Dentigerumycin: a bacterial mediator of an ant-fungus symbiosis

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1 Suppleentary nline Material for Dentigeruycin: a bacterial ediator of an ant-fungus sybiosis Dong-Chan h 1,3, Michael Poulsen 2,3, Caeron R. Currie 2, and Jon Clardy 1 1 Departent of Biological Cheistry and Molecular Pharacology, arvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA. 2 Departent of Bacteriology, University of Wisconsin-Madison, 1550 Linden Dr., Madison, Wisconsin 53706, USA. 3 These authors contributed equally to this work Correspondence should be addressed to C.R.C. (currie@bact.wisc.edu) or J.C. (jon_clardy@hs.harvard.edu).

2 Suppleentary Figures Fig. 1. An exaple of pairwise bioassay challenges of the fungal cultivar (left) to Pseudonocardia sp. (center). dentigeruycin Fig. 2. The PLC trace of the culture of the Pseudonocardia strain CC showing only one ajor secondary etabolite peak, dentigeruycin (1), at 18.3 in. S1

3 Fig. 3. MR spectra of 1, 6, 7, and 8. (a) 1 MR spectru of dentigeruycin (1). (b) 13 C MR spectru of dentigeruycin (1). S2

4 (c) gcsy MR spectru of dentigeruycin (1). (d) TCSY MR spectru of dentigeruycin (1). S3

5 (e) RESY MR spectru of dentigeruycin (1). (f) gsqc MR spectru of dentigeruycin (1). S4

6 (g) gmbc MR spectru of dentigeruycin (1). S5

7 (h) 1 MR spectru of S-MTPA ester (6) of dentigeruycin. (i) gcsy MR spectru of S-MTPA ester (6) of dentigeruycin. S6

8 (j) TCSY MR spectru of S-MTPA ester (6) of dentigeruycin. (k) 1 MR spectru of R-MTPA ester (7) of dentigeruycin. S7

9 (l) gcsy MR spectru of R-MTPA ester (7) of dentigeruycin. () TCSY MR spectru of R-MTPA ester (7) of dentigeruycin. S8

10 (n) 1 MR spectru of 15--deoxydentigeruycin (8) of dentigeruycin. (o) gcsy MR spectru of 15--deoxydentigeruycin (8) of dentigeruycin. S9

11 (p) TCSY MR spectru of 15--deoxydentigeruycin (8) of dentigeruycin. (q) gsqc MR spectru of 15--deoxydentigeruycin (8) of dentigeruycin. S10

12 (r) gmbc MR spectru of 15--deoxydentigeruycin (8) of dentigeruycin. (s) RESY MR spectru of 15--deoxydentigeruycin (8) of dentigeruycin. S11

13 (a) Pip-1 Pip-2 γ--pip β--leu --Ala Ala Acyl side chain (b) Fig. 4. Deterination of the planar structure of dentigeruycin (1). (a) The partial structures of dentigeruycin (1) identified by gcsy, TCSY, SQC, and MBC MR spectral analysis. (b) Key MBC correlations (arrows) in dentigeruycin (1) secure the connectivity of these partial structures. S12

14 (a) (b) 27 (c) Fig. 5. Deterination of relative configurations of dentigeruycin (1). (a) Key RESY correlations in the acyl side chain. (b) C-23-C-24 rotaer with key RESY correlations. The anti-relationship between -23 and -24 was established by J 2324 (11.5 z) 2 (c) Key RESY correlations in γ--pip. The 1-1 coupling constants of the γ--pip unit were copared with the literature values. 3 (a) (b) (c) , CF CF , S ,+0.15 S CF 3 Fig. 6. The odified Mosher ethod analysis of 1. 1 cheical shifts of γ-hydroxy piperazic acid (a) in S-MTPA ester (6) and (b) in R-MTPA ester (7) in CDCl 3. (c) δ S-R values in pp of γ- hydroxy piperazic acid pp for the S- and R-MTPA esters of dentigeruycin in CDCl 3, deterining S configuration of the secondary alcohol ε C wavelength (n) Fig. 7. Induced CD spectru by dentigeruycin and Mo 2 (Ac) 4 coplex. The negative sign at 305 n deterines the absolute configurations of 29S and 30R based on the preferred conforation of the coplex of the diol and the olybdenu ions shown with the spectru. Mo 2 4+ egative CD S13

