Bacteria and the growing threat of multidrug resistance for invasive cardiac interventions.

Invasive cardiovascular procedures which include heart transplantations, congenital heart surgery, coronary artery bypass grafts, cardiac valve repair and replacement, and interventional cardiac electrophysiology procedures represent common mechanisms to treat a variety of cardiovascular diseases across the globe. The majority of these invasive approaches employ antibiotics as a regular and obligatory feature of the invasive procedure. Although the growing incidence of bacterial resistance to currently used antibiotics threatens to curtail the use of all interventional surgical techniques, it remains an underappreciated threat within the arsenal of cardiovascular therapies.

Molecular dynamics modeling of the Vibrio cholera Na-translocating NADH:quinone oxidoreductase NqrB-NqrD subunit interface.

The Na-translocating NADH:quinone oxidoreductase (Na-NQR) is the major Na pump in aerobic pathogens such as Vibrio cholerae. The interface between two of the NQR subunits, NqrB and NqrD, has been proposed to harbor a binding site for inhibitors of Na-NQR. While the mechanisms underlying Na-NQR function and inhibition remain underinvestigated, their clarification would facilitate the design of compounds suitable for clinical use against pathogens containing Na-NQR.

Pharmacophore-based screening and modification of amiloride analogs for targeting the NhaP-type cation-proton antiporter in .

The genome of contains three structural genes for the NhaP-type cation-proton antiporter paralogues, Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3, mediating exchange of K and or Na for protons across the membrane. Based on phenotypic analysis of chromosomal , , and triple deletion mutants, we suggest that Vc-NhaP paralogues are primarily K/H antiporters and might play a role in the acid tolerance response of as it passes through the gastric acid barrier of the stomach. Comparison of the biochemical properties of Vc-NhaP isoforms revealed that Vc-NhaP2 was the most active among all three paralogues.

Physiological, Structural, and Functional Analysis of the Paralogous Cation-Proton Antiporters of NhaP Type from .

The transmembrane K/H antiporters of NhaP type of (Vc-NhaP1, 2, and 3) are critical for maintenance of K homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of at low pHs suggesting their possible role in the acid tolerance response (ATR) of . Our findings suggest that the Vc-NhaP123 group, and especially its major component, Vc-NhaP2, might be a promising target for the development of novel antimicrobials by narrowly targeting and other NhaP-expressing pathogens.

as Host for Expression of Multisubunit Membrane Protein Complexes.

is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing as a suitable expression host for membrane-bound proteins from . We achieved production of the primary Na pump, the NADH:quinone oxidoreductase (NQR), from in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed , and the sensitivity toward Ag, a specific inhibitor of the NQR.

A pathway leading to a cation-binding pocket determines the selectivity of the NhaP2 antiporter in .

The Vc-NhaP2 antiporter from exchanges H for K or Na but not for the smaller Li. The molecular basis of this unusual selectivity remains unknown. and Rosetta software were used to generate a structural model of the Vc-NhaP2.

Na+-NQR (Na+-translocating NADH:ubiquinone oxidoreductase) as a novel target for antibiotics.

The recent breakthrough in structural studies on Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae creates a perspective for the systematic design of inhibitors for this unique enzyme, which is the major Na+ pump in aerobic pathogens. Widespread distribution of Na+-NQR among pathogenic species, its key role in energy metabolism, its relation to virulence in different species as well as its absence in eukaryotic cells makes this enzyme especially attractive as a target for prospective antibiotics. In this review, the major biochemical, physiological and, especially, the pharmacological aspects of Na+-NQR are discussed to assess its 'target potential' for drug development.

Development of a novel rationally designed antibiotic to inhibit a nontraditional bacterial target.

The search for new nontraditional targets is a high priority in antibiotic design today. Bacterial membrane energetics based on sodium ion circulation offers potential alternative targets. The present work identifies the Na-translocating NADH:ubiquinone oxidoreductase (Na-NQR), a key respiratory enzyme in many microbial pathogens, as indispensible for the Chlamydia trachomatis infectious process.

Physiology of the Vc-NhaP paralogous group of cation-proton antiporters in Vibrio cholerae.

The genome of Vibrio cholerae encodes three cation-proton antiporters of NhaP-type, Vc-NhaP1, 2, and 3. To examine physiological roles of Vc-NhaP antiporters, triple ΔnhaP1ΔnhaP2ΔnhaP3 and single ΔnhaP3 deletion mutants of V. cholerae were constructed and characterized.

Effects of chromosomal deletion of the operon encoding the multiple resistance and pH-related antiporter in Vibrio cholerae.

