Bistable expression of virulence genes in salmonella leads to the formation of an antibiotic-tolerant subpopulation.

Phenotypic heterogeneity can confer clonal groups of organisms with new functionality. A paradigmatic example is the bistable expression of virulence genes in Salmonella typhimurium, which leads to phenotypically virulent and phenotypically avirulent subpopulations. The two subpopulations have been shown to divide labor during S. typhimurium infections. Here, we show that heterogeneous virulence gene expression in this organism also promotes survival against exposure to antibiotics through a bet-hedging mechanism. Using microfluidic devices in combination with fluorescence time-lapse microscopy and quantitative image analysis, we analyzed the expression of virulence genes at the single cell level and related it to survival when exposed to antibiotics. We found that, across different types of antibiotics and under concentrations that are clinically relevant, the subpopulation of bacterial cells that express virulence genes shows increased survival after exposure to antibiotics. Intriguingly, there is an interplay between the two consequences of phenotypic heterogeneity. The bet-hedging effect that arises through heterogeneity in virulence gene expression can protect clonal populations against avirulent mutants that exploit and subvert the division of labor within these populations. We conclude that bet-hedging and the division of labor can arise through variation in a single trait and interact with each other. This reveals a new degree of functional complexity of phenotypic heterogeneity. In addition, our results suggest a general principle of how pathogens can evade antibiotics: Expression of virulence factors often entails metabolic costs and the resulting growth retardation could generally increase tolerance against antibiotics and thus compromise treatment.

Robotically controlled microprey to resolve initial attack modes preceding phagocytosis.

Phagocytes, predatory cells of the immune system, continuously probe their cellular microenvironment on the hunt for invaders. This requires prey recognition followed by the formation of physical contacts sufficiently stable for pickup. Although immune cells must apply physical forces to pick up their microbial prey, little is known about their hunting behavior preceding phagocytosis because of a lack of appropriate technologies. To study phagocyte hunting behavior in which the adhesive bonds by which the prey holds on to surfaces must be broken, we exploited the use of microrobotic probes to mimic bacteria. We simulate different hunting scenarios by confronting single macrophages with prey-mimicking micromagnets using a 5-degree of freedom magnetic tweezers system (5D-MTS). The energy landscape that guided the translational and rotational movement of these microparticles was dynamically adjusted to explore how translational and rotational resistive forces regulate the modes of macrophage attacks. For translational resistive prey, distinct push-pull attacks were observed. For rod-shaped, nonresistive prey, which mimic free-floating pathogens, cells co-aligned their prey with their long axis to facilitate pickup. Increasing the rotational trap stiffness to mimic resistive or surface-bound prey disrupts this realignment process. At stiffness levels on the order of 105 piconewton nanometer radian-1, macrophages failed to realign their prey, inhibiting uptake. Our 5D-MTS was used as a proof-of-concept study to probe the translational and rotational attack modes of phagocytes with high spatial and temporal resolution, although the system can also be used for a variety of other mechanobiology studies at length scales ranging from single cells to organ-on-a-chip devices.

Construction and use of a microfluidic dissection platform for long-term imaging of cellular processes in budding yeast.

This protocol describes the production and operation of a microfluidic dissection platform for long-term, high-resolution imaging of budding yeast cells. At the core of this platform is an array of micropads that trap yeast cells in a single focal plane. Newly formed daughter cells are subsequently washed away by a continuous flow of fresh culture medium. In a typical experiment, 50-100 cells can be tracked during their entire replicative lifespan. Apart from aging-related research, the microfluidic platform can also be a valuable tool for other studies requiring the monitoring of single cells over time. Here we provide step-by-step instructions on how to fabricate the silicon wafer mold, how to produce and operate the microfluidic device and how to analyze the obtained data. Production of the microfluidic dissection platform and setting up an aging experiment takes ~7 h.

Continuous high-resolution microscopic observation of replicative aging in budding yeast.

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 µm). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.