay to fight the disease. The aim of current study was proteomic profiling of wild type (Ld-Wt) and arsenite-resistant (Ld-As20) L. donovani. Significant differences in protein profiles were observed between Ld-As20 and its parent Ld-Wt strain. Proteomic analysis of 158 spots from Ld-Wt and 144 spots from, Ld-As20 identified 77 and 74 protein entries, respectively, through MALDI-TOF/TOF based mass spectrometry and database search. A shift in the isoelectric point of few proteins was observed both in Ld-Wt and Ld-As20, which raises the possibility of continuous arsenite stress, resulting in the differences in the protein profiles of drug-resistant strain from its parent wild type strain. The comparative proteomic data holds the key for elucidation of the multifactorial and complex drug resistance mechanism, like arsenite resistance, in the parasite.     

A novel active DNA topoisomerase I in Leishmania donovani

A common feature shared by type I DNA topoisomerases is the presence of a "serine, lysine, X, X, tyrosine" motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.     

Isolation and diversity analysis of arsenite-resistant bacteria in communities enriched from deep-sea sediments of the Southwest Indian Ocean Ridge

Microorganisms play an important role in the geobiocycling of arsenic element. However, little is known about the bacteria involved in this process in oceanic environments. In this report, arsenite-resistant bacteria were detected in deep-sea sediments on the Southwest Indian Ridge. From arsenite enriched cultures, 54 isolates were obtained, which showed varied tolerance to arsenite of 2–80 mM. Phylogenetic analysis based on 16S rRNA showed that they mainly belonged to Proteobacteria and Actinobacteria. Denaturing gradient gel electrophoresis revealed that Microbacterium esteraromaticum was the dominant member in the arsenite enriched communities, and this was reconfirmed by 16S rRNA gene library analyses. Thus, M. esteraromaticum showed highest resistant to arsenite among the detected bacteria. These results indicate that there are quite diverse bacteria of arsenite resistance inhabiting the deep sea sediment, which may play a role in the geobiocycling of arsenic element in marine environments.     

Heavy metal resistance: a new role for P-glycoproteins in Leishmania.

P-glycoproteins are responsible for multidrug resistance in tumor cell lines and are thought to have a physiologic role in exporting cellular metabolites. We now report that a P-glycoprotein gene in the H region of the trypanosomatid protozoan Leishmania confers resistance to heavy metals when present in multiple copies. The Leishmania H region is frequently amplified in drug-resistant lines and is associated with metal resistance. Leishmania expression vectors were used to introduce multiple copies of segments of the Leishmania major H region into wild-type L. major promastigotes. Only constructs bearing a segment of L. major DNA containing the P-glycoprotein lmpgpA conferred arsenite resistance. Deletional analysis of the arsenite-resistant construct mapped resistance to the lmpgpA protein coding region. Lines expressing lmpgpA showed resistance to arsenite and trivalent antimonials, but not to pentavalent antimonials, zinc, cadmium, or the typical multidrug-resistant P-glycoprotein substrates vinblastine and puromycin. Transfection of the Leishmania tarentolae P-glycoprotein homologue ltpgpA resulted in a similar resistance profile. Thus, these pgpAs represent a functionally distinct group of P-glycoproteins which exhibit a substrate specificity similar to prokaryotic heavy metal pumps. Additionally, several arguments suggest that pgpAs may play a role in the susceptibility of Leishmania to clinically utilized antimonials.     

Effect of arsenite on the physiological and cytological properties of Pseudomonas putida strains capable of degrading polycyclic aromatic hydrocarbons]

Some physiological and cytological properties of Pseudomonas putida strains resistant to arsenite and capable of degrading polycyclic aromatic hydrocarbons were studied. The resistance of P. putida BS202 (NPL-1) to arsenite proved to be determined by chromosomal genes, while the arsenite resistance of P. putida BS238 (pBS2; pBS3031) was by plasmid-borne genes. Arsenite affected the pattern and rate of growth of strain BS202 (NPL-1) in media with naphthalene or salicylate as carbon sources; particularly, it lengthened the lag phase. Electron-microscope analysis of the strains studied did not reveal any arsenite-induced destructive changes in the cell envelope. At the same time, arsenite in the growth medium induced some alterations in the structure of the outer membrane of strain BS202 (NPL-1) and the cytoplasmic membrane of strain BS238 (pBS2; pBS3031) and, in both strains, led to an increase in the density of intramembrane particles on the EF face of the freeze-fractured cytoplasmic membrane. Arsenite resistance probably evidently protects cells of both strains from greater damage. Physiological and cytological data suggest that the mechanisms of arsenite resistance in the strains studied are different.     

