No abstract

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and ␣-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and ␣-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric ␣-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of ␣-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.

The concentration gradient of K across the cell membrane of a neuron determines its resting potential and cell excitability. During neurotransmission, the efflux of K from the cell via various channels will not only decrease the intracellular K content but also elevate the extracellular K concentration. However, it is not clear to what extent this change could be. In this study, we developed a multiple-parallel-connected silicon nanowire field-effect transistor (SiNW-FET) modified with K-specific DNA-aptamers (aptamer/SiNW-FET) for the real-time detection of the K efflux from cultured cortical neurons. The aptamer/SiNW-FET showed an association constant of (2.18 ± 0.44) × 10 M against K and an either less or negligible response to other alkali metal ions. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulation induced an outward current and hyperpolarized the membrane potential in a whole-cell patched neuron under a Na/K-free buffer. When neurons were placed atop the aptamer/SiNW-FET in a Na/K-free buffer, AMPA (13 μM) stimulation elevated the extracellular K concentration to ∼800 nM, which is greatly reduced by 6,7-dinitroquinoxaline-2,3-dione, an AMPA receptor antagonist. The EC of AMPA in elevating the extracellular K concentration was 10.3 μM. By stimulating the neurons with AMPA under a normal physiological buffer, the K concentration in the isolated cytosolic fraction was decreased by 75%. These experiments demonstrate that the aptamer/SiNW-FET is sensitive for detecting cations and the K concentrations inside and outside the neurons could be greatly changed to modulate the neuron excitability.