- Email: [email protected]
Comparative utilization of sterols by the OomycetesLagenidium giganteum and Lagenidium callinectes
ZXPERIMENTAL
MYCOLOGY
Comparative
7,
227-232 (1983)
Utilization
of Sterols by the Oomycetes
giganteum
and Lagenidium
S. A. WARNER,~G. Biochemistry
Laboratory,
W, SOVOCOOL,AND
~a~e~~~~~
callinectes A.J. DQMNA~~
Department of Botany, University of North Carolina, Chapel Hill, North Carolina 27514 Accepted for publication March 3 1, 1983
WARNER, S. A., SOVOCOOL, G. W., AND DOMNAS, A. .I. 1983. Comparative utilization of sterols by the Oomycetes Lagenidium giganteum and Lagenidium callinectes. Expezimentat Mycoi5gy 7, 227-232. The mosquito-parasitizing Oomycete Lagenidium giganteum requires exogenous sterols before it can produce the infective zoospores. In contrast, L. callinectes will synthesize small quantities of cholesterol and produce zoospores. Since the former is a promising biological control agent it was important to delineate the metabolic role(s) and fate(s) of these sterols in zoosporogenesis. Twenty-three different sterols were administered to growing cultures of both organisms. These compounds were reisolated after 7 days, along with any metabolites produced from them. This approach was designed so as to lead to the outline of some of the metabolic sequences which were present. The data show that both organisms possess very similar patterns of sterol trans?ormation. The apparent metabolic route leading to cholesterol is cycloartenol -+ --+ fucosterol -+ -+ cholesterol, which is similar to the overall metabolic sequence observed in many algae. Some transformations that were indicative of obvious intermediate biosynthetic steps were apparent. Both Lagenidium spp. transformed 24alkylidene but no? 24-alkyl sterols to cholesterol. The organisms reduced cholesta-5,7-dienol to cholesterol, metabolized cholestanol to cholesterol, and transformed 3-ketosteroids to 3@hydroxysterols. Coprostanol was changed to cholestanol. INDEX DESCRIPTORS: Lagenidium callinectes; Lagenidium giganteum; sterols; sterol metabolism; cycloartenol; fucosterol; Oomycetes.
The mosquito-parasitizing Oomycete Lagenidium giganteum Couch will not produce the infective zoospores when grown on a simple undefined medium such as peptone-yeast extract-glucose (PYG).3 Zoosporogenesis can be induced either by growth on aqueous extracts of hemp or soy beans, or on sterol-free media which has been supplemented with sterols or mevaionic acid (Domnas et al., 1977). L. giganteum is unable to incorporate precursors which occur prior to mevalonic acid into sterols (Warner, 1982), yet its congener, L. callinectes Couch, will synthesize ’ Current address: Department of Biology, University of North Carolina, Chapel Hill, N.C. 27514. 2 To whom correspondence should be addressed. 3Abbreviations used: PYG, peptone-yeast extractglucose; GC, gas chromatography; MS, mass spectrometry.
very small quantities of cholesterol (Warmer and Domnas, 1981) and produce zoospores under the above culture conditions (Han and Amerson, 1973). The apparent inability of only one wild-type organism within a genus to synthesize a class of metabolicahy important compounds is uncommon. Since the former organism is a very promising biological control agent it was important to attempt to delineate the metabolic role(s) and fate(s) of these sterols in order to elevate zoospore yields. We also wished to investigate possible similarities of the steroli synthetic pathways of Oomycetes with plants which have been alluded to in earlier work (Bu’Lock and Osagie, 1976; Warner and Domnas, 1981; Warner et al., 198%; Warner et al., 1983). This paper reports on aspects of sterol transformation by E~~Hzidium spp. and discusses the evolutionary and metabolic ramifications of our results.
227 0147-5975/83 $3.00 Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any faorm teserved.
228
WARNER,
MATERIALS
AND
SOVOCOOL,
METHODS
Minimization of chemical contamination. Highest commercial purity (HPLCgrade) solvents were used to reduce the levels of chemical impurities (Karasek et al., 1981), coupled with solvent prerinsing and bake-out at 100°C of all durable items which contacted the mycelia and the lipid extracts (Warner et al., 1981). Organisms and growth conditions. Lagenidium giganteum Couch from North Carolina was used for this study (Umphlett and Huang, 1972) and may be obtained from the American Type Culture Collection (Rockville, Md.) as ATCC No.36292. Lagenidium callinectes Couch, isolated from shrimp larvae (Lightner and Fontaine, 1973), was used as a sterol-synthesizing control and. may be obtained as isolate L-3-b from C. E. Bland of East Carolina University, Greenville, North Carolina. Both organisms were cultured for 7 days on a Gyrotory shaker at 60 rpm under continuous illumination at 23-2X. The culture vessels were 250~ml Erlenmeyer flasks capped with aluminum foil and filled with 100 ml of PYG (peptone-yeast extract-glucose) medium (Fuller, 1978) for growth of L. giganteum (Domnas et al., 1974). The same procedure was used for culture of L. callinectes except that 2% RILA artificial sea salts (Carolina Biological Co.) were also added to the culture medium (Warner and Domnas, 1981). Analyses of the culture media by the methods described below showed them to be sterol free to our limit of detection. Inoculation was with l-cm2 agar blocks from stock cultures on the same media. The culture media were sterilized by autoclaving at 121°C for 20 min. Zoosporogenesis in L. gigantewm was induced as previously described (Domnas et al., 1974), or by immersion of mycelia in sterile deionized, distilled water. A solution of 2% RILA sea salts in sterile deionized, distilled water was used for the induction of zoosporogenesis in L. callinectes.
