Yeast “contraceptives”

also novel drugsInvited video lecture for Translational Biomedicine

Prof. Lodewyk Kock, Dr. Chantel Swart,

Dr. Desmond Ncango and Dr. Carlien Pohl

kockjl@ufs.ac.za

Department of Microbial, Biochemical and Food Biotechnology

Audio Text

Talk 1

Good day Ladies and Gentlemen.

This presentation is hosted by Prof. Lodewyk

Kock, Dr. Chantel Swart, Dr. Desmond Ncango

and Dr. Carlien Pohl, all from the Department of

Microbial, Biochemical and Food Biotechnology, at

the University of the Free State in Bloemfontein,

South Africa. The lecture describes novel anti-

mitochondrial drugs, which also serve as selective

inhibitors of sexual reproduction in yeast.

Consequently, yeast sexual reproductive

structures may serve as indicators in novel bio-

assays to discover and develop new anti-mitochondrial antifungal and anticancer drugs.

Slide 1

Why do you think this yeast uses aspirin?

Certainly not for a headache because yeasts

simply do not get headaches, as far as we are

aware of. To tell you the truth, aspirin can act

as a “contraceptive” in yeasts.

Do you believe this?

Well let us see if we can convince you.

Some yeasts such as Dipodascopsis produce

spectacular birth sacs from which the offspring is

delivered through narrow openings, all of this in micron

space. The elongated structures are the birth sacs also

known as asci from which the “babies” or ascospores

are individually released by force. This is a video-

enhanced microscopic presentation (slowed down 10

times) showing the active individual delivery of oxylipin

lubricated “baby” yeast cells from the tip of a bottle-

shaped birth sac. This all happens while these cells

rotate at high speed of about 1200 rpm at approximately

110 length replacements per second. These ascospores

are invisible to the naked eye and about 1x2.5 µm small

while the birth sac is about, from the tip, 2 µm in

diameter.

Slide 2

This slide shows a mathematical model describing ascospore delivery in the

yeast Dipodascopsis. This is based on Nano Scanning Auger Microscopy

analysis. You can find the reference to this nanotechnology at the bottom of

the slide. The model shows that the higher turgor pressure (P1) at the base

of the ascus than at the tip (P2), causes fluid flow in the central channel

towards the tip with its velocity (vf) larger along the centre of the channel

than at the periphery. This velocity gradient causes differential pressure on

the elongated ascospores, causing them to rotate and spend a longer

duration of time in an orientation parallel to the fluid flow. Here the

ascospores encounter less resistance from drag force. The ascospores may

be linked by surface gears to maintain parallel orientation as the

ascospores move towards the ascus tip. Eventually the elongated

ascospores will be individually released from the tight fitting ascus opening.

With a higher ascospore density the surface gears will play a more

significant role in orientation.

Can you believe that when aspirin is added to this yeast, it prevents this

birth process!

Now how did we expose this phenomenon in yeast?

Slide 3

Slide 4

In 1988 we discovered that certain yeasts can

produce aspirin sensitive metabolites which are

probably produced in their mitochondria.

In an experiment conducted by us, Tritium labeled

arachidonic acid (AA) was fed to the yeast

Dipodascopsis uninucleata in liquid medium and the

lipid metabolites extracted and separated on silica gel

TLC plates. The plates were sprayed with a suitable

fluor and exposed at -70 0C for 6 days under X-ray

plates. From the results it is clear that the production

of one metabolite indicated as 3-HETE, was inhibited

in a dose dependent manner by aspirin.

Slide 5

Now how was this aspirin-sensitive metabolite identified as 3-

HETE, also known as 3-hydroxy eicosatetraenoic acid?

The aspirin-sensitive band was scraped off from repeated TLC

separations and then separated by HPLC. The solvent phase

was then removed by drying and the sample subjected to

spectra analyses and finally 1H NMR spectroscopy. The 2D-

COSY45 spectrum of the metabolite provided valuable

information about connectivities and facilitated assignment of

all signals in the 1H-spectrum. It was concluded that the

metabolite was in fact 3-HETE. This structure was verified with

FAB–MS and EI-MS of the methylated, methoximated and

trimethylsilylated purified metabolite. With this information in

hand, it was possible to devise a chemical synthesis method to

produce enough 3-HETE for antibody-probe development in

rabbits.

