Fatty Acids and Fungal Development: Structure ... - ACS Publications

Chapter 20

Fatty Acids and Fungal Development: Structure-Activity Relationships James L. Kerwin

Downloaded by UNIV OF NEW SOUTH WALES on August 26, 2015 | http://pubs.acs.org Publication Date: December 24, 1987 | doi: 10.1021/bk-1987-0325.ch020

Department of Entomology, University of California, Davis, CA 95616

Free and esterified fatty acids and their metabolites regulate fungal morphogenesis in diverse and poorly understood ways. Through use of structure-function relationships, the physiological bases for morphological development in fungi can be directly or indirectly investigated. Use of simple categories for classification, e.g. saturated vs. unsaturated or preferential partitioning in fluid or gel phases, can be misleading due to the presence of segregated lipid domains in cell membranes or to selective metabolism. Fatty acid composition has been shown to regulate a variety of membrane transport processes, oxidative phosphorylation, and a number of transferases and other enzymes. Fungal growth requirements for fatty acids can vary among genera, species or even isolates of the same species. Sporulation and spore germination are also affected by fatty acid composition as are the interactions between fungal parasites and their plant or insect hosts. Preliminary investigations support a role for oxygenated fatty acid derivatives in fungal morphogenesis. Fatty acids, which are i n t e g r a l components of c e l l u l a r and organelle membranes, primary storage products and substrates f o r secondary metabolism, are involved i n the regulation of fungal morphogenesis. Changes i n f a t t y acid composition and metabolism associated with fungal growth and d i f f e r e n t i a t i o n (reviewed i n 1-6) have been extens i v e l y documented; although a l t e r a t i o n s i n f a t t y acid components have been associated with the development of fungi, s p e c i f i c structurefunction relationships have r a r e l y been established. Secondary metabolism of these compounds has also l a r g e l y been ignored, espec i a l l y concerning possible regulatory roles of e.g. oxidized f a t t y acids i n fungi. The following review focuses on studies which attempt to more p r e c i s e l y r e l a t e f a t t y acid structure to p h y s i o l o g i c a l and morphological development i n fungi, often i n terms of t h e i r physical properties. I n i t i a l observations are also presented concerning potent i a l regulatory roles f o r f a t t y acid cyclooxygenase and lipoxygenase products i n fungal systems. 0097-6156/87/0325-0329$06.00/0 © 1987 American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Fatty Acid Structure Reviews of fungal f a t t y acid composition (5>7_>8) reveal that t h e i r primary constituents are 12- to 20-carbon chain length unbranched compounds, with even-numbered chains predominant. Both saturated and unsaturated compounds occur, with p a l m i t o l e i c (C-16:l), o l e i c (C-18.-1), l i n o l e i c (C-18:2) and l i n o l e n i c (C-18:3) acids the most common unsaturated moieties C5). As with most n a t u r a l l y occurring f a t t y acids (9), monounsaturated compounds usually contain a c i s o l e f i n i c bond and polyunsaturated acids have methylene-interrupted c i s double bonds. Although rare i n occurrence and subjected to l i m i t e d study, branched chains, hydroxy, oxo and epoxy acids are also synthesized (5). L i s t s of the structures of unusual f a t t y acids which can be employed i n structure a c t i v i t y studies are presented i n d e t a i l elsewhere (9-14). When discussing diverse f a t t y acid structures, i t i s convenient to group related compounds i n t o categories to f a c i l i t a t e the e l u c i d a t i o n of structure-function r e l a t i o n s h i p s . The most obvious approach i s to group compounds as being saturated or (poly) unsaturated; however, problems a r i s e i n instances where e.g. short chain saturated compounds and longer chain unsaturated acids have s i m i l a r e f f e c t s on membrane f l u i d i t y . An alternate approach involves grouping according to p r e f e r e n t i a l p a r t i t i o n i n g i n t o f l u i d or g e l phases (15,16), which separates cis-unsaturated f a t t y acids from saturated and trans-unsaturated compounds. In some developmental systems categorization may not be possible due to p r e f e r e n t i a l secondary metabolism of s p e c i f i c compounds, e.g. arachidonic acid, which subsequently regulates pivot a l events i n fungal morphogenesis. Quantitative s t r u c t u r e - a c t i v i t y studies using l i n e a r free energy-related parameters such as p a r t i t i o n c o e f f i c i e n t s , e l e c t r o n i c substituent constants and s t e r i c properties (17,18) would p r e c i s e l y define r e l a t i o n s h i p s among diverse f a t t y acids, but this approach has not been exploited i n fungal research. Physical Properties and Concepts A c y l carbon chains i n f u l l y saturated phospholipids occur i n extended a l l - t r a n s conformations below the phase t r a n s i t i o n temperature (19, 20). The presence of a s i n g l e c i s double bond disrupts the close packing of the a11-trans conformation and can profoundly a f f e c t physi c a l properties. The p o s i t i o n and configuration of double bonds a f f e c t basic c h a r a c t e r i s t i c s such as melting point, with lower melting points found f o r compounds i n c i s rather than trans configurations and i n f a t t y acids with double bonds near the center of the fatty acid chain (21-23). Carbon chain length, degree and p o s i t i o n of unsaturation and the presence of c y c l i c or oxygenated moieties a l l a f f e c t membrane f l u i d i t y which i n turn regulates a v a r i e t y of c e l l u l a r functions (24-26). Their e f f e c t s are mediated i n part by influencing the membrane l i p i d phase t r a n s i t i o n temperature (27-29) and related physical properties such as range and rate of a c y l chain motion; however, extreme changes i n l e v e l s of unsaturation often r e s u l t i n i n s i g n i f i c a n t changes i n f a t t y a c y l motional parameters i n both b i o l o g i c a l and model membrane systems (30). Recent observations on the properties of cyclopropane-containing membranes revealed that although the c y c l i c moiety perturbs membrane l i p i d packing, i t a c t u a l l y s t a b i l i z e s membrane structure by acting as a b a r r i e r to the



