Characterization of the Congeners in the Lantibiotic NAI-107 Complex

Jan 14, 2014 - and by the related Microbispora corallina NRRL 30420. These molecules differ by the presence of two, one, or zero hydroxyl groups at Pr...
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Characterization of the Congeners in the Lantibiotic NAI-107 Complex Sonia I. Maffioli,* Marianna Iorio, Margherita Sosio, Paolo Monciardini, Eleonora Gaspari, and Stefano Donadio NAICONS, Via Gaudenzio Fantoli 16/15, 20138, Milano, Italy S Supporting Information *

ABSTRACT: NAI-107, a lantibiotic produced by Microbispora sp. 107891, shows potent activity against multi-drug-resistant bacterial pathogens. It is produced as a complex of related molecules, which is unusual for ribosomally synthesized peptides. Here we describe the identification, characterization, and antibacterial activity of the congeners produced by Microbispora sp. 107891 and by the related Microbispora corallina NRRL 30420. These molecules differ by the presence of two, one, or zero hydroxyl groups at Pro-14, by the presence of a chlorine at Trp-4, and/or by the presence of a sulfoxide on the thioether of the first lanthionine.



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RESULTS AND DISCUSSION Analysis of NAI-107 Complex from Microbispora sp. 107891. Microbispora sp. 107891 produced, in addition to the main A1 and A2 congeners, four minor related compounds as shown by HPLC and LC-MS analysis. The latter form are the hydrophilic congeners F1 (1) and F2 (2) and the lipophilic B1 (3) and B2 (4) (Figure 2). Medium-pressure reverse-phase chromatography allowed the isolation of two purified fractions, containing F1 + F2 and B1 + B2, respectively. Congeners F1 + F2. Congeners F1 (1) and F2 (2) showed m/z signals of 1107 and 1099 as double-charge ions [M + 2H]2+, respectively, corresponding to masses 2212 and 2196. The 34 amu difference between F1 (1) and A1, and between F2 (2) and A2, suggested the absence of the chlorine atom, consistent with the different isotopic patterns observed for these minor congeners. 1H NMR analysis of the F1 + F2 sample highlighted the characteristic aromatic spin system of an unsubstituted Trp indole with all five aromatic protons clearly recognizable (Table 1; Figure S1). These data, together with the 16 amu difference between F1 (1) and F2 (2), strongly suggest that congeners F1 (1) and F2 (2) correspond to dechloro-A1 and -A2, respectively. Congeners B1 + B2. Congeners B1 (3) and B2 (4) showed MS signals at m/z 1132 and 1124 as double-charge ions [M + 2H]2+, respectively, corresponding to masses of 2262 and 2246.

anthipeptides (for lanthionine-containing peptides) are ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by the presence of mesolanthionine (Lan) and 3-methyllanthionine (MeLan) residues.1 Lan consists of two alanine residues cross-linked via a thioether linkage that connects their β-carbons, while MeLan contains one additional methyl group. Lanthipeptides with antimicrobial activity are called lantibiotics. The lantibiotic NAI-107, also known as 107891 or microbisporicin,2 exhibits potent activity against most Grampositive pathogens of medical importance and is effective in experimental models of infection.3 This compound is produced by Microbispora sp. 107891 as a complex of related molecules, with the most abundant congeners A1 and A2 differing by the presence of dihydroxy- or hydroxy-proline at position 14, respectively (Figure 1).2 The molecule also carries a halogenated Trp residue. While rings A and B share a similar topology in NAI-107 and nisin, where they have been demonstrated to be involved in lipid II binding,4 the Cterminal portions are clearly different, with NAI-107’s appearing more globular than nisin’s. Since halogenation and hydroxylation steps are rarely encountered in lantibiotics, we decided to characterize the congeners present in the NAI-107 complex, using either Microbispora sp. 107891 or M. corallina NRRL30420 as the most convenient producer strain. © 2014 American Chemical Society and American Society of Pharmacognosy

Received: August 29, 2013 Published: January 14, 2014 79

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Figure 1. NAI-107 congeners.

