Competition is the basis of the transport mechanism of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae

Abstract

Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.

Impact of Three Different Mutations in Ehrlichia chaffeensis in Altering the Global Gene Expression Patterns

“…We predict that the mutation in the anti-porter protein gene caused a metabolic depression. Antiporter and transport proteins play an important role in the transport of ions and solutes across the cell membranes of bacteria 45 . Antiporters are integral membrane proteins that perform secondary transport of Na + and/or K + for H + across a phospholipid membrane 5 .…”

Mutation of two key aspartate residues alters stoichiometry of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae

“…K m (pH) values are apparent affinities determined for the Na + substrate under different pH conditions. Na + affinity values for KpNhaB WT were determined previously 25 . n denotes the Hill coefficient of cooperativity where data where fitted with a Hill function.…”

“…Proteoliposomes of all prepared KpNhaB mutant variants were subjected to SSM-based electrophysiology in order to ascertain the effect of the mutations on the transport function. An electrophysiological characterization of the transport function has already been performed for KpNhaB WT 25 .…”

“…Since NhaB is an electrogenic transporter, it is well adapted to characterization by electrophysiological techniques, in particular solid supported membrane (SSM)-based electrophysiology 24 . We have recently characterized 25 a member of the NhaB family, NhaB from Klebsiella pneumoniae (KpNhaB) and were able to show that, despite the absence of sequence similarity to CPA exchangers, the function of KpNhaB can be described by a similar competition-based mechanism 25,26 .…”

“…As protonatable residues are essential for Na + /H + exchanger function 3 we sought to identify similarly charged residues in the putative TMs of KpNhaB, as determined by alignment 25 with the TMs established by the previous topological study on VaNhaB 17 . Two such residues are, in the sequence of KpNhaB, Asp146 and Asp404.…”