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propagation of perturbations (31). Unexpected r e s u l t s such as these emphasize the complex nature of lipid-mediated events, and care should be taken i n extrapolating properties exhibited by free f a t t y acids to e f f e c t s on b i o l o g i c a l systems. An added complication when attempting to i n t e r p r e t membrane-mediated phenomena i n terms of f a t t y a c y l composition i s the possible existence of l a t e r a l l y segregated l i p i d domains (32-35) which have been proposed to have both s t r u c t u r a l and f u n c t i o n a l s i g n i f i c a n c e . Previous discussion has emphasized membrane-mediated (phosphol i p i d ) phenomena; free f a t t y acids, which can act on membranes or have completely d i f f e r e n t modes of action, have also been used to perturb fungal morphogenesis. Although the same physical properties outlined above may regulate t h e i r e f f e c t s , s e l e c t i v e metabolism of a l i m i t e d number of s p e c i f i c compounds, e.g. by lipoxygenase enzymes, can often be of greater regulatory s i g n i f i c a n c e . This aspect of f a t t y acid metabolism i s presented i n greater d e t a i l i n appropriate sections. Membrane Transport Fungi have f a t t y acid c a r r i e r systems, often d i s p l a y i n g some degree of s p e c i f i c i t y e.g. f o r 12- and 14-C compounds vs. 16- and 18-C saturated or unsaturated f a t t y acids i n Saccharomyces uvarum and Saccharomyces l i p o l y t i c a (36). Exogenous f a t t y acids can be i n c o r porated or p a r t i t i o n e d i n t o membrane phospholipids and subsequently modify c a r r i e r systems f o r other classes of compounds. Short chain compounds, e.g. formate, acetate, butyrate, c a p r y l i c and caproic acids i n h i b i t phosphate (37) and thiamine (38) uptake by Saccharomyces cerevisiae, perhaps by blocking the use of energy from adenosine triphosphate. The k i n e t i c s of arginine uptake by anaerobically grown S* cerevisiae i s considerably a l t e r e d following incorporation of l i n o l e i c acid i n t o c e l l s when compared to o l e i c acid-enriched c e l l s (39). On the basis of comparative i n h i b i t i o n studies, i t was concluded that the a c t i v i t y of membrane-bound transport proteins was probably altered by the nature of surrounding f a t t y acids. Several related studies exploited d i f f e r i n g f a t t y acid compositions of thermophilic, mesophilic and psychrophilic species of Torul o p s i s (40) and several other species of yeast (41). Glucose and leucine uptake occurred at measurable rates only at temperatures at which the various fungi grew. The f a t t y acid unsaturation index was high for psychrophilic species, intermediate for mesophiles and low f o r thermo-tolerant yeasts, and t h i s was related to possible mediation of transport processes by membrane f l u i d i t y . Enzymic Regulation Fatty acid auxotrophs of j>. cerevisiae have been extensively u t i l i z e d to probe r e l a t i o n s h i p s between f a t t y a c i d unsaturation and enzymic a c t i v i t y . A major area of i n v e s t i g a t i o n has been e l u c i d a t i o n of the bases f o r loss of oxidative phosphorylation by yeast cultures characterized by less than ca. 20-30% unsaturated f a t t y acid (42-44). Much reduced or no a c t i v i t y i n mitochondrial membranes with highly saturated f a t t y a c y l compositions has been documented f o r cytochromes a, a3, b and c, mitochondrial ATPase, succinate oxidase and NADH oxidase (45-47). Fatty acids apparently mediate many mitochondrial functions