Figure 2. (A) HPLC analysis (λ 230 nm) of Microbispora sp. 107891 (up) and Microbispora corallina NRRL-30420 (down). (B) LC-MS analysis (λ 221 nm) of Microbispora sp. 107891 in minimal medium. (C) Precursor peptide.

pH in the medium rises above 7, suggesting an oxidative conversion of congeners A into B. Among the possible positions, oxidation of the Trp, or of the Dha, Dhb, and Avi residues, was excluded because of the presence in the 1H NMR

The 16 amu difference between the corresponding A congeners indicated the presence of an additional oxygen atom. It is worthwhile to note that congeners B usually appear when the Microbispora culture has entered into stationary phase and the 80

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Table 1. Selected NMR Spectroscopic Data (600 MHz) for NAI-107 Congeners 1, 2, 3, and 4 in Comparison with A1 and A2 NAI-107 F1 (1) + F2 (2) δH (J in Hz) 2 3 4

Dhb Ala Trpa

5 7 8 11 13 18 20 21 22 23 24

Dha Ala Abu Ala Ala Ala Ala Ala Phe Ala Avi

NAI-107 A1 + A2 δC

6.48

133

7.54, d (8); 7.40, d (8); 7.14, dd; 7.08, s; 7.03, dd; 9.47, NH 5.53, 6.22

106.8

7.20−7.30 m 6.92; 5.65

130; 105.5

δH (J in Hz) 6.48 overlapped 7.44, s, H-4; 7.11, s, H-2; 7.02, d (8.5); 7.27; 9.47, NH 5.57; 6.25 overlapped 3.60, β; 1.11, γc 3.93, α; 3.63, βc overlapped overlapped 8.64, NH; 4.53, αb; 3.01−3.71, βc overlapped 7.20−7.30 m 9.07, NH; 4.09 αb; 3.23−2.60, βc 6.92; 5.65

NAI-107 B1 (3) + B2 (4) δC

δH (J in Hz)

135

6.57 overlapped

105.5

5.64; 6.46 overlapped 3.60, β; 1.11, γc 3.93, α; 3.63, βc overlapped overlapped 8.64, NH; 4.53, αb; 3.01−3.71, βc overlapped

130; 105.5

9.07, NH; 4.09 αb; 3.23−2.60, βc 6.92; 5.65

5-Cl-Trp for congeners A1 + A2 and B1 (3) + B2 (4). bRecorded in 1:1 CD3CN−D2O + 10 μL of DCl at 400 and 125 MHz for 1H and 13C, respectively. cRecorded in dmso-d6 at 400 MHz. a

Figure 3. HSQC selected aliphatic region of congeners A1 + A2 (a) and B1 (3) + B2 (4) (b) in 6:4 MeCN-d3−D2O at 298 K. Arrows in (b) indicate the peaks in the regions containing the α (4.5−5.0 ppm) and β (2.5−3.5 ppm) amino acid signals, absent in (a) and consistent with the presence of an oxidized thioether bridge.

shifted downfield due to the negative β-effect of the S-oxide group, whereas the latter is shifted upfield by a strongly positive α-effect.5 In contrast, the 1H chemical shifts are not significantly affected. Comparing the HSQC spectra between the B (3 + 4) and A congeners in the region containing the α and β amino acid carbons highlighted the appearance of the expected up-

spectra of the NH proton and the double-bond protons, respectively (Table 1). Oxidation may thus involve one of the thioether bridges, as observed with other lantibiotics.5,6 It has been reported in the literature that oxidation to sulfoxide causes a diagnostic change in the chemical shifts of the β- and αcarbons of the lanthionine residue: the former is typically 81

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Table 2. MIC (μg/mL) of the Congeners F1 (1) + F2 (2), A1, A2, B1 (3) + B2 (4), GPA-A1 (5), + GPA-A2 (6) Isolated from Microbispora sp. 107891 and F0 (7) and A0 (8) Isolated from Microbispora corallina NRRL-30420 NAI-107 congeners

1 1 1 2 2 1 1 1 1 a

microrganism

F1 + F2 (1 + 2)

F0 (7)

