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Yeast “contraceptives” also novel drugs


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This lecture describes the translation of yeast "contraceptives" to new anti-mitochondrial drugs. Our nanotechnology research on yeast “contraceptives” suggests that “nanosurgery” and even …

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).

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  • 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 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 (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