Virulence factor secretion and assembly occurs at spatially restricted foci in some Gram-positive bacteria. Given the essentiality of the general secretion pathway in bacteria and the contribution of virulence factors to disease progression, the foci that coordinate these processes are attractive antimicrobial targets. In this study, we show in Enterococcus faecalis that SecA and Sortase A, required for the attachment of virulence factors to the cell wall, localize to discrete domains near the septum or nascent septal site as the bacteria proceed through the cell cycle. We also demonstrate that cationic human β-defensins interact with E. faecalis at discrete septal foci, and this exposure disrupts sites of localized secretion and sorting. Modification of anionic lipids by multiple peptide resistance factor, a protein that confers antimicrobial peptide resistance by electrostatic repulsion, renders E. faecalis more resistant to killing by defensins and less susceptible to focal targeting by the cationic antimicrobial peptides. These data suggest a paradigm in which focal targeting by antimicrobial peptides is linked to their killing efficiency and to disruption of virulence factor assembly.focal localization | immunofluorescence | microscopy | MprF

This paper presents a poly(dimethyl siloxane) (PDMS) polymer microfluidic device using alternating current (ac) dielectrophoresis (DEP) for separating live cells from interfering particles of similar sizes by their polarizabilities under continuous flow and for characterizing DEP behaviors of cells in stagnant flow. The ac-DEP force is generated by three-dimensional (3D) conducting PDMS composite electrodes fabricated on a sidewall of the device main channel. Such 3D PDMS composite electrodes are made by dispersing microsized silver (Ag) fillers into PDMS gel. The sidewall AgPDMS electrodes can generate a 3D electric field that uniformly distributes throughout the channel height and varies along the channel lateral direction, thereby producing stronger lateral DEP effects over the entire channel. This allows not only easy observation of cell/particle lateral motion but also using the lateral DEP force for manipulation of cells/particles. The former feature is used to characterize the frequency-dependent DEP behaviors of Saccharomyces cerevisiae (yeast) and Escherichia coli (bacteria). The latter is utilized for continuous separation of live yeast and bacterial cells from similar-size latex particles as well as live yeast cells from dead yeast cells. The separation efficiency of 97% is achieved in all cases. The demonstration of these functions shows promising applications of the microfluidic device.

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