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A broad-spectrum antifungal from the marine sponge Hyrtios erecta
International Journal of Antimicrobial Agents 9 (1998) 147 – 152
A broad-spectrum antifungal from the marine sponge Hyrtios erecta Robin K. Pettit a,*, Shane C. McAllister b, George R. Pettit a, Cherry L. Herald a, J. Morris Johnson b, Zbigniew A. Cichacz a a
Departments of Microbiology and Chemistry, Cancer Research Institute, Arizona State Uni6ersity, Tempe, Arizona, 85287 -1604, USA b Department of Biology, Western Oregon State Uni6ersity, Monmouth, Oregon, 97361, USA Accepted 25 August 1997
Abstract Spongistatin 1, a macrocyclic lactone polyether from the marine sponge Hyrtios erecta, was fungicidal for a variety of opportunistic yeasts and filamentous fungi, including strains resistant to amphotericin B, ketoconazole and flucytosine. In broth macrodilution assays, MICs ranged from 0.195 to 12.5 mg/ml, and minimum fungicidal concentrations ranged from 3.12 to 25 mg/ml. Initial disk diffusion screens with six related macrocyclic lactone polyethers from H. erecta and Spirastrella spinispirulifera, revealed that these polyethers were also antifungal. The fungicidal activity of spongistatin 1 was confirmed in killing kinetics studies, where killing of Candida albicans and Cryptococcus neoformans occurred within 6 and 12 h, respectively. During the killing kinetics experiments, non-treated C. albicans maintained the yeast morphology. However, elongated forms resembling germ tubes were the predominant morphologic form in spongistatin 1-treated C. albicans cultures. The spongistatins show promise as potential antifungal agents and as probes to study fungal morphogenesis and nuclear division. © 1998 Elsevier Science B.V. Keywords: Antifungal susceptibility; Spongistatin; Opportunistic fungi
1. Introduction Marine organisms are proving to be productive sources of new therapeutic agents. Some of the most promising antineoplastic drugs in clinical or preclinical trials were isolated from marine invertebrates or their associated microbes [1 – 3]. The spongistatins are a family of macrocyclic lactone polyethers isolated from marine Porifera (Fig. 1). Spongistatins 1 – 3 were discovered in the eastern Indian Ocean Hyrtios erecta [4,5], and spongistatins 4 – 7 in the southeast African marine sponge, Spirastrella spinispirulifera [6,7]. All of the spongistatins have exceptionally potent and selective inhibitory activity against a subset of the US National Cancer Institute’s human cancer cell lines * Corresponding author. Tel.: +1 602 9654907; fax: + 1 602 9658558; e-mail: [email protected] 0924-8579/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 9 2 4 - 8 5 7 9 ( 9 7 ) 0 0 0 4 4 - 7
[4–7]. In human melanoma and ovarian carcinoma xenografts, curative responses with no observed toxicities have been detected at 25 mg spongistatin 1/kg (personal communication Drs M. Boyd and R. Shoemaker, U.S.N.C.I.). We report here that spongistatin 1 also has broadspectrum antifungal activity. The paucity of effective antifungals, an expanding spectrum of disease caused by formerly innocuous fungi, and the increasing population of immunocompromised patients signal a critical need for novel antifungal agents. From 1980 to 1990 in the US, nosocomial fungal infections rose from 2.0 to 3.8 infections per 1000 discharges [8]. Of the six million cancer deaths that occur worldwide each year, 300 000 result from disseminated fungal infections [9], and fungal infections are increasing in patients infected with HIV-1 [10]. Candida and Cryptococcus, both susceptible to the spongistatins, are the most common fungal isolates from AIDS patients [10].
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2. Methods
2.5. Minimum fungicidal concentrations
2.1. Antifungal agents
Minimum fungicidal concentrations (MFCs) were determined by subculturing 0.1 ml from each tube with no visible growth in the MIC broth macrodilution series, onto drug-free YM plates. The plates were incubated at 35°C for 24 h for all yeast strains except Cryptococcus, which was incubated for 48 h. The MFC was defined as the lowest concentration of spongistatin 1 that completely inhibited growth on YM plates.
Spongistatins 1–7 were isolated from the marine sponges H. erecta and S. spinispirulifera as described elsewhere [4–7]. Spongistatins 1 – 7 were reconstituted in sterile dimethylsulfoxide (DMSO) immediately prior to all assays. DMSO alone had no detectable inhibitory effect on any of the tested microbes.
