Microfluidic-based transcriptomics reveal force-independent bacterial rheosensing

Abstract

Multiple cell types sense fluid flow as an environmental cue. Flow can exert shear force (or stress) on cells, and the prevailing model is that biological flow sensing involves the measurement of shear force^1,2. Here, we provide evidence for force-independent flow sensing in the bacterium Pseudomonas aeruginosa. A microfluidic-based transcriptomic approach enabled us to discover an operon of P. aeruginosa that is rapidly and robustly upregulated in response to flow. Using a single-cell reporter of this operon, which we name the flow-regulated operon (fro), we establish that P. aeruginosa dynamically tunes gene expression to flow intensity through a process we call rheosensing (as rheo- is Greek for flow). We further show that rheosensing occurs in multicellular biofilms, involves signalling through the alternative sigma factor FroR, and does not require known surface sensors. To directly test whether rheosensing measures force, we independently altered the two parameters that contribute to shear stress: shear rate and solution viscosity. Surprisingly, we discovered that rheosensing is sensitive to shear rate but not viscosity, indicating that rheosensing is a kinematic (force-independent) form of mechanosensing. Thus, our findings challenge the dominant belief that biological mechanosensing requires the measurement of forces.

Fig. 1:. Flow triggers induction of gene expression in Pseudomonas aeruginosa.

(A) Schematic of microfluidic devices used throughout this study. Channels are custom-fabricated of polydimethylsiloxane (PDMS) and glass. (B) The fold-change in transcript abundance of P. aeruginosa cells subjected to flow for 20 minutes relative to flow-naïve cells. Line length linearly corresponds to fold-change and is plotted as a function of genomic location on the P. aeruginosa chromosome. Only genes induced at least 3-fold are represented, and the raw data used to generate this graph is presented in Supplementary Table 2. The red line corresponds to the fro operon. (D) Schematic depicting the view from above the microchannel used in E. These channels are 50 μm tall by 500 μm wide. (E) Fluorescence and phase images of fro reporter strain in straight microfluidic channels before and after 4 hours of 10 μl/min flow. Images are representative of three independent experiments. Scale bars indicate 5 μm. (F) Schematic of the microchannel used in G. These channels are 90 μm tall by 100 μm wide. (G) A merged image of Phase, YFP, and mCherry from a single optical plane of a representative streamer biofilm projecting off the wall of a microchannel. Images are representative of three independent experiments. Streamers were cultured in 2 μl/min flow for 20 hours. Lower picture is zoomed in on cells that are not directly in contact with the channel surface. Scale bar of top image indicates 50 μm and scale bar of bottom image indicates 20 μm.

Fig. 2:. Shear rate rapidly and dynamically tunes rheosensing.

(A) Images of cells exposed to flow at a wall shear rate of 800 sec⁻¹ over 120 min. Top images show the fro reporter (YFP) channel, middle images show the mCherry normalization control channel, and bottom images show the phase contrast channel. Images are representative of three independent experiments. Scale bar indicates 5 μm. (B) fro expression over 2 hours of time in the presence of 8 (gray line), 80 (yellow line), and 800 sec⁻¹ (green line) shear rates. At 2 hours, 8/sec and 80/sec samples are statistically different from each other with P=0.03, calculated by a 2-sided T-test. At 2 hours, 80/sec and 800/sec samples are statistically different from each other with P=0.008, calculated by a 2-sided T-test. (C) fro expression over 4 hours of time in the presence of 80 sec⁻¹ (yellow line), 800 sec⁻¹ (green line), or an upshift from 80 sec⁻¹ to 800 sec⁻¹ (yellow-green line). The black arrow depicts the 2-hour time point where shear rate was increased from 80 sec⁻¹ to 800 sec⁻¹ for the upshifted sample. At 4 hours, the upshifted sample results in fro expression that is statistically different from the 80 sec⁻¹ sample with P=0.03, calculated by a 2-sided T-test. Error bars show SEM of three independent replicates. Each replicate represents quantification from 50 cells, fro expression at time 0 set is set to 1. Channels used for these experiments were 50 μm tall by 500 μm wide.

