Saibal Dey

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.

Orphan enzyme or patriarch of a new tribe: the arsenic resistance ATPase of bacterial plasmids

The plasmid‐determined arsenite and antimonite efflux ATPase of bacteria differs from other membrane transport ATPases, which are classified into several families (such as the F0F1‐type H+‐translocating ATP synthases, the related vacuolar H+‐translocating ATPases, the P‐type cation‐translocating ATPases, and the superfamily which includes the periplasmic binding‐protein‐dependent systems in Gram‐negative bacteria, the human multidrug resistance P‐glycoprotein, and the cystic fibrosis transport regulator). The amino acid sequences of the components of the arsenic resistance system are not similar to known ATPase proteins. New findings with the arsenic resistance operons of bacterial plasmids suggest that instead of being an orphan the Ars system will now be the first recognized member of a new class of ATPases. Furthermore, fundamental questions of energy‐coupling (ATP‐driven or chemiosmotic) have recently been raised and the finding that the arsC gene product is a soluble enzyme that reduces arsenate to arsenite changes the previous picture of the functioning of this widespread bacterial system.

Evolution of an ion-translocating ATPase.

Plasmid-encoded arsenical resistance (ars) operons are found on plasmids of both gram-negative and gram-positive organisms. Novick and Roth2 first reported plasmidmediated resistance to arsenite and arsenate in the Staphylococcus aureus plasmid pI258. The Escherichia coli plasmid R773 was shown to have a similar re~istance.~ Silver et showed that plasmid-bearing cells of either S. aureus or E. coli exhibit reduced uptake of 74As0,3, reflecting energy-dependent extrusion. As arsenicals are anions, these initial studies did not differentiate between secondary electrophoretic movement coupled to the outwardly positive membrane potential and a primary process coupled directly to a source of chemical energy. We showed that arsenical efflux from cells of E. coli expressing the ars genes was coupled to chemical but not electrochemical energy, indicative of a primary anion We cloned the genes for the anion pump from the original 90-kb R-factor R773.& From a combination of genetics and analysis of the nucleotide sequence, we determined that there are five genes in a single operon (FIG. 1). The first two, arsR and arsD, encode regulatory proteinsy. (J . Wu and B. P. Rosen, unpublished results). The products of the third and fourth genes, arsA and arsB, form an anion-translocating ATPase.] In E. cofi these two proteins are sufficient to provide resistance to arsenite and antimonite, which have the +3 oxidation state of the mctals.Z Resistance to arsenate (the +5 oxidation of arsenic) requires the arsC gene product.I2 Recent models of the Ars ATPase have included the ArsC protein as a component of the pump. However, recent evidence has shown that the ArsC protein exhibits arsenate reductase activity, converting arsenate to arsenite for subsequent extrusion through the pump.I3 As the pump does not require the ArsC protein, that protein will not be discussed further. The pump itself is composed of only the ArsA protein, the catalytic subunit, and the ArsB protein, an inner membrane protein in E. coli, which serves as the membrane anchor for the ArsA protein and forms the anion-conducting pathway (FIG. 2). A description of the two proteins and the complex that they form is the topic of this report. The ars operons of two plasmids, pI2.58 and pSX267, from the gram-positive staphyloccoci have also been sequenced and shown to have only three genes

A functional chimeric membrane subunit of an ion-translocating ATPase

A chimeric transport protein was made by expression of a fusion of thearsB genes fromEscherichia coli plasmid R773 andStaphylococcus aureus plasmid pI258. The two genes were fused to encode a functional protein with first eight membrane spanning α-helices of theS. aureus and the last four helices of theE. coli protein. The hybrid protein provided arsenite resistance and transport. When anarsA 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.

Anion-translocating ATPases

Publisher Summary This chapter describes the bacterial resistance to arsenicals and antimonials, other anion-translocating atpases, and arsenite resistance in eukaryotes. Arsenic is an active ingredient in a variety of commonly used insecticides, rodenticides, and herbicides and can be categorized into three groups: (1) the inorganic salts of the arsenical oxyanions arsenate (pentavalent) and arsenite (trivalent), (2) organic arsenicals, both trivalent and pentavalent, and (3) arsine gas. Bacterial resistance results from the activity of the ArsA, ArsB, and ArsC proteins. The ArsA and ArsB proteins form a membrane-bound oxyanion-translocating ATPase that confers resistance to arsenite and antimonite. Because of their toxicity, the use of arsenical and antimonial drugs is now limited to the treatment of a few topical protozoan diseases. Pentavalent antimony in the form of pentostam is still a widely used chemotherapeutic agent in the treatment of leishmaniasis. Treatment failure results from the emergence of pentostam-resistant Leishmani; Pentostam-resistant clinical isolates are cross-resistant to trivalent antimony, when cultured in macrophages.