Characterization of arsenic resistant and arsenopyrite oxidizing Acidithiobacillus ferrooxidans from Hutti gold leachate and effluents

Four arsenic resistant ferrous oxidizers were isolated from Hutti Gold Mine Ltd. (HGML) samples. Characterization of these isolates was done using conventional microbiological, biochemical and molecular methods. The ferrous oxidation rates with these isolates were 16, 48, 34 and 34 mg L(-1)h(-1) and 15, 47, 34 and 32 mg L(-1)h(-1) in absence and presence of 20 mM of arsenite (As3+) respectively. Except isolate HGM 8, other three isolates showed 2.9-6.3% inhibition due to the presence of 20 mM arsenite. Isolate HGM 8 was able to grow in presence of 14.7 g L(-1) of arsenite, with 25.77 mg L(-1)h(-1) ferrous oxidation rate. All the four isolates were able to oxidize iron and arsenopyrite from 20 g L(-1) and 40 g L(-1) refractory gold ore and 20 g L(-1) refractory gold concentrate. Once the growth was established pH adjustment was not needed inspite of ferrous oxidation, which could be due to concurrent oxidation of pyrite. Isolate HGM 8 showed the final cell count of as high as 1.12 x 10(8) cells mL(-1) in 40 g L(-1) refractory gold ore. The isolates were grouped into one haplotypes by amplified ribosomal DNA restriction analysis (ARDRA). The phylogenetic position of HGM 8 was determined by 16S rDNA sequencing. It was identified as Acidithiobacillus ferrooxidans and strain name was given as SRHGM 1.     

DNA amplification in arsenite-resistant Leishmania

Arsenite-resistant variants of a trypanosomatid protozoan, Leishmania mexicana amazonensis, were selected in vitro by stepwise increases of sodium arsenite concentrations up to 30 microM in the culture medium. These variants were found to contain amplified DNA as extrachromosomal supercoiled molecules of about 69 kb. They originate from a single chromosome in the wild-type cells. There is evidence of chromosomal changes in these cells associated with the selection for arsenite resistance. The apparent absence of these circular molecules in the wild type and their loss from the drug-sensitive revertants suggest amplification of chromosomal DNA into these extrachromosomal circles as the mechanism of arsenite resistance.     

Characterization of As(III) oxidizing <Emphasis Type="Italic">Achromobacter</Emphasis> sp. strain N2: effects on arsenic toxicity and translocation in rice

Achromobacter sp. strain N2 was isolated from a pyrite-cinder-contaminated soil and presented plant growth promoting traits (ACC deaminase activity, production of indole-3-acetic and jasmonic acids, siderophores secretion, and phosphate solubilization) and arsenic transformation abilities. Achromobacter sp. strain N2 was resistant to different metals and metalloids, including arsenate (100 mM) and arsenite (5 mM). The strain was resistant to ionic stressors (i.e., arsenate and NaCl), whereas bacterial growth was impaired by osmotic stress. Strain N2 was able to oxidize 1.0 mmol L^?1 of arsenite to arsenate in 72 h. This evidence was supported by the retrieval of an arsenite oxidase AioA gene highly homologous to arsenite oxidases of Achromobacter and Alcaligenes species. Rice seeds of Oryza sativa (var. Loto) were bio-primed with ACCD-induced and non-induced cells in order to evaluate the effect of inoculation on rice seedlings growth and arsenic uptake. The bacterization with ACCD-induced cells significantly improved seed germination and seedling heights if compared with the seeds inoculated with non-induced cells and non-primed seeds. Enhanced arsenic uptake was evidenced in the presence of ACCD-induced cells, suggesting a role of ACCD activity on the mitigation of the toxicity of arsenic accumulated by the plant. This kind of responses should be taken into account when proposing PGP strains for improving plant growth in arsenic-rich soils.     

