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High throughput 3D tracking of bacterial chemotaxis in complex environments
High throughput 3D tracking of bacterial chemotaxis in complex environments
Some bacteria are able to swim by rotating one or several flagella, propulsing them at speed up to the range of 100 micrometer per second (this is in the order of one hundred time their size! Imagine yourself running at 70 km/h or 45 mph.). Most of them are also able to bias this swimming in order to climb a chemical gradient: this mechanism known as chemotaxis is crucial for spreading, survival, and in certain cases for pathogenicity.
Chemotaxis has been studied extensively in the peritrichously flagellated model organism E. coli in liquid media, but much less attention has been devoted to the diversity of motility patterns displayed by other organisms with different flagellar architectures or to motility in viscous or porous environments that more closely mimic the complexity of natural habitats.
Figure above: A few exemples of flagellar architectures out of the hundreds in the Atlas of Bacterial Flagellation by Leifson (1960). We know they are here since the 60's, yet we have no idea how these flagellar architectures enable bacteria to swim, let alone chemotact!
We strive to understand how flagellar architecture and environmental properties relate to the resulting bacterial motility pattern, how bacteria achieve chemotaxis with these motility patterns, and how motility patterns might be adapted to specific environments.
My main work since November 2016 has been to develop a new chemotaxis assay to be able to tackle such questions. Existing population-level chemotaxis experiments such as capillary assays enable robust quantification [Adler, J. Gen. Microbiol. 1973], but have limited power for achieving a mechanistic understanding as they do not resolve the underlying individual behavior. On the other hand, 3D bacterial tracking in chemoattractant gradients resolves behavioral details [Berg & Brown, Nature 1972], but low throughput has constrained the statistical power which limits a full mechanistic understanding in the face of large variability between individuals.
I resolved this trade-off by combining a recently developed high-throughput 3D tracking method [Taute et al., Nat. Comm. 2015 or The Taute Lab Website] with microfluidically created chemical gradients to implement a simple yet powerful chemotaxis assay that enables access to the full 3D motility behavior of large numbers of bacteria within controlled chemoattractant gradients. Bacteria can be tracked in buffer, but also in soft agar or viscous polymer solutions.
We can directly determine chemotactic drift velocities in different types of environments while simultaneously resolving motility patterns and their modification in response to the gradient, thereby enabling unprecedented access to a mechanistic understanding of chemotactic mechanisms. You can find more information about the method in our (open access) article. The details on use of this assay with soft agar and viscous media can be found in this second article.
Figure above. Schematic of the Multiscale Assay (from Grognot et al., Communications Microbiology, 2022). In a microfluidic setup (top), a linear chemical gradient is established between two chambers where the chemical of interest is injected at different concentrations, along with bacteria. In this central channel, the 3D tracking method allow us to follow the bacteria navigating this gradient at a throughput of around 5,000 individual trajectories in less than 10 minutes (middle). All individual trajectories are accessible for mechanistics analysis (E. coli trajectories exemple at the bottom).
Application of our assay
Application of our assay
The first question I have tackled with this assay is the role of lateral flagella. Known for their role in attachment and swarming motility on surfaces, we also know they are expressed in certain viscous environments and upon contact with surfaces. In order to investigate their impact on swimming in liquid or complex environments (viscous, porous), I'm working with several flagellar mutants of the opportunistic pathogen Vibrio alginolyticus. Our work in extending their significance to complex environment is now available in PNAS.
An other demonstration made possible by our assay is that, as opposed to previous works conclusions, the freshwater bacterium C. crescentus (and likely many other singly-flagellated bacteria) doesn't follow the same chemotactic mechanism than the model organism E. coli. You can find more details here.
In a recent large-scale collaboration, we made use of our assay to demonstrate that Vibrio campbelli follows a run-reverse-flick motility and chemotacts towards the hormone epinephrine (adrenaline) in buffer. You can find more details here.
I am more and more interested by the navigation strategies of pathogens is complex environments relevant to the human host.
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