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including the rate of p r o t e o l y s i s of mitochondrial t r a n s l a t i o n prod ucts (48), dihydrolipoate-induced ATP synthesis i n promitochondria (49), mitochondrial p r o l i f e r a t i o n (50) and release from glucose r e pression (51). The Δ-9 isomer was the most e f f i c i e n t of the c i s octadecenoic acid compounds evaluated i n t h i s l a t t e r study, with eicosaenoic acid supporting 15% of the release induced by o l e i c a c i d . A l l of the l i s t e d processes were i n h i b i t e d by saturated f a t t y acids. L i n o l e n i c acid generally accelerates the a c t i v i t y of a number of enzymes i n anaerobic yeast cultures, including i s o c i t r a t e lyase, malate synthase and malate dehydrogenase (52), and s t e r o l synthesis (53). Linoleate and oleate had progressively less e f f e c t on synthesis of the three enzymes i n the former study (52). Oleic and l i n o l e i c acid supplementation at 0.01% i n anaerobic yeast cultures induced a 5- to 10-fold increase i n s t e r o l formation while e l a i d i c acid i n h i bited induction (54). Stearic and p a l m i t i c acids suppressed anaerobic growth while l a u r i c acid had no e f f e c t on e i t h e r parameter. In a related i n v e s t i g a t i o n on s t e r o l metabolism, o l e i c and l a u r i c acids abolished growth i n h i b i t i o n by hypocholesteremic compounds, with saturated acids having no e f f e c t (55). In other instances unsaturated f a t t y acids can strongly repress S^. cerevisiae enzymic a c t i v i t y e.g. alcohol acetyltransferase (56), acyltransferases involved i n g l y c e r o l i p i d synthesis (57), f a t t y acid biosynthesis ( i n both cerevisiae and Candida l i p o l y t i c a , 58) and a c e t y l CoA carboxylase (59). Some enzymes are repressed by long chain f a t t y acids (57-59), while unsaturated compounds have s p e c i f i c e f f e c t s i n others (56). Defined mechanisms f o r these diverse respon ses are not c l e a r , although nonspecific surfactant action i s probably rare (57) and f a t t y acid metabolites mediate some repressive f a t t y acid e f f e c t s (59). Temperature Adaptation and Fatty Acid Composition The f a t t y acid composition of microorganism membranes can be a l t e r e d by varying temperature during growth, with the usual pattern being an increase i n short chain and/or unsaturated compounds with decreasing growth temperature (60-64). Membrane f l u i d i t y appears to be under regulatory c o n t r o l (65) since i t must be maintained at a l e v e l com p a t i b l e with c e l l growth and function (60,66,67). A number of fungi produce more highly unsaturated f a t t y acids when grown or adapted to grow at low temperatures (68); however, tem perature adaptation can involve increased synthesis of d i s t i n c t 18-C monounsaturated or diunsaturated compounds by Mucor mucedo and Asper g i l l u s ochraceous, respectively (69); s i g n i f i c a n t s h i f t s from s t e a r i c to p a l m i t i c acid by IS. cerevisiae (70); increasing amounts of palmit o l e i c a c i d s p e c i f i c a l l y without changing the t o t a l quantity of unsat urated f a t t y acids by the psychrophilic yeast Candida u t i l i s (71); enhancement of l i n o l e n i c acid at the expense of mono- and diunsat urated 18-C compounds by three species of Leucosporidium (72); or the synthesis of rare highly unsaturated compounds, e.g. Δ-6,9,12,15octadecetetraenoic acid by the Zygomycete Thamnidium elegans (73). A related study documented greater amounts of unsaturated f a t t y acids i n spores and mycelium of several species of thermophilic and thermotolerant mucoralean fungi when grown at 25°C rather than 48°C (74). These authors suggested that perhaps low dissolved oxygen i n growth media at high temperatures would support only reduced l e v e l s