2.2. Fungal strains Yeast strains (Table 2) were maintained by single colony transfer on yeast morphology (YM) agar (Difco) at 35°C. ATCC c32354 is flucytosine resistant, ATCCc64124 is ketoconazole resistant and ATCCc 42720 is amphotericin B resistant. Issatchenkia orientalis was formerly classified as Candida krusei, and Rhodotorula mucilaginosa as Rhodotorula rubra (ATCC product literature). Filamentous fungi (Table 2) were maintained on potato dextrose agar (PDA) slants at 35°C.
2.3. Disk diffusion assay In our initial screen, antimicrobial activity was assayed by disk susceptibility tests according to the NCCLS [11]. Inocula were adjusted to a density of 0.10 at 625 nm in YM broth and spread on YM plates. Excess moisture was allowed to absorb for 10 min before applying dried disks containing 2-fold dilutions of the drugs. Test plates were incubated at 30°C, and zones of inhibition recorded after 48 h. The MIC was defined as the lowest concentration of drug resulting in a clear zone of growth inhibition. Disk susceptibility testing of Staphylococcus aureus (ATCC c 29213) and Neisseria gonorrhoeae (ATCC c 49226) was also performed.
2.4. Broth macrodilution method Spongistatin 1 was screened against yeasts by the broth macrodilution assay according to the NCCLS [12]. Antibiotic resistant strains were subcultured only once after acquisition, to ensure no loss of antibiotic resistance. Yeasts were suspended and diluted, as recommended, to yield final inocula ranging from 0.5– 2.5 ×103 cfu/ml. Tests were performed in sterile 12× 75 mm plastic tubes containing 2-fold dilutions of spongistatin 1 in 0.165 M morpholinepropanesulfonic acid (MOPS)-buffered RPMI 1640 medium (pH 7.0). One tube was left drug-free for a turbidity control. Tubes were incubated without agitation at 35°C. MICs were determined after 48 h for all yeast strains except Cryptococcus, which was read after 72 h. The MIC was defined as the lowest concentration of spongistatin 1 that inhibited all visible growth of the test organism.
Fig. 1. Structures of spongistatins 1 – 7, the macrocyclic lactone polyethers isolated from Hyrtios sp. (spongistatins 1 – 3) and Spirastrella spinispirulifera (spongistatins 4 – 7).
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Table 1 Antifungal activity of spongistatins 1–7 in the disk diffusion assay Compound Spongistatin Spongistatin Spongistatin Spongistatin Spongistatin Spongistatin Spongistatin
1 2 3 4 5 6 7
Candida albicans (ATCCc 90028) MIC (mg/disk)
Cryptococcus neoformans (ATCCc 90112) MIC(mg/disk)
6.25–12.5 25.0–50.0 25.0–50.0 12.5–25.0 25.0–50.0 25.0–50.0 6.25–12.5
6.25 – 12.5 50.0 – 100.0 50.0 – 100.0 6.25 – 12.5 6.25 – 12.5 12.5 – 25.0 12.5 – 25.0
2.6. Susceptibility testing of filamentous fungi
3. Results
Broth macrodilution susceptibility testing of Aspergillus fumigatus and Rhizopus oligosporus was performed according to a proposed standardized procedure [13] with a slight modification. To induce conidium and sporangiospore formation, A. fumigatus and R. oligosporus were grown on PDA slants at 35°C for 6 days. Fungal slants were covered with 1 ml sterile 0.85% NaCl, and suspensions made by gently probing the colonies with the tip of a sterile pipette. The resulting mixture of hyphal fragments and conidia or sporangiospores was withdrawn, transferred to a sterile clear microfuge tube, and heavy particles allowed to settle for 10 min. The upper homogeneous suspension was transferred to a sterile microfuge tube, vortexed 15 s, adjusted spectrophotometrically, and diluted in sterile 0.165 M MOPS-buffered RPMI 1640 medium, pH 7.0, to yield final inocula ranging from 0.5 – 2.5× 103 cfu/ ml. Susceptibility to spongistatin 1 was then determined by broth macrodilution assays, as described above for the yeast cultures. MICs for the filamentous fungi were read after 24 h, aliquots plated as above, and MFCs read 24 h later.
At concentrations up to 100 mg/disk, spongistatins 1–7 did not inhibit the tested bacterial strains. However, all of the spongistatins inhibited growth of the opportunistic fungi C. albicans and C. neoformans in disk diffusion assays (Table 1). Spongistatin 1 is the most abundant (typical yield 5 mg/400 kg of wet sponge) of the lactone polyethers isolated from H. erecta and S. spinispirulifera. Therefore, we chose this compound for further investigation. In broth macrodilution tests, spongistatin 1 was fungicidal for all yeast and filamentous fungi examined, including flucytosineresistant C. albicans, ketoconazole-resistant C. albicans and amphotericin B-resistant C. lusitaniae (Table 2). The fungicidal activity of spongistatin 1 for two of the opportunistic yeasts was confirmed in killing kinetics studies. These experiments demonstrated that spongistatin 1 was fungicidal for C. albicans and C. neoformans at 2 × MIC (Fig. 2 and Fig. 3). Spongistatin 1 at 2× MIC caused a 1 log10 reduction in the
2.7. Killing kinetics and microscopy
Organism
Overnight cultures of C. albicans (ATCC c 90028) and C. neoformans (ATCC c90112) in pH 7.0 MOPSbuffered RPMI 1640 medium, were inoculated into the same medium containing 1× or 2 × the broth macrodilution MIC of spongistatin 1, or an equivalent volume of DMSO. Cultures were shaken at 35°C, and aliquots aseptically removed at various times for dilution plating and microscopy. Cells were examined by light microscopy (Olympus BH2 microscope) and photographed using Kodak Technical Pan film aided by an Olympus PM10 AD autoexposure device. For each time point and treatment, 200 cells were counted and described. Two morphologic types were identified: yeast cells and elongated forms resembling germ tubes or hyphae. Approximately 90% of the elongated forms were germ tube-like in spongistatin-treated cultures. Killing kinetics studies were performed at least twice on separate days with similar results.
Table 2 Antifungal activity of spongistatin 1 in the broth macrodilution assay ATCC
MIC (mg/ml)
MFC (mg/ml)
c Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida lusitaniae Candida parapsilosis Candida tropicalis Candida glabrata Issatchenkia orientalis Cryptococcus neoformans Cryptococcus neoformans Rhodotorula mucilaginosa Aspergillus fumigatus Rhizopus oligosporus
90028 14053 32354 64124 60193 42720 22019 750 90030 6258
6.25 3.12 3.12 6.25 3.12 1.56 6.25 1.56 1.56 6.25
6.25 25 3.12 6.25 3.12 12.5 25 12.5 12.5 25
90112
3.12
3.12
66031
0.195
1.56
9449
1.56
12.5
96918 22959
12.5 6.25
12.5 6.25
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Fig. 2. Candida albicans killing kinetics of spongistatin 1. Killing determined with no drug (squares) and at 1 × (circles) and 2 × (triangles) the broth macrodilution MIC.
cfu/ml of C. albicans after 6 h of incubation, and after 12 h of incubation for C. neoformans. After 24 h at 2× MIC, there was a 2 log10 reduction in C. albicans cfu/ml, and no viable C. neoformans. Throughout the killing kinetics experiments, yeast cells were the predominant morphologic form in untreated C. albicans cultures (Fig. 4 and Fig. 5). However, from 6 h on, elongated structures predominated in 2× MIC treated cultures (Fig. 4 and Fig. 5). The most abundant elongated forms more closely resembled germ tubes than hyphae. No obvious morphological alterations in spongistatin 1-treated C. neoformans were observed.
Fig. 3. Cryptococcus neoformans killing kinetics of spongistatin 1. Killing determined with no drug (squares) and at 1 × (circles) and 2 × (triangles) the broth macrodilution MIC.
reliance on only a handful of effective antifungal drugs has led to clinical treatment failure and antifungal resistance [10]. We have described a promising antifun-
4. Discussion AIDS, cancer chemotherapy, and other medical conditions or procedures that cause immunosuppression, largely explain the recent exponential rise in fungal infections. The most common fungi responsible for human disease are C. albicans, C. tropicalis, C. glabrata, C. parapsilosis and C. neoformans [14]. Rhodotorula species, C. lusitaniae and C. krusei are considered important emerging yeasts [14]. The number of invasive infections caused by filamentous fungi is lower than those caused by yeasts, but new and less drug-susceptible mold pathogens are emerging [15]. Continued over-
Fig. 4. Morphology of spongistatin 1-treated Candida albicans over time. Morphologic types in untreated (solid lines), and in the presence of 1 × (dotted lines) and 2 × (dashed lines) the broth macrodilution MIC. Yeast (circles) and elongated forms (squares).