15 (a) (b) (c) Fig. 8. Plate antifungal assays. (a) An exaple of a plate antifungal assay progress against Escovopsis CC µl of 5 g/l pure dentigeruycin and DMS solution were applied to each disk. Dentigeruycin clearly inhibits the growth of Escovopsis CC (b) An exaple of a plate antifungal assay progress against the cultivar fungus CC µl of 5 g/l pure dentigeruycin and DMS solution were applied to each disk. Dentigeruycin does not inhibit the growth of the cultivar fungus CC (c) An exaple of a plate antifungal assay progress against the cultivar fungus CC with different doses. 10, 50, and 100 µl of 5 g/l (in DMS) pure dentigeruycin (1) and 100 µl of DMS were applied to each disk. Dentigeruycin does not inhibit the growth of the cultivar fungus CC even at higher doses. S14

16 Suppleentary Tables Table 1. MR Spectral Data for dentigeruycin (1) in DMS-d 6. C/ δ ult (J in z) δ C C C d (8.5) d (7.5) 17.1 C C dd (7.5, 1.5) 49.1 C 6a 2.26 br. d (13.0) 28.6 C 2 6b br C d (5.0) 52.8 C dd (12.0, 2.5) dd (6.5, 3.0) C C 11a C 2 11b C 2 13a 3.09 br. d (13.0) 46.0 C 2 13b dd (12.5, 1.5) C q (7.5) 53.2 C s d (7.5) 13.8 C dd (5.5, 1.5) C C 19a 2.07 br. d (12.5) 25.7 C 2 19b C 2 21a 3.00 br. d (12.5) 47.3 C 2 21b dd (12.5, 1.5) C dd (11.0, 1.0) d (11.0) 55.4 C dd (11.0, 1.5) 75.2 C d (7.0) C C d (7.0) 19.7 C C C s C 30-31a s 27.0 C 2 31b C C s C C 3 36a C 2 36b 37a C 2 37b t (7.0) 14.3 C C t (7.5) 8.2 C S15

17 Table 2. MR Spectral Data for reduction product (15--deoxydentigeruycin, 8) of dentigeruycin in CDCl 3. C/ δ ult (J in z) δ C C dq (8.5, 7.0) 48.5 C d (8.5) d (7.0) 18.4 C C br. d (7.0) 50.4 C 6a 2.32 br. d (15.0) 28.5 C 2 6b br C 8a 8b C br. d (13.0) dd (7.0, 1.5) C C 11a C 2 11b 12a C 2 12b 13a br. d (13.0) 47.0 C 2 13b dq (8.0, 7.5) C C d (8.0) d (7.5) C 3 C C C 2 C 2 21a C 2 21b C dd (11.0, 1.0) d (11.0) 55.6 C C d (6.5) C C d (6.5) 20.9 C C C 30 31a C C 2 31b C C C s 20.6 C 3 36a 36b C 2 37a C 2 37b t (7.0) 14.6 C C t (7.5) 8.6 C S16

