Remotely Controllable Engineered Bacteria for Targeted Therapy of Pseudomonas aeruginosa Infection.

Pseudomonas aeruginosa (P. aeruginosa) infection has become an intractable problem worldwide due to the decreasing efficacy of the mainstay therapy, antibiotic treatment. Hence, exploring new drugs and therapies to address this issue is crucial. Here, we construct a chimeric pyocin (ChPy) to specifically kill P. aeruginosa and engineer a near-infrared (NIR) light-responsive strain to produce and deliver this drug. Our engineered bacterial strain can continuously produce ChPy in the absence of light and release it to kill P. aeruginosa via remotely and precisely controlled bacterial lysis induced by NIR light. We demonstrate that our engineered bacterial strain is effective in P. aeruginosa-infected wound therapy in the mouse model, as it eradicated PAO1 in mouse wounds and shortened the wound healing time. Our work presents a potentially spatiotemporal and noninvasively controlled therapeutic strategy of engineered bacteria for the targeted treatment of P. aeruginosa infections.

Engineering Gac/Rsm Signaling Cascade for Optogenetic Induction of the Pathogenicity Switch in Pseudomonas aeruginosa.

Bacterial pathogens operate by tightly controlling the pathogenicity to facilitate invasion and survival in host. While small molecule inducers can be designed to modulate pathogenicity to perform studies of pathogen-host interaction, these approaches, due to the diffusion property of chemicals, may have unintended, or pleiotropic effects that can impose limitations on their use. By contrast, light provides superior spatial and temporal resolution. Here, using optogenetics we reengineered GacS of the opportunistic pathogen Pseudomonas aeruginosa, signal transduction protein of the global regulatory Gac/Rsm cascade which is of central importance for the regulation of infection factors. The resultant protein (termed YGS24) displayed significant light-dependent activity of GacS kinases in Pseudomonas aeruginosa. When introduced in the Caenorhabditis elegans host systems, YGS24 stimulated the pathogenicity of the Pseudomonas aeruginosa strain PAO1 in a brain-heart infusion and of another strain, PA14, in slow killing media progressively upon blue-light exposure. This optogenetic system provides an accessible way to spatiotemporally control bacterial pathogenicity in defined hosts, even specific tissues, to develop new pathogenesis systems, which may in turn expedite development of innovative therapeutics.

Imaging the Separation Distance between the Attached Bacterial Cells and the Surface with a Total Internal Reflection Dark-Field Microscope.

The attachment of bacterial cells to a surface is implicated in the formation of biofilms. Although the surface-related behaviors in this process, such as single cell motility and surface sensing, have been investigated intensively, the precise information of separation distance between the attached cells and the surface has remained unclear. Here, we set a prism-based total internal reflection dark-field microscope (p-TIRDFM) combined with the microfluidic method to image the separation distance of single attached cells. We directly observed that bacterial cells attached to the surface with one nearest touchpoint, and it gradually changed to two touchpoints, respectively, for the two offspring with the cell division. We first monitored the fluctuation of the relative distance on nanometer scale when cells twitch on a surface and further established the relationship between the twitching velocity and the separation distance. The results indicated that the moving cells are a considerable distance apart from the surface and the separation distance fluctuated more widely than immobile cells.

Influence of an Additive-Free Particle Spreading Method on Interactions between Charged Colloidal Particles at an Oil/Water Interface.

The assembly and manipulation of charged colloidal particles at oil/water interfaces represent active areas of fundamental and applied research. Previously, we have shown that colloidal particles can spontaneously generate unstable residual charges at the particle/oil interface when spreading solvent is used to disperse them at an oil/water interface. These residual charges in turn affect the long-ranged electrostatic repulsive forces and packing of particles at the interface. To further uncover the influence arising from the spreading solvents on interfacial particle interactions, in the present study we utilize pure buoyancy to drive the particles onto an oil/water interface and compare the differences between such a spontaneously adsorbed particle monolayer to the spread monolayer based on solvent spreading techniques. Our results show that the solvent-free method could also lead particles to spread well at the interface, but it does not result in violent sliding of particles along the interface. More importantly, this additive-free spreading method can avoid the formation of unstable residual charges at the particle/oil interface. These findings agree well with our previous hypothesis; namely, those unstable residual charges are triboelectric charges that arise from the violently rubbing of particles on oil at the interface. Therefore, if the spreading solvents could be avoided, then we would be able to get rid of the formation of residual charges at interfaces. This finding will provide insight for precisely controlling the interactions among colloidal particles trapped at fluid/fluid interfaces.