R.K. Pettit et al. / International Journal of Antimicrobial Agents 9 (1998) 147–152
Fig. 5. Morphological effects of spongistatin 1 on Candida albicans. Untreated yeast-like cells (a), and two elongated forms at 2× the broth macrodilution MIC: germ tube-like (b) and hyphae-like (c).
gal agent that is active against all of these clinically important fungi, including several antibiotic resistant strains. In addition, spongistatin 1 is exceptionally active (GI50 =2.5–3.5×10 − 11 M) against a subset of highly resistant tumour types [5]. Cell lines derived from human melanoma, lung, colon and brain cancers are particularly sensitive to spongistatin 1. In human melanoma and ovarian carcinoma xenografts, curative responses with no observed toxicities have been detected at 25 mg/kg (personal communication Drs M. Boyd and R. Shoemaker, U.S.N.C.I.). Thus, spongistatin 1 may have a future as an antifungal/antitumour agent. Tubulin, the major component of microtubules, is the target of many naturally occurring compounds that cause cells to arrest in mitosis [16]. Marine mollusks
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and sponges containing novel antimitotic agents have been described [17,18]. In mammalian cells, spongistatin 1 has been shown to be antimitotic [19,20]. In kangaroo rat kidney PtK1 cells, 3× 10 − 10 M spongistatin 1 caused the accumulation of cells arrested in mitosis, and 3×10 − 9 M spongistatin 1 caused the complete disappearance of microtubules from mitotic and interphase cells [19]. Spongistatin 1 (3.6×10 − 6 M) also inhibited the glutamate-induced polymerization of purified bovine brain tubulin [19]. Our spongistatin 1 MFC data, correspond to minimum fungicidal molarities of approximately 10 − 6 M. Fungal microtubules are involved in morphogenesis and nuclear division. In C. albicans, spongistatin 1 appears to induce a shift to the hyphal phase and then arrest the organism in a germ tube-like transition stage. The germ tube-like morphologies may represent cells inhibited in mitosis, and we intend to investigate this possibility using immunofluorescent antitubulin antibodies. The signals controlling morphogenesis of this dimorphic fungus have yet to be clearly defined, and spongistatin 1 may also find application in this arena. Spongistatin 1 inhibited growth of opportunistic fungi in liquid and solid phase susceptibility assays. As determined by the disk diffusion method, spongistatin 1 was the most potent of the spongistatins, and this is consistent with the relative cytotoxicities of the spongistatins against human cancer cell lines [4–7], and their antimitotic activities [19,20]. The spongistatin structures are unrelated to any antifungals in clinical use. If these compounds prove safe and effective in vivo, they would provide a much-needed alternative for treating patients infected with antibiotic-resistant fungi. Spongistatin 1 protection studies in mouse models of human infection await the synthesis, or further isolation from sponges, of this intriguing lactone polyether.
Acknowledgements With appreciation we acknowledge the financial assistance provided by a Western Oregon State College Foundation grant, Outstanding Investigator Grant CA44344-01-09 awarded by the Division of Cancer Treatment, the US National Cancer Institute, DHHS, The Arizona Disease Control Research Commission and the Fannie E. Rippel Foundation. We thank Melanie Filiatrault for performing the disk diffusion assays.
References [1] Flam F. Chemical prospectors scour the seas for promising drugs. Science 1994;266:1324 – 5.