AND
DOMNAS
Sterol supplements. Cycloartenol (9B, 19cycle-lanost-24-enol) was isolated by procedures previously described (Warner et al., 1982) from green jackfruit (Artocarpus integrifolia) which were a generous gift from Y. Sagawa of the Lyon Arboretum of the University of Hawaii (Manoa). Ostreasterol (24-methylenecholesterol, ergosta-5,24(28)dienol) was isolated by the same procedures from Saguaro cactus pollen (Carneigiea gigantea) obtained through the generosity of L. Standifer of the United States Department of Agriculture Honeybee Research Laboratory (Tucson, Ariz.). Cholesterol (cholest-5-enol), cholestanol (5a-cholestanol), coprostanol(5B-cholestanol), epicholesterol (cholest - 5 - en - 3a - ol), epicholestanol (ICY - cholestan -3a - ol), epicoprostanol (5B -cholestan - 3o - ol), 5a cholestan - 3 - one, 5B - cholestan - 3 - one, cholest - 5 - en - 3 - one, brassicasterol (24Bergosta - 5,22 - dienol), lophenol (4a - methylcholest - 7 - enol), campesterol (24a ergost - 5 - enol), lathosterol (cholest - 7 enol), and fucosterol (stigmasta - 5,24(28)E - dienol) were obtained from Steraloids Inc. (Wilton, N.H.). Poriferasterol (24B - stigmasta - 5,22 - dienol) and cholesta - 5,7 dienol were obtained from Research Plus Steroid Laboratories (Denville, N.J.). Desmosterol (cholesta - 5,24 - dienol) and lanosterol (lanosta -8,24 - dienol) were obtained from Applied Science Laboratories (State College, Pa.). Sitosterol (24a - stigmast - 5 - enol) and stigmasterol (24a - stigmasta - 5,22 - dienol) were obtained from Aldrich Chemical Company (Milwaukee, Wise.). Ergosterol (24B -ergosta - 5,7,22 trienol) was obtained from the Sigma Chemical Company (St. Louis, MO.). Where necessary, sterols were further purified by the methods of Thompson et al. (1980). The purity of all sterols was established through GC and. GC/MS as described below and exceeded 99% at use. Sterol (1 mg) was added to each culture flask in 1 ml of chloroform along with 1 ml of 2% aqueous Tween 80 (Sigma Chemical
STEROL
METABOLISM
Co.) prior to autoclaving (Warner and Domnas , 198 1). Uninoculated control cultures were examined for each sterol supplement after seven days so as to verify that any changes observed were due to metabolism by the organisms. Each experiment was replicated at least three times. Isolation of sterols. Vegetative mycelia were harvested and the total lipids isolated, saponified, and the total sterols isolated as described previously (Warner and Domnas, 1981; Warner et a/., 1982). Analysis ofsterols. Identification of each sterol reported below was based upon GC with four different liquid phases, GC/MS, and also uv spectroscopy, where applicable. Sterols were identified by GC with a Packard 417 chromatograph (Downer’s Grove, Ill.) equipped with dual quartz jet flame ionization detectors. The instrument was interfaced with a Perkin-Elmer Sigma 1OB chromatography data station (Norwalk, Conn.). Columns employed were 3% SE-30, N2 flow rate 20 ml/min; 1% QF-1, min; 3% Hi-Eff SBP, Nz NZ flow r flow rate ; and 2% PMPE, Nz flow rate 30 mlimin (Patterson, 1971). All columns were &mm x 2-m glass. Oven temperature was 240°C with detector and injector temperatures of 260°C. Ultraviolet spectra were obtained with a Perk&-Elmer 124D dual-beam spectrophotometer for sterols suspected of possessing conjugated double bonds. The sterols were fractionated (Warner et al., 1982), dissolved in absolute ethanol, and their spectra comared to those of authentic standards. CC/MS was performed with a Finnegan 4032 instrument (Sunnyvale, Calif.). A 13m flexible fused silica capillary column coated with QV-101 was programmed from 168 to 260°C at 1OYYmin with an initial 3min hold. Helium was the carrier gas and injection wa.s in the splitless mode. The quadrapole mass spectrometer was used in the electron-impact mode with an electron energy of 70 eV. The instrument was interfaced with an INCOS data system. This
229
BY Logenidium
configuration was sensitive to 100 ng per injection. Identifications were based u standards and references spectra. Corn isons were based upon m/e and relative intensity of M i , (M - CH3) + , ( (M - [Hz0 + CH,])+, and a uisite fragments. RESULTS
The products of sterol metabolism by the two Oomycetes were examined for the presence of any unusual compounds which could account for the induction of zoos rogenesis by exogenous sterols in L. gig teum. L. callinectes was used as a positive control in this study since it is capable sf de novo sterol synthesis (Warner and Domnas, 1981). Twenty-three different sterols were administered to cultures of both organisms and, after 7 days, reisolated, along with any metabolites produced from them. The results of these studies are shown in Table 1. L.gigankeum grown without sterol contained 6% (+- 0.01% variation for these determinations) lipid on a dry weight basis. Addition of sterol to the culture me&u increased the lipid content to 6.2% (1-2 p-lg total sterolJmg dry wt). L. callinectes jkp3\Nlar without sterol contained 2% lipid, 0.0003% of which was sterol (6-7 ng sterol/mg dry wt). Addition of sterol to its culture medium raised the total lipid content to 2.2% (1-2 kg total sterollmg dry weight). Free lipids, other than nnmetaboIize~ sterol supplements, were not present in the culture broth. Examination of the mycelia after induction of zoosporogenesis but zoospore release showed no ch values or in the results shown in Ta Addition of any of the above with the solubilizer Tween 80, affect the total dry weight of mycelium from the broth cultures, with growth elevated 2&40% in L. giganteum and inhibited 60% in L. callinectes when compared to cultures grown without sterol and Tween 80. These effects were independent of the particular
230
WARNER,
SOVOCOOL,
Sterols Isolated from Lagenidium Administered sterol None Brassicasterol Campesterol Cholestanol Sa-Cholestanone
SS-Cholestanone 5-Cholestenone Cholesterol Coprostanol CycloartenolC Cholesta-5,7-dienol Desmosterol Epicholestanol Epicholesterol Epicoprostanol Ergosterol Fucosterol LanosterolC Lathosterol
Lophenol Ostreasterol Poriferasterol Sitosterol Stigmasterol
AND DOMNAS
TABLE 1 spp. following Growth with Exogenous Sterols
L. giganteum Sterols isolated”
-
L. callinectes wt%b
-