To examine the possible physiological significance of Mrp, a multi-subunit cation/proton antiporter from Vibrio cholerae, a chromosomal deletion Δmrp of V. cholerae was constructed and characterized. The resulting mutant showed a consistent early growth defect in LB broth that became more evident at elevated pH of the growth medium and increasing Na+ or K+ loads.

The C-terminal cytoplasmic portion of the NhaP2 cation-proton antiporter from Vibrio cholerae affects its activity and substrate affinity.

In this work, we report the phenotypic and biochemical effects of deleting the C-terminal cytoplasmic portion of the NhaP2 cation/proton antiporter from Vibrio cholerae. While the deletion changed neither the expression nor targeting of the Vc-NhaP2 in an antiporter-less Escherichia coli strain, it resulted in a changed sensitivity of the host to sodium ions at neutral pH, indicating an altered Na(+) transport through the truncated variant. When assayed in inside-out sub-bacterial vesicles, the truncation was found to result in greatly reduced K(+)/H(+) and Na(+)/H(+) antiport activity at all pH values tested and a greater than fivefold decrease in the affinity for K(+) (measured as the apparent K m) at pH 7.

NhaP1 is a K+(Na+)/H+ antiporter required for growth and internal pH homeostasis of Vibrio cholerae at low extracellular pH.

Vibrio cholerae has adapted to a wide range of salinity, pH and osmotic conditions, enabling it to survive passage through the host and persist in the environment. Among the many proteins responsible for bacterial survival under these diverse conditions, we have identified Vc-NhaP1 as a K(+)(Na(+))/H(+) antiporter essential for V. cholerae growth at low environmental pH.

Insights into the biochemistry of the ubiquitous NhaP family of cation/H+ antiporters.

Na+/H+ antiporters are integral membrane proteins that exchange Na+ for H+ across the cytoplasmic or organellar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regulation of Na+ levels. Na+/H+ antiporters have become increasingly characterized and are now becoming important drug targets.

The putative Na+/H+ antiporter of Vibrio cholerae, Vc-NhaP2, mediates the specific K+/H+ exchange in vivo.

The existence of bacterial K(+)/H(+) antiporters that prevent the overaccumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost 50 years ago. The importance of K(+)/H(+) antiport for bacterial physiology is widely recognized, but its molecular mechanisms remain underinvestigated. Here, we demonstrate that a putative Na(+)/H(+) antiporter, Vc-NhaP2, protects cells of Vibrio cholerae growing at pH 6.

The past and present of sodium energetics: may the sodium-motive force be with you.

All living cells routinely expel Na(+) ions, maintaining lower concentration of Na(+) in the cytoplasm than in the surrounding milieu. In the vast majority of bacteria, as well as in mitochondria and chloroplasts, export of Na(+) occurs at the expense of the proton-motive force. Some bacteria, however, possess primary generators of the transmembrane electrochemical gradient of Na(+) (sodium-motive force).

The Vibrio cholerae Mrp system: cation/proton antiport properties and enhancement of bile salt resistance in a heterologous host.

The mrp operon from Vibrio cholerae encoding a putative multisubunit Na(+)/H(+) antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na(+) and Li(+) as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E.

Functional analysis of conserved polar residues in Vc-NhaD, Na+/H+ antiporter of Vibrio cholerae.

Vc-NhaD is a Na(+)/H(+) antiporter from Vibrio cholerae with a sharp maximum of activity at pH approximately 8.0. NhaD homologues are present in many bacteria as well as in higher plants.

Molecular characterization of PeNhaD1: the first member of the NhaD Na+/H+ antiporter family of plant origin.

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.

The sodium cycle in vibrio cholerae: riddles in the dark.

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.

Salt in the wound: a possible role of Na+ gradient in chlamydial infection.

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.

2-Aminoperimidine, a specific inhibitor of bacterial NhaA Na(+)/H(+) antiporters.

The diuretic drug amiloride and its numerous derivatives are competitive inhibitors of mammalian Na(+)/H(+) antiporters and other eukaryotic antiporters. Most prokaryotic antiporters, including the major NhaA family of enterobacteria, are resistant to these compounds. We show that 2-aminoperimidine (AP), a guanidine-containing naphthalene derivative with some similarity to amiloride, acts as a specific inhibitor of NhaA from Escherichia coli.

Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation.

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.

Experimental verification of a sequence-based prediction: F(1)F(0)-type ATPase of Vibrio cholerae transports protons, not Na(+) ions.

The membrane energetics of the intestinal pathogen Vibrio cholerae involves both H(+) and Na(+) as coupling ions. The sequence of the c subunit of V. cholerae F(0)F(1) ATPase suggested that this enzyme is H(+) specific, in contrast to the results of previous studies on the Na(+)-dependent ATP synthesis in closely related Vibrio spp.