Slide 6

In order to map the location of 3-OH oxylipins in yeasts, polyclonal

antibodies were raised in a rabbit against chemically synthesized 3-

HETE as shown on this slide. Here, 3R- and 3S-HETE were

synthesized from coupling a chiral aldehyde with a Wittig salt, which

was derived from 2-deoxy-D-ribose and arachidonic acid, respectively.

The chemically produced 3-HETE was then used to produce polyclonal

antibodies in rabbits. Yeast cells were centrifuged onto glass

microscope slides and fixed in acetone. The slides were then treated

with antibody against 3-HETE, washed with BSA and FITC anti-rabbit

IgG added followed again by washing with BSA. Immunofluorescence

micrographs were taken with a microscope equipped for

epifluorescence. Results were compared against appropriate controls.

The 3-HETE antibodies were found to be specific for 3-OH oxylipins

irrespective of desaturation or chain length. This served as the primary

antibody while fluorescing FITC-coupled secondary antibodies were

used to render 3-OH oxylipin primary antibodies fluorescent when also

using Confocal Laser Scanning Microscopy (CLSM). This fluorescing

system will from now on be referred to as OXYTRACK.

Slide 7

Using OXYTRACK we could prove that 3-OH

oxylipins are produced in and released from

mitochondria. This TEM slide shows 3-OH

oxylipin release from mitochondria in the yeast

Cryptococcus neoformans. This was verified

with OXYTRACK gold-labeling. In 1997, we

suggested the same in Dipodascopsis

uninucleata.

Slide 8

Next, the OXYTRACK probe was used to map the

distribution of 3-OH oxylipins over the life cycle of the

yeast Dipodascopsis uninucleata. The life cycle is

characterized by the delivery of haploid offspring or

ascospores from the birth sac also known as the

ascus. These ascospores then germinate to produce

vegetative cells or hyphae which eventually form

gametangia. These gametes conjugate to form an

ascus that is later filled with ascospores. Upon

maturity they are smartly delivered as described

previously. It was found that these oxylipins are

mainly associated with the birth sacs and especially

surrounding the offspring and not the asexual

vegetative growth stage.

Slide 9

Now that we know that aspirin inhibits mitochondrial activity

and that mitochondrial activity is elevated in sexual structures

probably to meet energy needs during ascus development, it

will be of interest to know what effect this non steroidal anti-

inflammatory drug or NSAID will have on the life cycle of this

yeast.

When Dipodascopsis uninucleata was grown in synchrony by

cultivating the ascospores only, we found that the most

susceptible stage to aspirin addition was the “labour” stage

especially ascospore delivery. This is indicated by the blue

bars in the graph on the left hand side (that is the control

without aspirin) compared to the graph on the right hand side

(that is in the presence of 1mM aspirin). Further research

shows that ascus formation and ascospore delivery were

inhibited in a dose dependent manner by aspirin.

Slide 10

Strikingly it was found that this phenomenon was

widespread amongst ascomycetous yeasts. This

collage shows some examples of yeast birth sacs

containing increased mitochondrial activity as is

indicated by increased fluorescence. This is also

observed in the asexual fruiting structures or

sporangia of Mucor. The same was reported for

Aspergillus and the distantly related Phytophthora. In

these cases the fruiting structures were most

susceptible to aspirin. This phenomenon seems

therefore to be highly conserved in fungi.

Slide 11

The yeast Galactomyces up close and

personal. This shows an animation of a Z-

stack of Galactomyces reessii obtained

with Confocal Laser Scanning Microscopy

after treatment with Rhodamine 123.

Here again asci showed increased mito-

chondrial activity indicated by increased

yellow fluorescence when compared to

the vegetative cells.

Slide 12

Ladies and Gentlemen, have you ever heard of hydrofoil and

boomerang movements during delivery? Well we are

convinced that such a phenomenon is present in the yeast

Eremothecium ashbyi. This yeast produces sickle-shaped

ascospores in elongated asci and is a notorious plant

pathogen. The top slide shows increased mitochondrial

activity inside asci of this yeast as indicated by increased

fluorescence. It is interesting to note that the fluorescence is

limited to the V-shaped fins situated on the broader blunt end

of the ascospore as indicated in the right hand bottom slide.

This implicates the presence of hydrophobic 3-OH oxylipins

coating the surfaces of these fins. On the left hand side, a 3-D

structure of the ascospore with fins and consisting of a blunt

end and very sharp spiky end is shown by Scanning Electron

Microscopy.

Slide 13

This figure shows a 3-D reconstruction of a

sickle shaped ascospore based on structural

research results. The hydrophobic V-shaped

fins in yellow were found to be mirror images

on both sides of the blunt end of each

ascospore in blue with the spiky tip indicated in

red.