18 Suppleentary Methods General cheical analysis procedures ptical rotation was easured on a Jasco P-2000 polarieter with a 5 c cell. IR spectra were obtained in a Perkin-Eler 1600 FT-IR spectroeter. CD spectra were collected in an AVIV odel 202 CD spectroeter with a 0.5 c long cuvette. 1 and two diensional MR spectra were acquired in a Varian Inova 600 Mz spectroeter. 13 C MR spectra were collected on a Varian Inova 150 Mz spectroeter. LC/MS analysis was perfored on an Agilent 1200 Series PLC / 6130 Series ass spectroeter. igh resolution ass spectra were obtained on a Waters Microass Q-Tof Ultia ESI-TF ass spectroeter. Isolations of icrobial sybionts fro Apterostiga dentigeru colony CC The colony was collected in 2001 in Gaboa, Panaa, and isolates of the utualistic fungus, the Escovopsis parasite, and the Pseudonocardia actinobacteriu were obtained. The utualistic fungus was isolated by applying sall tufts of fungus yceliu on Potato Dextrose Agar (PDA; 39 g/l) and subculturing until a pure culture of the sybiont was obtained. Escovopsis was obtained by placing larger fungus garden fragents on PDA allowing the parasite to grow and sporulate fro the garden piece. Pure cultures were obtained by transferring Escovopsis spores to new plates containing PDA. The Pseudonocardia isolate was obtained by scraping bacteria fro the ant cuticle onto chitin agar plates containing antifungals (nystatin 10,000 units/l and cyclohexiide 5% w/v), and subsequently sub-cultured onto yeast alt extract agar (YMEA; 10 g yeast extract, 4 g dextrose, 10 g alt extract and 20 g agar per liter of ediu) with antifungals (concentrations as above). 1 Cultivation of Pseudonocardia sp. CC The strain CC was initially cultured on the ediu YMEA (4 g yeast extract, 10 g alt extract, 4 g glucose, and 20 g agar per 1 L of deionized water) agar plates for 7 days. The bacterial cells were scraped fro the plate and transferred to a 125 L Erleneyer flask containing 25 L of YMEA liquid ediu (4 g yeast extract, 10 g alt extract, and 4 g glucose per 1 L of deionized water). The liquid culture was incubated at 30 C with shaking at 250 rp for 7 days. The 20 L of the YMEA culture was inoculated to 200 L of YMEA in 500 L Erleneyer flask. After incubating the 200 L culture at 30 C with shaking at 250 rp for 5 days, 25 L of the culture was transferred to 1 L of YMEA ediu in 4 L Erleneyer flask (8 L). The 8 L of YMEA culture was also incubated at 30 C with shaking at 250 rp for 14 days. S17

19 Extraction and isolation of dentigeruycin (1) The whole culture (8 L) was extracted with 12 L of ethyl acetate (EtAc) twice. The EtAc layer was separated fro the aqueous phase with a fractionating funnel. After fractionation, residual aount of water in the organic layer was reoved by adding sodiu sulfate anhydrous. The EtAc was reoved by rotavap and dry crude extract aterial was obtained. The crude aterial was resuspended in dichloroethane (DCM) / ethanol (Me) 1:1 solvent and celite was added to the ixture. The celite-extract ixture was dried in vacuo and the dry aterial was loaded on 2 g of a pre-packed C 18 Sepak resin. The crude extract was fractionated by eluting with a step gradient of water, Me, and DCM cobinations. The 80% Me fraction was ost active in inhibition of the parasitic fungus Escovopsis strain (CC ). The fraction was subsequently purified by reversed-phase PLC (Agilent 1100 Series PLC syste, Alltech seipreparative colun C 18, , 2 L/in, UV 210 n detection) with the isocratic solvent ixture 80% C 3 C in water. Dentigeruycin (1) was eluted at 16.4 in. The overall yield of the pure dentigeruycin through extraction and isolation is approxiately 3 g / L. Dentigeruycin (1): [α] 24 D -15 (c 0.59, CCl 3 ) ; IR (neat) ν ax 3372, 2936, 1752, 1648, 1503, 1452, 1322, 1249, 1192 c -1 ; MR spectral data, see Suppleentary Table 1; R-ESI-TFMS [M+a] + /z at , calcd [M+a] + at Deterination of the absolute configuration of the aino acid units in dentigeruycin 1.2 g of dentigeruycin was hydrolyzed in 0.5 L of 6 Cl at 115 C for 1 h, the Cl was reoved under vacuu. The dry aterial was resuspended in 0.5 L of water and dried three ties to reove residual acid. The hydrolysate was purified by colun chroatography using a C 18 Sepak colun (0.5 g). The free aino acids were eluted with 10 % C 3 C in water. The purified hydrolysate with the free aino acids was divided into two portions and dissolved in 1 ac 3 (100 µl). 50 µl of 10 g/l L-FDLA (2) and D-FDLA (3) 4 were added to the solution and the reaction ixtures were heated at 80 C for 3 in. The reaction was quenched by neutralization with 50 µl of 2 Cl. Aqueous 50 % C 3 C (300 µl) was added to the solution to dissolve the products, which were analyzed by LC/MS with a gradient solvent syste fro 20 % to 70 % C 3 C (0.1% foric acid) over 50 in (Agilent 1200 Series PLC / 6130 Series ass spectroeter, Phenoenex C 18 (2) 5 µ, , 0.7 L/in flow rate). The L- FDLA and D-FDLA derivatives of two piperazic acid units were eluted at retention tie 22.0 and 24.8 in, respectively. Both piperazic acid units in 1 were deterined to have R configuration based on the retention ties of the L-FDLA derivatives of synthetic R- and S-piperazic acids (4 and 5) (R: 22.3 in and S: 25.0 in). Alanine was eluted at 23.5 and 27.4 in with L-FDLA and S18

