Two cation transporters Ena1 and Nha1 cooperatively modulate ion homeostasis, antifungal drug resistance, and virulence of Cryptococcus neoformans via the HOG pathway

Maintenance of cation homeostasis is essential for survival of all living organisms in their biological niches. It is also important for the survival of human pathogenic fungi in the host, where cation concentrations and pH will vary depending on different anatomical sites. However, the exact role of diverse cation transporters and ion channels in virulence of fungal pathogens remains elusive. In this study we functionally characterized ENA1 and NHA1, encoding a putative Na(+)/ATPase and Na(+)/H(+) antiporter, respectively, in Cryptococcus neoformans, a basidiomycete fungal pathogen which causes fatal meningoencephalitis. Expression of NHA1 and ENA1 is induced in response to salt and osmotic shock mainly in a Hog1-dependent manner. Phenotypic analysis of the ena1Δ, nha1Δ, and ena1Δnha1Δ mutants revealed that Ena1 controls cellular levels of toxic cations, such as Na(+) and Li(+) whereas both Ena1 and Nha1 are important for controlling less toxic K(+) ions. Under alkaline conditions, Ena1 was highly induced and required for growth in the presence of low levels of Na(+) or K(+) salt and Nha1 played a role in survival under K(+) stress. In contrast, Nha1, but not Ena1, was essential for survival at acidic conditions (pH 4.5) under high K(+) stress. In addition, Ena1 and Nha1 were required for maintenance of plasma membrane potential and stability, which appeared to modulate antifungal drug susceptibility. Perturbation of ENA1 and NHA1 enhanced capsule production and melanin synthesis. However, Nha1 was dispensable for virulence of C. neoformans although Ena1 was essential. In conclusion, Ena1 and Nha1 play redundant and discrete roles in cation homeostasis, pH regulation, membrane potential, and virulence in C. neoformans, suggesting that these transporters could be novel antifungal drug targets for treatment of cryptococcosis.     

The Na(+)-dependence of alkaliphily in Bacillus

A Na(+) cycle plays a central role in the remarkable capacity of aerobic, extremely alkaliphilic Bacillus species for pH homeostasis. The capacity for pH homeostasis, in turn, appears to set the upper pH limit for growth. One limb of the alkaliphile Na(+) cycle consists of Na(+)/H(+) antiporters that achieve net H(+) accumulation that is coupled to Na(+) efflux. The major antiporter on which pH homeostasis depends is thought to be the Mrp(Sha)-encoded antiporter, first identified from a partial clone in Bacillus halodurans C-125. Mrp(Sha) may function as a complex. While this antiporter is capable of secondary antiport energized by an imposed or respiration-generated protonmotive force, the possibility of a primary mode has not been excluded. In Bacillus pseudofirmus OF4, at least two additional antiporters, including NhaC, have supporting roles in pH homeostasis. Some of these additional antiporters may be especially important for antiport at low [Na(+)] or at near-neutral pH. The second limb of the Na(+) cycle facilitates Na(+) re-entry via Na(+)/solute symporters and, perhaps, the ion channel associated with the Na(+)-dependent flagellar motor. The process of pH homeostasis is also enhanced, perhaps especially during transitions to high pH, by different arrays of secondary cell wall polymers in the two alkaliphilic Bacillus species studied most intensively. The mechanisms whereby alkaliphiles handle the challenge of Na(+) stress at very elevated [Na(+)] are just beginning to be identified, and a hypothesis has been advanced to explain the finding that B. pseudofirmus OF4 requires a higher [Na(+)] for growth at near-neutral pH than at very alkaline pH values.     

