This is a paper about quorum sensing. As such, it requires an introduction by Bonnie Bassler. She is not an author of this paper, but she is well known in this field and even did a TED Talk on it.
A biofilm is a community of bacteria that talk to each other in order to aggregate into a clump of mucus-protected film. Bacteria use chemicals to communicate. We call their communication quorum sensing (QS), and their chemical language is composed of QS signals. When they talk, they can build thick biofilms impenetrable to antibiotics, produce virulence factors, and engage in other group behaviors.
It turns out that there are many strategies for battling biofilms, and interfering with the communication system is one. One of the ways you can interfere is by overwhelming the signaling system with QS compounds of your own. Not only does this mess with bacterial messages, it does not (necessarily) affect cell growth or survival (Hennig, Rödel, & Ostermann, 2015).
The implication? There is little to no selection pressure. If you’ve heard of superbugs, you know that antibiotics set up a selection pressure for the bacteria to evolve resistance. But if these QS interferents work this way, they can be used to stop virulence without a strong selection for resistance.
That has huge implications for human medicine, and biofilm research is a current hot topic.
So, in studying a metabolite with QS signaling (QSS) properties, produced by a plant as a defense mechanism against infection, the authors are advancing a field that has potential to advance both agricultural and human health.
This work also relies on the fact that bacteria and plants communicate with each other, using QSS compounds to talk, eavesdrop, and compete with each other. A prokaryote and multicellular eukaryote produce chemicals that can be received and interpreted by each other – and that’s just plain cool.
The Logic.
Where did they begin tackling the problem?
This diagram depicts signaling systems in the context of quorum sensing, but generally, a signaling systems acts this way as well. A chemical in the environment binds to a protein. That protein is a regulator that, when it binds, activates transcription. The transcription is part of genes turning on that result in changes in behavior; it’s the cell’s response to the chemical signals in its environment. Here, those changes result in biofilm formation and virulence.
One of the most common QS signals is homoserine lactone (HSL). The plant (and human) pathogen Pseudomonas aeruginosa participates in group behaviors resulting in biofilm production and virulence (the virulent factors produced by P. aeruginosa include pyocyanin and elastase). This species produces its own QS signals, but in this study, the authors studied another compound, rosmarinic acid, produced only by plants, that acts as a QSS. How did they do this?
The paper builds off of prior studies showing that there are several plant-produced compounds (rosmarinic acid, chlorogenic acid, naringin, and others) that are predicted to interacted with QS regulators. So they chose two of these compounds to test on P. aeruginosa.
In Fig. 2, we can see some of these players. RhlR and LasR are the regulating proteins that activate transcription when they bind to certain molecules. These are bacterial regulators, of course, and part of P. aeruginosa’s QS system. The bacteria produce HSL signals: C4-HSL is a signal that binds to/activates RhlR, and 3-Oxo-C12-HSL is a signal that binds to/activates LasR. The two plant-based chemicals, which were predicted to bind to bacterial QSR’s, are also shown – rosmarinic acid and chlorogenic acid. You can see that the functional groups on the plant-based molecules (amino acids, alcohols, carboxylic acids) don’t really match the groups on the bacteria-based molecules (oxalanes, amino groups) – in other words, they’re totally different molecules. And yet, the authors are hypothesizing that they interact with the QS system.
To start with this, the authors did binding studies to see if chlorogenic acid and rosmarinic acid could actually interact with the RhlR and LasR regulatory proteins. Every chemical reaction carries entropy and enthalpy changes, and those changes can be detected via a lab technique called isothermal titration calorimetry.
The authors found that rosmarinic acid (RA) binds to RhlR, even better than the bacterial signal C4-HSL does! The rest of the experiment confirms that RA works at different levels: first as a transcription regulator in vivo, then in vitro, and then as an effect on group behaviors observable in cultures of P. aeruginosa. They were able to confirm that RA, a plant-produced molecule, simulates premature biofilm formation in a pathogenic bacteria. This suggests plants use RA as a defense mechanism, one that we can study to improve our understanding of biofilm treatment in human medicine.
In the next installment on this paper, we’ll go over the methods they used as they traced the logic from potential to actual plant-produced QS interferents.
References
Corral-Lugo, A., Daddaoua, A., Ortega, A., Espinosa-Urgel, M., & Krell, T. (2016). Rosmarinic acid is a homoserine lactone mimic produced by plants that activates a bacterial quorum-sensing regulator. Science Signaling, 9(409), 1–11. https://doi.org/10.1126/scisignal.aaa8271
Hennig, S., Rödel, G., & Ostermann, K. (2015). Artificial cell-cell communication as an emerging tool in synthetic biology applications. Journal of Biological Engineering, 9(1), 1–12. https://doi.org/10.1186/s13036-015-0011-2
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