Functionally Cloned <i>pdrM</i> from <i>Streptococcus pneumoniae</i> Encodes a Na⁺ Coupled Multidrug Efflux Pump

<div><p>Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from <i>Streptococcus pneumoniae</i> R6, and designated it <i>pdrM</i>. PdrM showed sequence similarity with NorM from <i>Vibrio parahaemolyticus</i>, YdhE from <i>Escherichia coli,</i> and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in <i>E. coli</i> KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na⁺- or Li⁺-dependent manner. Moreover, Na⁺ efflux via PdrM was observed when acriflavine was added to Na⁺-loaded cells expressing <i>pdrM</i>. Therefore, we conclude that PdrM is a Na⁺/drug antiporter in <i>S. pneumoniae</i>. In addition to <i>pdrM</i>, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the <i>S. pneumoniae</i> genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and <i>pdrM</i> was detected in <i>S. pneumoniae</i> R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.</p> </div

Salt concentration-dependent fluorescent dye efflux via PdrM.

<p>DAPI efflux activity (A). The concentration of NaCl or LiCl is (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 30 mM, (e) 50 mM, (f) 80 mM, and (g) 100 mM. Acriflavine efflux activity (B). The concentration of NaCl or LiCl is (a) 0 µM, (b) 10 µM, (c) 100 µM, (d) 1 mM, (e) 10 mM, and (f) 20 mM. Acriflavine fluorescence was quenched in cells because of its concentration. When an energy source was added, acriflavine was expelled and acriflavine fluorescence increased. Different from DAPI, the fluorescence of acriflavine does not change by DNA binding.</p

Drug susceptibility in cells transformed with genes predicted from genome information.

<p>DAPI: 4′,6-diamidino-2-phenylindole.</p

Na⁺ efflux from Na⁺-loaded cells elicited by an inwardly directed acriflavine gradient.

<p>Cells of <i>E. coli</i> KAM32/pHAP5 or <i>E. coli</i> KAM32/pHY300PLK (control) were diluted with 0.1 M MOPS-TMAH (pH7.0) until approximately 9 mg protein/ml. Na⁺ was loaded to cells via MelB by the addition of Mβgal (the first arrow), and acriflavine (final 200 µM) was added to the assay mixture at the time point indicated by the second arrow. Upward deflection of the curve indicates Na⁺ influx into cells and downward deflection indicates Na⁺ efflux from cells.</p

Drug susceptibility in cells transformed with <i>pdrM.</i>

<p>DAPI: 4′,6-diamidino-2-phenylindole, EtBr: ethidium bromide,</p><p>TPPCl: tetraphenylphosphonium chloride, ND: not determined.</p

RT-PCR analysis in <i>S. pneumoniae</i> R6.

<p>One ng total RNA was used for the template for one reaction of RT-PCR, and the reaction was repeated for 24 cycles. pUC19 digested with <i>Msp</i> I was used as a size marker (lane M). <i>pdrM</i> (lane a, e), spr1756 (lane b, f), spr1877 (lane c, g). The expression of the <i>atpB</i> was used as an internal control (lane d, h). Reverse transcriptional reactions were submitted on samples in lane a, b, c, and d, and were not on samples in lane e, f, g, and h.</p

Similarity of putative MATE transporters in <i>S. pneumoniae</i> R6 with characterized MATE-type transporters.

*<p>The classification was referred to the result of analysis with ClustalW and reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059525#pone.0059525-Omote1" target="_blank">[47]</a>.</p><p>Genetyx ver. 7.0.8 was used to estimate the scores of identity and similarity.</p><p> <i>Sp : Streptococcus pneumoniae, Ab : Acinetobacter baumannii, Hi : Haemophilus influenzae, Vc : Vibrio cholerae, At : Arabidopsis thaliana, Sc : Saccharomyces cerevisiae, Hs : Homo sapiens, Ec : Escherichia coli, Vp : Vibrio parahaemolyticus, Sa : Staphylococcus aureus.</i></p

Acriflavine efflux from cells elicited by an inwardly directed artificial Na⁺ gradient.

<p>An inwardly directed chemical gradient of Na⁺ was imposed by the addition of the cell suspension into the assay medium containing salt at the time point indicated by the arrow. (A) <i>E. coli</i> KAM32/pHAP5, (B) <i>E. coli</i> KAM32/pHY300PLK. The final concentration of the salt was 100 mM NaCl (curve a) or 100 mM KCl (curve b). The assay was performed at 37°C.</p

Validation of the diabetic phenotype of SDT rats.

<p>Changes in body weight (<b>A</b>) and blood glucose levels (<b>B</b>) in SD and SDT rats. □: SD/control, ■: SD/ZOL, ○: SDT/control, ●: SDT/ZOL. Data are expressed as means ± SD (n = 6). *<i>p</i><0.005 versus SD/control. Macroscopic opacity of the lens observed at approximately 32 weeks of age in SDT rats (<b>C</b>).</p

Macroscopic view of extraction sockets in SD/control (A-C), SD/ZOL (D-F), SDT/control (G-I), and SDT/ZOL (J-L) rats.

<p>Two representative specimens are shown. Normal healing after molar extraction was observed in SD/control (<b>A</b>) and SD/ZOL (<b>D</b>) rats by 2 weeks after extraction. Extraction sockets in both groups were covered with intact epithelium at 4 weeks (<b>B, E</b>) after extraction and at 8 weeks (<b>C, F</b>) after extraction. Apparent mucosal disruption with exposed bone (arrows) was seen at the extraction site at 2 (<b>G</b>) and 4 (<b>H</b>) weeks, but not at 8 weeks (<b>I</b>) after extraction in the SDT/control group and at 2 (<b>J</b>), 4 <b>(K</b>), and at 8 weeks (<b>L</b>) after extraction in the SDT/ZOL group.</p