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of f a t t y acid desaturase a c t i v i t y as had been proposed previously for other species of fungi (75,76). This may have occurred i n t h e i r ex perimental system; however, considering the wide range of fungal species examined under diverse c u l t u r a l conditions, i t i s u n l i k e l y that t h i s hypothesis can serve generally to explain r e l a t i o n s h i p s between unsaturation and temperature acclimation. Exceptions to cold temperature adaptation being accompanied by shorter chain or more unsaturated f a t t y acids include m y c e l i a l phos pholipids with greater unsaturation when grown at 36°C vs 20°C (77), and the lack of c o r r e l a t i o n of cold temperature acclimation with increased l i p i d unsaturation or membrane f l u i d i t y by four fungi representative of four d i f f e r e n t fungal classes (78). Many of these changes i n membrane f a t t y acid composition proba bly act by modulating enzymic a c t i v i t y by regulating l o c a l i z e d or bulk membrane f l u i d i t y ; however, since most studies have not measured rates or ranges of l i p i d or protein motion as a function of changes i n f a t t y acid composition, or even d i f f e r e n t i a t e d between t o t a l c e l l and phospholipid composition, there i s l i t t l e d i r e c t support f o r t h i s hypothesis. An a d d i t i o n a l complication i s the possible presence of l a t e r a l l y segregated l i p i d domains which could have profound e f f e c t s on protein assembly or enzyme a c t i v i t y with minimal changes i n bulk membrane f l u i d i t y . Fatty Acids and Fungal Growth There has been extensive use of anaerobic cultures or desaturase mu tants of S_. cerevisiae to probe the unsaturated f a t t y a c i d growth requirements of t h i s yeast (79). A number of i n v e s t i g a t i o n s have attempted to formulate empirical rules r e l a t i n g f a t t y a c i d structure to t h e i r a b i l i t y to support growth. Using a desaturase mutant of IS. cerevisiae, 23 f a t t y acids were evaluated and only those possessing a c i s double bond at C-9 supported growth (80). This claim was d i s puted using a d i f f e r e n t desaturase mutant of t h i s yeast i n which 16C to 22C f a t t y acids with double bonds i n positions M through Δ19 a l l supported growth (81). Arachidonic, eicosapentaenoic and docosahexaenoic acids had the highest e f f i c i e n c i e s ; i t was concluded that there was a d i r e c t c o r r e l a t i o n between greater number of double bonds and increased growth. A t h i r d study using a d i f f e r e n t desaturase mutant examined a number of cis-octadecenoate isomers and found a l l isomers between Δ7 and Δ12 able to support extensive growth (82). The Δ6 isomer was also e f f e c t i v e , which i s i n contrast to r e s u l t s from other studies (79,80) which showed no a c t i v i t y using t h i s compound. A more recent study using anaerobic cultures of J>. cerevisiae (79) concluded that those f a t t y acids which supported growth a l l had a maximum of 9 saturated C atoms before the occurrence of a c i s double bond or a hydroxyl group, or 7 saturated C atoms before the occurrence of a trans ethylenic bond. These rules are i n apparent general agreement with most published work although there are excep tions, e.g. the i n h i b i t o r y action of cis-All-octadecenoate re ported f o r one desaturase mutant (80) while supporting growth by S^. cerevisiae under anaerobic conditions (75,79). Requirements f o r s p e c i f i c f a t t y acids f o r S. cerevisiae growth can vary with s t e r o l structure (83) and i t i s probable that d i f f e r e n t desaturase mutants and d i f f e r e n t s t r a i n s of t h i s yeast grown anaerobically under d i f f e r ent culture conditions could also have varying f a t t y a c i d require-



