Arsenite Resistance Research Articles

Soft metal thiol chemistry is not involved in the transport of arsenite by the Ars pump

The single cysteine in the ArsB protein subunit of the arsenite resistance pump was changed to serine and alanine residues. Resistance in cells expressing the two mutant arsB genes was the same as in the wild type, and the serine substitution had no effect on the arsenite transport properties. These results eliminate possible thiol chemistry in translocation. Thus, the pump uses soft metal chemistry for metalloactivation and nonmetal chemistry for oxyanion transport.

Dual mode of energy coupling by the oxyanion-translocating ArsB protein

The arsA and arsB genes of the ars operon of R-factor R773 confer arsenite resistance in Escherichia coli by coding for an anion-translocating ATPase. Arsenite resistance and the in vivo energetics of arsenite transport were compared in cells expressing the arsA and arsB genes and those expressing just the arsB gene. Cells expressing the arsB gene exhibited intermediate arsenite resistance compared with cells expressing both the arsA and arsB genes. Both types of cells exhibited energy-dependent arsenite exclusion. Exclusion of 73AsO2- from cells expressing only the arsB gene was coupled to electrochemical energy, while in cells expressing both genes, transport was coupled to chemical energy, most likely ATP. These results suggest that the Ars anion transport system can be either an obligatory ATP-coupled primary pump or a secondary carrier coupled to the proton motive force, depending on the subunit composition of the transport complex.

Expression of heme oxygenase in arsenic-resistant human lung adenocarcinoma cells : Lee T-C, Ho I-C. Institute of Biomedical Sciences, Academia Sinica, Taipei 115. Cancer Res 1994;54:1660-4

We have established arsenic-resistant cells (CL3R) and their subclones from a human lung adenocarcinoma cell line (CL3). CL3R cells and their subclones were maintained in the presence of 4 microM sodium arsenite. They were 6-fold more resistant than CL3 cells to arsenite. Heme oxygenase was expressed in CL3R cells and their subclones, as demonstrated by electrophoretic analysis, Northern blotting, and enzyme activity assay. When CL3R15 cells were grown in arsenite-free medium, their arsenite resistance declined in parallel with their decreasing heme oxygenase activity. Tin-protoporphyrin, a heme oxygenase inhibitor, was found to increase the toxicity of arsenic to CL3R cells. Expression of heme oxygenase might therefore be involved in the mechanism of arsenic resistance. CL3R cells were also shown to be cross-resistant to oxygen-radical generating agents, such as menadione and Adriamycin. Furthermore, sodium arsenite treatment dose-dependently increased the dichlorofluorescein fluorescence in CL3 cells but not in CL3R15 cells. These results suggest that heme oxygenase plays an important role in reducing cellular oxidants that are enhanced by sodium arsenite treatment.

High level arsenite resistance in Leishmania tarentolae is mediated by an active extrusion system

Leishmania tarentolae cells selected for resistance to the oxyanions pentavalent or trivalent antimonials or to trivalent arsenicals exhibited cross-resistance to the other oxyanions. The basis for resistance in these mutants was studied by transport experiments using radioactive arsenite. All mutants exhibiting high level resistance to arsenite showed a marked decrease in the steady-state accumulation of arsenite. Decreased accumulation was also observed in antimonials-resistant mutants cross-resistant to various concentrations of arsenite. Cells depleted of endogenous energy reserves with metabolic inhibitors were loaded with radioactive arsenite; following addition of glucose, rapid efflux of arsenite was observed from arsenite mutant cells. Mutants resistant to high levels of arsenicals exhibited amplification of the P-glycoprotein related gene ltpgpA or of a linear amplicon of unknown function. However, the efflux-mediated arsenite resistance did not correlate with the amplification of the ltpgpA gene or with the presence of the linear amplicon. The calcium channel blocker verapamil and arsenite act in synergy in cells exhibiting the efflux system. Overall the oxyanion efflux system in Leishmania shares several properties with other resistance efflux systems mediated by transporters.

