cyathus helenae a cure for neurological diseases & new class of antibiotics! by david lieu

Cyathus helenaeA Cure for Neurological Diseases &

New Class of Antibiotics!

By David Lieu

Cyathus helenaeQuick Facts:• The fungus Cyathus helenae has many different species. They are part of the Class Basidiomycetes.

• They are very commonly referred to as the Bird’s nest Fungi since they produce their spores in a cup like mushroom.

• The asexual spores actually are dispersed by drops of water or rain that hit the egg-like sacs which can shoot spores up to a few feet away.

• They grow to about 5mm in diameter.

• Like most fungi, C. helenae prefer damp and shady areas.

• They tend to show up in the late winter or early spring.

• They produce a secondary metabolite called cyathin A3.

Cyathin A3

3D Chemical Structure of Cyathin A3

2D Chemical Structure

of Cyathin A3

So Why Do We Care?Neurological Diseases: There is great potential for cyathin A3 in future pharmaceutical drugs. It has been shown to induce production of nerve growth factor, which is secreted by neurons to help maintain brain and neural functions. Cyathin may be the answer to Alzheimer's, Parkinson’s and Huntington’s Disease where there is a massive lost of brain cells. These drugs could possibly slow down the diseases or even stop their progression all together.

Above: Normal Human BrainBelow: Human Brain Infected by Alzheimer'sPics from www.alzheimers.org/rmedia/graphicshighres.htm

Picture of Pharmaceutical Drugs From www.swisspharmaceutical.com/gifs/img.jpg

Antimicrobial Drugs: In previous research studies it has been shown that some species of Cyathus have antibacterial and even antifungal properties. These would be different from some of the more common antimicrobial drugs like penicillin because they don’t contain beta-lactam rings. This may prove useful against many new multi-drug resistant bacteria like Streptococcus pneumoniae and Mycobacterium tuberculosis.

Secondary Metabolite Production

How Did We Do It?~1st We grew the fungi Cyathus helenae on Brodie agar plates

for 2 weeks.

~2nd We then took 1X1cm slices of C. helenae from the agar plates and inoculated 150 mL flasks containing 50 mL of Brodie media.

~3rd These flasks are placed in appropriate environments in which they are being tested i.e. shaker, Incubator, bench...

~4th When the appropriate time we have assigned for each flask is reached we will induce them with E.coli. It is very unusual step for fungi metabolite production but the induction with E.coli actually stimulates the fungi to produce cyathin as a secondary metabolite.


How Did We Do It?~5th In my experiments I filtered the E.coli

cells from their metabolites in the seed cultures which were incubated on a shaker for 24 hrs at 28ºC before I induced the C. helenae. This is to test if the metabolites are causing the fungi to produce cyathin or if it is the E.coli cells themselves somehow stimulating the production.

~6th These E.coli induced flask are then set aside for 6 days for the fungi to react with the E.coli and produce secondary metabolites like cyathin which we can collect afterwards.

~7th After 6 days we extract the Cyathin A3 using an organic solvent ethyl acetate in a separatory funnel 3X50 mL. We then take the ethyl acetate and pour it into a 500mL round bottom flask.


How Did We Do It?~8th We concentrate the compound by running

the round bottom flask on a roto-vap. When we have a few mL left we transfer it to a smaller 10mL round bottom flask and run it to dry.

~9th Then the Cyathin is resuspended in acetyl nitrile and placed into a small vial, parafilmed and then in put into the fridge.

~10th When we are ready the vials are run through an HPLC and the retention times and UV Apex spectrums are compared to an authentic sample of Cyathin.

~11th The samples are then run on a LC-MS to verify the molecular weight of the compound at the matching retention time.

Authentic Cyathin A3 HPLC

Cyathin A3 HPLC Spectrogram at 230.2 nm Wavelength

Retention Time is 4.471 and Peak Area is 2573.23

Authentic Cyathin A3 HPLC

Cyathin A3 UV Apex Spectrum of Peak 4.471


Experimental Variables Tested to Maximize Production

Flask Numbers Fermentation Growth Variables

Flask # 1-5 Static at Room Temp.

Flask # 6-10 Shaken at 28ºC.

Flask # 11-15 Static with Large Inoculums at Room Temp.

Flask # 16-20 Static with Large Inoculums at 28ºC.

Flask # 21-25 Static with Large Inoculums at 28ºC.

Standard Flasks Set #1 Static with Large Inoculums at Room Temp.

Standard Flasks Set #2 Static with Large Inoculums at 28ºC.

Cyathin A3 ProductionSummary and Data Analysis of Flasks # 1-5

Static Fermentation with Small Cyathus Inoculums at Room Temp.

Flask Numbers

Fermentation Length

Inoculation Length

Cyathin A3 Peak Areas

Retention TIme

Flask # 1 1 Week 7 Days No Peak N/A

Flask # 2 2 Weeks 7 Days 39553.8* 4.480

Flask # 3 3 Weeks 7 Days No Peak N/A

Flask # 4 4 Weeks 7 Days 308.3* 4.675


* 3D Spectrum Doesn’t MatchCyathin A3 Retention Time is 4.471Cyathin A3 Peak Area is 2573.3

Cyathin A3 Production


Above: Authentic Cyathin A3

Below: Flask #2 Extraction Sample


Flask #2 UV Apex Spectrum of peak 4.480


Shaken Fermentation with Small Cyathus Inoculums at 28ºC.

