Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA.

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we use a combination of genetic and cell biological approaches to describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. Specifically, we show that V. cholerae cells retract MSHA pili and detach from a surface in a diffusion-limited, enclosed environment. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP to induce MSHA pilus retraction. CdpA contains a putative nitric oxide (NO)-sensing NosP domain, and we demonstrate that NO is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that the endogenous production of NO (or an NO-like molecule) in V. cholerae stimulates the retraction of MSHA pili. These results extend our understanding of how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

Characterization of Extracellular Vesicles by Resistive-Pulse Sensing on In-Plane Multipore Nanofluidic Devices.

Extracellular vesicles (EVs) are cell-derived, naturally produced, membrane-bound nanoscale particles that are linked to cell-cell communication and the propagation of diseases. Here, we report the design and testing of in-plane nanofluidic devices for resistive-pulse measurements of EVs derived from bovine milk and human breast cancer cells. The devices were fabricated in plane with three nanopores in series to determine the particle volume and diameter, two pore-to-pore regions to measure the electrophoretic mobility and zeta potential, and an in-line filter to prevent cellular debris and aggregates from entering the nanopore region. Devices were tested with and without the channels coated with a short-chain PEG silane to minimize electroosmotic flow and permit an accurate measurement of the electrophoretic mobility and zeta potential of the EVs. To enhance throughput of EVs, vacuum was applied to the waste reservoir to increase particle frequencies up to 1000 min-1. The nanopores had cross-sections 200 nm wide and 200 nm deep and easily resolved EV diameters from 60 to 160 nm. EVs from bovine milk and human breast cancer cells had similar particle size distributions, but their zeta potentials differed by 2-fold, -8 ± 1 and -4 ± 1 mV, respectively.

Integrated In-Plane Nanofluidic Devices for Resistive-Pulse Sensing.

Single-particle (or digital) measurements enhance sensitivity (10- to 100-fold improvement) and uncover heterogeneity within a population (one event in 100 to 10,000). Many biological systems are significantly influenced by rare or infrequent events, and determining what species is present, in what quantity, and the role of that species is critically important to unraveling many questions. To develop these measurement systems, resistive-pulse sensing is used as a label-free, single-particle detection technique and can be combined with a range of functional elements, e.g., mixers, reactors, filters, separators, and pores. Virtually, any two-dimensional layout of the micro- and nanofluidic conduits can be envisioned, designed, and fabricated in the plane of the device. Multiple nanopores in series lead to higher-precision measurements of particle size, shape, and charge, and reactions coupled directly with the particle-size measurements improve temporal response. Moreover, other detection techniques, e.g., fluorescence, are highly compatible with the in-plane format. These integrated in-plane nanofluidic devices expand the toolbox of what is possible with single-particle measurements.

The preparation and characterization of nitric oxide releasing silicone rubber materials impregnated with S-nitroso-tert-dodecyl mercaptan.

Recently, considerable research efforts have focused on increasing the biocompatibility a nd bactericidal activity of biomedical polymeric devices (e.g., catheters, etc.) through incorporation of nitric oxide (NO) releasing molecules. NO is an important endogenous molecule that is well known for enhancing blood flow via its vasodilatory activity, but it also exhibits potent antithrombotic and antimicrobial properties. In this work, we demonstrate that silicone rubber tubing can be impregnated with a tertiary S-nitrosothiol (RSNO), S-nitroso-tert-dodecylmercaptan, via a simple solvent swelling method. We further characterize the NO release and RSNO leaching from the tubing over time via use of chemiluminescence and UV/Vis spectroscopy, respectively. The tubing is shown to maintain an NO flux above the physiological levels released by endothelial cells, 0.5-4.0 × 10-10 molcm-2min-1, for more than 3 weeks while stored at 37 °C and exhibit minimal leaching. Finally, the RSNO impregnated tu bing exhibits significant antimicrobial activity over a 21 d period (vs. controls) during incubation in a CDC bioreactor after inoculation of media with S. aureus bacteria. The use of such lipophilic RSNO impregnated silicone rubber tubing could dramatically reduce the risk of catheter-related infections, which are a common problem associated with placement of intravascular or urinary catheters.