Working through this paper (Part 1 and Part 2) has introduced us to the painstaking process a group of scientists took to prove plants are no wimps when it comes to chemical warfare. Pathogens like P. aeruginosa produce chemicals to talk to each other (technical term: quorum sensing, or QS) and conspire when they should start engaging in pathogenic behaviors (defensive biofilm structures, offensive virulence factors like pyocyanin and elastase). But many plants fight back by producing a chemical called rosmarinic acid (RA), which apparently interferes with bacteria’s QS system by overwhelming the signals.
How did they test their hypothesis?
We have reached the final test: biofilm formation and virulence factor production. We know that RA:
- Binds to RhlR.
- Stimulates transcription (in multiple genes controlled by RhlR).
- Stimulates transcription in P. aeruginosa better than and earlier than the native RhlR activating substrate (that is, C4-HSL).
- Does not harm cell growth rates at concentrations necessary to have a transcriptional affect.
So – does all this increased transcription actually increase biofilm formation? Does it result in pyocyanin and elastase production?
To measure biofilm formation, pyocyanin production, and elastase production, various dyes could be added and then OD measured. You can visually see the affects of RA on biofilm production in Fig. 7. Fig. 7B contains a photograph of three cell cultures: a control, a culture exposed to RA, and a culture exposed to chlorogenic acid. There is some biofilm formation dyed purple in the control, but more in the RA-exposed culture, and less in the chlorogenic acid-exposed culture. This was quantified by OD measurements in Fig. 7B’s graph, where we see no statistically significant difference between control and CA-exposed cultures, but RA stimulates biofilm formation. Of course, at 24 hours, all cultures have the same amount of biofilms; RA stands out as producing more biofilms earlier in the culture. It’s a strong, fast-acting biofilm stimulator.
Fig. 7A shows a detail of RA exposure, this time on pyocyanin production (which is green). The photograph shows the increase in cell greenness as RA is increased from 0 to 2mM. OD measurements quantify the effects of RA on the increase in pyocyanin, with CA exposure at various concentrations again showing little to no affect.
Replicating the literature was again a concern, however; other studies had shown that RA was a biofilm-inhibitor. So They again amped up the concentration of RA exposure. The photograph under Fig. 7B shows an even more fine-tuned gradient with a larger range of RA exposure in cell cultures, from 0 to 15mM. We can see the biofilm inhibition start at 5mM. A natural question to ask is: at what level are plants releasing this chemical into their environment? Are they producing mM or μM amounts? Either way, stimulation or inhibition counts as interfering with P. aeruginosa’s QSS system.
Finally, in Fig. 7C, we see an increase in elastase when cells are exposed to RA vs. the control. We could have guessed RA did that much, since we already saw that RA increased transcription of the lasB promoter; lasB codes for elastase.
Rosmarinic acid is a plant-based chemical that can manipulate bacterial signaling, transcription, and group behaviors by interfering with its quorum sensing signaling system. While not the only compound known to do this, RA may possess other health benefits that make it an attractive chemical to use in human medicine. While more research would be needed to test its effectiveness for human medical applications, RA is a promising candidate.
How exactly, one might ask, does a chemical that stimulates biofilm production – better and faster and stronger than native QS signals – count as a defense mechanism? If biofilms and virulent factors are induced before the bacteria are at the population level that naturally activates these group behaviors, then they are expressing pathogenicity before they have reached the number to be a more dangerous threat (de Kievit & Iglewski, 2000). Premature biofilm formation, then, signals to the plants that the enemy is coming, and exposes the enemy before they’ve reached a size that makes them a more formidable threat.
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
de Kievit, T. R., & Iglewski, B. H. (2000). Bacterial quorum sensing in pathogenic relationships. Infection and Immunity, 68(9), 4839–4849. https://doi.org/10.1128/IAI.68.9.4839-4849.2000