Brassicasterol Campesterol Cholesterol Cholestanol Cholestanol Cholesta-5,7-dienol Soi-Cholestanone Cholesterol Cholestanol
100 100 90 16 48 28 12 12 100
5-Cholestenone Cholesterol Cholesterol Coprostanol Cholesterol Cycloartenol Cholesterol Fucosterol Cholesterol Cholesta-5,7-dienol Desmosterol Epicholestanol Epicholesterol Epicoprostanol Ergosterol Cholesterol Lanosterol Cholesterol Lathosterol Mt = 414 (1.03) So-Cholestanone Lophenol Ostreasterol Cholesterol Poriferasterol Sitosterol Stigmasterol
61 39 100 90 10 48 43 9 90 10 100 100 100 100 100 100 100 54 35 11
NQ
100 70 30 100 100 100
Sterols isolated”
wt%b
Cholesterol Brassicasterol Campesterol Cholesterol Cholestanol Cholestanol So-Cholestanone Cholesterol Cholesta-5,7-dienol Cholestanol SB-Cholestanone Cholesterol Cholestanol Cholesterol Cholesterol Coprostanol Cholesterol Cycloartenol Cholesterol Fucosterol Cholesterol Cholesta-5,7-dienol Desmosterol Epicholestanol Epicholesterol Epicoprostanol Ergosterol Cholesterol Lanosterol Lathosterol Sa-Cholestanone Cholesterol
100 loo 100 90 10 91 4 4 1 94 3 3 75 25 100 97 3 73 18 9 90 10 100 100 100 100 100 100 100 70 15 15
Lophenol Ostreasterol Cholesterol Poriferasterol Sitosterol Stigmasterol
100 84 16 100 100 100
a Unknown sterols are designated by their molecular ion. Values in parentheses refer to retention time relative to cholesterol on OV:IO¶. The C-24 configuration of the apparently unmetabolized 24-alkyI sterols was assumed to be unchanged. b The area normalization for the weight percent (+ 0.5%) was determined on SE-30. NQ = detected, but not at quantifiable levels. Values represent the mean of at least three determinations with a standard deviation of 1%. c Warner and Domnas, 1981.
sterol tested. Tween 80 alone inhibited the growth of L. callinectes by 50% but did not affect the growth of L. giganteum in comparison to cultures grown without Tween 80.
DISCUSSION
The inhibition of growth of L. callinectes by Tween 80 alone is unusual, since this solubilizer does not effect the growth of L.
STEROL
METABOLISM
and has actually been shown to stimulate growth in Phytophthora cactorum (Nes et al., 1979), another Oomycete which does not synthesize sterols. Similarly, all of the sterols uniformly stimulated the growth of L. giganteum and inhibited the growth of L. callinectes. In comparison, different sterols have varying effects on the growth of P. cactorum (Nes et al., 1979; Nes and Patterson, 1981). The exact reasons for these observations are not apparent, but may reflect major differences between the unknown compositions of the membranes of the three organisms. Both Lagenidium spp. exhibited an almost identical behavior in their metabolism of sterols. Neither organism appeared to metabolize 24-alkyl sterols of either the “CX” or “P” configuration; however, 24-alkylidene sterols (fucosterol and ostreasterol) were transformed. This is of particular interest since 24-alkyl sterols maximally induced zoosporogenesis in L. giganteum (Domnas et al., 1977). These apparently nonmetabolizable sterols may be more readily available for the unknown metabolic processes which are involved in the initiation of zoosporogenesis, and may be transformed to products not isolated and identified by the above methods. Neither organism was capable of the reduction of the 2%double bond. Both organisms metabolized cholestanol and cholesta-5,7-dienol to cholesterol; however, the only other sterol observed in the metabolism of lathosterol to cholesterol was cholestanol, which suggests that in de ytovo synthesis the 7-double bond may be reduced before the introduction of the 5-double bond. This result may ‘be contrasted with that which occurs in animal systems, where the S-double bond is usually introduced prior to the reduction of the 7-double bond (Dvornik et al., 1963). The 3a-hydroxy sterols (episterols) were not transformed, but the SP-hydro steroids, coprostanol and SF-cholestanone, were metabolized to cholestanol and cholesterol. Both organisms metabolized steroid 3-ketones to the corresponding 3P-hydroxy
giganteum
BY
Lagenidium
231
sterols. No evidence was obtained for the presence of any steroids analogous to the hormones found in Achlya spp. (Pup stone and Unrau, 1974). The metabolism of cycloartenol but not lanosterol by both species of Lagenidium is of particular interest (Warner and Domnas, 1981>, since these results suggest that the former is the first sterol naturally produced by the cyclization of 2,3-oxidosqualene; an the cyclization of this compound to cycloartenol but not lanosterol clearly divides photosynthetic (or photosynthetically-~~rived) organisms from all others (Nes and McKean, 1977; Nes and Nes, 1980). These data suggest an ancestral affinity to photosynthetic organisms, which is ~~rt~gr COProborated by studies with ~a~roleg~~a j&ax (Bu’Lock and Osagie, 1976) and rn~.~b~r~ of the Peronosporales (Warner et al.) 1982) ~ The sterol compositions of the ~~~yc~t~s differ from other fungi and are akin to those of the algae (McCorkindale et al., Bean et al., 1972; Warner et al., 1 which further suggests that their mode sterol synthesis may be akin to t mode, The synthesis and metaboli costerol, a typical algal sterol (Nes and McKean, 1977), which we have o~$~~ed here strengthens this argument. Lan~st~rol may also be formed by the isomerization of cycloartenol in some plants (Sekula an Nes, 1980), members of the P~r~~~s~~r~~es (Warner et al., 19621, and pro Saprolegnia ferax (Bu’Lock an 1976). We have recently presen inary data which show milfordensis, also of the utilize cyclaartenol b (Warner el al., X983), thus we propos,e that the utilization of only cy~~oarte~~~ versus both cyoloartenol and lanosterol ‘may represent a distinction of this order from at least the Peronosporales and Saprole ales. Further work is obviously require prove or disprove this hypothesis. The ability to metabolize cer$aia skroli structural features must reflect .the h metabolic capabilities of an or~a~is,~,
232
WARNER,
SOVOCOOL,
our approach has led to the outline of some of the metabolic sequences which are present. The data presented suggest that both Lagenidium spp. possess very similar postcycloartenol sterol synthetic pathways. The apparent metabolic route leading to cholesterol is cycloartenol terol.
