Screening microbiota for effects on host tissues.

The assembly and function of microbial communities depends on many factors including the local environment and the metabolic properties of the colonizing organisms. Chemical communications or other secreted factors also play a role and are used by different microbial strains both cooperatively and competitively. The spectrum of microbial secretions have various effects on the microbe's respective hosts, both positive and negative. Thus, characterizing the roles of microbial community members and their secretions can yield key mechanistic insights into microbiome function and can lead to new intervention strategies. Focusing on the simple, yet important functional impact of toxicity, we quantify supernatant dosage responses with image data and examine the morphological effects of microbial secretions on skin-associated host cells. Since the diversity of microbial communities, coupled with the multiplicity of host tissues requires scalable methods, we develop and demonstrate a microfluidic device that enables high-content screening of microbial secretion effects on adherent cell types.

A Microfluidics and Agent-Based Modeling Framework for Investigating Spatial Organization in Bacterial Colonies: The Case of Pseudomonas Aeruginosa and H1-Type VI Secretion Interactions.

The factors leading to changes in the organization of microbial assemblages at fine spatial scales are not well characterized or understood. However, they are expected to guide the succession of community development and function toward specific outcomes that could impact human health and the environment. In this study, we put forward a combined experimental and agent-based modeling framework and use it to interpret unique spatial organization patterns of H1-Type VI secretion system (T6SS) mutants of P. aeruginosa under spatial confinement. We find that key parameters, such as T6SS-mediated cell contact and lysis, spatial localization, relative species abundance, cell density and local concentrations of growth substrates and metabolites are influenced by spatial confinement. The model, written in the accessible programming language NetLogo, can be adapted to a variety of biological systems of interest and used to simulate experiments across a broad parameter space. It was implemented and run in a high-throughput mode by deploying it across multiple CPUs, with each simulation representing an individual well within a high-throughput microwell array experimental platform. The microfluidics and agent-based modeling framework we present in this paper provides an effective means by which to connect experimental studies in microbiology to model development. The work demonstrates progress in coupling experimental results to simulation while also highlighting potential sources of discrepancies between real-world experiments and idealized models.

Quantitative profiling of innate immune activation by viral infection in single cells.

Cells infected by viruses can exhibit diverse patterns of viral and cellular gene expression. The patterns arise in part from the stochastic or noisy reaction kinetics associated with the small number of genomes, enzymes, and other molecules that typically initiate virus replication and activate cellular anti-viral defenses. It is not known what features, if any, of the early viral or cellular gene expression correlate with later processes of viral replication or cell survival. Here we used two fluorescent reporters to visualize innate immune activation of human prostate cancer (PC3) cells against infection by vesicular stomatitis virus. The cells were engineered to express green-fluorescent protein under control of the promoter for IFIT2, an interferon-sensitive component of the anti-viral response, while red-fluorescent protein was expressed as a byproduct of virus infection. To isolate and quantitatively analyze single-cells, we used a unique microwell array device and open-source image processing software. Kinetic analysis of viral and cellular reporter profiles from hundreds of cells revealed novel relationships between gene expression and the outcome of infection. Specifically, the relative timing rather than the magnitude of the viral gene expression and innate immune activation correlated with the infection outcome. Earlier viral or anti-viral gene expression favored or hindered virus growth, respectively. Further, analysis of kinetic parameters estimated from these data suggests a trade-off between robust antiviral signaling and cell death, as indicated by a higher rate of detectable cell lysis in infected cells with a detectable immune response. In short, cells that activate an immune response lyse at a higher rate. More broadly, we demonstrate how the intrinsic heterogeneity of individual cell behaviors can be exploited to discover features of viral and host gene expression that correlate with single-cell outcomes, which will ultimately impact whether or not infections spread.

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

Correction: Stochastic Assembly of Bacteria in Microwell Arrays Reveals the Importance of Confinement in Community Development.

[This corrects the article DOI: 10.1371/journal.pone.0155080.].