A1

A2

A0 (8)

B1 + B2 (3 + 4)

GPA-A1+ GPA-A2 (5 + 6)

GENa

S. aureus MSSA L819/ATCC 19636 S. aureus MRSA ND035208 S. aureus GISA L3798 Streptococcus pneumoniae ATCC 49619 S. pyogenes L49 Enterococcus faecalis VanS L559 E. faecalis VanA L560 E. faecium VanS L568 E. faecium VanA L569

1 4 8 ≤0.125

4 8 16 ≤0.03

0.25 1 1 0.015

0.25 1 2 0.03

0.50 4 4 0.03

2 8 8 0.5

>1.7 nt nt nt

1 >128 0.02 8

≤0.125 64 32 32 32

≤0.03 64 32 32 32

≤0.001 2 1 4 4

≤0.001 2 2 4 4

≤0.001 8 4 8 8

0.015 16 8 16 32

0.22 nt nt nt nt

16 4 4 16 16

GEN: gentamicin.

and down-shifted 13C signals in the former, consistent with a sulfoxide (Figure 3). Among the five thioether links present in NAI-107, changes in bridges 8−11, 21−24, 13−20, and 18−23 were excluded since the spectra showed at least one of the methylene-forming bridges unaltered with respect to the A congeners (Table 1). By exclusion, oxidation should then have occurred in the first thioether bridge 3−8. Consistent with this hypothesis, the 1H and 13C chemical shifts for the Dha-5 and Dhb-2 olefinic signals were also significantly affected (Table 1 and Figure S2). It should be noted that a less pronounced upfield shift was also detected for the olefinic protons of Dha-5 in the dechlorinated congeners (Table 1 and Figure S1). All these data strongly suggest that congeners B have the structure reported in Figure 1. The 16 amu difference between B1 (3) and B2 (4) again suggests that they represent the oxidized derivatives of congeners A1 and A2, respectively. Finally, the pH dependence of congeners B formation during fermentation and their formation upon storage of purified A1 and A2 under alkaline conditions point to a spontaneous oxidation process. This is in contrast with actagardine oxidation, where the garO gene has been involved in the oxidation process.7 The occurrence of the oxidation at the first lanthionine bridge might suggest a better accessibility of this sulfur.8 N-Terminally Extended Congeners. While using a minimal medium for NAI-107 production, we observed the appearance of two new congeners in approximately equal amounts with m/z signals at 1229 and 1237 [M + 2H]2+ (Figure 2b). The masses of the new congeners were consistent with the presence of the extra tripeptide Gly-Pro-Ala at the NAI-107 N-terminus, as expected from the sequence of the precursor peptide (Figure 2c). Again, the 16 amu difference suggested that the m/z 1229 and 1237 signals represented the GPA-extended analogues of A1 (5) and A2 (6), respectively. The small amounts made by the strain prevented NMR characterization of the GPA-A congeners. Congeners A0 and F0. The HPLC traces of the NAI-107 complex from Microbispora sp. 107891 (Figure 2a) highlighted the presence of additional minor congeners, but their low amounts prevented further characterization. We thus investigated whether M. corallina NRRL30420, which also produces NAI-107,9,10 showed any difference in the complex. Figure 2a compares the HPLC traces of the two Microbispora strains, and Table S1 lists the corresponding distribution of congeners observed in the two NAI-107 producing strains. In addition to A1 and A2, M. corallina produced two additional peaks that did

