Falcarindiol and its Effectiveness as an Antifungal Agent

Lizzie Schousek Author

08/03/2021 Added

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[00:00:01.250]Hello, my name is Lizzie Schousek,
[00:00:03.287]and this summer I worked on falcarindiol
[00:00:05.670]and its effectiveness as an antifungal agent.
[00:00:09.480]A current fungal pathogen of note is Candida auris,
[00:00:12.490]which has had enough outbreaks recently to make the news.
[00:00:15.740]It is harmful to immunocompromised individuals,
[00:00:18.400]and more than 1/3 of patients infected with it
[00:00:21.710]die within 30 days.
[00:00:23.270]The reason we need new antifungals
[00:00:25.100]is because fungal pathogens are becoming immune
[00:00:27.350]to current antifungals.
[00:00:29.230]There are three major classes of antifungal drugs:
[00:00:31.923]azoles, polyenes, and echinocandins.
[00:00:35.400]Strains can be resistant to one, two, or all three classes.
[00:00:42.060]Falcarindiol is a polyacetylene compound
[00:00:44.410]found in plants from the Apiaceae family,
[00:00:47.280]like carrots, celery, and parsnips.
[00:00:49.910]It is hypothesized to have antifungal properties.
[00:00:52.840]Anecdotally, this makes sense,
[00:00:55.110]because when these foods are stored in the refrigerator
[00:00:57.130]for extended periods of time,
[00:00:58.840]they tend to end up slimy,
[00:01:00.060]which indicates bacterial growth,
[00:01:01.540]and never moldy, which would indicate fungal growth.
[00:01:04.700]Falcarindiol has been shown to reverse the effects
[00:01:06.970]of induced colon cancer in rats.
[00:01:09.570]Previous research in our lab has shown that falcarindiol
[00:01:12.660]is a stronger antifungal agent
[00:01:14.470]than the counterpart falcarinol,
[00:01:17.540]which has only one alcohol group.
[00:01:20.360]My summer's research focuses on the hypothesis
[00:01:23.210]that falcarindiol inhibits fatty acid synthesis.
[00:01:26.600]We used Saccharomyces cerevisiae, baker's yeast,
[00:01:29.370]as a model organism for this project.
[00:01:33.550]There were three main objectives
[00:01:35.080]in my summer research project.
[00:01:37.050]With help for my labmates,
[00:01:38.210]I had to first determine the best source material
[00:01:42.150]and extraction method for falcarindiol.
[00:01:44.890]After that, we needed to verify the extract's efficacy.
[00:01:48.680]Finally, we would run the genetic screen
[00:01:50.960]to find the drug's gene targets,
[00:01:54.370]which would indicate
[00:01:55.203]whether or not our hypothesis was correct.
[00:01:59.980]In order to determine the best source material,
[00:02:02.630]We tested three different Apiaceae species,
[00:02:05.250]carrot, celery, and parsnip.
[00:02:07.570]Since we had celery leaves in stock as well,
[00:02:10.070]we tested them too.
[00:02:13.060]First, we chopped up five grams of each fresh material
[00:02:16.120]and extracted it with ethyl acetate.
[00:02:18.820]The ethyl acetate was decanted off
[00:02:20.650]and evaporated under nitrogen gas.
[00:02:23.170]The extract was then resuspended
[00:02:26.060]in a lesser amount of ethyl acetate
[00:02:27.870]for storage and TLC testing.
[00:02:30.800]To verify that falcarindiol was in these extracts
[00:02:33.470]and determine which one had the most,
[00:02:35.200]I ran a thin layer chromatography plate, TLC.
[00:02:38.980]The green box shows us where the falcarindiol should be.
[00:02:41.950]Above the box are non-polar carotinoids and other pigments.
[00:02:45.620]Below the box are polar lipids,
[00:02:47.150]such as phospholipids and glycolipids.
[00:02:50.100]The celery stalk and leaves both have no falcarindiol.
[00:02:55.280]The carrot root does not have as much falcarindiol
[00:02:57.950]when compared to the other roots.
[00:02:59.630]The parsnip root contains more falcarindiol
[00:03:01.860]than the celeriac, although we determined
[00:03:04.100]that the best source material of falcarindiol is celeriac,
[00:03:08.900]because it contains the least amount of contaminants.
[00:03:11.950]To confirm that celeriac extract
[00:03:14.250]contain mostly falcarindiol,
[00:03:16.220]we had Wonmi perform a gas chromatogram
[00:03:19.000]and mass spectrometer samples.
[00:03:21.810]The peaks of 73, 147, and 319,
[00:03:25.090]confirmed that it was falcarindiol,
[00:03:27.240]when compared to the mass spec found in the Busta paper.
[00:03:33.050]After determining that we would use celeriac
[00:03:35.780]as our source material,
[00:03:37.200]we had to scale up the extraction process.
[00:03:39.510]This time around, we chopped up 100, 200,
[00:03:41.970]or 500 grams of celeriac.
[00:03:44.320]We again extracted with ethyl acetate
[00:03:46.210]and used a rotovap to evaporate a liter of solvent.
[00:03:49.230]The extract was then again resuspended
[00:03:50.980]in a lesser amount of ethyl for storage and TLC.
[00:03:55.000]We ran another TLC plate with our different extracts
[00:03:57.620]next to a falcarindiol standard
[00:03:59.670]produced previously in our lab.
[00:04:03.230]To verify that the extract actually inhibited cell growth,
[00:04:06.220]we ran some serial dilutions.
[00:04:08.460]After 24 hours of growth,
[00:04:10.410]the wells would be read by a plate reader
[00:04:12.010]which would tell us the optical density.
[00:04:14.410]High optical density indicates cell growth.
[00:04:17.520]When the optical density is low,
[00:04:19.170]that means that the cells did not grow,
[00:04:20.970]and that the celeriac extract inhibited growth.
[00:04:23.930]The celeriac extract was stored in the freezer,
[00:04:26.040]suspended in ethyl acetate.
[00:04:28.010]For the experiments,
[00:04:29.020]we dried that down and resuspended it initially in DMSO.
[00:04:32.650]As you can see in the graph on the right,
[00:04:34.420]at the highest percent extract,
[00:04:36.150]the optical density and apparent cell growth increases.
[00:04:39.290]This is because the extract (indistinct) absolution
[00:04:41.550]at high concentrations, making the media appear turbid,
[00:04:45.060]and increasing the optical density.
[00:04:47.510]This issue was resolved by switching the extract solvent
[00:04:50.120]from DMSO to 1% Tergitol.
[00:04:52.710]This switch in solvent
[00:04:53.600]also decreased the minimal inhibitory concentration, MIC,
[00:04:57.560]of the celeriac extract.
[00:05:01.040]To confirm that we are on the right track
[00:05:02.520]with our hypothesis
[00:05:03.810]that falcarindiol inhibits fatty acid synthesis,
[00:05:06.470]We did a serial dilution with (indistinct)
[00:05:08.250]and palmitic fatty acids, to rescue the cells
[00:05:10.490]when falcarindiol would have otherwise inhibited growth.
[00:05:13.510]The difference in optical density at zero hours
[00:05:15.820]is due to the 1% Tergitol,
[00:05:17.860]which led us to switching solvents from DMSO
[00:05:20.020]to the Tergitol as described on the previous slide.
[00:05:23.500]If the fatty acid rescue did not work,
[00:05:25.320]the graph on the right would have had two lines
[00:05:27.050]on top of each other.
[00:05:28.220]The fatty acid rescue did work,
[00:05:31.480]allowing for the rescued cells
[00:05:32.640]to have higher growth rates.
[00:05:34.280]At higher concentrations of celeriac extract,
[00:05:37.060]there is not as much rescue.
[00:05:39.830]After verifying the extract efficacy,
[00:05:42.010]we moved on to the genetic screen.
[00:05:43.900]Up until now, we had used liquid media
[00:05:45.690]to culture cell growth,
[00:05:46.950]and the genetic screen used solid media.
[00:05:49.490]We tested different concentrations of extract
[00:05:51.890]on both wild types and mutants.
[00:05:54.000]We decided to use 0.003% of extract number three
[00:05:58.190]for the genetic screen to test for sensitive strains.
[00:06:04.610]the genetic screen utilized
[00:06:06.470]the Mat-alpha knockout collection.
[00:06:08.410]The knockout collection contains mutants
[00:06:10.160]that each have one of each of these genes knocked out.
[00:06:14.410]The mutant gene grows on the control plate.
[00:06:16.660]If the mutant grows in the control plate
[00:06:18.860]but not the celeriac extract plate,
[00:06:21.110]we can conclude that the gene the mutant is lacking
[00:06:23.490]is necessary for yeast to grow
[00:06:25.250]in the presence of falcarindiol,
[00:06:27.560]the major compound in our celeriac extract.
[00:06:30.490]Knowing the necessary genes for yeast survival
[00:06:32.800]in presence of falcarindiol gives us a glimpse
[00:06:34.800]into the falcarindiol as mode of action.
[00:06:37.300]The big square plates picture here
[00:06:39.130]have 564 mutants stamped on each.
[00:06:42.350]I circled one mutant here that grew on the controls
[00:06:45.110]but not the celeriac plate.
[00:06:48.130]The preliminary results of our genetic screen
[00:06:50.370]show 164 mutants, or 164 target genes
[00:06:54.850]that did not grow on the celeriac extract plates.
[00:06:58.690]The genes boxed together have similar function.
[00:07:01.060]For now, we can tell
[00:07:02.160]that falcarindiol affects the ERMES complex,
[00:07:04.390]ergosterol biosynthesis, amino acid auxotrphies,
[00:07:08.290]fatty acid chain elongation, and vacuolar proton pumps.
[00:07:13.740]In the future,
[00:07:14.573]I need to redo some portions of the genetic screen,
[00:07:16.990]because unfortunately, some of my plates were contaminated.
[00:07:19.830]I also need to verify the sensitive mutants
[00:07:22.300]from the genetic screen.
[00:07:24.010]After I have those results,
[00:07:25.170]I will be able to continue my work on determining the genes
[00:07:27.670]and/or pathways that falcarindiol targets.
[00:07:31.510]I would like to thank UCARE for the funding,
[00:07:33.170]and all the help I received from the Riekhof Lab,
[00:07:35.780]as well as my collaborators
[00:07:37.010]and those who did work on this project before me.
[00:07:40.240]And thank you for listening to my presentation.
[00:07:42.410]If you have any questions,
[00:07:43.470]feel free to reach out to me or the Riekhof Lab.

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Falcarindiol and its Effectiveness as an Antifungal Agent

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