Stochastic Assembly of Bacteria in Microwell Arrays Reveals the Importance of Confinement in Community Development.

The structure and function of microbial communities is deeply influenced by the physical and chemical architecture of the local microenvironment and the abundance of its community members. The complexity of this natural parameter space has made characterization of the key drivers of community development difficult. In order to facilitate these characterizations, we have developed a microwell platform designed to screen microbial growth and interactions across a wide variety of physical and initial conditions. Assembly of microbial communities into microwells was achieved using a novel biofabrication method that exploits well feature sizes for control of innoculum levels. Wells with incrementally smaller size features created populations with increasingly larger variations in inoculum levels. This allowed for reproducible growth measurement in large (20 µm diameter) wells, and screening for favorable growth conditions in small (5, 10 µm diameter) wells. We demonstrate the utility of this approach for screening and discovery using 5 µm wells to assemble P. aeruginosa colonies across a broad distribution of innoculum levels, and identify those conditions that promote the highest probability of survivial and growth under spatial confinement. Multi-member community assembly was also characterized to demonstrate the broad potential of this platform for studying the role of member abundance on microbial competition, mutualism and community succession.

Tools for Single-Cell Kinetic Analysis of Virus-Host Interactions.

Measures of cellular gene expression or behavior, when performed on individual cells, inevitably reveal a diversity of behaviors and outcomes that can correlate with normal or diseased states. For virus infections, the potential diversity of outcomes are pushed to an extreme, where measures of infection reflect features of the specific infecting virus particle, the individual host cell, as well as interactions between viral and cellular components. Single-cell measures, while revealing, still often rely on specialized fluid handling capabilities, employ end-point measures, and remain labor-intensive to perform. To address these limitations, we consider a new microwell-based device that uses simple pipette-based fluid handling to isolate individual cells. Our design allows different experimental conditions to be implemented in a single device, permitting easier and more standardized protocols. Further, we utilize a recently reported dual-color fluorescent reporter system that provides dynamic readouts of viral and cellular gene expression during single-cell infections by vesicular stomatitis virus. In addition, we develop and show how free, open-source software can enable streamlined data management and batch image analysis. Here we validate the integration of the device and software using the reporter system to demonstrate unique single-cell dynamic measures of cellular responses to viral infection.

High-Throughput Single-Cell Kinetics of Virus Infections in the Presence of Defective Interfering Particles.

UNLABELLED: Defective interfering particles (DIPs) are virus mutants that lack essential genes for growth. In coinfections with helper virus, the diversion of viral proteins to the replication and packaging of DIP genomes can interfere with virus production. Mounting cases of DIPs and DIP-like genomes in clinical and natural isolates, as well as growing interest in DIP-based therapies, underscore a need to better elucidate how DIPs work. DIP activity is primarily measured by its inhibition of virus infection yield, an endpoint that masks the dynamic and potentially diverse individual cell behaviors. Using vesicular stomatitis virus (VSV) as a model, we coinfected BHK cells with VSV DIPs and recombinant helper virus carrying a gene encoding a red fluorescent protein (RFP) whose expression correlates with the timing and level of virus release. For single cells within a monolayer, 10 DIPs per cell suppressed the reporter expression in only 1.2% of the cells. In most cells, it slowed and reduced viral gene expression, manifested as a shift in mean latent time from 4 to 6 h and reduced virus yields by 10-fold. For single cells isolated in microwells, DIP effects were more pronounced, reducing virus yields by 100-fold and extending latent times to 12 h, including individual instances above 20 h. Together, these results suggest that direct or indirect cell-cell interactions prevent most coinfected cells from being completely suppressed by DIPs. Finally, a gamma distribution model captures well how the infection kinetics quantitatively depends on the DIP dose. Such models will be useful for advancing a predictive biology of DIP-associated virus growth and infection spread. IMPORTANCE: During the last century, basic studies in virology have focused on developing a molecular mechanistic understanding of how infectious viruses reproduce in their living host cells. However, over the last 10 years, the advent of deep sequencing and other powerful technologies has revealed in natural and patient infections that viruses do not act alone. Instead, viruses are often accompanied by defective virus-like particles that carry large deletions in their genomes and fail to replicate on their own. Coinfections of viable and defective viruses behave in unpredictable ways, but they often interfere with normal virus growth, potentially enabling infections to evade host immune surveillance. In the current study, controlled levels of defective viruses are coinfected with viable viruses that have been engineered to express a fluorescent reporter protein during infection. Unique profiles of reporter expression acquired from thousands of coinfected cells reveal how interference acts at multiple stages of infection.