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merits. There i s no compelling reason to assume d i f f e r e n t i s o l a t e s should have i d e n t i c a l metabolic requirements; t h i s alone i s s u f f i cient to explain occasional discrepancies i n the l i t e r a t u r e concerning unsaturated f a t t y acid needs. Saturated f a t t y acids are also required f o r growth by :S. cerev i s i a e (84,85), with a negative c o r r e l a t i o n of growth demonstrated for high l e v e l s of dodecenoic and tridecenoic acids and a p o s i t i v e c o r r e l a t i o n with the presence of m y r i s t i c , pentadecenoic and p a l m i t i c acids (86). Fatty acid supplementation a f f e c t s the growth of a number of fungi. Among the documented e f f e c t s are the promotion of growth of the crustacean parasite Haliphthoros milfordensis by o l e i c a c i d , t r i o l e i n and t r i p a l m i t o l e i n . Saturated f a t t y acids were not e f f e c t i v e while polyunsaturated f a t t y acids were i n h i b i t o r y (87). When s i x thermophilic fungi were grown at 45°C on various f a t t y acids, optimum y i e l d s were obtained for a l l species on m y r i s t i c , p a l m i t i c , s t e a r i c and o l e i c acids except for one i s o l a t e of Chaetomium which could not grow on m y r i s t i c a c i d (88). Capric, c a p r y l i c , l a u r i c , l i n o l e i c and arachidic acids were not u t i l i z e d by any of the fungi. Comparative studies of several i s o l a t e s of saprobic s k i n fungi provided d i r e c t evidence that d i f f e r e n t s t r a i n s of the same species can have diverse growth requirements as speculated previously concerning S_. c e r e v i s i a e . A l l three s t r a i n s of Pityrosporum ovale u t i l i z e d o l e i c acid, but i n addition one used m y r i s t i c acid only, one used l a u r i c and m y r i s t i c acids, and a t h i r d could use those two acids plus p a l m i t i c and s t e a r i c (89,90). A related species, Pityrosporum orbiculare, u t i l i z e d only m y r i s t o l e i c and p a l m i t o l e i c acids (90). It i s apparent from t h i s b r i e f o u t l i n e that fungi have diverse f a t t y acid requirements to sustain growth, r e f l e c t i n g t h e i r adaptation to a v a r i e t y of saprobic and p a r a s i t i c habits. E l u c i d a t i o n of the precise r o l e of s p e c i f i c f a t t y acids w i l l probably r e f l e c t some of the enzymic a c t i v i t i e s outlined above, and requires research directed towards s p e c i f i c morphogenetic controls before more than the simplest of generalizations can be made. Sporulation Changes i n d i s t r i b u t i o n and abundance of l i p i d s have been documented i n fungi undergoing asexual or sexual reproduction (91-93); however, r a r e l y have s p e c i f i c structure-function r e l a t i o n s h i p s been establ i s h e d . Recently an exogenous requirement f o r s p e c i f i c f a t t y acids has been documented f o r the induction and maturation of the sexual oospore stage of Lagenidium giganteum, a f a c u l t a t i v e parasite of mosquito larvae (94,95). Free f a t t y acids and t r i a c y l g l y c e r o l s cont a i n i n g saturated f a t t y acids from 14C to 20C i n length induce l i m i ted numbers of v i a b l e oospores i n l i q u i d c u l t u r e . P a l m i t o l e i c acid added to basal growth media containing appropriate s t e r o l s induced s i g n i f i c a n t oosporogenesis, while o l e i c acid was less e f f e c t i v e and l i n o l e i c and l i n o l e n i c acids were t o x i c at r e l a t i v e l y low concentrations; however, o l e i c and l i n o l e i c acids added as mono-, d i - or t r i a c y l g l y c e r o l s produced the maximum number of oospores. T r i l i n o l e n i n , arachidonic and 11,14,17-eicosatrienoic acids induced no or few oopores (95). Analysis of whole c e l l f a t t y acids confirmed that a l l exogenous f a t t y acids were taken up from the media, although saturated f a t t y acids were incorporated more slowly. Metabolism of the



