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Twenty years ago, V. P. Skulachev put forward the revolutionary concept of the chemiosmotic sodium cycle which is an integral of the paradigm of modern bioenergetics. This fundamental concept stimulated studies in many areas and yielded plenty of sometimes quite unexpected (and thus most valuable) discoveries. In particular, variations of the sodium cycle have been found in a surprisingly large number of pathogenic microorganisms, raising the question about the possible link of sodium energetics and virulence. This brief review discusses some paradoxes related to the Na(+) cycle in an important human pathogen, Vibrio cholerae.

PeNhaD1 encodes a putative Na+/H+ antiporter from the salt-resistant tree Populus euphratica. It is the first characterization of a member of the NhaD type ion transporter family of plant origin. Homology searches revealed its close relation to functionally characterized microbial Na+/H+ antiporters VpNhaD and VcNhaD. Na+/H+ antiporters have proven to play a key role in salt resistance, both in plants and bacteria. Under salt stress transcript levels of PeNhaD1 were maintained only in the salt-resistant P. euphratica, but collapsed in Populus x canescens, a salt-sensitive species. To address the function of PeNhaD1, complementation studies with the salt-sensitive Escherichia coli EP432 mutant strain, lacking activity of the two Na+/H+ antiporters EcNhaA and EcNhaB were carried out. PeNhaD1 was able to restore growth of EP432 under stress imposed by up to 400 mM NaCl demonstrating its protective function. Growth rates of EP432 were always highest at pH 5.5 while growth was suppressed under salt stress at pH 7.0 and pH 8.0 suggesting that the antiporter activity is strongly pH dependent. Element analyses of EP432 cells complemented with PeNhaD1 growing under salt stress showed that salt resistance was correlated with a significant reduction in sodium accumulation. These results suggest that PeNhaD1 might play a role in the salt resistance of P. euphratica.

Vibrio cholerae is the infectious agent of the deadly diarrheal disease, cholera. Na+ ion homeostasis is believed to play a key role in both physiology and pathogenicity of this bacterium. However, molecular mechanisms of sodium exchange in V. cholerae are still poorly understood. In the present work a gene encoding an unusual Na+/H+ antiporter, nhaD, was identified in the V. cholerae genome. nhaD was cloned from Vibrio cholerae and expressed in Escherichia coli. The antiporter functioned in an E. coli nhaAnhaB mutant strain to confer resistance to LiCl and NaCl. When assayed in inside-out subbacterial vesicles, V. cholerae NhaD demonstrated high affinity for Na+ ions (1.1 mM Na+ was required for the half-maximal response at the pH-optimum). The most striking feature of Vc-NhaD is a unique pH-profile of its activity with a sharp maximum at pH 8.0, different from that of any bacterial sodium-proton antiporter described so far. The difference is rationalized as being the result of a His to Arg substitution in a putative pH sensing residue.

Genome analysis has revealed the presence of key components of the Na(+) chemiosmotic cycle, including the primary Na(+) pump (Na(+)-translocating NADH:ubiquinone oxidoreductase), in the cytoplasmic membrane of two ubiquitous human pathogens, Chlamydia trachomatis and Chlamydiophyla pneumoniae. This observation seemed paradoxical in the case of obligatory intracellular parasites because the Na(+) cycle is thought to be primarily a mechanism that enhances the adaptive potential in free-living bacteria that are often facing drastic changes in the salinity and pH of the environment. We present a model suggesting that operation of the Na(+) cycle may play an important role in the course of chlamydial infection, when the Na(+) and H(+) homeostasis of the host cell become severely impaired. This introduces the intriguing possibility of the application of drugs targeting Na(+)-transporting enzymes to chlamydial infections, which are notoriously difficult to treat.

The Na+/H+ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na+/H+ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na+/H+ exchanger were examined for their ability to translocate protons as assessed by a Cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na+/H+ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na+/H+ exchanger. These results confirm that polar amino acids are important in proton flux activity of Na+/H+ exchangers.