Toward Microfluidic Reactors for Cell-Free Protein Synthesis at the Point-of-Care.

Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro and nanofluidic architectures, CFPS can be optimized for point-of-care use. Here, the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care, is described. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel "reactor" and "feeder" channels. This engineered membrane facilitates the exchange of metabolites, energy, and inhibitory species, and can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. It has been shown that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.

Fabrication of nanoporous membranes for tuning microbial interactions and biochemical reactions.

New strategies for combining conventional photo- and soft-lithographic techniques with high-resolution patterning and etching strategies are needed in order to produce multiscale fluidic platforms that address the full range of functional scales seen in complex biological and chemical systems. The smallest resolution required for an application often dictates the fabrication method used. Micromachining and micropowder blasting yield higher throughput, but lack the resolution needed to fully address biological and chemical systems at the cellular and molecular scales. In contrast, techniques such as electron beam lithography or nanoimprinting allow nanoscale resolution, but are traditionally considered costly and slow. Other techniques such as photolithography or soft lithography have characteristics between these extremes. Combining these techniques to fabricate multiscale or hybrid fluidics allows fundamental biological and chemical questions to be answered. In this study, a combination of photolithography and electron beam lithography are used to produce two multiscale fluidic devices that incorporate porous membranes into complex fluidic networks in order to control the flow of energy, information, and materials in chemical form. In the first device, materials and energy were used to support chemical reactions. A nanoporous membrane fabricated with e-beam lithography separates two parallel, serpentine channels. Photolithography was used to pattern microfluidic channels around the membrane. The pores were written at 150 nm and reduced in size with silicon dioxide deposition from plasma enhanced chemical vapor deposition and atomic layer deposition. Using this method, the molecular weight cutoff of the membrane can be adapted to the system of interest. In the second approach, photolithography was used to fabricate 200 nm thin pores. The pores confined microbes and allowed energy replenishment from a media perfusion channel. The same device can be used for study of intercellular communication via the secretion and uptake of signal molecules. Pore size was tested with 750 nm fluorescent polystyrene beads and fluorescein dye. The 200 nm polydimethylsiloxane pores were shown to be robust enough to hold 750 nm beads while under pressure, but allow fluorescein to diffuse across the barrier. Further testing showed that extended culture of bacteria within the chambers was possible. These two examples show how lithographically defined porous membranes can be adapted to two unique situations and used to tune the flow of chemical energy, materials, and information within a microfluidic network.

Kinetics of virus production from single cells.

The production of virus by infected cells is an essential process for the spread and persistence of viral diseases, the effectiveness of live-viral vaccines, and the manufacture of viruses for diverse applications. Yet despite its importance, methods to precisely measure virus production from cells are lacking. Most methods test infected-cell populations, masking how individual cells behave. Here we measured the kinetics of virus production from single cells. We combined simple steps of liquid-phase infection, serial dilution, centrifugation, and harvesting, without specialized equipment, to track the production of virus particles from BHK cells infected with vesicular stomatitis virus. Remarkably, cell-to-cell differences in latent times to virus release were within a factor of two, while production rates and virus yields spanned over 300-fold, highlighting an extreme diversity in virus production for cells from the same population. These findings have fundamental and technological implications for health and disease.

A quantitative comet infection assay for influenza virus.

The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2-6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells.