Morphological Effects of Lipopeptides Against Aspergillus Fumigatus Correlate with Activities Against (1,3)-1-D-Glucan Synthase M

Home , Echinocandin, Triazole

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1994, p. 1480-1489 Vol. 38, No. 7 0066-4804/94/$04.00+0 Copyright C 1994, American Society for Microbiology Morphological Effects of Lipopeptides against Aspergillus fumigatus Correlate with Activities against (1,3)-1-D-Glucan Synthase M. B. KURTZ,1* I. B. HEATH,2 J. MARRINAN,' S. DREIKORN,' J. ONISHI,' AND C. DOUGLAS' Merck Research Laboratories, Rahway, New Jersey 07065,1 and York University, North Yorkl Ontario, Canada2 Received 19 November 1993/Returned for modification 25 February 1994/Accepted 28 April 1994 The lipopeptide antifungal agents, echinocandins, papulacandins, and pneumocandins, kill Candida albicans by inhibiting glucan synthesis. For this fungus, there is a good correlation of in vitro enzyme inhibition with in vitro assays of MICs. Semisynthetic lipopeptides such as cilofungin, LY303366, L-693,989, and L-733,560 have activity in vivo against Aspergillus infections but appear to be inactive in broth dilution in vitro tests (MICs, > 128 ,ug/ml). To understand how compounds which lack activity in vitro can have good in vivo activity, we monitored the effect of pneumocandins on the morphology ofAspergillusf migatus and A. flavus strains by light microscopy and electron microscopy and related the changes in growth to inhibition of glucan synthesis. Pneumocandin Bo caused profound changes in hyphal growth; light micrographs showed abnormally swollen germ tubes, highly branched hyphal tips, and many cells with distended balloon shapes. Aspergillus electron micrographs confirmed that lipopeptides produce changes in cell walls; drug-treated germlings showed very stubby growth with thick walls and a conspicuous dark outer layer which was much thicker in the subapical regions. The rest of the hyphal tip ultrastructure was unaffected by the drug, indicating considerable specificity for the primary target. The drug-induced growth alteration produced very compact clumps in broth dilution wells, making it possible to score the morphological effect macroscopically. The morphological changes could be assayed quantitatively by using conventional broth microdilution susceptibility assay conditions. We defined the endpoint as the lowest concentration required to produce the morphological effect and called it the minimum effective concentration to distinguish it from the no-growth endpoints used in MIC determinations. The minimum effective concentration assay was related to inhibition of glucan synthase activity in vitro and may provide a starting point for development of susceptibility testing methods for lipopeptides.

Echinocandins, pneumocandins, and papulacandins are an- antifungal susceptibility, compounds such as echinocandins tifungal agents which inhibit the synthesis of 1,3-p-D-glucan and pneumocandins are active against only a relatively narrow (22, 39, 43, 44). ,B-Glucans are vital cell wall polymers in range of Candida species; Aspergillus species are insensitive to clinically important pathogenic fungi, including Candida and the lipopeptide class of antifungal agents in the MIC test (21, Aspergillus spp. (8). The proportion of this polysaccharide in 22, 26, 33). However, other tests for efficacy illustrate that the walls of different organisms varies, but at least 10% of the echinocandins do affect filamentous fungi. One sensitive bio- dry weight of the walls of these organisms is in the form of assay of echinocandins uses inhibition ofAspergillus growth on 1,3-p-D-glucan (4). This polysaccharide is synthesized by a an agar plate as the test system (21). Denning and Stevens (15) membrane-associated activity which has been partially charac- and Beaulieu et al. (5) have described the in vivo efficacy of terized in yeasts such as Candida albicans and Saccharomyces cilofungin against a systemic Aspergillus fumigatus infection in cerevisiae (31, 40, 46). The enzyme uses UDP-glucose as a animal models, despite its poor activity in an MIC assay. More substrate and catalyzes the polymerization of a linear polymer recently, new semisynthetic analogs of echinocandin (LY303366 of 1,3-p-D-glucan reported to be 60 U (40) or as much as 700 and cilofungin) and water-soluble pneumocandin derivatives U (35) long. The Km for UDP-glucose is approximately 2 mM, (L-693,989 and L-733,560) have been shown to have in vivo and the reaction is stimulated by nucleoside triphosphates, efficacy in murine models of systemic