not match any of the NAI-107 congeners described above. These metabolites, designated F0 (7) and A0 (8), originally claimed to contain an unsubstituted Trp residue,10 were subsequently reported on the basis of MS data only to correspond to the F and A congeners, respectively, devoid of hydroxyls at Pro-14.9 In the work reported herein, the 1H NMR characterization of the A0 congener, with the presence of diagnostic singlets at δ 7.45 and 7.10 ppm belonging to H-4 and H-2, respectively, unambiguously define the presence of a 5-chlorine atom on the indolic tryptophan ring in A0 (Figure S3). While the amount of purified 7 was insufficient for a full 1 H NMR characterization, its HPLC retention time, MS isotopic pattern,9 and the congener distribution observed for the two producing strains (Table S1) are consistent with its identification as the dehalogenated variant of 8. Interestingly, LC-MS analysis confirmed that strain 107891 also produces traces of compounds 7 and 8 (Figure 2a, Table S1). Overall, our data indicate that Microbispora sp. 107891 can produce a complex with all possible permutations at residues Trp-4 (no modification or chlorination) and Pro-14 (no modification, mono- or dihydroxylation). Further diversity is introduced by sulfoxide formation of the first thioether. These results suggest that Trp halogenation and Pro hydroxylation can occur independently during NAI-107 biosynthesis. As seen with other ribosomally synthesized peptides,11,12 variations in N-terminal processing have also been observed with NAI-107, although it remains to be determined whether the GPA extension can be observed only under poor growth of the strain, as in a minimal medium. Antibiotic Activity. The purified congeners A0 (8) and F0 (7), the mixture of F1 (1) + F2 (2), the mixture of B1 (3) +B2 (4), and GPA-A as a mixture of the 1 and 2 forms (5 + 6) were tested for their antibacterial activity in comparison with A1 and A2 (Table 2). The three A congeners showed comparable activities, although A0 (8) tended to be slightly less active than A1 and A2. In contrast, the B and F congeners were less active, particularly against staphylococci and enterococci. The GPA-A congener was only weakly active. Thus, the hydroxylation state at Pro-14 seems to have a minor effect on activity. In contrast, the observed lower activity of congeners B and F, both carrying alterations in the first ring, is in agreement with the mechanism of action of nisin-like lantibiotics, in which the two N-terminal rings bind with the lipid II target.4 The increased hydrophobicity provided by the chlorine atom on Trp-4 may strengthen this interaction, as observed for other lipid II82

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binding antibiotics.13 The oxidation state of the first thioether ring may also affect target binding. Nonetheless, the observation that the activity of congeners B and F is only marginally affected against streptococci suggests that more than just lipid II binding contributes to NAI-107 activity.



concentrated under vacuum, and lyophilized from water. Purified congeners A1 and A2 were obtained as described.2 Antimicrobial Assay. MIC assays were performed by the broth microdilution methodology in sterile 96-well microtiter plates according to CLSI guidelines. Media used were Müller Hinton broth containing 20 mg/L CaCl2 and 10 mg/L MgCl2 for all strains, except for Streptococcus spp., which were grown in Todd Hewitt broth (Difco Laboratories, Detroit, MI, USA) and in RPMI 1640 (Sigma Aldrich) supplemented with 0.165 M MOPS (pH 7), respectively. Bacteria were inoculated at 5 × 105 cfu mL−1. After 24 h incubation at 37 °C, MIC was defined as the lowest drug concentration causing complete suppression of visible growth. Compounds were dissolved in DMSO. Appropriate dilutions were made with the required culture medium immediately before testing. All strains were either from the ATCC or the NAICONS collection. The latter are indicated by an L-prefix.