+ + fucosterol + + choles-
Some transformations that were indicative of obvious intermediate biosynthetic steps were apparent from this study. The results of investigations in progress with specific inhibitors of sterol biosynthesis and their effect on sterol intermediate accumulation in cell-free extracts will aid in the ultimate interpretation of many of the above results. ACKNOWLEDGMENTS We wish to thank S. M. Fagan for excellent technical assistance. This work was supported in part by NIH Grant AI17024 to A.J.D. and a Mycological Society of America Fellowship to S.A.W. REFERENCES BEAN, G. A., PATTERSON, G. W., AND MOTTA, J. J. 1972. Sterols and fatty acids of some aquatic phycomycetes. Camp. B&hem. Physiol. B 43, 935939. BLAND, C. E., AND AMERSON, H. V. 1973. Observations on Lagenidium callinectes: Isolation and sporangial development. Mycologia 65, 310-320. BU’LOCK J. D., AND OSAGIE, A. U. 1976. Sterol biosynthesis via cycloartenol in Saprolegnia. Phytochemistry 15, 1249-1251. DOMNAS, A. J., GIEBEL, P. E., AND MCINNIS, T. M. 1974. Biochemistry of mosquito infection: Preliminary studies of biochemical change in Culex pipiens quinquefasciatus. J. Znvertebr. Pathol. 24, 293-304. DOMNAS, A. J., SREBRO, J. P., AND HICKS, B. F. 1977. Sterol requirement for zoospore formation in the mosquito-parasitizing fungus Lagenidium giganteam. Mycologia 69, 87.5-886. DVORNIK, D., KRAML, M., DUBUC, J., GIVNER, M., AND GAUDRY, R. 1963. A novel mode of inhibition of cholesterol biosynthesis. J. Amer.Chem. Sot. 85, 3309. FULLER, M. S. 1978. Lower Fungi in the Laboratory. University of Georgia Press, Athens. KARASEK, F. W., CLEMENT, R. E., AND SWEETMAN, J. A. 1981. Preconcentration for trace analysis of organic compounds. Anal. Chem. 53, 1050A-1058A.
AND DOMNAS
LIGHTNER, D. V., AND FONTAINE, C. T. 1973. A new fungus disease of the white shrimp Penaeus setiferus. J. Znvertebr. Pathol. 22, 94-99. MC~ORKINDALE, N. J., HUTCHINSON, S. A., PURSEY, B. A., SCOTT, W. T., AND WHEELER, R. 1969. A comparison of the types of sterol found in species of the Saprolegniales and Leptomitales with those found in some other phycomycetes. Phytochemistry 8, 861-867. NES W. D., PATIXRSON, G. W., AND BEAN, G. A. 1979. The effect of steroids and their solubilizing agents on mycelial growth of Phytophthoru cactorum. Lipids 14, 458-462. NES, W. D., AND PATTERSON, G. W. 1981. Effects of tetracyclic and pentacyclic triterpenoids on growth of Phytophthora cactorum. J. Nat. Prod. 44, 215220. NES, W. R., AND MCKEAN, M. L. 1977. Biochemistry of Steroids and Other Zsopentenoids. Univ. Park Press, Baltimore. NES, W. R., AND NES, W. D. 1980. Lipids in Evolution. Plenum, New York. PATERSON, G. W. 1971. Relation between structure and retention time of sterols in gas chromatography. Anal. Chem. 43, 116.5-1170. POPPLESTONE,C. R., AND UNRAU, A. M. 1974. Studies on the biosynthesis of antheridiol. Canad. J. Chem. 52, 462-468. SEKULA, B. C., AND NES, W. R. 1980. The identification of cholesterol and other steroids in Euphorbia pulcherrima. Phytochemistry 19, 1509-1512. THOMPSON, M. J., PATTERSON, G. W., DUTKY, S. R., SVOBODA, J. A., AND KAPLANIS, J. N. 1980. Techniques for the isolation and identification of steroids in insects and algae. Lipids 15, 719-733. UMPHLETT, C. J., AND HUANG, C. S. 1972. Experimental infection of mosquito larvae by a species of the aquatic fungus Lagenidium. J. Znvertebr. Pathol. 20, 326-331. WARNER, S. A. 1982. Biosynthesis and Metabolism of Sterols by Pantonemic Fungi. Ph.D. dissertation, The University of North Carolina at Chapel Hill. WARNER, S. A., AND DOMNAS, A. J. 1981. Evidence for a cycloartenol-based sterol synthetic pathway in Lagenidum spp. Exp. Mycol. 5, 184-188. WARNER, S. A., EIERMAN, D. F., SOVOCOOL, G. W., AND DOMNAS, A. J. 1982. Cycloartenol-derived sterol biosynthesis in the Peronosporales. Proc. Nat. Acad. Sci. USA 79, 3769-3772. WARNER, S. A., GRAHAM, M. S., SOVOCOOL, G. W., AND DOMNAS, A. J. 1981. Alkane contamination of lipids extracted from Lagenidium giganteum and Lagenidium callinectes. Lipids 16, 628-630. WARNER, S. A., SOVOCOOL,G. W., AND DOMNAS, A. J. 1983. Sterols of selected species of Oomycetes and Hyphochytridiomycetes. Mycologia 75, 285-291.
