Characterization of a Multigene-Encoded Sodium/Hydrogen Antiporter (Sha) from Pseudomonas aeruginosa : Its Involvement in Pathogenesis

Abstract

Sha (also known as Mrp/Mnh/Pha) is a Na؉ /H ؉ antiporter encoded by a cluster of six or seven genes that probably form a multisubunit transport complex. The Sha system is important for the homeostasis of H ؉ , Na ؉ , and other monovalent cations and plays a critical role in various functions, including alkaliphily, sporulation, and symbiosis. Here, we characterized the sha homologue genes from the opportunistic pathogen Pseudomonas aeruginosa, which exist as a cluster of six genes (PA1054 to PA1059). The gene cluster PA1054 to PA1059, but not the cluster with a deletion of PA1054, complemented a growth defect in the presence of 0.2 M NaCl and a defect in Na ؉ /H ؉ antiport activity of the Escherichia coli TO114 mutant lacking the three major Na ؉ /H ؉ antiporters, indicating that genes PA1054 to PA1059 are responsible for Na ؉ /H ؉ antiport activity. We disrupted PA1054 (a shaA homologue gene) and determined its effect on Na ؉ tolerance during growth, Na ؉ efflux, and pathogenicity in mice. Disruption of PA1054 resulted in severe Na ؉ sensitivity during growth and decreased Na ؉ efflux activity. In mice, the deletion mutant of PA1054 also exhibited an attenuated virulence in systemic, pulmonary, and urinary tract infections and also a decrease in colonization of the infected organs. From these results, we conclude that the genes PA1054 to PA1059 encode a Na ؉ /H ؉ antiporter that is largely responsible for Na ؉ extrusion in P. aeruginosa and has a role in the infection of the pathogen. We propose to designate PA1054 to PA1059 as the sha (sodium hydrogen antiporter) genes, shaABCDEFG.

Catalytic Properties of Staphylococcus aureus and Bacillus Members of the Secondary Cation/Proton Antiporter-3 (Mrp) Family Are Revealed by an Optimized Assay in an Escherichia coli Host

“…The standard fluorescence-based antiporter assays of these properties are usually conducted with everted (inside-out) membrane vesicles from antiporter-deficient Escherichia coli strains expressing the test antiporter (12, 52). Such assays of Mrp systems have thus far yielded signals that were too low for kinetic analyses or assays of transmembrane electrical potential (⌬⌿) consumption (17,21,33,34).…”

“…Members of the family of monovalent cation/H ϩ antiporters referred to here as the Mrp family have diverse designations, including Mrp (19), Mnh (17), Pha (48), Sha (31), and Sno (3), and are widely distributed among physiologically diverse prokaryotes, including numerous pathogenic bacteria (56). Important physiological functions have been attributed to the monovalent cation/H ϩ activity of Mrp antiporters, since mutational loss or compromise of this activity has been associated with: alkali and Na ϩ sensitivity in alkaliphilic Bacillus (14) and Anabaena (4); Na ϩ sensitivity, alkali sensitivity, and a sporulation defect in Bacillus subtilis (19,20,31,32); a growth defect in Staphylococcus aureus (3, 24); and Na ϩ sensitivity and decreased virulence in Pseudomonas aeruginosa (33). Several bacterial strains have more than one Mrp system.…”

Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter

“…Studies of seven-subunit Bacillus Mrp antiporters show that they form hetero-oligomeric complexes containing all seven subunits, and the active form of Mrp appears to be a dimer of the hetero-oligomeric Mrp complex (15,16). Mrp-type antiporters have been found in bacteria and archaea from diverse ecological niches and genera and are among the antiporters that have critical roles in bacterial pathogens (11,12,(17)(18)(19)(20)(21)(22). However, the structural complexity of Mrp antiporters has made it difficult to unravel the antiport mechanism, which is needed to gain an understanding of the advantages conferred by its unusual complexity in settings in which it is important and often essential.…”

Single Gene Deletions of mrpA to mrpG and mrpE Point Mutations Affect Activity of the Mrp Na + /H + Antiporter of Alkaliphilic Bacillus and Formation of Hetero-Oligomeric Mrp Complexes

“…It is widespread in both gram-positive and gramnegative bacteria (39). Roles for Mrp antiporters have already been shown in alkaline pH homeostasis and Na ϩ resistance (10,15,39), sporulation (22), symbiotic nitrogen fixation (30), pathogenesis (21), arsenite resistance (19), and bile salt resistance (5,15,16). Mrp antiporters are classified in their own family, the cation/proton antiporter-3 family of the transporter classification system, because of their unique complexity (34,39).…”