20 D-FDLA derivatizations, respectively, establishing its S configuration. The products derived fro β-hydroxy leucine unit were detected at 23.2 and 29.5 in for L and D-FDLA derivatization, deterining S configuration at its α-carbon chiral center. The L-FDLA and D- FDLA derivates of γ-hydroxy piperazic acid unit were eluted at 19.6 in and 18.1 in but the absolute configuration could not be established only with the Marfey analysis. The products originated fro -hydroxy alanine were not detected. S- and R-MTPA esters (6 and 7) of dentigeruycin Dry dentigeruycin (0.8 g) was prepared in two separate vials and several dry crystals of diethylainopyridine were added to the two reaction vials. The ixture was dissolved in freshly distilled dry pyridine (2 L) and stirred for 15 in under argon at roo teperature. 50 µl of R- and S-MTPA chloride (5.36 µol/µl) were added respectively to the separate reaction vials and the reaction ixtures were stirred at roo teperature for 1 h. Then the reaction vials were heated at 65 C for 3 h with onitoring the reaction by LC/MS. After 3 hours, 100 µl of Me were added and the acylation products were purified by reversed-phase PLC (Agilent PLC 1100 Series, Alltech C 18 seipreparative colun, , 2 L/in, gradient 0-10 in: 20% aqueous C 3 C / in: % aqueous C 3 C). S-MTPA ester (6) and R-MTPA ester (7) were eluted at 43.3 and 43.5 in respectively. The olecular forulas for S- and R-MTPA esters were confired as C F by ESI-RMS analysis ([M- 2 +] + /z at for S-MTPA ester and at for R-MTPA ester, calculated at ). The 1 cheical shifts of S- and R-MTPA esters were assigned by 1, gcsy, and TCSY MR spectral analysis. The δ values in Fig. 5 deterined the absolute configuration of the secondary alcohol at C-7 as S. 5 S-MTPA ester (7-S-ethoxy-trifluoroethyl-phenyl acetyl dentigeruycin ester, 6): 1 MR (600 Mz, CDCl 3 ) δ 0.79 (3, d, J = 7.0 z) 0.80 (3, t, J = 7.5 z), 0.87 (3, t, J = 7.5 z), 0.93 (3, d, J = 7.0 z), 0.99 (2, ), 1.22 (1, ), 1.28 (3, d, J = 7.5 z), 1.37 (3, s), 1.38 (2, ), 1.48 (2, ), 1.51 (3, d, J = 7.0 z), 1.53 (2, ), 1.61 (2, ), 1.71 (4, ), 1.82 (1, ), 1.94 (2, ), 1.95 (1, ), 1.96 (1, ), 2.02 (2, ), 2.76 (1, ), 2.78 (1, ), 3.02 (1, ), 3.04 (1, ), 3.13 (1, d, J = 12.0 z), 3.22 (1, d, J = 12.0 z), 3.49 (3, s), 3.66 (1, ), 4.18 (1, d, J = 12.5 z), 4.24 (1, ), 4.76 (1, dd, J = 11.0, 11.0 z), 5.04 (1, br. d, J = 7.5 z), 5.08 (1, ), 5.10 (1, ), 5.23 (1, dd, J = 11.0, 1.5 z), 5.63 (1, ), 5.64 (1, ), 5.74 (1, q, J = 7.0), 5.84 (1, d, J = 12.0 z), 6.22 (1, d, J = 9.0 z), 7.41 (3, ), 7.52 (2, ), 8.68 (1, d, J = 11.0 z). S19