MYCOLOGY
Comparative
7,
227-232 (1983)
Utilization
of Sterols by the Oomycetes
giganteum
and Lagenidium
S. A. WARNER,~G. Biochemistry
Laboratory,
W, SOVOCOOL,AND
~a~e~~~~~
callinectes A.J. DQMNA~~
Department of Botany, University of North Carolina, Chapel Hill, North Carolina 27514 Accepted for publication March 3 1, 1983
WARNER, S. A., SOVOCOOL, G. W., AND DOMNAS, A. .I. 1983. Comparative utilization of sterols by the Oomycetes Lagenidium giganteum and Lagenidium callinectes. Expezimentat Mycoi5gy 7, 227-232. The mosquito-parasitizing Oomycete Lagenidium giganteum requires exogenous sterols before it can produce the infective zoospores. In contrast, L. callinectes will synthesize small quantities of cholesterol and produce zoospores. Since the former is a promising biological control agent it was important to delineate the metabolic role(s) and fate(s) of these sterols in zoosporogenesis. Twenty-three different sterols were administered to growing cultures of both organisms. These compounds were reisolated after 7 days, along with any metabolites produced from them. This approach was designed so as to lead to the outline of some of the metabolic sequences which were present. The data show that both organisms possess very similar patterns of sterol trans?ormation. The apparent metabolic route leading to cholesterol is cycloartenol -+ --+ fucosterol -+ -+ cholesterol, which is similar to the overall metabolic sequence observed in many algae. Some transformations that were indicative of obvious intermediate biosynthetic steps were apparent. Both Lagenidium spp. transformed 24alkylidene but no? 24-alkyl sterols to cholesterol. The organisms reduced cholesta-5,7-dienol to cholesterol, metabolized cholestanol to cholesterol, and transformed 3-ketosteroids to 3@hydroxysterols. Coprostanol was changed to cholestanol. INDEX DESCRIPTORS: Lagenidium callinectes; Lagenidium giganteum; sterols; sterol metabolism; cycloartenol; fucosterol; Oomycetes.
The mosquito-parasitizing Oomycete Lagenidium giganteum Couch will not produce the infective zoospores when grown on a simple undefined medium such as peptone-yeast extract-glucose (PYG).3 Zoosporogenesis can be induced either by growth on aqueous extracts of hemp or soy beans, or on sterol-free media which has been supplemented with sterols or mevaionic acid (Domnas et al., 1977). L. giganteum is unable to incorporate precursors which occur prior to mevalonic acid into sterols (Warner, 1982), yet its congener, L. callinectes Couch, will synthesize ’ Current address: Department of Biology, University of North Carolina, Chapel Hill, N.C. 27514. 2 To whom correspondence should be addressed. 3Abbreviations used: PYG, peptone-yeast extractglucose; GC, gas chromatography; MS, mass spectrometry.
very small quantities of cholesterol (Warmer and Domnas, 1981) and produce zoospores under the above culture conditions (Han and Amerson, 1973). The apparent inability of only one wild-type organism within a genus to synthesize a class of metabolicahy important compounds is uncommon. Since the former organism is a very promising biological control agent it was important to attempt to delineate the metabolic role(s) and fate(s) of these sterols in order to elevate zoospore yields. We also wished to investigate possible similarities of the steroli synthetic pathways of Oomycetes with plants which have been alluded to in earlier work (Bu’Lock and Osagie, 1976; Warner and Domnas, 1981; Warner et al., 198%; Warner et al., 1983). This paper reports on aspects of sterol transformation by E~~Hzidium spp. and discusses the evolutionary and metabolic ramifications of our results.
227 0147-5975/83 $3.00 Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any faorm teserved.
228
WARNER,
MATERIALS
AND
SOVOCOOL,
METHODS
Minimization of chemical contamination. Highest commercial purity (HPLCgrade) solvents were used to reduce the levels of chemical impurities (Karasek et al., 1981), coupled with solvent prerinsing and bake-out at 100°C of all durable items which contacted the mycelia and the lipid extracts (Warner et al., 1981). Organisms and growth conditions. Lagenidium giganteum Couch from North Carolina was used for this study (Umphlett and Huang, 1972) and may be obtained from the American Type Culture Collection (Rockville, Md.) as ATCC No.36292. Lagenidium callinectes Couch, isolated from shrimp larvae (Lightner and Fontaine, 1973), was used as a sterol-synthesizing control and. may be obtained as isolate L-3-b from C. E. Bland of East Carolina University, Greenville, North Carolina. Both organisms were cultured for 7 days on a Gyrotory shaker at 60 rpm under continuous illumination at 23-2X. The culture vessels were 250~ml Erlenmeyer flasks capped with aluminum foil and filled with 100 ml of PYG (peptone-yeast extract-glucose) medium (Fuller, 1978) for growth of L. giganteum (Domnas et al., 1974). The same procedure was used for culture of L. callinectes except that 2% RILA artificial sea salts (Carolina Biological Co.) were also added to the culture medium (Warner and Domnas, 1981). Analyses of the culture media by the methods described below showed them to be sterol free to our limit of detection. Inoculation was with l-cm2 agar blocks from stock cultures on the same media. The culture media were sterilized by autoclaving at 121°C for 20 min. Zoosporogenesis in L. gigantewm was induced as previously described (Domnas et al., 1974), or by immersion of mycelia in sterile deionized, distilled water. A solution of 2% RILA sea salts in sterile deionized, distilled water was used for the induction of zoosporogenesis in L. callinectes.