Functional characterization of a NapA Na(+)/H(+) antiporter from Thermus thermophilus

Na(+)/H(+) antiporters are ubiquitous membrane proteins and play an important role in cell homeostasis. We amplified a gene encoding a member of the monovalent cation:proton antiporter-2 (CPA2) family (TC 2.A.37) from the Thermus thermophilus genome and expressed it in Escherichia coli. The gene product was identified as a member of the NapA subfamily and was found to be an active Na(+)(Li(+))/H(+) antiporter as it conferred resistance to the Na(+) and Li(+) sensitive strain E. coli EP432 (DeltanhaA, DeltanhaB) upon exposure to high concentration of these salts in the growth medium. Fluorescence measurements using the pH sensitive dye 9-amino-6-chloro-2-methoxyacridine in everted membrane vesicles of complemented E. coli EP432 showed high Li(+)/H(+) exchange activity at pH 6, but marginal Na(+)/H(+) antiport activity. Towards more alkaline conditions, Na(+)/H(+) exchange activity increased to a relative maximum at pH 8, where by contrast the Li(+)/H(+) exchange activity reached its relative minimum. Substitution of conserved residues D156 and D157 (located in the putative transmembrane helix 6) with Ala resulted in the complete loss of Na(+)/H(+) activity. Mutation of K305 (putative transmembrane helix 10) to Ala resulted in a compromised phenotype characterized by an increase in apparent K(m) for Na(+) (36 vs. 7.6 mM for the wildtype) and Li(+) (17 vs. 0.22 mM), In summary, the Na(+)/H(+) antiport activity profile of the NapA type transporter of T. thermophilus resembles that of NhaA from E. coli, whereas in contrast to NhaA the T. thermophilus NapA antiporter is characterized by high Li(+)/H(+) antiport activity at acidic pH.     

Effects of nonpolar mutations in each of the seven Bacillus subtilis mrp genes suggest complex interactions among the gene products in support of Na(+) and alkali but not cholate resistance

The Bacillus subtilis mrp (multiple resistance and pH) operon supports Na(+) and alkali resistance via an Na(+)/H(+) antiport, as well as cholate efflux and resistance. Among the individual mutants with nonpolar mutations in each of the seven mrp genes, only the mrpF mutant exhibited cholate sensitivity and a cholate efflux defect that were complemented by expression of the deleted gene in trans. Expression of mrpF in the mrp null (VKN1) strain also restored cholate transport and increased Na(+) efflux, indicating that MrpF does not require even low levels of other mrp gene expression for its own function. In contrast to MrpF, MrpA function had earlier seemed to depend upon at least modest expression of other mrp genes, i.e., mrpA restored Na(+) resistance and efflux to strain VK6 (a polar mrpA mutant which expresses low levels of mrpB to -G) but not to the null strain VKN1. In a wild-type background, each nonpolar mutation in individual mrp genes caused profound Na(+) sensitivity at both pH 7.0 and 8.3. The mrpA and mrpD mutants were particularly sensitive to alkaline pH even without added Na(+). While transport assays in membrane vesicles from selected strains indicated that MrpA-dependent antiport can occur by a secondary, proton motive force-dependent mechanism, the requirement for multiple mrp gene products suggests that there are features of energization, function, or stabilization that differ from typical secondary membrane transporters. Northern analyses indicated regulatory relationships among mrp genes as well. All the mrp mutants, especially the mrpA, -B, -D, -E, and -G mutants, had elevated levels of mrp RNA relative to the wild type. Expression of an upstream gene, maeN, that encodes an Na(+)/malate symporter, was coordinately regulated with mrp, although it is not part of the operon.     

Yeast plasma membrane Ena1p ATPase alters alkali-cation homeostasis and confers increased salt tolerance in tobacco cultured cells

In plants, the plasma membrane Na(+)/H(+) antiporter is the only key enzyme that extrudes cytosolic Na(+) and contributes to salt tolerance. But in fungi, the plasma membrane Na(+)/H(+) antiporter and Na(+)-ATPase are known to be key enzymes for salt tolerance. Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na(+) and Li(+) efflux across the plasma membrane during salt stress and for K(+) efflux at high pH and high K(+). To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells. Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na(+) and Li(+) than nonexpressing BY2 clones. Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K(+) than nonexpressing cells under no-stress conditions. These results suggest that the yeast Ena1p can also function as an alkali-cation (Na(+), Li(+), and K(+)) ATPase and alter alkali-cation homeostasis in plant cells. We conclude that, even with K(+)-ATPase activity, Na(+)-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress.