Vibrio natriegens as Host for Expression of Multisubunit Membrane Protein Complexes

Escherichia coli 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 Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae. We achieved production of the primary Na+ pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens, and the sensitivity toward Ag+, a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae, another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li+ transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens

Sodium/Proton Antiporter Activity is Essential for Virulence of Yersinia pestis

We found that a strains of Yersinia pestis (KIM5) which lacked the nhaA gene was fully attenuated in a plague model. This gene produces a protein of the sodium-proton antiporter family which expel sodium ions from the bacterial cytoplasm in exchange for hydrogen ions, or protons, from the surrounding environment. A Y. pestis strain that contained the nhaA mutation showed a significant decrease in its ability to survive in both sheep’s blood and serum. Decreased growth rates were also observed when the nhaA deficient strain was tested in the artificial serum media Opti-MEM® when compared to the wild type strain. A similar growth phenotype was observed when wild type and nhaA mutant strains were tested in LB media set to mimic pH and salt conditions of blood. These observations indicate that sodium-proton antiporter activity of Y. pestis is essential for the survival of the bacterium in certain environments, such as the blood of an infected host. 2-aminopyrimidine was used to inhibit NhaA activity, and when tested in Opti-MEM®, bacterial growth rates decreased. These findings lead us to propose that sodium-proton antiporter inhibition is a novel way of treating bacterial blood-borne diseases

Na +/H + Antiport Is Essential for Yersinia pestis Virulence

Na⁺/H⁺ antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na⁺/H⁺ antiport in Yersinia pestis virulence and found that Y. pestis strains lacking the major Na⁺/H⁺ antiporters, NhaA and NhaB, are completely attenuated in an in vivo model of plague. The Y. pestis derivative strain lacking the nhaA and nhaB genes showed markedly decreased survival in blood and blood serum ex vivo. Complementation of either nhaA or nhaB in trans restored the survival of the Y. pestis nhaA nhaB double deletion mutant in blood. The nhaA nhaB double deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na⁺ levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na⁺/H⁺ antiport is indispensable for the survival of Y. pestis in the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused by Y. pestis and possibly for those caused by other blood-borne bacterial pathogens.Keywords: Vibrio cholerae, Inhibitors, Expression, Ph, Escherichia coli, nhaB, Low calcium response, Homeostasis, Bacteria, resistanc

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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. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-exponential phase, after 4-6 h of culture. Using a pH-sensitive GFP, cytosolic pH was shown to be dependent on K + in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless Escherichia coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K + or Na + ions for H + within a broad pH range (7.25-9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K + (Na + )/H + antiporter of the CPA1 family that is required for bacterial pH homeostasis and growth in an acidic environment. INTRODUCTION Vibrio cholerae is a Gram-negative pathogen which causes cholera, a dangerous disease that remains a public health concern (Enserink, 2010). As it transitions between the infectious state and its environmental reservoir, the bacterium encounters a dynamic range of osmotic and pH conditions. During human infection, V. cholerae produces a potent enterotoxin, cholera toxin, which promotes accumulation of Na + and Cl 2 ions in the host intestinal lumen and, in turn, causes rapid osmotic dehydration of host tissue and profuse diarrhoea. In the environment, V. cholerae is found in many coastal and estuarine waters, where it is exposed to severe periodic changes in salinity, pH and osmolarity as variable ratios of brackish and fresh water mix at different rates A number of V. cholerae proteins have been described that generate, maintain or use a transmembrane gradient of cations such as Na + (Häse et al., 2001). These proteins are predicted to help the bacterium survive hypo-and hyperosmolar states in addition to exploiting the Na + gradient for solute transport, pH regulation and motility. For example, the NQR complex couples Na + export to electron transport, resulting in the generation of a sodium-motive force that can then be used for various types of membrane wor

Development of a novel rationally-designed antibiotic to inhibit a non-traditional bacterial target

The search for new non-traditional 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. Infection by Chlamydia trachomatis significantly increased first H+ and then Na+ levels within the host mammalian cell. A newly designed furanone Na+-NQR inhibitor, PEG-2S, blocked the changes in both H+ and Na+ levels induced by Chlamydia trachomatis infection. It also inhibited intracellular proliferation of Chlamydia trachomatis with a half-minimal inhibitory concentration in the submicromolar range but did not affect the viability of mammalian cells or bacterial species representing benign intestinal microflora. At low nanomolar concentrations (IC50 value = 1.76 nM), PEG-2S inhibited the Na+-NQR activity in sub-bacterial membrane vesicles isolated from Vibrio cholerae. Taken together, these results show, for the first time, that Na+-NQR is critical for the bacterial infectious process and is susceptible to a precisely targeted bactericidal compound in situ. The obtained data have immediate relevance for many different diseases caused by pathogenic bacteria that rely on Na+-NQR activity for growth, including sexually transmitted, pulmonary, oral/gum, and ocular infections.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Physiological, Structural, and Functional Analysis of the Paralogous Cation–Proton Antiporters of NhaP Type from <i>Vibrio cholerae</i>

The transmembrane K+/H+ antiporters of NhaP type of Vibrio cholerae (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 V. cholerae at low pHs suggesting their possible role in the acid tolerance response (ATR) of V. cholerae. 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 V. cholerae and other NhaP-expressing pathogens. On the basis of Vc-NhaP2 in silico structure modeling, Molecular Dynamics Simulations, and extensive mutagenesis studies, we suggest that the ion-motive module of Vc-NhaP2 is comprised of two functional regions: (i) a putative cation-binding pocket that is formed by antiparallel unfolded regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, known as the NhaA fold; and (ii) a cluster of amino acids determining the ion selectivity

Chemiosmotic Mechanism of Antimicrobial Activity of Ag(+) in Vibrio cholerae

Although the antimicrobial effects of silver salts were noticed long ago, the molecular mechanism of the bactericidal action of Ag(+) in low concentrations has not been elucidated. Here, we show that low concentrations of Ag(+) induce a massive proton leakage through the Vibrio cholerae membrane, which results in complete deenergization and, with a high degree of probability, cell death