AND
DOMNAS
Sterol supplements. Cycloartenol (9B, 19cycle-lanost-24-enol) was isolated by procedures previously described (Warner et al., 1982) from green jackfruit (Artocarpus integrifolia) which were a generous gift from Y. Sagawa of the Lyon Arboretum of the University of Hawaii (Manoa). Ostreasterol (24-methylenecholesterol, ergosta-5,24(28)dienol) was isolated by the same procedures from Saguaro cactus pollen (Carneigiea gigantea) obtained through the generosity of L. Standifer of the United States Department of Agriculture Honeybee Research Laboratory (Tucson, Ariz.). Cholesterol (cholest-5-enol), cholestanol (5a-cholestanol), coprostanol(5B-cholestanol), epicholesterol (cholest - 5 - en - 3a - ol), epicholestanol (ICY - cholestan -3a - ol), epicoprostanol (5B -cholestan - 3o - ol), 5a cholestan - 3 - one, 5B - cholestan - 3 - one, cholest - 5 - en - 3 - one, brassicasterol (24Bergosta - 5,22 - dienol), lophenol (4a - methylcholest - 7 - enol), campesterol (24a ergost - 5 - enol), lathosterol (cholest - 7 enol), and fucosterol (stigmasta - 5,24(28)E - dienol) were obtained from Steraloids Inc. (Wilton, N.H.). Poriferasterol (24B - stigmasta - 5,22 - dienol) and cholesta - 5,7 dienol were obtained from Research Plus Steroid Laboratories (Denville, N.J.). Desmosterol (cholesta - 5,24 - dienol) and lanosterol (lanosta -8,24 - dienol) were obtained from Applied Science Laboratories (State College, Pa.). Sitosterol (24a - stigmast - 5 - enol) and stigmasterol (24a - stigmasta - 5,22 - dienol) were obtained from Aldrich Chemical Company (Milwaukee, Wise.). Ergosterol (24B -ergosta - 5,7,22 trienol) was obtained from the Sigma Chemical Company (St. Louis, MO.). Where necessary, sterols were further purified by the methods of Thompson et al. (1980). The purity of all sterols was established through GC and. GC/MS as described below and exceeded 99% at use. Sterol (1 mg) was added to each culture flask in 1 ml of chloroform along with 1 ml of 2% aqueous Tween 80 (Sigma Chemical
STEROL
METABOLISM
Co.) prior to autoclaving (Warner and Domnas , 198 1). Uninoculated control cultures were examined for each sterol supplement after seven days so as to verify that any changes observed were due to metabolism by the organisms. Each experiment was replicated at least three times. Isolation of sterols. Vegetative mycelia were harvested and the total lipids isolated, saponified, and the total sterols isolated as described previously (Warner and Domnas, 1981; Warner et a/., 1982). Analysis ofsterols. Identification of each sterol reported below was based upon GC with four different liquid phases, GC/MS, and also uv spectroscopy, where applicable. Sterols were identified by GC with a Packard 417 chromatograph (Downer’s Grove, Ill.) equipped with dual quartz jet flame ionization detectors. The instrument was interfaced with a Perkin-Elmer Sigma 1OB chromatography data station (Norwalk, Conn.). Columns employed were 3% SE-30, N2 flow rate 20 ml/min; 1% QF-1, min; 3% Hi-Eff SBP, Nz NZ flow r flow rate ; and 2% PMPE, Nz flow rate 30 mlimin (Patterson, 1971). All columns were &mm x 2-m glass. Oven temperature was 240°C with detector and injector temperatures of 260°C. Ultraviolet spectra were obtained with a Perk&-Elmer 124D dual-beam spectrophotometer for sterols suspected of possessing conjugated double bonds. The sterols were fractionated (Warner et al., 1982), dissolved in absolute ethanol, and their spectra comared to those of authentic standards. CC/MS was performed with a Finnegan 4032 instrument (Sunnyvale, Calif.). A 13m flexible fused silica capillary column coated with QV-101 was programmed from 168 to 260°C at 1OYYmin with an initial 3min hold. Helium was the carrier gas and injection wa.s in the splitless mode. The quadrapole mass spectrometer was used in the electron-impact mode with an electron energy of 70 eV. The instrument was interfaced with an INCOS data system. This
229
BY Logenidium
configuration was sensitive to 100 ng per injection. Identifications were based u standards and references spectra. Corn isons were based upon m/e and relative intensity of M i , (M - CH3) + , ( (M - [Hz0 + CH,])+, and a uisite fragments. RESULTS
The products of sterol metabolism by the two Oomycetes were examined for the presence of any unusual compounds which could account for the induction of zoos rogenesis by exogenous sterols in L. gig teum. L. callinectes was used as a positive control in this study since it is capable sf de novo sterol synthesis (Warner and Domnas, 1981). Twenty-three different sterols were administered to cultures of both organisms and, after 7 days, reisolated, along with any metabolites produced from them. The results of these studies are shown in Table 1. L.gigankeum grown without sterol contained 6% (+- 0.01% variation for these determinations) lipid on a dry weight basis. Addition of sterol to the culture me&u increased the lipid content to 6.2% (1-2 p-lg total sterolJmg dry wt). L. callinectes jkp3\Nlar without sterol contained 2% lipid, 0.0003% of which was sterol (6-7 ng sterol/mg dry wt). Addition of sterol to its culture medium raised the total lipid content to 2.2% (1-2 kg total sterollmg dry weight). Free lipids, other than nnmetaboIize~ sterol supplements, were not present in the culture broth. Examination of the mycelia after induction of zoosporogenesis but zoospore release showed no ch values or in the results shown in Ta Addition of any of the above with the solubilizer Tween 80, affect the total dry weight of mycelium from the broth cultures, with growth elevated 2&40% in L. giganteum and inhibited 60% in L. callinectes when compared to cultures grown without sterol and Tween 80. These effects were independent of the particular
230
WARNER,
SOVOCOOL,
Sterols Isolated from Lagenidium Administered sterol None Brassicasterol Campesterol Cholestanol Sa-Cholestanone
SS-Cholestanone 5-Cholestenone Cholesterol Coprostanol CycloartenolC Cholesta-5,7-dienol Desmosterol Epicholestanol Epicholesterol Epicoprostanol Ergosterol Fucosterol LanosterolC Lathosterol
Lophenol Ostreasterol Poriferasterol Sitosterol Stigmasterol
AND DOMNAS
TABLE 1 spp. following Growth with Exogenous Sterols
L. giganteum Sterols isolated”
-
L. callinectes wt%b
-