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[2] Pettit GR. Marine animal and terrestrial plant anticancer constituents. Pure Appl Chem 1994;66:2271–81. [3] Pettit GR. Progress in the discovery of biosynthetic anticancer agents. J Nat Prod 1996;59:812–21. [4] Pettit GR, Cichacz ZA, Gao F, Herald CL, Boyd MR. Isolation and structure of the remarkable human cancer cell growth inhibitors spongistatins 2 and 3 from an Eastern Indian Ocean Spongia sp. J Chem Soc Chem Commun 1993;14:1166–8. [5] Pettit GR, Cichacz ZA, Herald CL, Boyd MR, Schmidt JM, Hooper JNA. Isolation and structure of spongistatin 1. J Org Chem 1993;58:1302 –4. [6] Pettit GR, Herald CL, Cichacz ZA, et al. Antineoplastic agents 293. The exceptional human cancer cell growth inhibitors spongistatins 6 and 7. Nat Prod Lett 1993;3:239–44. [7] Pettit GR, Herald CL, Cichacz ZA, et al. Isolation and structure of the powerful human cancer cell growth inhibitors spongistatins 4 and 5 from an African Spirastrella spinispirulifera (Porifera). J Chem Soc Chem Commun 1993;24:1805–7. [8] The National Nosocomial Infections Surveillance System, BeckSague CM, Jarvis WR. Secular trends in the epidemiology of nosocomial infections in the US, 1980–1990. J Infect Dis 1993;167:1247– 51. [9] McCann J. Deadly fungal infections spreading in cancer patients. J Natl Cancer Inst 1995;87:1434–6. [10] Ampel NM. Emerging disease issues and fungal pathogens associated with HIV infection. Emerg Infect Dis 1996;2:109– 16. [11] National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests, 6th edn: Approved Standard M2-A6. NCCLS, Wayne, PA, 1997. [12] National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Test-
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A broad-spectrum antifungal from the marine sponge Hyrtios erecta Robin K. Pettit a,*, Shane C. McAllister b, George R. Pettit a, Cherry L. Herald a, J. Morris Johnson b, Zbigniew A. Cichacz a a
Departments of Microbiology and Chemistry, Cancer Research Institute, Arizona State Uni6ersity, Tempe, Arizona, 85287 -1604, USA b Department of Biology, Western Oregon State Uni6ersity, Monmouth, Oregon, 97361, USA Accepted 25 August 1997
Abstract Spongistatin 1, a macrocyclic lactone polyether from the marine sponge Hyrtios erecta, was fungicidal for a variety of opportunistic yeasts and filamentous fungi, including strains resistant to amphotericin B, ketoconazole and flucytosine. In broth macrodilution assays, MICs ranged from 0.195 to 12.5 mg/ml, and minimum fungicidal concentrations ranged from 3.12 to 25 mg/ml. Initial disk diffusion screens with six related macrocyclic lactone polyethers from H. erecta and Spirastrella spinispirulifera, revealed that these polyethers were also antifungal. The fungicidal activity of spongistatin 1 was confirmed in killing kinetics studies, where killing of Candida albicans and Cryptococcus neoformans occurred within 6 and 12 h, respectively. During the killing kinetics experiments, non-treated C. albicans maintained the yeast morphology. However, elongated forms resembling germ tubes were the predominant morphologic form in spongistatin 1-treated C. albicans cultures. The spongistatins show promise as potential antifungal agents and as probes to study fungal morphogenesis and nuclear division. © 1998 Elsevier Science B.V. Keywords: Antifungal susceptibility; Spongistatin; Opportunistic fungi
1. Introduction Marine organisms are proving to be productive sources of new therapeutic agents. Some of the most promising antineoplastic drugs in clinical or preclinical trials were isolated from marine invertebrates or their associated microbes [1 – 3]. The spongistatins are a family of macrocyclic lactone polyethers isolated from marine Porifera (Fig. 1). Spongistatins 1 – 3 were discovered in the eastern Indian Ocean Hyrtios erecta [4,5], and spongistatins 4 – 7 in the southeast African marine sponge, Spirastrella spinispirulifera [6,7]. All of the spongistatins have exceptionally potent and selective inhibitory activity against a subset of the US National Cancer Institute’s human cancer cell lines * Corresponding author. Tel.: +1 602 9654907; fax: + 1 602 9658558; e-mail: [email protected] 0924-8579/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 9 2 4 - 8 5 7 9 ( 9 7 ) 0 0 0 4 4 - 7
[4–7]. In human melanoma and ovarian carcinoma xenografts, curative responses with no observed toxicities have been detected at 25 mg spongistatin 1/kg (personal communication Drs M. Boyd and R. Shoemaker, U.S.N.C.I.). We report here that spongistatin 1 also has broadspectrum antifungal activity. The paucity of effective antifungals, an expanding spectrum of disease caused by formerly innocuous fungi, and the increasing population of immunocompromised patients signal a critical need for novel antifungal agents. From 1980 to 1990 in the US, nosocomial fungal infections rose from 2.0 to 3.8 infections per 1000 discharges [8]. Of the six million cancer deaths that occur worldwide each year, 300 000 result from disseminated fungal infections [9], and fungal infections are increasing in patients infected with HIV-1 [10]. Candida and Cryptococcus, both susceptible to the spongistatins, are the most common fungal isolates from AIDS patients [10].