21 R-MTPA ester (7-R-ethoxy-trifluoroethyl-phenyl acetyl dentigeruycin ester, 7): 1 MR (600 Mz, CDCl 3 ) δ 0.80 (3, t, J = 7.5 z), 0.81 (3, d, J = 7.0 z), 0.87 (3, t, J = 7.5 z), 0.91 (3, d, J = 7.0 z), 0.99 (2, ), 1.17 (3, d, J = 7.5 z), 1.22 (1, ), 1.37 (3, s), 1.38 (2, ), 1.48 (2, ), 1.50 (3, d, J = 7.0 z), 1.52 (2, ), 1.61 (2, ), 1.72 (4, ), 1.83 (1, ), 1.91 (1, ), 1.92 (2, ), 1.93 (1, ), 2.05 (2, ), 2.74 (1, ), 2.78 (1, ), 2.93 (1, ), 3.00 (1, ), 3.06 (1, ), 3.12 (1, d, J = 14.0 z), 3.52 (3, s), 3.66 (1, ), 3.88 (1, d, J = 12.5 z), 4.23(1, ), 4.75 (1, dd, J = 11.0, 11.0 z), 5.05 (1, br. d, J = 7.5 z), 5.08 (1, br. d, J = 13.0 z), 5.20 (1, ), 5.23 (1, dd, J = 11.0, 1.5 z), 5.53 (1, dd, J = 6.5, 1.5 z), 5.63 (1, ), 5.71 (1, q, J = 7.0 z), 5.84 (1, d, J = 12.0 z), 6.13 (1, d, J = 9.0 z), 7.43 (3, ), 7.51 (2, ), 8.67 (1, d, J = 11.0 z). Reduction of - in dentigeruycin and deterination of the absolute configuration of -hydroxy alanine Dentigeruycin (1.0 g) was dissolved in 2 L of tetrahydrofuran (TF) under argon. 1 L of 4.5 M aqueous aoniu acetate and 0.5 L of a 10 % TiCl 3 solution in wt. % Cl were added to the solution in sequence. The ixture was stirred at roo teperature for 2 h. The product was extracted with EtAc (10 L) and the organic layer was washed with saturated ac 3 (15 L) and acl (15 L) solutions. 6 The reduction product (15-deoxydentigeruycin, 8) was purified at 46.7 in by reversed-phase PLC (Agilent PLC 1100 Series, Alltech C 18 seipreparative colun, , 2 L/in, gradient 0-10 in: 30% aqueous C 3 C / in: 30-90% aqueous C 3 C). The olecular forula was confired as C by ESI-RMS analysis ([M+a] + /z at , calculated at ). The 1 and 13 C cheical shifts of 15--deoxydentigeruycin (8) were copletely assigned by 1, gcsy, TCSY, gsqc, gmbc, and RESY MR spectral analysis (Table 2). As the result of reduction, two alanine units were observed at (1) C-1, C-2, C-3, 2- and (2) C-14, C-15, C-16, deoxydentigeruycin (8) was hydrolyzed in 6 Cl at 115 C for 1 h, derivatized with L-FDLA, and the L-FDLA derivatives were analyzed by LC/MS as described above. The single L-FDLA derivative originated fro two alanine units eluted at 23.9 in, deterining that the alanine unit derived fro -hydroxy alanine possesses S configuration. Deterination of the 29, 30-diol oiety using CD spectroscopy Dentigeruycin (5.8 g) was dissolved in 1.50 L of anhydrous diethyl sulfoxide (DMS) and the solution was divided exactly to two 0.75 L aliquots M DMS solution of Mo 2 (Ac) 4 was prepared and 0.75 L of the solution was added to 0.75 L of prepared dentigeruycin solution to ake 2.19 M dentigeruycin and Mo 2 (Ac) 4 ixture solution. S20

22 After 40 in, a CD spectru was recorded for induced CD. The other 0.75 L of dentigeruycin solution was diluted to 1.50 L with 0.75 L of anhydrous DMS and an inherent CD spectru was obtained. The inherent olar CD spectru was subtracted fro the induced olar CD spectru. The negative sign of the diagnostic band at 305 n is correlated to the absolute configuration of the 29, 30-diol oiety, deterining 29S and 30R absolute configurations based on the preferred conforation of the coplex. 7 Plate antifungal assay The parasitic fungal strain Escovopsis CC was plated on YMEA agar plates. The plate cultures were incubated for 2 days at 30 C. Sterile paper disks (2.5 diaeter) were placed on the agar plates, not touching the fungal culture. Ethyl acetate extracts and fractions of Pseudonocardia sp. CC cultures and pure dentigeruycin (1) were dissolved in DMS at various concentrations and applied to the paper disks on the plates. For negative control, pure DMS was also applied to a disk on each assay plate. The cultures were incubated at 30 C and onitored for inhibition. The fungal cultivar CC was cultivated fro the center of YMEA plates. Since the fungal cultivar grows uch slowly, the plates were incubated at 30 C for 14 days before dentigeruycin was applied on paper disks. After applying dentigeruycin, the cultures were placed in a 30 C incubator and onitored for inhibition. Liquid antifungal assay The Escovopsis strain CC was cultivated on potato dextrose agar plates. The ycelia were collected and hoogenized in potato dextrose broth ediu. The suspension was transferred to the wells in 96-well plates and various concentrations of pure dentigeruycin were applied. The plates were incubated at 37 C for 48 h and alaarblue (Soretec Ltd.) was added. The fluorescence was easured with excitation at 540 n and eission at 590 n by Wallac Vector 2 plate reader after h. A liquid antifungal assay was not applicable to the utualistic fungus because it grows extreely slow in liquid culture. Bioassays against Candida albicans strains (wild type, ATCC10231, ATCC200955) were perfored as described above but without cell hoogenization. Suppleentary References 1. Cafaro, M.J. & Currie, C.R. Can. J. Microbiol. 51, (2005). 2. Matsuori,., Kaneno, D., Murata, M., akaura,. & Tachibana, K. J. rg. Che. 64, (1999). 3. Miller, E.D., Kauffan, C.A., Jensen, P.R. & Fenical, W. J. rg. Che. 72, (2007). S21

23 4. Fujii, I.K., Ikai, Y., ka,., Suzuki, M. & arada, K. Anal. Che. 69, (1997). 5. Séco, J.M., Quiñoa, E. & Riguera, R. Tetrahedron: Asyetry 12, (2001). 6. Pennings, M.L.M., Reinhoudt, D.., arkea, S. & van uel, G.J. J. rg. Che. 48, (1983). 7. Snatzke, G., Wagner, U. & Wolff,.P. Tetrahedron 37, (1981). S22