Structures and Antibacterial Properties of Isorugosins H–J, Oligomeric Ellagitannins from <i>Liquidambar formosana</i> with Characteristic Bridging Groups between Sugar Moieties

Three new ellagitannin oligomers, isorugosins H (<b>1</b>), I (<b>2</b>), and J (<b>3</b>), together with 11 known hydrolyzable tannins were isolated from an aqueous acetone extract of the fresh leaves of <i>Liquidambar formosana</i>. Their chemical structures were elucidated based on spectroscopic data and chemical conversion into known hydrolyzable tannins. The bridging mode of the valoneoyl groups between their sugar moieties has been identified only in this plant species. Additionally, the effects of the isorugosins isolated from this species on drug-resistant bacteria were evaluated and showed that isorugosin A (<b>4</b>) exhibited the most potent antibacterial activity against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). The isorugosins also had a suppressing effect on pigment formation in <i>Pseudomonas aeruginosa</i>. The isorugosin–protein complexes were analyzed using size-exclusion chromatography and polyacrylamide gel electrophoresis to clarify the relationship of their antibacterial properties with their protein interaction potency as hydrolyzable tannins. The results suggested that the antibacterial properties of hydrolyzable tannins are not simply a result of their binding activity to proteins, but are due to other factors such as the accessibility of polyphenolic acyl groups to bacterial membranes

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

mRNA expression of deduced MATE-type transporter genes in <i>K</i>. <i>pneumoniae</i>.

<p>Gene expression was investigated by RT-PCR. Panel A: <i>ketM</i>, Panel B: <i>dinF</i>, Panel C: <i>yeeO</i>, and Panel D: <i>uncB</i>. The amplification of <i>uncB</i> was used as a standard control. Amplification was performed without a reverse-transcriptase reaction for samples in the RT (-) lanes. The experiment was repeated more than five times and the most reproducible result was shown. Lane 1: MGH78578, Lane 2: ATCC10031, Lane 3: NCTC9632, Lane 4: SKYM</p