Figure Legends
Figure 1. A microfluidic device for bacterial nanoliter cultureA) Schematic shows dimensions of microchamber. Each egg-shaped device consists of a central 200 µm diameter, 50 µm high cylindrical microchamber connected to the outer chamber by a 125 µm long, 10 µm wide, 10 µm high connecting channel. B) Depending on how the device is loaded, it can be configured for mono-culture, co-culture, or for studying innate immune responses.
Figure 2. Species-specific formation of “exclusion zones” in co-cultures of Actinomyces with Streptococcal species. A) Color map shows the results of co-culture experiments with combinations of five strains of actinomyces and three strains of schaalia with seven strains of streptococcus. B) Representative image at 8 hours showing co-culture of A. naeslundii FCC36 (full yellow arrowheads) andS. salivarius A64PA33 showing no exclusion zone formation. C) Representative image at 8 hours showing co-culture of A. graevenitzii FO582 (Empty yellow arrowheads) and S. salivarius A64PA33 showing formation or large exclusion zones around the A. graevenitzii colonies (red dashed circles).
Figure 3. Exclusion zones are formed in co-cultures of A. graevenitzii and S. aureus. A) Time-lapse images show the growth of GFP-expressing S. aureus in the presence of A. graevenitzii FO582 microcolonies. Over 8 hours, S. aureusproliferate to fill the chamber (bright green fluorescence), except for the regions bordering A. graevenitzii microcolonies (GFP-negative regions). B) Magnified view of co-culture chamber (left) showing growth of exclusion of S. aureus from a region containing a cluster ofA. graevenitzii microcolonies. The cartoon on the right depicts the area of GFP fluorescence measured as a percentage of the chamber. C) Average coverage of microchamber area over time by S. aureusgrowth in presence of different species of Actinomyces.Suppression of S. aureus growth, corresponding to formation of exclusion zones, was only observed in co-culture with A. graevenitzii . Error bars: mean ± SEM. N ≥ 5 chambers measured per condition. D) Graph shows the negative correlation between S. aureus growth and the number of A. graevenitzii microcolonies in each co-culture. Linear regression. Dashed lines are 95% confidence intervals. N = 152 chambers scored. E) Graph shows no significant correlation between S. aureus coverage and the initial ratio of S. aureus: A. graevenitzii loaded into each microchamber. N = 178 chambers scored.
Figure 4. Exclusion zones form around stressed A. graevenitzii colonies . A) Magnified micrograph showing details ofA. graevenitzii FO582 microcolony structure and exclusion zone formation. B) Cartoon depicting colony structure. C) Graph showing the positive correlation between the size of the A. graevenitziimicrocolony core and the size of the exclusion zone. Linear regression, dashed lines show 95% confidence intervals. N = 75 chambers scored. D) Graph shows negative correlation between the length of A. graevenitzii radial filaments and exclusion zone size. Linear regression, dashed lines show 95% confidence intervals. N = 70 chambers scored.
Figure 5. Clustering of A. graevenitzii microcolonies does not increase the exclusion zone in co-culture with S. aureus. A) Graph shows increase in number of clustered A. graevenitzii FO582 microcolonies compared to monocultures or co-culture with S. cristatus . Mean ± SEM. data pooled from at least 2 experiments. B) Cartoon depicts individual versus clusteredA. graevenitzii microcolonies in co-culture with S. aureusmeasured in (C). Exclusion edge radius measurement is shown in red. C) Scatterplot shows no clear increase in exclusion edge radius, even bordering very large A. graevenitzii clusters. N = 161 individual colonies scored. N = 89 clustered colonies scored.
Figure 6. Modulation of human neutrophil responses to co-cultures of A. graevenitzii and S. aureus. A) Representative micrograph showing experimental setup for testing neutrophil recruitment to co-cultures of A. graevenitzii FO582and S. aureus . B) Bubble plots showing human neutrophil recruitment towards S. aureus and A. graevenitzii , alone and in co-culture for different loading ratios. The diameter of each bubble represents the number of neutrophils inside each microchamber at the end of experiment. N = 18 chambers scored per condition from 2 independent experiments.