hi im currently conducting a research on inhibition of quorum sensing of pseudomonas aeruginosa, and one of its virulence is the proteolytic and elastolytic activity, how can i show the inhibition of these two using my sample which is a plant extract?
You can follow this paper: http://aac.asm.org/content/52/1/198
Thank you for the question, but would you be more specific? Do you want to test the inhibition by determining expression of these enzymes? Do you want to do RT-qPCR to determine transcript levels? Do you want to purify the enzymes in conjunction? Are you gathering empirical data related to phenotypic traits? There are so many ways to do this, so what specifically are you trying to accomplish?
Dear RA Tan Caraan, If you are unsure whether your plant extract has any significant impact on the QS system of your particular P. aeuriginosa strain, I'd suggest doing a simple experiment first : drop-inoculate 5 - 10 ul of and over-night culture of P. aeruginosa onto the dry surface of milk-agar plates containing a range of concentrations of your plant extract. P. aeruginosa, like many other pseuodmonads, secretes a protease that will degrade milk proteins leaving a cleared halo around the colonies. If the plant extract does effect quorum sensing regulation of this protease, you should get a simple quantitative indication of this without too much difficulty (Milk agar plates can be made with 20g milk powder, 1.5g Yeast extract and 12 -15g Technical agar per litre). If you found an effect using this method, you could then consider more involved or expensive means at looking at gene expression changes etc. Of course, you can buy labelled proteins which when degraded, loose their colour (which can be measured spectrophometrically; elastin degradation is often measured using this approach). Regards, Andrew.
You may consider using a zymography analysis with the introduction of specific substrates (gelatin, for example) into gel. Use conditioned media that contains secreted products for the analysis. This will allow you to identify conditions (quorum sensing, inducing factors) that promote the expression of proteolytic phenotype in P. aeruginosa. The sequence analysis of the eluted protein will help you to identify the protease that is inhibited by the plant extract. Specifically to elastase, you can use its capacity to degrade collagen. Using commercially available fluorescein-labeled collagens, you can perform high throughput screening analysis in 96 well plates (see Shogan et al., STM 2015, Collagen degradation and MMP9 activation by Enterococcus faecalis contribute to intestinal anastomotic leak) for the analysis of your plant extracts
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