Blocking of Single α-Hemolysin Pore by Rhodamine Derivatives.

Measurements of ion conductance through α-hemolysin pore in a bilayer lipid membrane revealed blocking of the ion channel by a series of rhodamine 19 and rhodamine B esters. The longest dwell closed time of the blocking was observed with rhodamine 19 butyl ester (C4R1), whereas the octyl ester (C8R1) was of poor effect. Voltage asymmetry in the binding kinetics indicated that rhodamine derivatives bound to the stem part of the aqueous pore lumen. The binding frequency was proportional to a quadratic function of rhodamine concentrations, thereby showing that the dominant binding species were rhodamine dimers. Two levels of the pore conductance and two dwell closed times of the pore were found. The dwell closed times lengthened as the voltage increased, suggesting impermeability of the channel for the ligands. Molecular docking analysis revealed two distinct binding sites within the lumen of the stem of the α-hemolysin pore for the C4R1 dimer, but only one binding site for the C8R1 dimer. The blocking of the α-hemolysin nanopore by rhodamines could be utilized in DNA sequencing as additional optical sensing owing to bright fluorescence of rhodamines if used for DNA labeling.

Oxacillin alters the toxin expression profile of community-associated methicillin-resistant Staphylococcus aureus.

The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a growing cause for concern. These strains are more virulent than health care-associated MRSA (HA-MRSA) due to higher levels of toxin expression. In a previous study, we showed that the high-level expression of PBP2a, the alternative penicillin binding protein encoded by the mecA gene on type II staphylococcal cassette chromosome mec (SCCmec) elements, reduced toxicity by interfering with the Agr quorum sensing system. This was not seen in strains carrying the CA-MRSA-associated type IV SCCmec element. These strains express significantly lower levels of PBP2a than the other MRSA type, which may explain their relatively high toxicity. We hypothesized that as oxacillin is known to increase mecA expression levels, it may be possible to attenuate the toxicity of CA-MRSA by using this antibiotic. Subinhibitory oxacillin concentrations induced PBP2a expression, repressed Agr activity, and, as a consequence, decreased phenol-soluble modulin (PSM) secretion by CA-MRSA strains. However, consistent with other studies, oxacillin also increased the expression levels of alpha-toxin and Panton-Valentine leucocidin (PVL). The net effect of these changes on the ability to lyse diverse cell types was tested, and we found that where the PSMs and alpha-toxin are important, oxacillin reduced overall lytic activity, but where PVL is important, it increased lytic activity, demonstrating the pleiotropic effect of oxacillin on toxin expression by CA-MRSA.

Two-step purification and in vitro characterization of a hemolysin from the venom of jellyfish Cyanea nozakii Kishinouye.

Hemolysin is one of the most hazardous components in the venom of Cyanea nozakii Kishinouye. Here we describe the purification and in vitro characterization of the hemolysin, which we named CnPH. The CnPH was isolated by anion-exchange and size-exclusion chromatography from the nematocyst venom. Two protein bands with molecular masses of 20 kDa, 60 kDa respectively were shown in the reducing SDS-PAGE analysis of the CnPH. And Approximately 5 µg/mL of the CnPH resulted in 50% hemolysis of the erythrocyte suspension. The hemolytic activity of the CnPH was both temperature and pH dependent. Moreover, it was significantly inhibited in the presence of divalent metal cations, including Cu(2+), Mg(2+), Mn(2+), Zn(2+) and Ca(2+), but enhanced in the presence of EDTA. However, how CnPH performs its hemolytic activity is not yet clear, therefore the mechanism of the hemolytic activity of the CnPH is under research.