Paromomycin affects translation and vesicle-mediated trafficking as revealed by proteomics of paromomycin -susceptible -resistant Leishmania donovani

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL) and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr) strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC) followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr) strain identified a total of 226 proteins at ≥ 95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani.     

A genome-wide screen in Saccharomyces cerevisiae reveals pathways affected by arsenic toxicity

We have used Saccharomyces cerevisiae to identify toxicologically important proteins and pathways involved in arsenic-induced toxicity and carcinogenicity in humans. We performed a systemic screen of the complete set of 4733 haploid S. cerevisiae single-gene-deletion mutants to identify those that have decreased or increased growth, relative to wild type, after exposure to sodium arsenite (NaAsO₂). IC₅₀ values for all mutants were determined to further validate our results. Ultimately we identified 248 mutants sensitive to arsenite and 5 mutants resistant to arsenite exposure. We analyzed the proteins corresponding to arsenite-sensitive mutants and determined that they belonged to functional categories that include protein binding, phosphate metabolism, vacuolar/lysosomal transport, protein targeting, sorting, and translocation, cell growth/morphogenesis, cell polarity and filament formation. Furthermore, these data were mapped onto a protein interactome to identify arsenite-toxicity-modulating networks. These networks are associated with the cytoskeleton, ubiquitination, histone acetylation and the MAPK signaling pathway. Our studies have potential implications for understanding toxicity and carcinogenesis in arsenic-induced human conditions, such as cancer and aging.     

Mutational alteration of the breakage/resealing subunit of bacteriophage T4 DNA topoisomerase confers resistance to antitumor agent m-AMSA

Summary Bacteriophage T4 provides a simple model system in which to examine the mechanism of action of antitumor agents that have been proposed to attack type II DNA topoisomerases. Prior results demonstrated that T4 type II DNA topoisomerase is the target of antitumor agent 4-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in phage-infected Escherichia coli: a point mutation in topoisomerase structural gene 39 was shown to confer both m-AMSA-resistant phage growth and m-AMSA-insensitive topoisomerase activity. We report here that a point mutation in T4 topoisomerase structural gene 52 can also independently render both phage growth and topoisomerase activity resistant to m-AMSA. The DNA relaxation and DNA cleavage activities of this newly isolated mutant topoisomerase were significantly insensitive to m-AMSA. The drug-resistance mutation in gene 52, as well as that in gene 39, alters the DNA cleavage site specificity of wild-type T4 topoisomerase. This fording is consistent with a mechanism of drug action in which both topoisomerase and DNA participate in formation of the drug-binding site.     

Leishmania major LmACR2 is a pentavalent antimony reductase that confers sensitivity to the drug pentostam

Arsenicals and antimonials are first line drugs for the treatment of trypanosomal and leishmanial diseases. To create the active form of the drug, Sb(V) must be reduced to Sb(III). Because arsenic and antimony are related metalloids, and arsenical resistant Leishmania strains are frequently cross-resistant to antimonials, we considered the possibility that Sb(V) is reduced by a leishmanial As(V) reductase. The sequence for the arsenate reductase of Saccharomyces cerevisiae, ScAcr2p, was used to clone the gene for a homologue, LmACR2, from Leishmania major. LmACR2 was able to complement the arsenate-sensitive phenotype of an arsC deletion strain of Escherichia coli or an ScACR2 deletion strain of Saccharomyces cerevisiae. Transfection of Leishmania infantum with LmACR2 augmented Pentostam sensitivity in intracellular amastigotes. LmACR2 was purified and shown to reduce both As(V) and Sb(V). This is the first report of an enzyme that confers Pentostam sensitivity in intracellular amastigotes of Leishmania. We propose that LmACR2 is responsible for reduction of the pentavalent antimony in Pentostam to the active trivalent form of the drug in Leishmania.