Brassicasterol Campesterol Cholesterol Cholestanol Cholestanol Cholesta-5,7-dienol Soi-Cholestanone Cholesterol Cholestanol
100 100 90 16 48 28 12 12 100
5-Cholestenone Cholesterol Cholesterol Coprostanol Cholesterol Cycloartenol Cholesterol Fucosterol Cholesterol Cholesta-5,7-dienol Desmosterol Epicholestanol Epicholesterol Epicoprostanol Ergosterol Cholesterol Lanosterol Cholesterol Lathosterol Mt = 414 (1.03) So-Cholestanone Lophenol Ostreasterol Cholesterol Poriferasterol Sitosterol Stigmasterol
61 39 100 90 10 48 43 9 90 10 100 100 100 100 100 100 100 54 35 11
NQ
100 70 30 100 100 100
Sterols isolated”
wt%b
Cholesterol Brassicasterol Campesterol Cholesterol Cholestanol Cholestanol So-Cholestanone Cholesterol Cholesta-5,7-dienol Cholestanol SB-Cholestanone Cholesterol Cholestanol Cholesterol Cholesterol Coprostanol Cholesterol Cycloartenol Cholesterol Fucosterol Cholesterol Cholesta-5,7-dienol Desmosterol Epicholestanol Epicholesterol Epicoprostanol Ergosterol Cholesterol Lanosterol Lathosterol Sa-Cholestanone Cholesterol
100 loo 100 90 10 91 4 4 1 94 3 3 75 25 100 97 3 73 18 9 90 10 100 100 100 100 100 100 100 70 15 15
Lophenol Ostreasterol Cholesterol Poriferasterol Sitosterol Stigmasterol
100 84 16 100 100 100
a Unknown sterols are designated by their molecular ion. Values in parentheses refer to retention time relative to cholesterol on OV:IO¶. The C-24 configuration of the apparently unmetabolized 24-alkyI sterols was assumed to be unchanged. b The area normalization for the weight percent (+ 0.5%) was determined on SE-30. NQ = detected, but not at quantifiable levels. Values represent the mean of at least three determinations with a standard deviation of 1%. c Warner and Domnas, 1981.
sterol tested. Tween 80 alone inhibited the growth of L. callinectes by 50% but did not affect the growth of L. giganteum in comparison to cultures grown without Tween 80.
DISCUSSION
The inhibition of growth of L. callinectes by Tween 80 alone is unusual, since this solubilizer does not effect the growth of L.
STEROL
METABOLISM
and has actually been shown to stimulate growth in Phytophthora cactorum (Nes et al., 1979), another Oomycete which does not synthesize sterols. Similarly, all of the sterols uniformly stimulated the growth of L. giganteum and inhibited the growth of L. callinectes. In comparison, different sterols have varying effects on the growth of P. cactorum (Nes et al., 1979; Nes and Patterson, 1981). The exact reasons for these observations are not apparent, but may reflect major differences between the unknown compositions of the membranes of the three organisms. Both Lagenidium spp. exhibited an almost identical behavior in their metabolism of sterols. Neither organism appeared to metabolize 24-alkyl sterols of either the “CX” or “P” configuration; however, 24-alkylidene sterols (fucosterol and ostreasterol) were transformed. This is of particular interest since 24-alkyl sterols maximally induced zoosporogenesis in L. giganteum (Domnas et al., 1977). These apparently nonmetabolizable sterols may be more readily available for the unknown metabolic processes which are involved in the initiation of zoosporogenesis, and may be transformed to products not isolated and identified by the above methods. Neither organism was capable of the reduction of the 2%double bond. Both organisms metabolized cholestanol and cholesta-5,7-dienol to cholesterol; however, the only other sterol observed in the metabolism of lathosterol to cholesterol was cholestanol, which suggests that in de ytovo synthesis the 7-double bond may be reduced before the introduction of the 5-double bond. This result may ‘be contrasted with that which occurs in animal systems, where the S-double bond is usually introduced prior to the reduction of the 7-double bond (Dvornik et al., 1963). The 3a-hydroxy sterols (episterols) were not transformed, but the SP-hydro steroids, coprostanol and SF-cholestanone, were metabolized to cholestanol and cholesterol. Both organisms metabolized steroid 3-ketones to the corresponding 3P-hydroxy
giganteum
BY
Lagenidium
231
sterols. No evidence was obtained for the presence of any steroids analogous to the hormones found in Achlya spp. (Pup stone and Unrau, 1974). The metabolism of cycloartenol but not lanosterol by both species of Lagenidium is of particular interest (Warner and Domnas, 1981>, since these results suggest that the former is the first sterol naturally produced by the cyclization of 2,3-oxidosqualene; an the cyclization of this compound to cycloartenol but not lanosterol clearly divides photosynthetic (or photosynthetically-~~rived) organisms from all others (Nes and McKean, 1977; Nes and Nes, 1980). These data suggest an ancestral affinity to photosynthetic organisms, which is ~~rt~gr COProborated by studies with ~a~roleg~~a j&ax (Bu’Lock and Osagie, 1976) and rn~.~b~r~ of the Peronosporales (Warner et al.) 1982) ~ The sterol compositions of the ~~~yc~t~s differ from other fungi and are akin to those of the algae (McCorkindale et al., Bean et al., 1972; Warner et al., 1 which further suggests that their mode sterol synthesis may be akin to t mode, The synthesis and metaboli costerol, a typical algal sterol (Nes and McKean, 1977), which we have o~$~~ed here strengthens this argument. Lan~st~rol may also be formed by the isomerization of cycloartenol in some plants (Sekula an Nes, 1980), members of the P~r~~~s~~r~~es (Warner et al., 19621, and pro Saprolegnia ferax (Bu’Lock an 1976). We have recently presen inary data which show milfordensis, also of the utilize cyclaartenol b (Warner el al., X983), thus we propos,e that the utilization of only cy~~oarte~~~ versus both cyoloartenol and lanosterol ‘may represent a distinction of this order from at least the Peronosporales and Saprole ales. Further work is obviously require prove or disprove this hypothesis. The ability to metabolize cer$aia skroli structural features must reflect .the h metabolic capabilities of an or~a~is,~,
232
WARNER,
SOVOCOOL,
our approach has led to the outline of some of the metabolic sequences which are present. The data presented suggest that both Lagenidium spp. possess very similar postcycloartenol sterol synthetic pathways. The apparent metabolic route leading to cholesterol is cycloartenol terol.