Contribution of the Leishmania P-glycoprotein-related gene ltpgpA to oxyanion resistance.

Oxyanions in the form of pentavalent antimony compounds are currently the drug of choice for treating leishmaniasis. Leishmania mutants resistant to high concentrations of the oxyanion arsenite were obtained in a stepwise selection procedure. Amplification of the H locus P-glycoprotein-related gene ltpgpA, as part of extrachromosomal circles, is a frequent event in arsenite-resistant cells, but was observed only in cells resistant to high concentrations of the metalloid salts. Revertants grown in the absence of the drug lost their ltpgpA-containing amplicon and part of their resistance. The results of previous transfection experiments in Leishmania had suggested that ltpgpA is only involved in low level resistance to arsenite and antimonite. The results of this study using transfection of ltpgpA alleles isolated from arsenite-resistant mutants or of whole circular amplicons containing ltpgpA demonstrate clearly that this P-glycoprotein-related gene is involved in low level resistance to oxyanions, including the pentavalent antimony-containing drug Pentostam. By site-directed mutagenesis, the LtpgpA protein was shown to require an intact nucleotide-binding site to confer arsenite resistance. Under the experimental conditions used, decreased accumulation of the 73AsO2- drug was not observed in the ltpgpA transfectants. One possibility is that LtpgpA-mediated arsenite resistance could result from sequestration of the toxic anion in an intracellular compartment. The results indicate that despite the fact that ltpgpA amplification is a frequent event in oxyanion-resistant mutants, it contributes only slightly to the overall resistance.

Identification and characterization of a transmissible linear plasmid from Rhodococcus erythropolis BD2 that encodes isopropylbenzene and trichloroethene catabolism.

Rhodococcus erythropolis BD2, which is able to utilize isopropylbenzene as a sole carbon and energy source, was shown to contain a conjugative linear plasmid, pBD2. The estimated size of pBD2 is 208 to 212 kb. Linear plasmid-deficient strains had lost both the isopropylbenzene degradation and trichloroethene degradation characteristics, as well as the arsenite resistance and mercury resistance phenotypes. Reintroduction of pBD2 restored all four characteristics. Conjugational transfer of pBD2 to a plasmidless mutant of strain BD2 and other R. erythropolis strains occurred at frequencies between 3.5 x 10(-5) and 2.6 x 10(-3) transconjugants per recipient. R. erythropolis BD2 degrades isopropylbenzene via 3-isopropylcatechol and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate. Both isopropylbenzene-oxidizing and meta-cleavage activities were shown to correspond with the presence of pBD2. Southern hybridizations with DNA encoding the toluene dioxygenase structural genes (todC1C2BA) from Pseudomonas putida F1 revealed homology to linear plasmid DNA. These results indicate that the isopropylbenzene degradation pathway encoded by linear plasmid pBD2 is initiated by an isopropylbenzene dioxygenase analogous to toluene dioxygenase.

Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana.

Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite- or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-type minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type- or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.

A functional chimeric membrane subunit of an ion-translocating ATPase.

A chimeric transport protein was made by expression of a fusion of the arsB genes from Escherichia coli plasmid R773 and Staphylococcus aureus plasmid pI258. The two genes were fused to encode a functional protein with first eight membrane spanning alpha-helices of the S. aureus and the last four helices of the E. coli protein. The hybrid protein provided arsenite resistance and transport. When an arsA gene was expressed in trans with the ArsB proteins encoded by the R773, pI258 and fusion genes, arsenite efflux was dependent on chemical but not electrochemical energy. The Ars system is hypothesized to be a novel transport system that functions as a primary ATP-driven pump or a secondary carrier, depending on the subunit composition of the complex.

Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana.

Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite- or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-type minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type- or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.