aspergillosis (1, 7, 15, 50). particularly GTP (31, 42). For both C. albicans and S. cerevi- The synthesis of 1,3-3-D-glucan by crude microsomal prepara- siae, the in vitro product, which is synthesized without a tions from A. fumigatus is inhibited by pneumocandin B0 and requirement for an exogenous primer to initiate the reaction, is cilofungin (5, 16, 45). Therefore, the MIC test is not effective an acid-insoluble, alkali-soluble polymer (26) sensitive to di- as a means to assess the anti-Aspergillus activity of these gestion with exo-p-D-glucanase but not a-amylase or chitinase compounds, and we sought another whole-cell assay which (31, 37, 40, 41). would reflect the efficacy seen in animals and correlate with the Inhibitors of 1,3-p-D-glucan synthesis have potential as a inhibition of glucan synthesis. The results of this study show new therapeutic class for treating serious fungal infections, but that pneumocandins produce profound morphological changes their apparently limited spectrum of activity has tempered in Aspergillus hyphae which correlate with inhibition of in vitro enthusiasm for clinical development (26). When conventional 1,3-4-D-glucan synthase. broth microdilution methods are used to evaluate in vitro MATERIALS AND METHODS * Corresponding author. Mailing address: Department of Infectious Disease Research, Merck Research Laboratories, RY80Y-220, P.O. Antifungal compounds. Figure 1 shows the chemical struc- Box 2000, Rahway, NJ 07065-0900. Phone: (908) 594-5124. Fax: (908) tures of pneumocandin Bo (2), pneumocandin Ao (49), and 594-1399. Electronic mail address: [email protected]. L-693,989 (38) provided by scientists at Merck Research 1480 VOL. 38, 1994 MORPHOLOGICAL EFFECTS OF LIPOPEPTIDES ON A. FUMIGATUS 1481

OH pellet was stored frozen in 1-ml aliquots at -80°C. Protein OH OH OH H NH NH OR determinations were performed by the dye-binding ..\ - method ,QH OR NH> (11). 0 NHO CH The assay measures the formation of radiolabeled trichloro- acetic acid (TCA)-precipitable material formed from [3H]UDP- H2NCO -2<°N O<;HHa H2NCO N O..(-HH glucose. Thirty micrograms of Aspergillus membrane protein '+ adjusted to 2 mg/ml in buffer B (Dulbecco's phosphate- O buffered saline, 8% glycerol, 0.5 mM phenylmethylsulfonyl R 0H fluoride, 1 mM dithiothreitol) were incubated for 5 min with a HO _ HO range of concentrations of pneumocandin Bo diluted in di- . Pneumocaindin Bo rneurimocandin AO methyl sulfoxide (DMSO). The reaction was initiated by OH OH OH addition of a cocktail containing the substrate and ot-amylase H i HNHNH COR (Sigma). The complete reaction mixture contained 0.125 M Tris (pH 7.0), 30 mM KF, 1 mM EDTA, 40% phosphate- buffered saline, 5% glycerol, 0.3 mM phenylmethylsulfonyl

H2NC OH H OH fluoride, 0.5 mM dithiothreitol, 3 ,uM GTP--y-S, 0.25% bovine serum albumin, 0.6 mM UDP-glucose, and 1 puCi of [3H]UDP- glucose (TRK-385; 8.5 Ci/mmol; Amersham). After incubation O for 150 min at room temperature (-22°C), the reaction was OR = CNH terminated with an equal volume of ice-cold 20% TCA. The HO - < reactions were harvested onto a prewetted glass fiber filter mat L-693,989 with a cell harvester (Cambridge Scientific). The filter mat was FIG. 1. Chemical structures of 1,3-l3-D-glucan synthesis inhibi- washed with water, blown dry, sealed in plastic with 10 ml of tors. scintillant, and counted in a Betaplate liquid scintillation counter (LKB Wallac). Each assay was run in duplicate. Activity is expressed as nanomoles of product formed per Laboratories, Rahway, N.J. All compounds were shown by minute per milligram of crude protein. high-performance liquid chromatography to be >95% pure. Product characterization. Reactions from the 1,3-,3-D-glu- Fluconazole was obtained from Pfizer Central Research, Gro- can synthesis assay described above were boiled for 2 min after ton, Conn., and amphotericin B was from Bristol Myers- 150 min of synthesis. Five separate pairs of reactions were Squibb. treated as follows to characterize the radiolabeled product: 1, In vitro antifungal activity. The in vitro antifungal suscep- addition of 2 volumes of 10% TCA; 2, addition of 5 volumes of tibility of the pneumocandins was determined for a panel of 1 N NaOH; 3, addition of 5 volumes of 0.1 M acetate buffer Candida and Aspergillus species (see Table 1). Yeast nitrogen (pH 5.3); 4, addition of 4 volumes of 0.1 M acetate buffer and base (Difco) with 2% glucose was used for MIC and minimum then 0.25 U of laminarinase (Sigma L-5272); 5, addition of 4 fungicidal concentration determinations with a broth microdi- volumes of 0.1 M acetate buffer. Samples 2 and 3 were lution assay described previously (3). Briefly, 104 yeast cells or incubated at 22°C for 30 min and then centrifuged for 10 min conidia were inoculated into 0.15-ml volumes of media con- at 11,000 x g at 4°C. We added 2 volumes of 10% TCA to the taining twofold serial dilutions of the test compounds. Growth pellet. Samples 4 and 5 were incubated overnight at 37°C and was monitored visually after incubation for 48 h at 30°C for the centrifuged, and TCA was added as described above. The MIC assay. For minimum fungicidal concentration determina- precipitate from all reactions was collected on GFC glass fiber tions, 1.5-pl aliquots were taken from the microtiter dishes filters (Millipore), washed three times with water, dried, and after 24 h at 30°C and inoculated onto solidified Sabouraud counted in a liquid scintillation counter. Curdlan, a linear medium with a Dynatech 2000 inoculator (Dynatech, Inc.). 1,3-0-D-glucan isolated from Alcaligenes faecalis (Accurate The morphological assay (see Results) used the same media, Chemical & Scientific Corp., Westbury, N.Y.), was also used as inocula, and growth conditions as the standard MIC determi- a standard. Total hexose was measured by phenol-sulfonic acid nation. For some experiments, we used RPMI 1640 medium assay (17a). (Gibco) with L-glutamine, without sodium bicarbonate, and Electron microscopy. Colonies of A. flavus MF0383 and A. buffered with morpholinepropanesulfonic acid (MOPS), Anti- fumigatus MF4839 were prepared for transmission electron biotic Medium III (AMIII; Difco), or Sabouraud medium. microscopy (TEM) by freeze-substitution fixation. Conidia Membrane preparation and 1,3-j0-D-glucan synthesis assay. were harvested and suspended in liquid AMIII at a density of A glucan synthase assay was established with microsomal 8 x 102/ml. One milliliter of this suspension was spread over membranes prepared from A. fumigatus MF4839, a mouse- the surface of a 6-cm-diameter petri dish containing AMIII virulent strain maintained for many years at Merck. After 36 h agar with 8 pug of pneumocandin Bo per ml or 15 [il of DMSO of growth in 2 liters of Sabouraud broth at 370C, the hyphae as the control. The spores settled onto the surfaces of dialysis were harvested on Whatman filters, washed with breakage membrane rectangles (4 by 2 cm) coated with a 0.05% solution buffer (0.1 M phosphate buffer [pH 7.0], 1 mM dithiothreitol, of locust bean gum in liquid AMIII lying on the surface of the 1 mM phenylmethylsulfonyl fluoride), and broken in a Bead agar. Following incubation at 37°C for 18 h, pieces of mem- Beater homogenizer (BioSpec Products, Bartlesville, Okla.) brane bearing isolated colonies were rapidly plunge-frozen in with 100 g of acid-washed glass beads and 40 ml of breakage liquid propane, freeze-substituted in 1% OS04 in acetone, and buffer. The crude homogenate was centrifuged at 2,000 x g for embedded in epoxy resin as described previously (29). Blocks 10 min at 4°C to collect unbroken cells. Membranes and were trimmed to single colonies, or parts of colonies, and microsomes in the low-speed supernatant were collected by serially sectioned. The sections were collected on Formvar- centrifugation at 100,000 x g for 1 h at 4°C, and the pellet was coated single-slot grids, stained in uranyl acetate and lead resuspended in 15 ml of breakage buffer with 25% glycerol. citrate, and examined in a transmission electron microscope. After 20 strokes with a Dounce homogenizer, the resuspended Colonies for scanning electron microscopy were grown in a 1482 KURTZ ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. Susceptibility of Candida and Aspergillus spp. to pneumocandin Bo and pneumocandin AO A Pneumocandin Bo Pneumocandin A0 Strain MIC MFC or MIC MFC or (g/ml)((g/m1)(MEC (,ug/ml)p.g/ml) MECMEG C. albicans MY1028 1 1 4 4 C. albicans MY1055 1 1 2 0.125 C. albicans MY1750 1 1 4 2 C. guillermnondii MY1019 8 32 8 16 C. parapsilosis MY1010 4 1 2 1 C. pseudotropicalis MY2099 2 4 4 0.5 C. tropicalis MY1012 0.5 0.125 1 0.5 A. flavus MF0383 >128 0.03 >128 0.015 A. fiumigatus MF4839 >128 1 >128 0.5 A. fiumigatus MF5668 >128 2 >128 2 A. fumigatus MF5669 >128 1 >128 0.5 a For Candida spp., the MFC is given, and for Aspergillus spp., the MEC is given. manner similar to that used for TEM, fixed in phosphate- B buffered glutaraldehyde followed by OS04, impregnated with uranyl acetate during ethanol dehydration, critical point dried, sputter coated with gold-palladium, and examined with a scanning electron microscope with a resolution of approximately 7 nm.