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exogenous f a t t y acids occurred but to a l i m i t e d extent. Delayed addition of t r i o l e i n to developmentally synchronized cultures r e s u l ted i n a gradual loss of oospore induction and maturation, which varied according to the concentration of added c h o l e s t e r o l (95). Exogenous f a t t y acids are incorporated by L. giganteum i n t o a l l major classes of l i p i d s including phospholipids (unpubl. observ.). Membrane-mediated metabolism may p a r t l y explain the p o s i t i v e e f f e c t of c e r t a i n unsaturated f a t t y acids on oosporogenesis, which would increase b i l a y e r f l u i d i t y since the major f a t t y acid synthesized by L^. giganteum on unsupplemented media i s p a l m i t i c acid (95). A regu l a t o r y r o l e i n oospore induction has been documented f o r c y c l i c nucleotides (96) and b i l a y e r f l u i d i t y i s known to modify the a c t i v i t y of adenylate cyclase (30). Neurospora crassa mutants characterized by an aberrant colony morphology and a corresponding c y c l i c AMP de f i c i e n c y reverted to normal c y c l i c nucleotide l e v e l s and colony morphology when l i n o l e i c , o r l i n o l e n i c or arachidonic acid was added to basal growth media (97). P a l m i t o l e i c , o l e i c or 16C to 20C satur ated compounds had no e f f e c t . I t i s possible that a comparable pro cess i s operating i n the induction of L^. giganteum oosporogenesis. I t i s i n t e r e s t i n g to note that the Myxomycete Dictyostelium d i s c o i deum, whose morphogenesis i s also regulated by c y c l i c AMP, dramati c a l l y increases i t s c e l l u l a r unsaturated f a t t y acids, e s p e c i a l l y octadeca-5,11-dienoic a c i d , during i t s development from yeast to mature sporocarp (98). Arachidonic acid and i t s methyl, e t h y l and propyl esters a f f e c t the movements of the m u l t i c e l l u l a r migratory slug stage of t h i s organism at micromolar concentrations (99). Methyl esters of o l e i c , l i n o l e i c and l i n o l e n i c acids also disoriented slug phototaxis at 100-200 μΜ concentrations. An unspecified prostaglandin at 10 μΜ and 0.1 μΜ leukotriene B4 had no e f f e c t . Two other i s o l a t e s of L. giganteum (ATCC nos. 48336 and 48337) do not produce oospores i n v i t r o using the protocol b r i e f l y described above for the C a l i f o r n i a s t r a i n (ATCC no. 52675) of the fungus. The two former i s o l a t e s also r a r e l y produce oospores following i n f e c t i o n of laboratory-reared Culex t a r s a l i s mosquito larvae; however, large numbers of oospores are induced following i n f e c t i o n of an alternate l a r v a l host, Chaoborus astictopus, the Clear Lake gnat. They also produce oospores i n media supplemented with cod l i v e r o i l (unpubl. observ.). Preliminary i n v e s t i g a t i o n of t h i s phenomenon has implicated 5,8,11,14,17-eicosapentaenoic acid (EPA) as the s p e c i f i c compound responsible f o r oospore induction. EPA i s present i n low q u a n t i t i e s i n laboratory-reared 0· t a r s a l i s larvae, which are incapable of syn thesizing t h i s f a t t y acid and must obtain i t from t h e i r d i e t (R. Dadd, unpubl. observ.); however, i t i s present i n r e l a t i v e l y high concentrations i n cod l i v e r o i l and fourth i n s t a r diapausing C_. astictopus larvae (unpubl. observ.). This phenomenon i s discussed i n more d e t a i l below i n r e l a t i o n to lipoxygenase mediation of fungal morphogenesis. Spore Germination Regulation of spore germination by s p e c i f i c f a t t y acids has been documented f o r several fungi. Macroconidial germination by Microsporum gypseum i s stimulated by o l e i c , l i n o l e i c and arachidonic acids, while short chain saturated compounds were i n h i b i t o r y and longer chain f a t t y acids had no e f f e c t (100). An even more s p e c i f i c



