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Double-stranded RNA (dsRNA) induces production of pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEK) by specific binding to endosomal Toll-like receptor-3 (TLR3). Recently, it has been shown that hyperactivation of TLR3 in psoriatic keratinocytes by dsRNA can occur in the presence of human antimicrobial peptide (AMP) LL37. Here, we combine synchrotron X-ray scattering, microscopy, computer simulations, and measurements of NHEK cytokine production to elucidate a previously unanticipated form of specific molecular pattern recognition. LL37 and similar α-helical AMPs can form pro-inflammatory nanocrystalline complexes with dsRNA that are recognized by TLR3 differently than dsRNA alone. dsRNA complexes that activate IL-6 production in NHEK and those that do not are both able to enter cells and co-localize with TLR3. However, the crystallinity of these AMP-dsRNA complexes, specifically the geometric spacing between parallel dsRNA and the repeat number of ordered dsRNA, strongly influences the level of TLR3 activation. Crystalline complexes that present dsRNA at a spacing that matches with the steric size of TLR3 can recruit and engage multiple TLR3 receptors, driving receptor clustering and immune amplification, whereas crystalline complexes that exhibit poor steric matching do not. Reverse-transcription quantitative PCR of IL-6 during siRNA knockdown of TLR3 confirms that cytokine production is due to TLR3: High levels of IL-6 transcription are observed for sterically matched complexes without TLR3 knockdown, whereas such activity is abrogated with TLR3 knockdown.

Bacteria exhibit surface motility modes that play pivotal roles in early-stage biofilm community development, such as type IV pili-driven twitching motility and flagellum-driven spinning and swarming motility. Appendage-driven motility is controlled by molecular motors, and analysis of surface motility behavior is complicated by its inherently 3D nature, the speed of which is too fast for confocal microscopy to capture. Here, we combine electromagnetic field computation and statistical image analysis to generate 3D movies close to a surface at 5 ms time resolution using conventional inverted microscopes. We treat each bacterial cell as a spherocylindrical lens and use finite element modeling to solve Maxwells equations and compute the diffracted light intensities associated with different angular orientations of the bacterium relative to the surface. By performing cross-correlation calculations between measured 2D microscopy images and a library of computed light intensities, we demonstrate that near-surface 3D movies of Pseudomonas aeruginosa translational and rotational motion are possible at high temporal resolution. Comparison between computational reconstructions and detailed hydrodynamic calculations reveals that P. aeruginosa act like low Reynolds number spinning tops with unstable orbits, driven by a flagellum motor with a torque output of ∼2 pN μm. Interestingly, our analysis reveals that P. aeruginosa can undergo complex flagellum-driven dynamical behavior, including precession, nutation, and an unexpected taxonomy of surface motility mechanisms, including upright-spinning bacteria that diffuse laterally across the surface, and horizontal bacteria that follow helicoidal trajectories and exhibit superdiffusive movements parallel to the surface.

The electrical conductivity measured in Shewanella and Geobacter spp. is an intriguing physical property that is the fundamental basis for possible extracellular electron transport (EET) pathways. There is considerable debate regarding the origins of the electrical conductivity reported in these microbial cellular structures, which is essential for deciphering the EET mechanism. Here, we report systematic on-chip nanoelectronic investigations of both Shewanella and Geobacter spp. under physiological conditions to elucidate the complex basis of electrical conductivity of both individual microbial cells and biofilms. Concurrent electrical and electrochemical measurements of living Shewanella at both few-cell and the biofilm levels indicate that the apparent electrical conductivity can be traced to electrochemical-based electron transfer at the cell/electrode interface. We further show that similar results and conclusions apply to the Geobacter spp. Taken together, our study offers important insights into previously proposed physical models regarding microbial conductivities as well as EET pathways for Shewanella and Geobacter spp.

Cell size control and homeostasis are fundamental features of bacterial metabolism. Recent work suggests that cells add a constant size between birth and division ("adder" model). However, it is not known how cell size homeostasis is influenced by the existence of heterogeneous microenvironments, such as those during biofilm formation. Shewanella oneidensis MR-1 can use diverse energy sources on a range of surfaces via extracellular electron transport (EET), which can impact growth, metabolism, and size diversity. Here, we track bacterial surface communities at single-cell resolution to show that not only do bacterial motility appendages influence the transition from two- to three-dimensional biofilm growth and control postdivisional cell fates, they strongly impact cell size homeostasis. For every generation, we find that the average growth rate for cells that stay on the surface and continue to divide (nondetaching population) and that for cells that detach before their next division (detaching population) are roughly constant. However, the growth rate distribution is narrow for the nondetaching population, but broad for the detaching population in each generation. Interestingly, the appendage deletion mutants (ΔpilA, ΔmshA-D, Δflg) have significantly broader growth rate distributions than that of the wild type for both detaching and nondetaching populations, which suggests that Shewanella appendages are important for sensing and integrating environmental inputs that contribute to size homeostasis. Moreover, our results suggest multiplexing of appendages for sensing and motility functions contributes to cell size dysregulation. These results can potentially provide a framework for generating metabolic diversity in S. oneidensis populations to optimize EET in heterogeneous environments.

The microbiome presents great opportunities for understanding and improving the world around us and elucidating the interactions that compose it. The microbiome also poses tremendous challenges for mapping and manipulating the entangled networks of interactions among myriad diverse organisms. Here, we describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond. These technical needs will provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering. We anticipate that the new tools and methods developed will also be more broadly useful in environmental monitoring, medicine, forensics, and other areas.