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2. Methods
2.5. Minimum fungicidal concentrations
2.1. Antifungal agents
Minimum fungicidal concentrations (MFCs) were determined by subculturing 0.1 ml from each tube with no visible growth in the MIC broth macrodilution series, onto drug-free YM plates. The plates were incubated at 35°C for 24 h for all yeast strains except Cryptococcus, which was incubated for 48 h. The MFC was defined as the lowest concentration of spongistatin 1 that completely inhibited growth on YM plates.
Spongistatins 1–7 were isolated from the marine sponges H. erecta and S. spinispirulifera as described elsewhere [4–7]. Spongistatins 1 – 7 were reconstituted in sterile dimethylsulfoxide (DMSO) immediately prior to all assays. DMSO alone had no detectable inhibitory effect on any of the tested microbes.
2.2. Fungal strains Yeast strains (Table 2) were maintained by single colony transfer on yeast morphology (YM) agar (Difco) at 35°C. ATCC c32354 is flucytosine resistant, ATCCc64124 is ketoconazole resistant and ATCCc 42720 is amphotericin B resistant. Issatchenkia orientalis was formerly classified as Candida krusei, and Rhodotorula mucilaginosa as Rhodotorula rubra (ATCC product literature). Filamentous fungi (Table 2) were maintained on potato dextrose agar (PDA) slants at 35°C.
2.3. Disk diffusion assay In our initial screen, antimicrobial activity was assayed by disk susceptibility tests according to the NCCLS [11]. Inocula were adjusted to a density of 0.10 at 625 nm in YM broth and spread on YM plates. Excess moisture was allowed to absorb for 10 min before applying dried disks containing 2-fold dilutions of the drugs. Test plates were incubated at 30°C, and zones of inhibition recorded after 48 h. The MIC was defined as the lowest concentration of drug resulting in a clear zone of growth inhibition. Disk susceptibility testing of Staphylococcus aureus (ATCC c 29213) and Neisseria gonorrhoeae (ATCC c 49226) was also performed.
2.4. Broth macrodilution method Spongistatin 1 was screened against yeasts by the broth macrodilution assay according to the NCCLS [12]. Antibiotic resistant strains were subcultured only once after acquisition, to ensure no loss of antibiotic resistance. Yeasts were suspended and diluted, as recommended, to yield final inocula ranging from 0.5– 2.5 ×103 cfu/ml. Tests were performed in sterile 12× 75 mm plastic tubes containing 2-fold dilutions of spongistatin 1 in 0.165 M morpholinepropanesulfonic acid (MOPS)-buffered RPMI 1640 medium (pH 7.0). One tube was left drug-free for a turbidity control. Tubes were incubated without agitation at 35°C. MICs were determined after 48 h for all yeast strains except Cryptococcus, which was read after 72 h. The MIC was defined as the lowest concentration of spongistatin 1 that inhibited all visible growth of the test organism.
Fig. 1. Structures of spongistatins 1 – 7, the macrocyclic lactone polyethers isolated from Hyrtios sp. (spongistatins 1 – 3) and Spirastrella spinispirulifera (spongistatins 4 – 7).
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Table 1 Antifungal activity of spongistatins 1–7 in the disk diffusion assay Compound Spongistatin Spongistatin Spongistatin Spongistatin Spongistatin Spongistatin Spongistatin
1 2 3 4 5 6 7
Candida albicans (ATCCc 90028) MIC (mg/disk)
Cryptococcus neoformans (ATCCc 90112) MIC(mg/disk)
6.25–12.5 25.0–50.0 25.0–50.0 12.5–25.0 25.0–50.0 25.0–50.0 6.25–12.5
6.25 – 12.5 50.0 – 100.0 50.0 – 100.0 6.25 – 12.5 6.25 – 12.5 12.5 – 25.0 12.5 – 25.0
2.6. Susceptibility testing of filamentous fungi
3. Results
Broth macrodilution susceptibility testing of Aspergillus fumigatus and Rhizopus oligosporus was performed according to a proposed standardized procedure [13] with a slight modification. To induce conidium and sporangiospore formation, A. fumigatus and R. oligosporus were grown on PDA slants at 35°C for 6 days. Fungal slants were covered with 1 ml sterile 0.85% NaCl, and suspensions made by gently probing the colonies with the tip of a sterile pipette. The resulting mixture of hyphal fragments and conidia or sporangiospores was withdrawn, transferred to a sterile clear microfuge tube, and heavy particles allowed to settle for 10 min. The upper homogeneous suspension was transferred to a sterile microfuge tube, vortexed 15 s, adjusted spectrophotometrically, and diluted in sterile 0.165 M MOPS-buffered RPMI 1640 medium, pH 7.0, to yield final inocula ranging from 0.5 – 2.5× 103 cfu/ ml. Susceptibility to spongistatin 1 was then determined by broth macrodilution assays, as described above for the yeast cultures. MICs for the filamentous fungi were read after 24 h, aliquots plated as above, and MFCs read 24 h later.