Now why does this yeast produce such strange

looking ascospores? Definitely not for our

curiosity!

Slide 14

In order to answer this question, mathematical modeling was attempted to describe a possible

function of the curiously shaped ascospores with attached hydrophobic V-shaped fins.

This model suggests a sharp built-up of pressure between fins with a flow of water towards the

blunt-end and across the fins (from left to right) causing a boomerang movement. The forces

exerted on the fins in (a) due to the pressure will be perpendicular to the fins. Because of the

hydrophobic behavior of the fins, there should be no viscose effects that is, no forces parallel

to the fins. Consequently, these forces will culminate into a resultant force across the spore

from left to right and slightly downwards indicated by force vector F, thereby causing

movement of the spore to the right. Since the line of force passes below the centre of mass at

+C, the spore will also tend to rotate anticlockwise that is, in the direction of the spiky tip due to

an anticlockwise moment of force about +C. In addition, there should be a tendency for water

pressure to be more at the left of the spore than on the right since the spore is gradually

tapered towards the spike i.e. from approx. 3 µm in diameter at the blunt-end to approx. 2 nm

diameter at the spike. This should also enhance a boomerang movement effect. Furthermore,

the shape of the fins in (a) is such that they will also act as hydrofoils when movement (left to

right and boomerang) is initiated, causing a lifting force (as a result of the backward force on

the slanted lower fins) on the spore, similar to the wings of an aircraft. Thus, the spore will

start drifting to the right and slightly upwards (i.e. closer to the cell wall), rotating anticlockwise

until the spike reaches the ascus wall where it may be ruptured and the spore pushed out by

water pressure.

Continues on next slide.

Slide 14 Continued

In addition, fins will lend stability to the blunt-end. It will resist rotation when pushed

by water-flow causing the spike-tip to reach the cell wall at a speed required for

rupturing. Fins are also constructed in such a way that upon release through a self

inflicted narrow opening, the spear-end of the hydrophobic V will first exit thereby

preventing spores becoming easily stuck to cell wall. The relative small height and

width dimensions of fins also support this argument although the effective water

resistance area is probably increased by their hydrophobic nature. We propose the

formation of “nanobubbles” through drying at the fin-water interface thereby

increasing the relative flat and thin fin surface area on the otherwise non-

hydrophobic spore surface. This in turn would increase the resistance of fins to

water movement thereby increasing overall spore stability and boomerang speed.

Scaled–up models (10 000 times), simulating sickle spore shape and subjected to

water movement from the blunt-end side, support our proposed boomerang

movement hypothesis. The hydrophobic water-resistant properties of the fins could

not be tested since these forces would only become significant when exerted on

small objects in small environments. Furthermore, the many spores crowding the

micron-scale asci, may also lead to altered physical behaviour.

Now is this a novel way of labour delivery or what!

Slide 15

This movie hypothesizes movement of a sickle-shaped ascospore of

Eremothecium ashbyi within an elongated birth sac during delivery.

Here we see ascospore movement inside the birth sac with spiked tip in red

moving towards the screen. Hydrophobic V-shaped fins are indicated in

yellow. This is followed by ascospore movement along the length of the

birth sac with spiked tip leading the way. The direction of the ascospore

movement is in the same direction as water flow indicated by bubble

movement. We also see a side view of the ascospore movement with the

spiked tip gliding towards the inside wall of the container and eventually

piercing the wall of the birth sac. Finally we observe forced ascospore

delivery with spiked tip piercing first through the wall of the birth sac in

boomerang style. Sometimes parts of the birth sac can remain attached to

the spiked tip, while the fins can tear as a result of moving through a tight-

fitting torn wall.

Similar to the yeast Dipodascopsis, the production of 3-OH oxylipins that

coat the V-shaped fins, was sensitive to aspirin resulting in ascospore

delivery to be the most sensitive stage towards this “contraceptive”.

Slide 16

Follow up research indicates that many anti-mitochondrial drugs such as

aspirin and other NSAIDs may act as yeast “contraceptives” in general as

well as inhibiting other asexual fruiting structures in other fungi and fungi-

like organisms. As a result of these research findings, Kock and co-workers

proposed in 2007 the following hypothesis with regards to the sensitivity of

yeasts and other fungi to anti-mitochondrial drugs. In this schematic

representation the yeasts are divided into two groups. Those that can only

aerobically respire, and those that can aerobically respire and also ferment.