Stable and Inducible Arsenite Resistance in Chinese Hamster Cells

A number of Chinese hamster V79 cell sublines that are arsenite resistant or arsenite sensitive were isolated. After more than 6 months of growth in the absence of arsenite, these sublines still maintain their arsenite-resistant or -sensitive phenotypes. At least some arsenite-resistant cell lines are also cross-resistant to sodium arsenate and potassium antimonyl tartrate. Wild-type or arsenite-resistant sublines show a further increase in resistance to toxic concentrations of arsenite after pretreatment with a nontoxic concentration. Pretreatment of cells with a nontoxic dose of arsenite also increased their survival to toxic doses of antimonite. Likewise, pretreatment with antimonite increased survival to arsenite as well as to antimonite. The inducible arsenite resistance increases with pretreatment time, reaching a plateau after 8 hr of pretreatment. Fusion of arsenite-resistant cells with arsenite-sensitive cells demonstrated a clear dominance of arsenite resistance. These results suggest that mammalian cells contain an arsenite/antimonite pump whose activity may be modulated by prior exposure to arsenite or antimonite.

Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pI258.

The arsenic resistance operon of Staphylococcus aureus plasmid pI258 consists of three genes, arsR (encoding the repressor regulatory protein), arsB (the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells), and arsC (the small gene whose protein product is required for arsenate resistance only, not for arsenite resistance). ArsC has now been shown to be an arsenate reductase, converting intracellular arsenate [As(V)] to arsenite [As(III)], which is then exported from the cells by an energy-dependent efflux process. The arsenate reductase activity was found in the soluble cytoplasmic fraction in Escherichia coli (and not associated with the periplasmic fraction or the sedimentable cell envelope). Purified ArsC protein coupled in vitro with thioredoxin plus dithiothreitol (but not 2-mercaptoethanol or reduced glutathione) to reduce arsenate to arsenite.

Expression and regulation of the antimonite, arsenite, and arsenate resistance operon of Staphylococcus xylosus plasmid pSX267.

The arsenate, arsenite, and antimonite resistance region of the Staphylococcus xylosus plasmid pSX267 was subcloned in Staphylococcus carnosus. The sequenced DNA region revealed three consecutive open reading frames, named arsR, arsB, and arsC. Expression studies in Escherichia coli with the bacteriophage T7 RNA polymerase-promoter system yielded three polypeptides with apparent molecular weights of 8,000, 35,000, and 15,000, which very likely correspond to ArsR, ArsB, and ArsC, respectively. ArsB was distinguished by its overall hydrophobic character, suggesting a membrane association. The arsenate, arsenite, and antimonite resistance was shown to be inducible by all three heavy metal ions. Inactivation of the first gene, arsR, resulted in constitutive expression of resistance. Similar results were obtained with transcriptional fusions of various portions of the ars genes with a lipase reporter gene, indicating a function of ArsR as a negative regulator of a putative promoter in front of arsR. The inactivation of arsR also resulted in reduction of resistance to arsenite and antimonite, while arsenate resistance was unaffected. The three ars genes conferred arsenite resistance in E. coli and arsenite as well as arsenate resistance in Bacillus subtilis.

Restriction endonuclease mapping of the HI2 incompatibility group plasmid R478

A restriction map of the 272-kb IncHI2 plasmid R478 was constructed by using the enzymes ApaI, XbaI, SalI, and XhoI. The map was derived from cloned restriction fragments from R478 inserted into cosmid and plasmid vectors as well as from double-digestion analysis of R478 and R478 miniplasmids. All previously known resistance determinants were cloned from R478, and their positions were located on the restriction map. A region involved in incompatibility was cloned and mapped. The location of a previously unreported arsenite resistance gene was also determined. The genes encoding tellurite resistance, colicin B resistance, and phage inhibition were found to be associated with a 6.7-kb SalI fragment of R478.

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.