RESULTS In vitro antifungal susceptibility assays. In standard MIC methods, the endpoint for the assay is complete absence of growth. By this criterion, the lipopeptides shown in Fig. 1 are inactive against all of the Aspergillus strains tested (Table 1). However, microscopic examination of the standard MIC microdilution wells showed that drug treatment produced profound morphological alterations (Fig. 2). In the presence of the lipopeptides, hyphae grew abnormally, with highly branched tips, swollen germ tubes, and many distended, balloon-like cells. The effect was medium similar independent; morpholog- FIG. 2. Light micrograph of drug-induced morphology changes in ical changes were observed with RPMI medium, AMIII, and A. fumigatus MF4839. Panels: A, DMSO solvent control; B, 5 p,g of Sabouraud medium. This morphological effect was dose de- pneumocandin Bo per ml. pendent, with more severe effects seen at the highest concentrations. However, the cutoff for this effect was quite sharp and was usually within a twofold dilution and within the experi- mental error we have experienced with MIC microtiter assays icin B produced clear-cut MICs, with a defined endpoint of no for C. albicans. We call the minimum drug concentration which growth, while the triazole fluconazole did not inhibit growth produced this morphological change the minimum effective either macroscopically or microscopically under the conditions concentration (MEC) to distinguish it from traditional MICs. used in these experiments. As a result of the drug-induced altered growth, the short, Ultrastructure. Hyphae of both A. flavus and A. fumigatus highly branched filaments produce very compact clumps in treated for 18 h with 8 ,ug of pneumocandin Bo per ml showed dilution wells, making it possible to score the MEC macroscop- major ultrastructural differences from the controls treated with ically (Fig. 3). The endpoint could easily be determined; the DMSO. Tips from DMSO-treated hyphae showed the normal very compact clumps correspond to the morphology seen in organization expected for an ascomycete, with an apical con- Fig. 2B, while diffuse growth corresponds to normal growth centration of predominantly darkly stained vesicles accumu- (Fig. 2A). As mentioned above, the dose-dependent interme- lated around a Spitzenkorper composed of a dense cluster of diate effects are limited and generally do not represent a smaller vesicles (Fig. 4). Subapical to the vesicular zone were significant problem in trailing endpoints. We found the results many mitochondria interspersed with circular cisternae known quite reproducible from test to test. In 25 tests, the MEC of as Golgi body equivalents (Fig. 4A). The Golgi body equiva- pneumocandin Bo for MF 4839 was 0.71 (standard deviation, lents in the controls were typically associated with numerous 0.74). However, different Aspergillus strains varied in suscepti- darkly stained vesicles comparable to those concentrated in the bility; the MECs of some compounds were the same for all four tips and are probably the source of the vesicles. strains (data not shown), but A. flavus was approximately The cell walls at the tips of the control cells were even in 30-fold more susceptible to pneumocandin AO than was A. thickness at about 35 nm and were lightly stained with a fine, fumigatus (Table 1). In control assays, the polyene amphoter- darker outer layer (Fig. 4). This wall structure pattern did not VOL. 38, 1994 MORPHOLOGICAL EFFECTS OF LIPOPEPTIDES ON A. FUMIGATUS 1483 1 2 3 4 5 6 7 8 9 10 11 12 A B C

D E F FIG. 3. MEC assay plate ofA. flavus MF383 (rows A to C) and A. fumigatus MF4839 (rows D to F). Rows A and D contained pneumocandin AO; rows B and E contained pneumocandin Bo. For both panels, the drug was serially diluted twofold, from the highest concentration in column 1 (32 ,ug/ml) to the lowest concentration in column 12 (0.015 ,ug/ml). Rows C and F contained no drug. vary through the transition to the subapical regions, although shorter compartments by the more frequent insertion of septae the walls were somewhat thicker subapically. In contrast, the (Fig. SC). Because the interseptal distances in control cells treated tips had much thicker walls, with a lighter inner layer of were highly variable and the highly branched growth form of about 90 nm and a thick, darkly stained outer layer of about 40 the treated colonies made it difficult to obtain accurate mea- to 70 nm (Fig. 5A and B). The inner layer did not increase surements, we did not attempt a statistically rigorous analysis much subapically, but the darkly stained outer layer often of interseptal lengths. Those shown in Fig. SC, at 6.8 and 8.3 became much thicker and more variable in thickness (Fig. 5A ,um, respectively, are typical, whereas the values for randomly and C). The appearance of the dark layer varied between the selected control hyphae had a mean of 67.4 ,um (n = 25; two species, being primarily homogeneous in A. flavus and standard deviation, 34.6 ,um) and a range of 32 to 178 ,um for more reticulate and fibrous inA. fumigatus. In both species, the A. flavus. A. fumigatus gave similar values. There were no dark layer was sometimes mixed with material which looked detectable differences between treatment and control septae. like senescent cytoplasm in the older regions of the colonies, Because the treated hyphae were coated with the thick layer of but this material was variable in extent, was rare at the darkly stained material, it seemed likely that their surfaces periphery of the colonies, and was not always present. While it would show a different appearance in scanning electron mi- was not measured, the cytoplasm-like material clearly did not croscopy. However, no differences in the appearance of the make up a very significant percentage of the total darkly surfaces of treated and control hyphae were observed (data not stained material in the colonies. shown). In the cytoplasm, the treated tips contained very few darkly In vitro glucan synthesis. In view of the known activity of stained vesicles (Fig. SA and B) and barely detectable Spitzen- pneumocandins against the glucan synthase of C. albicans and korpers (Fig. SB). The Golgi body equivalents present in the the aberrant hyphal morphology that these compounds pro- treated tips (Fig. 5A and B) appeared to be less abundant than duce in Aspergillus spp., it seemed likely that pneumocandins in controls, and there seemed to be fewer vesicles associated would inhibit the enzyme in these organisms. Our previous with them. However, other organelles appeared to be essen- findings obtained with the natural product pneumocandin Bo tially normal, with no overt signs of cytological damage (Fig. (16) and more recent results reported for cilofungin (5) 5). In both treated and control hyphae of both species, the demonstrated that pneumocandins and echinocandins inhibit plasmalemma was always rather smooth and appressed to the A. fumigatus in vitro glucan synthesis. We have characterized cell wall (Fig. 4 and 5), with no evidence of the presence of an in vitro enzyme assay for glucan synthase from strain plasmalemmasomes or myelin-like figures at the cell surface or MF4839. Incorporation of radiolabel from [3H]UDP-glucose in the cytoplasm. into a TCA-precipitable product proceeded linearly for several In addition to showing the stubby, highly branched growth hours and was dependent on the protein concentration (data form (Fig. 2), the treated hyphae were also divided into much not shown). The standard assay was terminated at 150 min, at ,ifQ ' r k - FIG. 4. TEM of control A. flavus hyphal tips, showing the abundant, darkly staining vesicles which enclose an aggregation of smaller vesicles, the Spitzenkdrper (S), a smooth plasmalemma, mitochondria (m), and circular profiles of the Golgi body equivalents (G). Note the thin cell wall with a very thin, darkly stained outer layer. 1484 FIG. 5. (A and B) TEM of treated A. flavus hyphal tips, showing the thick cell wall with an extensive outer layer of darkly stained material and very few darkly stained vesicles (arrows). Golgi body equivalents (G), mitochondria (m), and a generally smooth plasmalemma are also evident. (C) TEM of the central region of a treated colony, showing the abundant septae (s) which divide the hyphae into very short segments. Note that the septae look normal for an ascomycete. 1485 1486 KURTZ ET AL. ANTIMICROB. AGENTs CHEMOTHER.

140- 120-

0ow 100- G) C') x 0a 80- a X x 60- coo_ 0 > 40Q 0Q E 20- C

12 vI[S] [min-1 x 10(5)] 0.0 ., . , FIG. 6. Km for UDP-glucose. The in vitro glucan synthase reaction .0001 .001 .01 .1 1 10 was run at six different concentrations of UDP-glucose, ranging from 0.3 to io mM. The reaction product precipitated with 10% TCA was Pneumocandin Bo (gM) counted, and the nanomoles of [3H]glucose incorporated were calcu- FIG. 7. Pneumocandin Bo inhibition of glucan synthesis. A 40 ,uM lated. Assays were run in triplicate, and error bars are shown for each stock of pneumocandin Bo in DMSO was serially diluted five times and datum point. The Eadie-Hofstee plot of the results was used to added to in vitro glucan synthase reactions such that the final calculate the Km (absolute value of the slope of the line, 1.1 mM) and concentration ranged from 0.6 nM to 2 ,uM, and the final DMSO the maximum reaction rate (y intercept; 155 nmol liter- min-) for the concentration was 5%. After synthesis, the product from each reaction reaction. S, substrate (UDP-glucose); v, velocity. was harvested and quantitated as described in Materials and Methods. The upper line indicates synthesis in 5% DMSO alone; the lower line indicates incorporation in the presence of 150 FM pneumocandin AO. which time less than 10% of the substrate had been consumed. We measured synthesis at several concentrations of UDP- glucose and used an Eadie-Hofstee plot to estimate the 7. Inhibition was dose dependent, reaching a maximum of ca. apparent Km (Fig. 6). The value of 1.1 mM we obtained agrees 80%. Pneumocandin Bo and several echinocandins were un- well with those calculated for other fungal 1,3-13-D-glucan able to inhibit synthesis more than 80%, even at concentrations synthases in general (12) and the value of 1.9 mM reported for as high as 150 ,iM (data not shown). Therefore, if we define A. fumigatus (6). the IC50 as the midpoint between uninhibited synthesis and the To ensure that the product of the reaction was 1,3-0-D- level of maximal inhibition produced by a reference compound glucan and not another polymer, the TCA precipitate was (pneumocandin AO) added at 150 ,uM, pneumocandin Bo is a characterized on the basis of acid-base solubility and sensitivity potent inhibitor, with an IC50 of 15 nM. A value of 50 nM is to enzymatic digestion. As shown in Table 2, the product of the obtained with a conventional IC50 calculation, i.e., by using the Aspergillus reaction was partially insoluble at acidic pH, soluble midpoint between the uninhibited reaction and 100% inhibi- in dilute alkali, and sensitive to digestion with exo-13-D-glu- tion. For A. fumigatus MF4839, these IC50 values against a canase. Curdlan, an authentic 1,3-0-D-glucan, had the same crude enzyme preparation are at least 20- to 50-fold lower than properties (Table 2). the MEC against whole cells (approximately 1,000 nM; Table Inhibition of glucan synthesis. The 50% inhibitory concen- 1). From these measurements, the inhibition of glucan synthe- tration (IC50) curve for pneumocandin Bo is presented in Fig. sis is sufficient to account for the morphological effects seen in drug-treated cells. We measured synthesis at different pneumocandin Bo and TABLE 2. Characterization of reaction product from in vitro A. substrate concentrations to further evaluate the kinetics of fumigatus glucan assay inhibition. The reaction velocity was calculated, and plots of [substrate] versus [substrate]/volume at the three drug concen- Amt (pmol) of Concn ( g/ml) trations are illustrated in Fig. 8. The nonparallel lines which A. fumigatus Sample product of curdlan converge at the x axis are consistent with noncompetitive (% soluble) (%slbe%slbe inhibition. Total precipitable product' 389 100 Pellet treated with 1 N NaOH 30(92) 5.2 (95) DISCUSSION Pellet treated with 0.1 M acetate 225 (42) 80.8 (19) Pellet treated with exo-P-D-glucanase 23 (94) 0.1 (99) For a long time, the potential of glucan synthesis inhibitors Pellet treated without enzymec 408 (0) 64.6 (35) for in vivo efficacy against Aspergillus spp. was overlooked, in part because of the method of in vitro antifungal testing used. a Each sample was treated as indicated and centrifuged, and the pellet was assayed for total hexose as described in Materials and Methods. Although an accepted standard procedure for susceptibility b For A. fumigatus samples, the radiolabeled TCA-insoluble pellet was the testing of molds is in the making (19), in this work and as total precipitable product. For curdlan, a 100-p,g/ml suspension in the glucan reported by others (5, 15), broth dilution MIC tests modeled synthesis reaction cocktail (without A. fumigatus membranes) was the total on precipitable product. yeast testing procedures (18, 30) have shown thatAspergillus cThe A. fumigatus samples were TCA precipitated prior to counting. The spp. are not susceptible to echinocandins, papulacandins, or curdlan samples were centrifuged, and the pellet was assayed as described in pneumocandins. However, such tests do not give an accurate Materials and Methods. portrayal of the antifungal activity of these agents. Light VOL. 38, 1994 MORPHOLOGICAL EFFECTS OF LIPOPEPTIDES ON A. FUMIGATUS 1487

5 (unpublished data). Like cilofungin and echinocandin B, the pneumocandins are noncompetitive inhibitors of both C. albicans and A. fumigatus glucan synthase (37, 44, 45, 46; Fig. 8). 4 Our preliminary results suggest that in vitro drug susceptibility varies with the age of the hyphae used as a source of the crude 1-1 0 enzyme, a LO 3 phenomenon seen with C. albicans (31). Consider- x ing the susceptibility of the Aspergillus enzyme to pneumocandins, it unclear why the inhibitors do not produce lysis in these 2 organisms but do so in Candida species. We are studying .C, whether differences in cell wall composition, uptake, or media can account for the differences in susceptibility. The dramatic difference in cell wall structure following drug treatment is consistent with the in vitro enzyme assays and colony and hyphal morphology results in showing that the drug alters the cell wall. However, the increase in wall thickness was -2 0 2 4 6 8 10 unexpected; a thinner and weaker wall was predicted. Presum- [S] M x 10(-3) ably, the inhibition of 1,3-p-D-glucan synthesis inhibits the FIG. 8. Kinetics of pneumocandin Bo inhibition. Glucan synthase formation of one of the growth rate-limiting components of the reactions were run with different concentrations of UDP-glucose cell wall, so that the rate of hyphal growth is reduced but the (ranging from 0.3 to 7.5 mM), with pneumocandin Bo added at a final rate of formation of other wall components may be reduced, concentration of 8, 40, or 100 nM. The 3H-labelled product was unaffected, or increased to yield a thicker wall. Consistent with collected, and the reaction velocity was calculated. The Hanes-Woolf this suggestion is the accumulation of darkly stained wall plot of the results produced lines for each drug concentration which material; it is presumably an excess quantity of the ususal outer converge at they axis, which is indicative of noncompetitive inhibition. wall layer, a conclusion supported by the similarity in surface S, substrate (UDP-glucose); v, velocity. appearance between the treated and control hyphae in light micrographs and scanning electron microscopy. The uncou- pling of the synthesis of the different wall components may micrographs of yeast cells treated with these compounds have indicate a lack of regulatory coordination or a highly devel- shown severe effects on cell shape and the integrity of the cell oped compensatory mechanism for an essential structural wall (10, 14, 24). The results shown here demonstrate that component. The change in colony morphology following inhi- pneumocandins produce profound morphological changes in bition of glucan synthase implicates this polymer in morpho- aspergilli that can be quantitated as easily as an MIC (Fig. 2 genesis, especially branch formation patterns. However, the and 3). In this respect, the assay is similar to the MIC-1 or requirement for normal wall composition for hyphal formation MIC-2 being considered for use by the National Committee for is clearly not critical since treated colonies do indeed form Clinical Laboratory Standards for standardized antifungal sus- tubular hyphae despite alterations in branching and septation ceptibility tests (20, 30). MIC-1 and MIC-2 were defined as the patterns. This supports the recent suggestion that tip morpho- lowest drug concentrations that reduced growth to 1 + or 2+ genesis may be controlled more by the cytoskeleton than by the turbidity on a scale of 0 to 4+ estimated visual turbidity. This wall (25, 27, 48). The apparent normality of the septae in the multicenter evaluation showed that agreement was maximized treated hyphae suggests that their form and synthesis are also by using the criterion of the lowest drug concentration re- independent of the 1,3-4-D-glucan. quired to reduce growth to a turbidity of 1+. For research The changes in the cytoplasm of the treated hyphae are most purposes, the MEC assay may have potential as a high- likely a consequence of the reduced growth rate of the hyphae. throughput test to rank the activity of related compounds and The vesicles accumulated around the Golgi body equivalents analogs. By using the MEC, representatives ofAspergillus spp. and in the tips are almost certainly in transit to the tip; thus, tested in this study (A. fumigatus andA. flavus) and others such reduced growth (assuming an effective regulatory feedback as A. nidulans (unpublished data) are at least as susceptible as system) yields a reduced rate of production of wall polymers Candida species to the pneumocandin analogs used. Further and fewer vesicles to transport their precursors. The concom- work is necessary to confirm the general applicability of this itant reduction in the Spitzenkorpers suggests that they too method for lipopeptides with a large number of Aspergillus represent a "snapshot" of a population of microvesicles in species and isolates. We view these observations as a starting transit, a point not clear in previous studies of tip growth (23). point for the development of susceptibility assays for a class of Perhaps one of the more surprising aspects of the ultrastruc- agents which have promise for treatment of candidiasis and ture results is the generally normal appearance of the treated aspergillosis. hyphae. Previous studies of the effects of the lipopeptides on The in vitro enzyme assay data and electron micrographs ultrastructure have reported gross damage, including the for- presented here support the idea that pneumocandins inhibit mation of plasmalemmasomes and myelin-like bodies, vacuo- the growth ofAspergillus species by inhibiting glucan synthesis. lation, and general necrosis in as little as 10 min of treatment Earlier reports of glucan synthase enzymes from a number of (13, 17). Some of these changes may have arisen from differ- fungi (47) including the filamentous fungus Neurospora crassa ences in the handling of the cells prior to fixation (17) but also (43) are consistent with this hypothesis. The product of in vitro may reflect differences between Aspergillus and Candida spe- glucan synthesis assays using S. cerevisiae (28, 40), C. albicans cies. (31, 37, 46), or N. crassa (34) enzyme preparations has the Efficacy in animal models is clearly the most important step same properties. We have not characterized the small percent- towards introducing an echinocandin-like compound into clin- age of material which is alkali insoluble and/or resistant to ics for treatment of fungal infections. We have been studying glucanase digestion. The enzymes from Candida and Aspergil- L-693,989 and, more recently, L-733,560 (1, 7) as model lus spp. have similar substrate requirements, optimal assay compounds for the pneumocandin class of inhibitors. The conditions, and relative susceptibilities to lipopeptide analogs phosphate ester prodrug version of pneumocandin Bo circum- 1488 KURTZ ET AL. ANTIMICROB. AGENTS CHEMOTHER.