At concentrations up to 100 mg/disk, spongistatins 1–7 did not inhibit the tested bacterial strains. However, all of the spongistatins inhibited growth of the opportunistic fungi C. albicans and C. neoformans in disk diffusion assays (Table 1). Spongistatin 1 is the most abundant (typical yield 5 mg/400 kg of wet sponge) of the lactone polyethers isolated from H. erecta and S. spinispirulifera. Therefore, we chose this compound for further investigation. In broth macrodilution tests, spongistatin 1 was fungicidal for all yeast and filamentous fungi examined, including flucytosineresistant C. albicans, ketoconazole-resistant C. albicans and amphotericin B-resistant C. lusitaniae (Table 2). The fungicidal activity of spongistatin 1 for two of the opportunistic yeasts was confirmed in killing kinetics studies. These experiments demonstrated that spongistatin 1 was fungicidal for C. albicans and C. neoformans at 2 × MIC (Fig. 2 and Fig. 3). Spongistatin 1 at 2× MIC caused a 1 log10 reduction in the
2.7. Killing kinetics and microscopy
Organism
Overnight cultures of C. albicans (ATCC c 90028) and C. neoformans (ATCC c90112) in pH 7.0 MOPSbuffered RPMI 1640 medium, were inoculated into the same medium containing 1× or 2 × the broth macrodilution MIC of spongistatin 1, or an equivalent volume of DMSO. Cultures were shaken at 35°C, and aliquots aseptically removed at various times for dilution plating and microscopy. Cells were examined by light microscopy (Olympus BH2 microscope) and photographed using Kodak Technical Pan film aided by an Olympus PM10 AD autoexposure device. For each time point and treatment, 200 cells were counted and described. Two morphologic types were identified: yeast cells and elongated forms resembling germ tubes or hyphae. Approximately 90% of the elongated forms were germ tube-like in spongistatin-treated cultures. Killing kinetics studies were performed at least twice on separate days with similar results.
Table 2 Antifungal activity of spongistatin 1 in the broth macrodilution assay ATCC
MIC (mg/ml)
MFC (mg/ml)
c Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida lusitaniae Candida parapsilosis Candida tropicalis Candida glabrata Issatchenkia orientalis Cryptococcus neoformans Cryptococcus neoformans Rhodotorula mucilaginosa Aspergillus fumigatus Rhizopus oligosporus
90028 14053 32354 64124 60193 42720 22019 750 90030 6258
6.25 3.12 3.12 6.25 3.12 1.56 6.25 1.56 1.56 6.25
6.25 25 3.12 6.25 3.12 12.5 25 12.5 12.5 25
90112
3.12
3.12
66031
0.195
1.56
9449
1.56
12.5
96918 22959
12.5 6.25
12.5 6.25
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Fig. 2. Candida albicans killing kinetics of spongistatin 1. Killing determined with no drug (squares) and at 1 × (circles) and 2 × (triangles) the broth macrodilution MIC.
cfu/ml of C. albicans after 6 h of incubation, and after 12 h of incubation for C. neoformans. After 24 h at 2× MIC, there was a 2 log10 reduction in C. albicans cfu/ml, and no viable C. neoformans. Throughout the killing kinetics experiments, yeast cells were the predominant morphologic form in untreated C. albicans cultures (Fig. 4 and Fig. 5). However, from 6 h on, elongated structures predominated in 2× MIC treated cultures (Fig. 4 and Fig. 5). The most abundant elongated forms more closely resembled germ tubes than hyphae. No obvious morphological alterations in spongistatin 1-treated C. neoformans were observed.