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response has been demonstrated f o r asexual conidia produced by Ernyia v a r i a b i l i s , a parasite of small dipteran insects (101). Conidia (asexual spores) are f o r c i b l y discharged by the fungus and subse quently develop i n one of four ways: d i r e c t vegetative germination which leads to i n f e c t i o n of a p o t e n t i a l insect host; formation and discharge of a smaller secondary conidium with the same developmental p o s s i b i l i t i e s as primary conidia; mycelial growth dependent s o l e l y on endogenous reserves; or death. In the presence of a basal agar medium containing yeast extract, c h i t i n or chitosan, vegetative c o n i d i a l germination i s induced by o l e i c a c i d . Oleic acid on water agar alone resulted i n secondary sporulation or death of discharged c o n i d i a . P a l m i t o l e i c , l i n o l e i c and l i n o l e n i c acids were t o x i c to conidia over a wide range of concentrations, and an excess of o l e i c acid could mitigate these t o x i c e f f e c t s . P a l m i t o l e i c acid was unique among the f a t t y acids which k i l l e d conidia i n i t s promotion of mycelial growth subsequent to spore germination. Saturated f a t t y acids from 14C to 20C induced p r i m a r i l y secondary sporulation (101). In a related study i t was shown that E. v a r i a b i l i s conidia ger minate vegetatively on the c u t i c u l a r surface of adults of the lesser housefly, Fannia c a n i c u l a r i s , and on basal media containing yeast extract plus l i p i d extracts of adult f l y c u t i c l e s . In contrast, conidia developed p r i m a r i l y i n t o secondary spores when discharged onto puparia of Έ_· c a n i c u l a r i s or basal media supplemented with puparial c u t i c u l a r l i p i d s . Although the r e l a t i v e free f a t t y acid compositions of the two d i f f e r e n t stages of housefly were nearly i d e n t i c a l , adult c u t i c l e s contained nearly f i v e times as much free f a t t y acid as puparial c u t i c u l a r surfaces. I t was concluded that the l i m i t a t i o n of host range of E_. v a r i a b i l i s to adult dipterans was due i n part to c h a r a c t e r i s t i c s of the f a t t y acids of t h i s order of i n sects, i . e . s u f f i c i e n t o l e i c acid to induce spore germination; high l e v e l s of p a l m i t o l e i c acid to enhance mycelial growth; and r e l a t i v e l y low l e v e l s of i n h i b i t o r y polyunsaturated 18C acids (102). Subsequent research has shown that of several s t r a i n s of a re lated entomophthoralean fungus, Erynia radicans, i s o l a t e d from a diverse group of insects, only c e r t a i n s t r a i n s respond to o l e i c a c i d by vegetative c o n i d i a l germination, and t h i s can be related to host range (A. U z i e l , R. Kenneth and I . Ben-Ze'ev, pers. communication). I t appears, therefore, that c u t i c u l a r f a t t y acid composition may have a d e f i n i t i v e r o l e i n regulating host range by a number of entomopathogenic fungi. Host-pathogen i n t e r a c t i o n s In addition to the entomophthoralean fungi discussed above, other laboratories have documented a r o l e for f a t t y acids i n the regulation of host-parasite i n t e r a c t i o n s (103). Research on a number of physio l o g i c a l races of Phytophthora infestans, some of which i n f e c t c u l t i vars of potato, Solanum tuberosum, has shown that both v i r u l e n t and a v i r u l e n t s t r a i n s of the fungus contain chemical e l i c i t o r s of hyper s e n s i t i v e resistance, including induction of sesquiterpenoid phytoa l e x i n accumulation (104). A f t e r a number of inconclusive reports, the e l i c i t o r s were i d e n t i f i e d as arachidonic (AA) and eicosapentaenoic (EPA) acids (105-107). Methyl esters of AA and EPA also e l i c t e d sesquiterpene accumulation (106) a f t e r a s l i g h t delay, while the propyl ester was much less active (103). A l l saturated f a t t y


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acids, 18C compounds and arachidonyl cyanide f a i l e d to induce hyper s e n s i t i v e responses (103,108). Among the e i c o s a t r i e n o i c compounds tested, the Δ11,14 and Δ11,14,17 isomers were weakly active while the Δ5,8,11 and Δ5,11,14 compounds were highly active (103,108). The Δ5 bond appears to be of importance f o r a c t i v i t y , perhaps due to enzymic substrate s p e c i f i c i t y , leading to a more metabolically active compound. Such enzymic modification could be due to l i p o x y genase or cyclooxygenase a c t i v i t y , which i s discussed i n the f i n a l section of t h i s review. Regulation of fungal morphogenesis by lipoxygenase and cyclooxygenase products Using the P^. infestans-potato tuber system outlined above, e l i c i t o r induced accumulation of phytoalexins has been i n h i b i t e d by s a l i c y l hydroxaraic acid and d i s u l f i r a m (103,109,110); these compounds i n h i b i t both cyanide-resistant r e s p i r a t i o n and lipoxygenase enzymes, these l a t t e r acting s p e c i f i c a l l y on polyunsaturated f a t t y acids with a c i s 1,4-pentadiene system to form hydroperoxides (111). I t has been speculated without further data that lipoxygenase a c t i v i t y may be involved i n e l i c i t i n g the hypersensitive response i n potato tubers (103). Direct i m p l i c a t i o n of cyclooxygenase enzymes i n fungal morpho genesis has been obtained using the Oomycetes Achlya caroliniana, Achlya ambisexual!s and Saprolegnia p a r a s i t i c a . A s p i r i n and indomethacin, both cyclooxygenase i n h i b i t o r s , reduced or eliminated mycelial growth by these fungi, often inducing abnormal colony mor phology i n l i q u i d culture (112). Inhibited colonies i n the presence of 0.1 mM indomethacin, when allowed to grow f o r more than 10 days, assume normal colony morphology, but do not undergo sexual reproduc t i o n (oosporogenesis). Addition of prostaglandin F i ( P G F i ) at 2 μg/ml p a r t l y overcame indomethacin induced growth i n h i b i t i o n , while PGF2 and PGE had no e f f e c t . The authors suggested growth and oosporogenesis may be regulated by the i n t e r a c t i o n of a prosta glandin or PG-like substance produced v i a cyclooxygenase f a t t y acid metabolism with s t e r o i d or other hormones (112). Using a developmentally synchronized L. giganteum culture system, our laboratory has documented that stage-specific addition of l i p o x y genase i n h i b i t o r s (nordihydroguaiaretic acid, esculetin, ornaphthol, propyl g a l l a t e , salicylhydroxamic acid), at concentrations which do not a f f e c t asexual reproduction, blocks the induction and maturation (gametangial fusion, meiosis, spore c e l l w a l l formation, v e s i c u l a r fusion w i t h i n the immature spore) of oospores by t h i s fungus. Cyclo oxygenase i n h i b i t i o n using ibuprofen and to a lesser extent indo methacin had comparable s p e c i f i c effects on oosporogenesis. The cyclooxygenase i n h i b i t o r s a s p i r i n , s a l i c y l i c acid and phenylbutazone, however, had minimal effect on L. giganteum oosporogenesis at concen trations less than those which had a deleterious effect on mycelium (unpubl. observ.). This does not preclude a r o l e f o r cyclooxygenase metabolism i n oosporogenesis by t h i s fungus since the L. giganteum enzymes may be r e l a t i v e l y i n s e n s i t i v e to i n h i b i t i o n by these com pounds. Also, the e f f e c t s of these i n h i b i t o r s on L. giganteum oxygenases, which may be d i f f e r e n t from those documented i n mammal ian systems, have not been investigated. Tentative confirmation by t h i n layer chromatography (114) has a