Charging and discharging of single colloidal particles at oil/water interfaces.

The physical behavior of solid colloids trapped at a fluid-fluid interface remains in itself an open fundamental issue. Here, we show that the gradients of surface tension can induce particles to jet towards the oil/water interface with velocities as high as ≈ 60 mm/s when particle suspensions come in contact with the interface. We hypothesize that rubbing between the particles and oil lead to the spontaneous accumulation of negative charges on the hemisphere of those interfacial particles that contact the oil phase by means of triboelectrification. The charging process is highly dependent on the sliding distances, and gives rise to long-ranged repulsions that protect interfacial particles from coagulating at the interface by the presence of electrolyte. These triboelectric charges, however, are compensated within several hours, which affect the stability of interfacial particles. Importantly, by charging different kinds of colloidal particles using various spreading solvents and dispersion methods, we have demonstrated that charging and discharging of single colloidal particles at oil/water interfaces impacts a broad range of dynamical behavior.

Stabilization of colloidal suspensions: competing effects of nanoparticle halos and depletion mechanism.

Bimodal colloidal mixtures of nanoparticles and microparticles may show different phase behaviors depending upon the interparticle interaction of both species. In the present work, we examined the stabilization of spherical microparticles using highly charged, spherical nanoparticles. Total internal reflection microscopy (TIRM) was used to measure the interaction forces between a charged microparticle and flat glass substrate in aqueous solutions at varying volume fractions of nanoparticles of the same sign. We found that, in the system containing of highly charged nanoparticles, microparticle, and glass substrate, non-adsorbing charged nanoparticles in solution did not lead to depletion attraction. Instead, the addition of nanoparticles was to consistently create a repulsive force between the microparticle and glass substrate even at a very low nanoparticle volume fraction. This result might attributed to the formation of thin shells (halos) with a high local nanoparticle volume fraction to the region near the glass surface, resulting in electrostatic repulsion between the decorated surfaces. This study demonstrates that nanoparticle halos can also arise in binary systems of mutually but highly repulsive microparticle/nanoparticle dispersions.

Depletion attraction between a polystyrene particle and a hydrophilic surface in a pluronic aqueous solution.

In practice, many colloidal suspensions also contain polymers where their presence has a major effect on the stability of colloidal particles. In this work, we use total internal reflection microscopy to directly measure the interactions between a approximately 6.0 microm polystyrene spherical particle and a hydrophilic flat surface with the presence of an triblock copolymer, poly(ethylene oxide-block-propylene oxide-block-ethylene oxide) (Pluronic PE10500), in an aqueous solution with low ionic strength. A discernible attractive force between the particle and surface is observed with the measurable range of up to approximately 100 nm. Dynamic laser light scattering study reveals that monomers, micelles, and larger nanobubbles (approximately 166 nm) coexist when PE10500 triblock copolymer is spontaneously dissolved in the low ionic strength aqueous solution. We attribute this measured long-range attractive force to the creation of a significant depletion force between the particle and surface as caused by the existence of relatively large nanobubbles free in solution. Replacement of the PE10500 copolymer solution with salt solution removes the nanobubbles, which is reflected in disappearing of the attractive force. Moreover, we find that the adsorbed PE10500 chains at both charged particle and flat surface may cause a redistribution of counterions and colons that make up the electric double layer of the surfaces, thus displacing the repulsive potential between the particle and surface.