The influence of mitochondrial inhibitors on fungal life cycles

Abstract

English: In 2007, the Kock group published the Aspirin Antifungal Hypothesis showing a clear link between oxylipin production, mitochondrial activity and acetylsalicylic acid (ASA, aspirin) sensitivity in respiring as well as non-respiring yeasts. This hypothesis suggests that mitochondrial inhibitors such as ASA selectively inhibits parts of yeast life cycles, especially the sexual stage. According to the hypothesis, mitochondrial β-oxidation products such as 3-OH oxylipins are present in elevated amounts in yeast sexual structures (asci) and lesser amounts in vegetative asexual structures (hyphae and single cells). This suggests increased mitochondrial activity in asci. Consequently, ascomycetous yeast sexual structures should be more sensitive to mitochondrial inhibitors compared to vegetative asexual structures. The purpose of the study became to assess if the Aspirin Antifungal Hypothesis could be expanded to also include other mitochondrial inhibiting drugs as well as other structures present in fungal life cycles where increased mitochondrial activities are expected. In this study, the anti-inflammatory drugs ASA, ibuprofen, indomethacin, salicylic acid and benzoic acid as well as anticancer drugs such as Lonidamine, also known for inhibiting mitochondrial activity in mammalian cells, were found to be antifungal and specifically target the sexual stage of yeast. This is shown by a unique yeast bioassay, with the mitochondrion-dependent sexual dispersal structure producing many ascospores, riboflavin production, and hyphal morphology of the notorious yeast plant pathogen Eremothecium ashbyi serving as indicators. These drugs affect this yeast in a similar way as found under oxygen limitation conditions by inhibiting sexual structure development (most sensitive), riboflavin production, and yielding characteristically wrinkled and granular hyphae, presenting a unique “anoxic” morphological pattern. Only drugs associated with mitochondrial inhibiting activity presented such a pattern. This bio-assay may find application in the preliminary screening for novel drugs from various sources with possible mitochondrial inhibiting actions. In another part of the study, the effects of antimitochondrial compounds on asexual fungal spore dispersal structures in the pathogens Aspergillus fumigatus and Rhizopus oryzae were investigated. When anti-mitochondrial ASA and other anti-mitochondrial non-steroidal antiinflammatory drugs (NSAIDs) were added to A. fumigatus and R. oryzae, asexual fungal spore-releasing structures were targeted first at lower concentrations. Similar results were obtained when oxygen was limited. These asexual fungal spore-releasing structures contained increased levels of mitochondrial activity compared to hyphae. Increased mitochondrial activity may be necessary for the formation of asexual fungal spore dispersal structures of these fungi. Consequently, mitochondrial inhibitors may serve as effective antifungals to combat asexual fungal spore dispersal of these pathogenic fungi. In this study, the Aspirin Antifungal Hypothesis is expanded to also include various antiinflammatory compounds, anticancer drugs, plant extracts, traditional medicines and others – many showing anti-mitochondrial activity. These compounds should be further investigated to determine their minimum inhibitory concentrations (MICs) and application to combat plant and human fungal pathogens. In this study, the hypothesis is also expanded to include asexual fungal dispersal structures with increased mitochondrial activity.

Afrikaans: Die Kock-groep het in 2007 die Aspirien Antifungale Hipotese gepubliseer en daarmee ʼn duidelike verband tussen oksilipienproduksie, mitochondriale aktiwiteit en sensitiwiteit vir asetielsalisielsuur (ASS, aspirien) in respirerende en nie-respirerende giste aangetoon. Die hipotese stel dit dat mitochondriale inhibeerders soos ASS dele van gislewensiklusse, veral die geslagtelike fase, selektief inhibeer. Volgens die hipotese kom mitochondriale β-oksidasieprodukte, soos 3-OH oksilipiene, teen verhoogde vlakke in giste se geslagtelike strukture (askusse) en teen verlaagde vlakke in vegetatiewe ongeslagtelike strukture (hifes en enkel selle) voor. Dit stel voor dat daar verhoogde mitochondriale aktiwiteit in askusse is. Dus behoort askomisete giste se geslagtelike strukture meer sensitief te wees vir mitochondriale inhibeerders in vergeleke met vegetatiewe ongeslagtelike strukture. Die doel van die studie was om te bepaal of die Aspirien Antifungale Hipotese uitgebrei kan word om ook ander mitochondriale inhibeerders asook ander strukture in fungale lewensiklusse waar verhoogde mitochondriale aktiwiteit verwag word, in te sluit. Hierdie studie het gevind dat die anti-inflammatoriese middels ASS, ibuprofen, indometasien, salisiensuur en bensoësuur asook antikankermiddels soos Lonidamien wat ook mitochondriale aktiwiteit in soogdierselle inhibeer, antifungaal is en spesifiek die geslagtelike fase van giste teiken. Dit is aangetoon deur ʼn unieke gisgebaseerde biotoets met die mitochondrionafhanklike geslagtelike verspreidingstrukture wat baie askospore vorm, riboflavienproduksie en hife morfologie van die berugte plant patogeniese gis, Eremothecium ashbyi, as aanduiders. Hierdie middels beïnvloed die gis op soortgelyke wyse as wat onder suurstofbeperkende toestande gevind is, deur die ontwikkeling van geslagtelike strukture (mees sensitief) en riboflavienproduksie te inhibeer, en om kenmerkende gekreukelde en granulêre hifes te veroorsaak wat ʼn unieke “anoksiese” morfologiese patroon daarstel. Slegs middels geassosieer met inhibisie van mitochondriale aktiwiteit het so ʼn patroon veroorsaak. Hierdie biotoets mag toepassing vind in die soektog na nuwe middels met moontlike anti-mitochondriale aktiwiteit vanuit verskeie bronne. In ʼn ander deel van die studie is die effek van anti-mitochondriale middels op ongeslagtelike spoorverspreidingstrukture van die patogene Aspergillus fumigatus en Rhizopus oryzae ondersoek. Toe anti-mitochondriale ASS en ander anti-mitochondriale nie-steroïed anti-inflammatoriese middels (NSAIMs) by A. fumigatus en R. oryzae gevoeg is, is ongeslagtelike spoorverspreidingstrukture eerste teen laer konsentrasies geteiken. Soortgelyke resultate is verkry onder suurstofbeperking. Hierdie ongeslagtelike spoorverspreidingstrukture bevat verhoogde vlakke van mitochondriale aktiwiteit in vergeleke met die hifes. Mitochondriale aktiwiteit mag nodig wees vir die vorming van ongeslagtelike spoorverspreidingstrukture van hierdie fungi. Gevolglik mag mitochondriale inhibeerders dien as effektiewe antifungale om ongeslagtelike spoorverspreiding van hierdie patogene fungi te bekamp. In hierdie studie is die Aspirien Antifungale Hipotese uitgebrei om ook verskeie antiinflammatoriese verbindings, antikankermiddels, plantekstrakte, tradisionele medisyne en ander – baie met anti-mitochondriale aktiwiteit – in te sluit. Hierdie verbindings behoort verder ondersoek te word om hulle minimum inhibitoriese konsentrasies (MIKs) en hul toepassing om plant- en menslike patogene fungi te beveg, te bepaal. In hierdie studie is die hipotese ook uitgebrei om ongeslagtelike spoorverspreidingstrukture met verhoogde mitochondriale aktiwiteit in te sluit.