a

a



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been made for the production of both cyclooxygenase and lipoxygenase products by L . giganteum, with more of both types of fatty acid metabolites i s o l a t e d from the l i q u i d culture medium than from the my celium (unpubl. observ.). This implicates these compounds i n some type of i n t e r c e l l u l a r communication r o l e . An i n t r a c e l l u l a r regulatory r o l e i s also suggested for the lipoxygenase metabolites by the i n h i b i t o r y effects on oospore maturation. The i n h i b i t o r y effects of nordihydroguaiaretic acid on oospore induction could be reversed using p a r t l y p u r i f i e d eicosanoid extracts from growth media (113). Further e l u c i d a t i o n of the nature and stage-specific synthesis of these prod ucts i s i n progress. Arachidonic a c i d , which i s synthesized by L . giganteum (95), and eicosapentaenoic acid, which i s not, are suitable substrates for cy clooxygenase and lipoxygenase. Since two strains of t h i s f a c u l t a t i v e parasite appear to i n i t i a t e oosporogenesis only i n the presence of exogenous EPA (unpubl. observ.), t h i s compound i s h i g h l y suspect as a p o t e n t i a l regulatory molecule, perhaps after i t s lipoxygenase-raediated metabolism to a more active product. I t i s noteworthy that EPA i s present, often as the p r i n c i p a l fatty acid, i n a number of Oomy cetes (103,107,112,114). Further evaluation of the r o l e of EPA i n sexual reproduction by t h i s p r i m i t i v e class of fungi i s warranted. Leukotrienes are a recently discovered class of lipoxygenase metabo l i t e s with highly potent b i o l o g i c a l effects (116-118). I t i s possible that these or related metabolites play a central regulatory role i n fungal morphogenesis. Lipoxygenase regulation of morphogenesis may not be l i m i t e d to the oomycetous fungi described above. As an extension of a previous hypothesis regarding possible fatty acid hydroperoxide involvement i n c o n i d i a l germination by the Zygomycete Erynia v a r i a b i l i s (101), a s t r a i n of Erynia delphacis, a related parasite of small adult d i p teran insects, was investigated. Conidia discharged by 15. delphacis, which germinate vegetatively on yeast extract supplemented with t r i o l e i n , undergo nearly 100% secondary sporulation when 150 μΜ n o r d i hydroguaiaretic acid i s added to the germination medium (unpubl. observ.) These i n i t i a l observations suggest that lipoxygenase-mediated morphogenesis may be widespread among fungi. Research on fungal eicosanoids w i l l provide insights into t h e i r basic developmental biology and serve as useful models for the role of these metabolites i n mammalian systems. Acknowledgments Writing of t h i s review was supported i n part by USDA research grant CR 806771-02, RF-4148A, R. K. Washino, P r i n c i p a l Investigator. I thank Dr. R. Herman for the preprint of h i s cyclooxygenase a r t i c l e .

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