As the anti-mitochondrial drug concentration increases, the mitochondrial

activity and 3-OH oxylipin production decreases. Also, fungi that can only

aerobically respire are more sensitive to these drugs than fungi that can

also ferment. The fruiting sexual and asexual stages in both groups are

more sensitive to anti-mitochondrial drugs than the normal yeast and hyphal

asexual vegetative stages. Also, the accumulation of 3-OH oxylipins as well

as mitochondrial activity measured as transmembrane potential or Δψm,

decreases from the fruiting (FRUIT) to asexual vegetative (VEG.) stage.

Similar results have been obtained for other NSAIDs, such as Ibuprofen as

well as many known anti-mitochondrial drugs.

Slide 17

The next step in the research program was to apply this Anti-mitochondrial Antifungal Hypothesis to

the construction of a practical bio-assay that can screen for new anti-mitochondrial drugs.

Consequently it was decided to evaluate the development of sexual reproductive phases or asci as

indicators to track such compounds in the yeasts Lipomyces in top orange row, Eremothecium in the

middle green row and Nadsonia in the bottom purple row using the diffusion plate method.

To affect this, agar media in Petri dishes were fitted with central wells for testing acetylsalicylic acid,

also known as aspirin, for possible anti-mitochondrial activity. This is illustrated in plates 2, 3, 6, 7, 10

and 11. Each well of the control plates – slides 2, 6 and 10, each contained 46 µl ethanol compared

to the experimental plates 3, 7 and 11, which contained 4%, 8% and again 8% aspirin in ethanol

solution respectively. Prior to filling the wells with these aspirin solutions, yeast cells of Lipomyces,

Eremothecium and Nadsonia that develop easily distinguishable colored asci were first streaked out

on these agar surfaces before the wells were filled with the aspirin solutions. The cultures were

incubated until the sexual cycle could be observed by the development of a distinguishable color that

is brown in Lipomyces –see plates 2 and 3; yellow in Eremothecium – see plates 6 and 7 as well as

again brown in Nadsonia – see plates 10 and 11. Aspirin was regarded as possibly anti-

mitochondrial, also referred to as a positive hit, if a zone could be detected that only developed light

or pale colored asexual cells with no concomitant change in color as depicted on plate 3 with

Lipomyces, plate 7 with Eremothecium and plate 11 with Nadsonia. The zones with mainly asexual

cells were formed closer to the well at higher aspirin concentrations than the colored zones

containing asci. This illustrated that the asci were more sensitive towards aspirin than the vegetative

cells as was expected from the Anti-mitochondrial Antifungal Hypothesis described earlier. The

ultrastructure of the asci in the colored zones – see plates 1, 5 and 9 as well as the asexual

vegetative cells in the light zones- see plates 4, 8 and 12 are shown for each yeast, respectively.

Continues on next slide.

Slide 17 Continued

It is interesting to note that the bio-assay using Lipomyces was

about two times more sensitive to aspirin than bio-assays using the

other two yeasts. This is shown by the fact that a 4% aspirin solution

presented a similar sized light asexual zone compared to the other

yeasts confronted with 8% aspirin solutions. Also, ethanol alone had

very small inhibitory effects on growth and not on asci formation

thereby illustrating the inhibitory effect of aspirin alone on growth and

asci formation in plates 3, 7 and 11.

One should however keep in mind that such apparent positive hits

may be false due to the inhibition of other stages in sexual

reproduction development. Therefore these bio-assays should only

be regarded as an up-stream preliminary screening method for novel

drugs. Positive hits should therefore be followed up by further

detailed research. Here in vitro, in vivo and in silico tools as well as

“omics” technologies may be applied.

Slide 18

Next a wide variety of compounds with known and unknown anti-

mitochondrial activity, were screened using the three yeast bio-

assays. The results are shown in this table with each bio-assay

depicted with a similar color as in the previous slide. From the

results it is clear that all NSAIDs and antifungal compounds yielded

positive hits as well as the classical mitochondrial inhibitors namely

Antimycin A, Rotenone and when oxygen was limited. In addition, a

good correlation was found between positive hits with compounds

that also pose a mitochondrial liability as per Black Box Warnings by

the FDA. However some exceptions were found. Diflunisal and

fenoprofen tested negative for Black Box FDA Warnings, while the

bio-assays yielded positive hits. Interestingly, according to literature,

these NSAIDs in fact show anti-mitochondrial activity. These

discrepancies should now be followed up and antifungals subjected

to detailed rigorous tests for anti-mitochondrial activities. The

anticancer drugs all showed anti-mitochondrial activities some of

which has also been reported for this inhibitory activity in literature.