Flask Numbers

Fermentation Length

Inoculation Length


Retention Time

Flask # 6 7 Days 7 Days 12711.6 3.806

Flask # 7 14 Days 7 Days 10131.4* 4.508

Flask # 8 21 Days 7 Days 9206.1 4.485

Flask # 9 28 Days 7 Days 699.3 4.368

Flask # 10 35 Days 7 Days 1582.9 4.426



Flask #6 Shaken at 28ºCUV Apex Spectrum of peak 3.806






Static Fermentation with Large Cyathus Inoculums at Room Temp.

Flask Numbers

Fermentation Length

Inoculation Length


Retention TIme

Flask # 11 7 Days 7 Days 69.1* 3.828

Flask # 12 14 Days 7 Days 18414.8* 4.471

Flask # 13 21 Days 7 Days 219.8* 4.598

Flask # 14 28 Days 7 Days 61.6* 4.773

Flask # 15 35 Days 7 Days 154.0* 4.684



Flask #11 Static Large Rm Temp.

UV Apex Spectrum of peak 3.828










Static Fermentation with Large Cyathus Inoculums at 28ºC.

Flask Numbers

Fermentation Length

Inoculation Length


Retention Time

Flask # 16 1 Week 7 Days 117.6* 4.656


Flask # 18 3 Weeks 7 Days 5414.1* 4.256

Flask # 19 4 Weeks 7 Days 79.4* 4.162

Flask # 20 5 Weeks 7 Days 220.7* 4.706



Flask #16 Static Large 28ºC









Static Fermentation with Large Cyathus Inoculums at 28ºC.

Flask Numbers

Fermentation Length

Inoculation Length


Retention Time

Flask # 21 1 Week 7 Days 207.5* 4.596


Flask # 23 3 Weeks 7 Days 66.1* 4.262

Flask # 24 4 Weeks 7 Days 494.6* 4.770

Flask # 25 5 Weeks 7 Days 831.6* 4.604


Cyathin A3 ProductionSummary and Data Analysis of Standard Flasks

Static Fermentation with Large Cyathus Inoculums.

Flask Number

Fermentation Length

Inoculation Length

Temp. Peak Areas

Retention TIme

Standard #1 18 Days 7 Days Room Temp

422.6* 4.693

Standard #2 18 Days 7 Days Room Temp

255.3* 4.726

Standard #1 18 Days 7 Days 28ºC 842.2 4.430

Standard #2 18 Days 7 Days 28ºC 499.0* 4.620



Standard #1 Static Rm Temp.




Standard #1 Static 28ºC





Top Left:


Bottom Left:


Top Right:


Bottom Right:


Summary Cyathin A3 Production

Results & ConclusionsSo What Does It All Mean? Based on the current data it seems that the shaken flask have the maximum cyathin production with the correct UV Apex Spectra as the authentic sample.

The static flasks tend to have a different UV Apex Spectra which were similar to each other and have a later retention time. This may mean that the cyathin became glycosalated in storage which would change it’s retention time as well as its UV Apex Spectra.

The LC-MS shows nothing with a similar molecular weight at this particular retention time with any of the samples that had the different UV Spectra.

The static fermentation seemed to be creating a anaerobic environment in the flasks while the shaken flasks got more oxygen but tend to create large ball like pellets with anaerobic-like centers.

Since the data has no apparent trends and was very inconsistent it can be concluded that the E.coli metabolite is not what is stimulating the fungi to produce cyathin but the E.coli cells themselves.

ProblemsSo What Went Wrong? The fungus Cyathus helenae has been known to have sectoring problems. This can be seen on the pictures to the right. Different parts of the fungi will express different genes for no apparent reason and this may interfere with metabolite production by not being able to standardize our cultures inoculums.

Another problem is unknown bacterial contamination. There are a number of ways that some other forms of bacteria may have contaminated our cultures on the agar plates or in the media flasks. A contaminated fungal agar plate is shown below.

Another problem is storage. After a few days the samples dry and precipitate when resuspended. We don’t know what effects this has on the compound itself.

Future PlansWhere To Go From Here? Right now we are in the middle of “Operation Mass Production”.

Hopefully next we will try to locate the specific genes that code for cyathin production and try to knock them out and create a new fungi that can produce cyathin in much larger quantities at a much faster rate.

Another possibility is to try and find out what part of the E.coli cell itself is stimulating the metabolite production. Since it can be concluded that it is not extracellular it may be something intracellular. We would lyse the cell and separate the intracellular contents by centrifuge and induce the fungal flasks with different cellular components. This would help us understand the mechanism of the metabolite production and would aid us in controlling the expression of the particular genes.

Acknowledgements

cyathus helenae a cure for neurological diseases & new class of antibiotics! by david lieu

Documents

fungi cyathus helenae

fungi metabolite production

coli cells

fungus cyathus helenae

secondary metabolites

cyathus helenae quick

species of cyathus

coli induced flask