EXPERIMENTAL SECTION

General Experimental Procedures. Analytical HPLC was performed on a Shimadzu LC 2010A-HT (Shimadzu Corporation, Japan) equipped with a HiQ sil C18HS, 5 μm (250 × 4.6 mm) column eluted at 1 mL/min and at 50 °C. Elution was with a multistep program: time = 0−5 min (32% phase B); time = 26 min (42% phase B); time = 27−30 min (90% phase B); and time = 31−40 min (32% phase B). Phase A was 80 mM ammonium formate, pH 4.5, 5% acetonitrile, and phase B was acetonitrile. The UV detector was set at 230 and 270 nm. LC-MS was performed on an Agilent 1100 series liquid chromatograph equipped with an Ascentis Express Supelco RP18, 2.7 μm (50 × 4.6 mm) column eluted at 1 mL/min and at 40 °C. Elution was performed with a multistep program: time = 0 (5% phase B); time = 6 min (95% phase B); time = 7 min (100% phase B); time = 7.2 min (5% phase B); and time = 10 min (5% phase B). Phases A and B were 0.05% (v/v) trifluoroacetic acid in water and acetonitrile, respectively. The effluent from the column was split in a ratio of 50:50, and one part (500 μL/min) was diverted to a photodiode array detector, while the remaining was diverted to the ESI interface of a Bruker Esquire3000 Plus ion trap mass spectrometer. The mass spectrometric analysis was performed under the following conditions: sample inlet conditions: sheath gas (N2) 50 psi; dry gas 10 L/min; capillary heater 365 °C; sample inlet voltage settings: polarity: positive; capillary voltage −4000 V; end plate offset −500 V; scan conditions: maximum ion time 200 ms; ion time 5 ms; full micro: scan 3; segment: duration 10 min, scan events positive (100−2400 m/z). 1 H, 13C, and 1D and 2D NMR spectra (COSY, TOCSY, HSQC) were measured in 6:4 MeCN−D2O-d3 (or H2O) mixture at 298 K using an AMX 600 MHz spectrometer (Bruker, Karlsruhe, Germany). Proton and carbon chemical shifts were referenced to the residual solvent signal (CD3CN) at δ 1.94 and 1.32 ppm, respectively Strain Cultivation. Microbispora sp. 107891 was cultivated essentially as described2 and using a minimal medium containing 20 g/L glucose, 5 g/L NH4Cl, 0.5 g/L K2HPO4, 0.5 g/L NaCl, 0.2 g/L MgSO4, 0.09 g/L FeSO4, 10 g/L MOPS, 1 mL/L vitamins (25 μg/mL thiamin-HCl, 250 μg/mL Ca-pantothenate, 250 μg/mL nicotinic acid, 500 μg/mL biotin, 1.25 mg/mL riboflavin, 6 μg/mL vitamina B12, 25 μg/mL p-aminobenzoic acid, 500 μg/mL folic acid, 500 μg/mL pyridoxal-HCl), and 1 mL/L trace elements (40 mg/L ZnCl2, 200 mg/L FeCl3·6H2O, 10 mg/L CuCl2·2H2O, 10 mg/L MnCl2·4H2O, 10 mg/L Na2B4O7·10H2O, 10 mg/L (NH4)6Mo7O24·4H2O). The culture was harvested after 9 days in a shaking incubator at 30 °C. M. corallina NRRL30240 was cultured in VSP medium (soluble starch 24 g/L, dextrose 1 g/L, meat extract 3 g/L, yeast extract 5 g/L, triptose 5 g/L, agar 1 g/L, sucrose 50 g/L, L-proline 0.5 g/L; pH 7.2). NAI-107 production was monitored by withdrawing 1 mL of culture and extracting with two volumes of MeOH at 50 °C with shaking. The sample was then centrifuged (3000 rpm for 10 min), and the supernatant analyzed by HPLC and LC-MS. Congeners Purification. The culture broth was centrifuged at 3000 rpm for 10 min. The resulting mycelium was extracted with 2 vol of methanol under shaking at room temperature overnight. The solution obtained after centrifugation (3000 rpm, 10 min) was then evaporated to dryness. The mixtures of congeners B1 + B2 (3 + 4) and F1 + F2 (1 + 2) and the single congeners A0 (8) and F0 (7) were purified by medium-pressure chromatography on 86 g of reverse-phase C18 RediSep RF column (40−63 μm particle size, 60 Å pore size, 230−400 mesh) by using a CombiFlash RF Teledyne Isco mediumpressure chromatography system. The resin was previously conditioned with a mixture of phase A−phase B, 8:2 (v/v), and was then eluted at 60 mL/min with a 20 min linear gradient from 20% to 45% phase B. Phase A was water with 0.1% TFA, and phase B was acetonitrile. The congeners-containing fractions were pooled,



ASSOCIATED CONTENT

S Supporting Information *

Additional data and selected 1H NMR and HSQC spectra. This material is available free of charge via the Internet at http:// pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*E-mail: smaffi[email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was has been supported by the European Commission under the Seventh Framework Programme. We thank G. del Gatto and E. Mazzei for help with compound purification and strain fermentation, respectively.



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