vents the water solubility problem of the parent compound, 11. Bradford, M. 1976. A rapid and sensitive method for the quanti- while L-733,560 is intrinsically water soluble. L-733,560 has a tation of microgram quantities of protein utilizing the principle of broader intrinsic spectrum of in vitro anti-Candida activity protein-dye binding. Anal. Biochem. 72:248-254. 12. Cabib, E., and M. S. Kang. 1987. Fungal 1,3-0-glucan synthase. than the natural pneumocandins, with MICs of <1 against C. Methods Enzymol. 138:637-642. albicans, C. tropicalis, C. parapsilosis, C. pseudotropicalis, C. 13. Cassone, A., R E. Mason, and D. Kerridge. 1981. Lysis of growing guillermondii, C. glabrata, and C. krusei (9, 32). Water-soluble yeast-form cells of Candida albicans by echinocandin: a cytological pneumocandins also have potent in vivo activity in a rat model study. Sabouraudia 19:97-110. of Pneumocystis carinii pneumonia (3, 38, 39). Results obtained 14. Davila, T., B. G. San, and B. F. San. 1986. Effect of papulacandin recently with rat models of either disseminated (1) or pulmo- B on glucan synthesis in Paracoccidioides brasiliensis. J. Med. Vet. nary (7) aspergillosis have shown that L-693,989 and L-733,560 Mycol. 24:193-202. significantly prolong survival after a lethal dose ofA. fumigatus. 15. Denning, D. W., and D. A. Stevens. 1991. Efficacy of cilofungin These compounds are fungicidal in animal models for candi- alone and in combination with amphotericin B in a murine model of disseminated aspergillosis. Antimicrob. Agents Chemother. diasis and significantly less toxic than amphotericin B. The 35:1329-1333. anti-Aspergillus activity of pneumocandins and other glucan 16. Douglas, C., J. Marrinan, J. Curotto, J. Onishi, and M. Kurtz. synthase inhibitors increases the opportunities for a new class 1992. Activity of a new echinocandin, L-688,786, against filamen- of safe and effective broad-spectrum antifungal agents. tous fungi, abstr. 1845. Abstr. 92nd Annu. Meet. Am. Soc. Microbiol., New Orleans, La. ACKNOWLEDGMENTS 17. Drouhet, E., B. Dupont, L. Improvisi, M. Lesourd, and M. C. Prevost. 1990. Activity of cilofungin (LY121019), a new lipopep- We thank our colleagues at Merck (K. Bartizal, S. Ponticas- tide antibiotic, on the cell wall and cytoplasmic membrane of Dufresne, K. Nollstadt, J. Milligan, J. Thompson, J. Balkovec, R. Candida albicans. Structural modifications in scanning and trans- Fromtling, and R. Schwartz) for sharing their expertise, providing mission electron microscopy. J. Med. Vet. Mycol. 28:425-436. compounds, and critically reading the manuscript. The excellent 17a.Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. technical assistance of Mary-Lou Ashton is gratefully acknowledged. Smith. 1956. Colorimetric method for determination of sugars and The financial support of I.B.H. by the Natural Sciences and Engi- related substances. Anal. Chem. 28:350-356. neering Research Council of Canada is gratefully acknowledged. 18. 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