Fig. 3. Cryptococcus neoformans killing kinetics of spongistatin 1. Killing determined with no drug (squares) and at 1 × (circles) and 2 × (triangles) the broth macrodilution MIC.
reliance on only a handful of effective antifungal drugs has led to clinical treatment failure and antifungal resistance [10]. We have described a promising antifun-
4. Discussion AIDS, cancer chemotherapy, and other medical conditions or procedures that cause immunosuppression, largely explain the recent exponential rise in fungal infections. The most common fungi responsible for human disease are C. albicans, C. tropicalis, C. glabrata, C. parapsilosis and C. neoformans [14]. Rhodotorula species, C. lusitaniae and C. krusei are considered important emerging yeasts [14]. The number of invasive infections caused by filamentous fungi is lower than those caused by yeasts, but new and less drug-susceptible mold pathogens are emerging [15]. Continued over-
Fig. 4. Morphology of spongistatin 1-treated Candida albicans over time. Morphologic types in untreated (solid lines), and in the presence of 1 × (dotted lines) and 2 × (dashed lines) the broth macrodilution MIC. Yeast (circles) and elongated forms (squares).
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Fig. 5. Morphological effects of spongistatin 1 on Candida albicans. Untreated yeast-like cells (a), and two elongated forms at 2× the broth macrodilution MIC: germ tube-like (b) and hyphae-like (c).
gal agent that is active against all of these clinically important fungi, including several antibiotic resistant strains. In addition, spongistatin 1 is exceptionally active (GI50 =2.5–3.5×10 − 11 M) against a subset of highly resistant tumour types [5]. Cell lines derived from human melanoma, lung, colon and brain cancers are particularly sensitive to spongistatin 1. In human melanoma and ovarian carcinoma xenografts, curative responses with no observed toxicities have been detected at 25 mg/kg (personal communication Drs M. Boyd and R. Shoemaker, U.S.N.C.I.). Thus, spongistatin 1 may have a future as an antifungal/antitumour agent. Tubulin, the major component of microtubules, is the target of many naturally occurring compounds that cause cells to arrest in mitosis [16]. Marine mollusks
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and sponges containing novel antimitotic agents have been described [17,18]. In mammalian cells, spongistatin 1 has been shown to be antimitotic [19,20]. In kangaroo rat kidney PtK1 cells, 3× 10 − 10 M spongistatin 1 caused the accumulation of cells arrested in mitosis, and 3×10 − 9 M spongistatin 1 caused the complete disappearance of microtubules from mitotic and interphase cells [19]. Spongistatin 1 (3.6×10 − 6 M) also inhibited the glutamate-induced polymerization of purified bovine brain tubulin [19]. Our spongistatin 1 MFC data, correspond to minimum fungicidal molarities of approximately 10 − 6 M. Fungal microtubules are involved in morphogenesis and nuclear division. In C. albicans, spongistatin 1 appears to induce a shift to the hyphal phase and then arrest the organism in a germ tube-like transition stage. The germ tube-like morphologies may represent cells inhibited in mitosis, and we intend to investigate this possibility using immunofluorescent antitubulin antibodies. The signals controlling morphogenesis of this dimorphic fungus have yet to be clearly defined, and spongistatin 1 may also find application in this arena. Spongistatin 1 inhibited growth of opportunistic fungi in liquid and solid phase susceptibility assays. As determined by the disk diffusion method, spongistatin 1 was the most potent of the spongistatins, and this is consistent with the relative cytotoxicities of the spongistatins against human cancer cell lines [4–7], and their antimitotic activities [19,20]. The spongistatin structures are unrelated to any antifungals in clinical use. If these compounds prove safe and effective in vivo, they would provide a much-needed alternative for treating patients infected with antibiotic-resistant fungi. Spongistatin 1 protection studies in mouse models of human infection await the synthesis, or further isolation from sponges, of this intriguing lactone polyether.
Acknowledgements With appreciation we acknowledge the financial assistance provided by a Western Oregon State College Foundation grant, Outstanding Investigator Grant CA44344-01-09 awarded by the Division of Cancer Treatment, the US National Cancer Institute, DHHS, The Arizona Disease Control Research Commission and the Fannie E. Rippel Foundation. We thank Melanie Filiatrault for performing the disk diffusion assays.
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