+ + fucosterol + + choles-
Some transformations that were indicative of obvious intermediate biosynthetic steps were apparent from this study. The results of investigations in progress with specific inhibitors of sterol biosynthesis and their effect on sterol intermediate accumulation in cell-free extracts will aid in the ultimate interpretation of many of the above results. ACKNOWLEDGMENTS We wish to thank S. M. Fagan for excellent technical assistance. This work was supported in part by NIH Grant AI17024 to A.J.D. and a Mycological Society of America Fellowship to S.A.W. REFERENCES BEAN, G. A., PATTERSON, G. W., AND MOTTA, J. J. 1972. Sterols and fatty acids of some aquatic phycomycetes. Camp. B&hem. Physiol. B 43, 935939. BLAND, C. E., AND AMERSON, H. V. 1973. Observations on Lagenidium callinectes: Isolation and sporangial development. Mycologia 65, 310-320. BU’LOCK J. D., AND OSAGIE, A. U. 1976. Sterol biosynthesis via cycloartenol in Saprolegnia. Phytochemistry 15, 1249-1251. DOMNAS, A. J., GIEBEL, P. E., AND MCINNIS, T. M. 1974. Biochemistry of mosquito infection: Preliminary studies of biochemical change in Culex pipiens quinquefasciatus. J. Znvertebr. Pathol. 24, 293-304. DOMNAS, A. J., SREBRO, J. P., AND HICKS, B. F. 1977. Sterol requirement for zoospore formation in the mosquito-parasitizing fungus Lagenidium giganteam. Mycologia 69, 87.5-886. DVORNIK, D., KRAML, M., DUBUC, J., GIVNER, M., AND GAUDRY, R. 1963. A novel mode of inhibition of cholesterol biosynthesis. J. Amer.Chem. Sot. 85, 3309. FULLER, M. S. 1978. Lower Fungi in the Laboratory. University of Georgia Press, Athens. KARASEK, F. W., CLEMENT, R. E., AND SWEETMAN, J. A. 1981. Preconcentration for trace analysis of organic compounds. Anal. Chem. 53, 1050A-1058A.
AND DOMNAS
LIGHTNER, D. V., AND FONTAINE, C. T. 1973. A new fungus disease of the white shrimp Penaeus setiferus. J. Znvertebr. Pathol. 22, 94-99. MC~ORKINDALE, N. J., HUTCHINSON, S. A., PURSEY, B. A., SCOTT, W. T., AND WHEELER, R. 1969. A comparison of the types of sterol found in species of the Saprolegniales and Leptomitales with those found in some other phycomycetes. Phytochemistry 8, 861-867. NES W. D., PATIXRSON, G. W., AND BEAN, G. A. 1979. The effect of steroids and their solubilizing agents on mycelial growth of Phytophthoru cactorum. Lipids 14, 458-462. NES, W. D., AND PATTERSON, G. W. 1981. Effects of tetracyclic and pentacyclic triterpenoids on growth of Phytophthora cactorum. J. Nat. Prod. 44, 215220. NES, W. R., AND MCKEAN, M. L. 1977. Biochemistry of Steroids and Other Zsopentenoids. Univ. Park Press, Baltimore. NES, W. R., AND NES, W. D. 1980. Lipids in Evolution. Plenum, New York. PATERSON, G. W. 1971. Relation between structure and retention time of sterols in gas chromatography. Anal. Chem. 43, 116.5-1170. POPPLESTONE,C. R., AND UNRAU, A. M. 1974. Studies on the biosynthesis of antheridiol. Canad. J. Chem. 52, 462-468. SEKULA, B. C., AND NES, W. R. 1980. The identification of cholesterol and other steroids in Euphorbia pulcherrima. Phytochemistry 19, 1509-1512. THOMPSON, M. J., PATTERSON, G. W., DUTKY, S. R., SVOBODA, J. A., AND KAPLANIS, J. N. 1980. Techniques for the isolation and identification of steroids in insects and algae. Lipids 15, 719-733. UMPHLETT, C. J., AND HUANG, C. S. 1972. Experimental infection of mosquito larvae by a species of the aquatic fungus Lagenidium. J. Znvertebr. Pathol. 20, 326-331. WARNER, S. A. 1982. Biosynthesis and Metabolism of Sterols by Pantonemic Fungi. Ph.D. dissertation, The University of North Carolina at Chapel Hill. WARNER, S. A., AND DOMNAS, A. J. 1981. Evidence for a cycloartenol-based sterol synthetic pathway in Lagenidum spp. Exp. Mycol. 5, 184-188. WARNER, S. A., EIERMAN, D. F., SOVOCOOL, G. W., AND DOMNAS, A. J. 1982. Cycloartenol-derived sterol biosynthesis in the Peronosporales. Proc. Nat. Acad. Sci. USA 79, 3769-3772. WARNER, S. A., GRAHAM, M. S., SOVOCOOL, G. W., AND DOMNAS, A. J. 1981. Alkane contamination of lipids extracted from Lagenidium giganteum and Lagenidium callinectes. Lipids 16, 628-630. WARNER, S. A., SOVOCOOL,G. W., AND DOMNAS, A. J. 1983. Sterols of selected species of Oomycetes and Hyphochytridiomycetes. Mycologia 75, 285-291.