Arsenite resistance in Listeria monocytogenes

A total of 74 isolates of Listeria (53 L. monocytogenes, ten L. innocua, three L. ivanovii, six L. welshimeri and two L. seligeri) were evaluated for their ability to grow in the presence of 100 μg ml −1 sodium arsenite. Six L. monocytogenes and one L. innocua strains were arsenite-resistant. There appeared to be a relationship between arsenite resistance and serotype, with five of the six arsenite-resistant strains of L. monocytogenes being serotype 4, and the incidence of arsenite resistance being substantially higher in type 4 L. monocytogenes than type 1 (63% vs 3%). Plasmid profiles of selected strains indicated that arsenite resistance is not a plasmid-linked trait in Listeria.

Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P-glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.

Drug resistance and P-glycoprotein gene amplification in the protozoan parasite Leishmania

Amplification of the H circle is often associated with methotrexate (MTX) selection in Leishmania species. We have shown that the H circle of Leishmania tarentolae contains an open reading frame, ItpgpA, that has the attributes of P-glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). H region amplification was also noted in some mutants selected for resistance to arsenite and vinblastine. Mutants having the complete 68-kb circles were cross-resistant to MTX, but two arsenite mutants having only part of the H region amplified, but including ItpgpA, were not cross-resistant to MTX. These results suggest that the putative determinant for MTX resistance present on the H circle is not ItpgpA. We have also determined how ItpgpA-containing plasmids were generated from the chromosomal copy. The H circle contains a 30-kb inverted duplication separated by two unique DNA segments. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We have shown that the two unique segments in chromosomal DNA are flanked by inverted repeats suggesting that H circles could be formed by a foldback mechanism (see fig. 2). Unexpectedly, a plasmid present in cells selected for arsenite resistance lacked part of the H region and the long inverted repeats. It appears to have been formed by intrachromosomal recombination between two P-glycoprotein genes, ItpgpA and ItpgpB, located adjacent to the H region. Our results show that under drug pressure, the same P-glycoprotein-encoding region in Leishmania may be amplified by very different mechanisms and yield different amplicons.

Nocardioform arsenic resistance plasmids and construction of Rhodococcus cloning vectors

One of a number of large nocardioform plasmids previously obtained by a primarily genetic approach was reduced in size to about ~ 11 kb. This smaller plasmid possessed determinants for resistance to sodium arsenate and sodium arsenite, as well as immunity to nocardiophage Q4. It was joined to an Escherichia coli-positive selection vector constructed by M. Zabeau and colleagues, which had the EcoR1 endonuclease gene placed under the control of the P R promoter of λ as well as a bla determinant. The resulting shuttle vector of about 14.6 kb was maintained in E. coli and in several strains of Rhodococcus. The vector was efficient in cloning DNA without prior alkaline phosphatase treatment, as a result of the presence of the positive selection function. This function was not significantly expressed in Rhodococcus, and the presence of the nocardioform resistance determinants led to no increase in arsenate or arsenite resistance in E. coli. The presence of the bla gene resulted in an increase of about threefold in ampicillin resistance in Rhodococcus strains.

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 μ M 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.

A plasmid-encoded arsenite pump produces arsenite resistance in [formula omitted

The arsenate resistance operon of R-factor R773, a conjugative resistance plasmid, has two functional regions, a promoter-proximal region encoding resistance to arsenite and antimonate, and a promoter-distal one encoding arsenate resistance. Cells bearing arsenite resistance plasmids exhibited reduced accumulation of 74 AsO 2 − . When resistant cells were depleted of endogenous energy reserves and then loaded with 74 AsO 2 − , active extrusion of the ion was observed when an energy source was supplied. Intracellular ATP was required for extrusion, but a proton motive force was neither necessary nor sufficient. An arsenite-sensitive mutant was unable to extrude arsenite, while an arsenate-sensitive mutant had normal arsenite transport. These results suggest that the action of a plasmid-encoded primary arsenite efflux pump is the mechanism of arsenite resistance.