Slide 19

It is interesting to note that the anti-malarial drug chloroquine

showed a stimulatory effect on asci formation in the Lipomyces

bio-assay and not in the other yeast bio-assays. This may

probably be ascribed to the fact that this yeast is more

sensitive to mitochondrial activity intervention. With

chloroquine the brown zone next to the well was darker in color

and contained a significant higher percentage of mature asci

compared to the lighter brown zone at the periphery of the bio-

assay plate and exposed to lower concentrations of this drug.

The stimulatory effect of chloroquine on mitochondrial activity

has also been reported in literature.

Can this compound therefore be regarded as a “fertility” drug in

some yeasts?

Slide 20

A flyer with more information regarding

this presentation can be obtained from

the authors free of charge (see contact e-

mail elsewhere). This also describes

NSAIDs as antifungal drugs in combating

Candida albicans infections, a protocol

which has been patented.

Slide 21

This apparatus is a Nano Scanning Auger

Microscope, which has been applied

successfully for the first time in Biology in 2010.

Here, this apparatus was applied to study the

effects of the yeast “contraceptive” fluconazole

on the sexual cells of the yeast Nadsonia. This

can be accessed in another TBM video lecture

at

http://videolectures.transbiomedicine.com/

(Title: A new Nanotechnology for Trans-

lational Medicine; presented by Kock and

Swart, 2011).

Slide 22

To conclude:

• Yeast sexual reproductive structures may

serve as indicators in novel bio-assays to

discover and develop new antifungal and

anticancer drugs.

• These bio-assays may be used as preliminary

up-stream screening methods for novel

drugs.

• Positive hits to be followed up by in vitro, in

vivo and in silico as well as “omics” research.

• Yeast “contraceptives” also novel drugs.

AcknowledgementsSlide 23

M.Sc. Students (1982-2011)BC Viljoen

M Cottrell

HB Muller

HG Tredoux

A Oosthuizen

DJ Coetzee

M Miller

T Pearson

EL Jansen van Rensburg

L van der Berg

J Jeffery

D Jansen van Vuuren

A Mothibeli

CH Pohl

PD Venter

T Strauss

G Morakile

J Lekekiso

I Paul

TR Pelesane

S Bareetseng

T Venter

M Kalorizas

OM Sebolai

NJ Leeuw

A van Heerden

DM Ncango

CW Swart

M Goldblatt

R Ells

AcknowledgementsSlide 24

Ph.D. Students (1982-2011)BC Viljoen

M Cottrell

OPH Augustyn

EJ Smit

DJ Coetzee

MS Smit

A Botha

JPJ van der Westhuizen

E Blignaut

E Jansen van Rensburg

MP Roux

J Badenhorst

CH Pohl

P Venter

LECM Anelich

T Strauss

GI Morakile

DP Smith

M Joseph

S Tarr

S Bareetseng

CJ Strauss

OM Sebolai

NJ Leeuw

DM Ncango

CW Swart

Acknowledgements

• The National Research Foundation, South Africa

• The Claude Leon Foundation, South Africa

• The University of the Free State, South Africa

• Prof. H.C. Swart, Physics, University of the FreeState (UFS), South Africa

• Prof. P.W.J. van Wyk, Centre for Microscopy,UFS, South Africa

• Prof. S.W. Schoombie & J. Smit, Mathematicsand Applied Mathematics, UFS, South Africa

• Stephen Collett, Digipix, South Africa

Slide 25

Main References

• Kock, J.L.F., Sebolai, O.M., Pohl, C.H., van Wyk, P.W.J. andLodolo, E.J. (2007) Oxylipin studies expose aspirin asantifungal. FEMS Yeast Research 7: 1207-1217.

• Kock, J.L.F., Swart, C.W., Ncango, D.M., Kock (Jr), J.L.F.,Munnik I.A., Maartens M.M.J., Pohl, C.H. and van Wyk,P.W.J. (2009) Development of a yeast bio-assay to screenanti-mitochondrial drugs. Current Drug DevelopmentTechnologies 6(3): 186-191.

• Kock, J.L.F., Swart, C.W. and Pohl, C.H. (2011) The anti-mitochondrial antifungal assay for the discovery anddevelopment of new drugs. Expert Opinion on DrugDiscovery (In Press).

Slide 26

Research HighlightsSlide 27