Sod2 is the sodium-proton antiporter on the plasma membrane of the fission yeast Schizosaccharomyces pombe. It is vitally important for sodium export and pH homeostasis in this organism. Recently, the sod2 gene has been cloned and sequenced. However, initial attempts to express sod2 in Escherichia coli using the T7 promoter failed. In the present work we examined physiological consequences of expression of sod2 in E. coli. To alleviate problems caused by expression of sod2 we: (i) used sodium-free media at all steps; (ii) used the moderate tac promoter for expression and; (iii) used E. coli strain MH1 which has impaired sodium exchange. The effect of sod2 expression on E. coli varied depending on the E. coli genotype. When sod2 was expressed in BL21 cells which have normal Na+/H+ antiporters, the result was a Li+ sensitive phenotype. LiCl completely arrested or prevented growth of BL21 E. coli transformed with the sod2 gene. The effect on growth was pronounced in media of low external pH. Sod2 was then expressed in E. coli MH1 which is devoid of endogenous Na+/H+ antiporters. These cells became more resistant to external LiCl, but only in Na+ containing media. In the absence of external Na+, the presence of sod2 reduced growth. The results are explained in a model which demonstrates the physiological consequences of interference by expression of a foreign electroneutral Na+/H+ antiporter in conjunction with different housekeeping systems of E. coli host cells.

The Na+/H+ exchanger is a ubiquitous protein present in all mammalian cell types that functions to remove one intracellular H+ for one extracellular Na+. Several isoforms of the protein exist, which are referred to as NHE1 to NHE6 (for Na+/H+ exchanger one through six). The NHE1 protein was the first isoform cloned and studied in a variety of systems. This review summarizes recent papers on this protein, particularly those that have examined regulation of the protein and its expression and activity.

Genome analysis has revealed the presence of key components of the Na super(+) chemiosmotic cycle, including the primary Na super(+) pump (Na super(+)-translocating NADH:ubiquinone oxidoreductase), in the cytoplasmic membrane of two ubiquitous human pathogens, Chlamydia trachomatis and Chlamydiophyla pneumoniae. This observation seemed paradoxical in the case of obligatory intracellular parasites because the Na super(+) cycle is thought to be primarily a mechanism that enhances the adaptive potential in free-living bacteria that are often facing drastic changes in the salinity and pH of the environment. We present a model suggesting that operation of the Na super(+) cycle may play an important role in the course of chlamydial infection, when the Na super(+) and H super(+) homeostasis of the host cell become severely impaired. This introduces the intriguing possibility of the application of drugs targeting Na super(+)-transporting enzymes to chlamydial infections, which are notoriously difficult to treat.

Dietary flaxseed has cardioprotective effects that may be achieved through its rich content of the omega-3 fatty acid, alpha linolenic acid (ALA). Because ALA can be stored in adipose tissue, it is possible that some of its beneficial actions may be due to effects it has on the adipose tissue. We investigated the effects of dietary flaxseed both with and without an atherogenic cholesterol-enriched diet to determine the effects of dietary flaxseed on the expression of the adipose cytokines leptin and adiponectin. Rabbits were fed one of four diets: a regular (RG) diet, or a regular diet with added 0.5% cholesterol (CH), or 10% ground flaxseed (FX), or both (CF) for 8 weeks. Levels of leptin and adiponectin expression were assessed by RT-PCR in visceral adipose tissue. Consumption of flaxseed significantly increased plasma and adipose levels of ALA. Leptin protein and mRNA expression were lower in CH animals and were elevated in CF animals. Changes in leptin expression were strongly and positively correlated with adipose ALA levels and inversely correlated with levels of en face atherosclerosis. Adiponectin expression was not significantly affected by any of the dietary interventions. Our data demonstrate that the type of fat in the diet as well as its caloric content can specifically influence leptin expression. The findings support the hypothesis that the beneficial cardiovascular effects associated with flaxseed consumption may be related to a change in leptin expression.