This lecture describes the translation of yeast "contraceptives" to new anti-mitochondrial drugs. Our nanotechnology research on yeast “contraceptives” suggests that “nanosurgery” and even “nanotransplantation” of cell parts on different viable cell types, should now be explored using Focused Ion Beam (FIB) technologies, References can be obtained from the authors (Prof. J.L.F. Kock et al).

1. Audio Text
Yeast “contraceptives”
also novel drugs
Invited 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

2. 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.

3. 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.

4. Slide 2
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.

5. Slide 3
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?

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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!

16. 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.

17. 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!

18. 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”.

19. 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.

20. 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.

21. 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.

22. 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.

23. 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?

24. 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.

25. 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).

26. 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.

27. Slide 23
Acknowledgements
M.Sc. Students (1982-2011)
BC Viljoen
M Cottrell
I Paul
HB Muller
TR Pelesane
HG Tredoux
S Bareetseng
A Oosthuizen
T Venter
DJ Coetzee
M Kalorizas
M Miller
OM Sebolai
T Pearson
NJ Leeuw
EL Jansen van Rensburg
A van Heerden
L van der Berg
DM Ncango
J Jeffery
CW Swart
D Jansen van Vuuren
M Goldblatt
A Mothibeli
R Ells
CH Pohl
PD Venter
T Strauss
G Morakile
J Lekekiso

28. Slide 24
Acknowledgements
Ph.D. Students (1982-2011)
BC Viljoen
M Cottrell
OPH Augustyn
EJ Smit
M Joseph
DJ Coetzee
S Tarr
MS Smit
S Bareetseng
A Botha
CJ Strauss
JPJ van der Westhuizen
OM Sebolai
E Blignaut
NJ Leeuw
E Jansen van Rensburg
DM Ncango
MP Roux
CW Swart
J Badenhorst
CH Pohl
P Venter
LECM Anelich
T Strauss
GI Morakile
DP Smith

29. Slide 25
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 Free
State (UFS), South Africa
• Prof. P.W.J. van Wyk, Centre for Microscopy,
UFS, South Africa
• Prof. S.W. Schoombie & J. Smit, Mathematics
and Applied Mathematics, UFS, South Africa
• Stephen Collett, Digipix, South Africa

30. Slide 26
Main References
• Kock, J.L.F., Sebolai, O.M., Pohl, C.H., van Wyk, P.W.J. and
Lodolo, E.J. (2007) Oxylipin studies expose aspirin as
antifungal. 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 screen
anti-mitochondrial drugs. Current Drug Development
Technologies 6(3): 186-191.
• Kock, J.L.F., Swart, C.W. and Pohl, C.H. (2011) The anti-
mitochondrial antifungal assay for the discovery and
development of new drugs. Expert Opinion on Drug
Discovery (In Press).

31. Slide 27
Research Highlights