Fig. 3:. fro induction requires the sigma factor FroR and anti-sigma factor FroI but not known surface sensors.

(A) fro expression levels in wild-type cells, ΔfroI mutant cells, ΔfroR mutant cells, froI++ overexpressing cells, and froR++ overexpressing cells either subjected to no flow (gray bars) or 2 hours of flow at a shear rate of 800 sec⁻¹ (green bars). In no flow conditions, wild-type expression is significantly different from ΔfroI (P=0.04), ΔfroR (P=0.008) and froR++ expression (P=0.02), but statistically indistinguishable from froI++ expression (P=0.18), as calculated by a 2-sided T-test. In flow conditions, wild-type expression is significantly different from ΔfroR (P=0.002), froI++ expression (P=0.0005) and froR++ expression (P=0.01), but statistically indistinguishable from ΔfroI (P=0.14), as calculated by a 2-sided T-test. (B) fro expression levels in wild-type cells, ΔpilA mutant cells, ΔpilB mutant cells, ΔpilTU mutant cells, and ΔpilY1 mutant cells either subjected to no flow (gray bars) or 2 hours of flow at a shear rate of 800 sec⁻¹ (green bars). Error bars show SD of three independent replicates and points indicate values for each replicate. In flow conditions, wild-type expression is statistically indistinguishable from ΔpilA (P=0.88), ΔpilB (P=0.28), ΔpilTU (P=0.76), and ΔpilY1 (P=0.95), as calculated by a 2-sided T-test. Values normalized to WT in no flow, which is set to 1 for each replicate. Channels used for these experiments were 50 μm tall by 500 μm wide.

Fig. 4:. Rheosensing is a force-independent sensory modality.

(A) Schematic of the microchannel used in B. (B) Image of fro reporter cells in flow-exposed and flow-shielded regions of the channel after treatment with a shear rate of 800 sec⁻¹ for 4 hours. Phase image is on the bottom and merged YFP/mCherry images are on the top. Smaller square boxes are zoomed in on images depicted by red squares. The scale bar indicates 50 μm. The image is representative of two independent replicates. (C) Schematic showing how the flow profile corresponds to shear rate in a microfluidic device. Equations showing that shear stress is the product of shear rate and fluid viscosity and shear force is the product of shear stress and surface area. (D) Microscopic viscosity of Ficoll solutions as measured with micron-scale beads and optical tweezers. Error bars show SD of three independent replicates. (E, F) Expression of the fro reporter in response to two hours of flow at the defined shear rates and shear forces. Shear forces were calculated by multiplying shear stress by cell surface area, which was estimated at 2.5 μm². Error bars show SD of four independent replicates and points indicate values for each replicate. fro expression at 80 sec⁻¹ is significantly different from fro expression at 800 sec⁻¹ with treatments of 0% Ficoll (P=0.003), 5% Ficoll (P=0.002), 10% Ficoll (P=0.002), and 15% Ficoll (P=0.006), calculated by a 2-sided T-test. fro expression at 80 sec⁻¹ with no Ficoll is significantly indistinguishable from fro expression at 80 sec⁻¹ with 5% Ficoll (P=0.79), 10% Ficoll (P=0.67), and 15% Ficoll (P=0.37), calculated by a 2-sided T-test. fro expression at 800 sec⁻¹ with no Ficoll is significantly indistinguishable from fro expression at 800 sec⁻¹ with 5% Ficoll (P=0.33), 10% Ficoll (P=0.66), and 15% Ficoll (P=0.10), calculated by a 2-sided T-test. fro expression of cells before flow treatment is set to 1. Channels used for these experiments were 50 μm tall and 500 μm wide.