Introduction

Gram-negative bacterial strains of the genus Xenorhabdus are known to be symbiotically associated with soil dwelling nematodes of the Steinernematidae family.1, 2, 3 After entering the insect larvae through natural openings, nematodes release bacteria from their intestine to the host's hemocoel.4 Bacteria are involved in killing the insect host by producing insecticidal proteins5 and inhibitors of the insect immune system.6, 7, 8 The bacteria proliferate in the killed host and favor the reproduction of the nematode by degrading the insect biomass9 and by producing antibiotics that inhibits the development of the other microorganisms present in the insects corpse (bacteria, fungi).10 Boemare et al.11 classified the antibiotic activities of Xenorhabdus into two categories: (i) antimicrobial molecules with broad spectrum and (ii) bacteriocins with very narrow spectrum and active only against bacteria closely related to X. nematophila. Only a few families of antimicrobial compounds have been described from Xenorhabdus in the literature: xenocoumacins,12 xenorhabdins,13 indole derivatives,14, 15 puromycin,16 benzylidenacetone,17 proteinaceous bacteriocins,11, 18 and xenortide and xenematide.19 All Xenorhabdus strains spontaneously produce two distinct physiological states in vitro,20 phase I and II variants.21 Phase I variants produce several antibiotics and secrete a variety of proteins, whereas these properties are apparently absent or greatly reduced in phase II variants.

In our screening program, we found new cyclolipopeptidic antimicrobial compounds in the culture supernatant of the X. nematophila F1/1. These compounds possess significant activity against fungi and moderate activity against few Gram-negative and Gram-positive bacteria.

Nonribosomaly antimicrobial lipopetides are produced in bacteria and fungi during cultivation.22, 23, 24 They are composed of a cationic or an anionic peptide covalently bound to a specifically modified aliphatic chain. Most of the peptidic moieties have complex cyclic structures. Some of these molecules are highly active against bacteria including multiresistant strains.25, 26, 27, 28 Others display solely antifungal activity22, 29 and a few both antifungal and antimicrobial activities.22 Members of this family were approved for clinical use by the Food and Drug Administration: daptomycine, polymixine, echinochandine.30, 31

This article describes the fermentation of the X. nematophila F1/1 strain, the isolation and biological activities of these active compounds named PAX (for peptide antimicrobial from Xenorhabdus), and the chemical structure elucidation of PAX 3. This is the first example of lysine-rich cyclolipopetide characterized from the genus Xenorhabdus.

Results and discussion

Fermentation

Xenorhabdus nematophila F1/1 was cultivated for 48 h, at 28 °C with shaking in a 5 l Erlenmeyer flask containing 1 l of LB broth. The culture was inoculated with 0.1% (v/v) of a 24 h preculture in the same medium. The antibiotic production was controlled and quantified by diffusion test agar against Micrococcus luteus, and by analytical HPLC.

Isolation

Bacterial cells were removed by low-speed centrifugation (6000 g, 10 min at 4 °C) and supernatant was sterilized on 0.22 μm pore size filter. Supernatant was added (1:1; v/v) to a 0.1 M NaCl, 0.02 M Tris buffer (pH 9) and subjected to cation-exchange chromatography on a Sep Pack CarboxyMethyl cartridge (Acell Plus CM, Waters, Milford, MA, USA). Unbound material was removed by washes with a 0.1 M NaCl, 0.02 M Tris buffer (pH 9) and the antibiotic eluted with 0.5 M NaCl, 0.02 M Tris buffer (pH 9). This eluate was acidified with 0.1% (v/v) trifluoroacetic acid (TFA) and was then subjected to reversed-phase chromatography on a Sep Pack C18 cartridge (Sep-Pak Plus C18, Waters). Unbound material was removed by washing with H2O-TFA 0.1%, and the antibiotic pool was eluted with acetonitrile. The eluate was concentrated by evaporation under reduced pressure and diluted in water (1:5; v/v). Pure compounds were isolated from the crude extract by reverse-phase HPLC using a semi-preparative C18 column (Waters; Symmetry Prep C18; 7 μm; 7.8 × 300 mm), a linear gradient of H2O, 0.1% TFA-acetonitrile, 0.1% TFA starting from 20 to 80% in 30 min, a flow rate of 5 ml min−1 and an UV detection at 214 nm, yielding pure PAX compounds with the following HPLC-retention times: PAX 1=19.9 min, PAX 2=20.94 min, PAX 3=21.1 min, PAX 4=21.3 min and PAX 5=22.3 min with roughly the 30/10/50/9/1 percentages. The collected fractions were freeze-dried.

Biological properties

The PAX compounds show antifungal and antibacterial activities. They were tested for antimicrobial activity against a wide range of bacteria and fungi involved in nosocomial infection and phytopathologies (Tables 1 and 2). Regarding human pathogens (Table 1), PAX 1 and 2 did not show antibacterial activity except against Staphylococcus epidermidis and M. luteus. PAX 3 and 4 have weak activity against only few Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium), moderate activity against S. epidermidis and B. cereus and high activity against M. luteus. PAX 5 has moderate activity against few Gram-negative (E. coli, P. aeruginosa and S. typhimurium) including multiresistant strains and against few Gram-positive bacteria. However, high activity against the fungus Fusarium oxysporum was observed for PAX 1–5, whereas weak activity was observed against Candida albicans. The fungi belonging to the genus Fusarium are well-known plant pathogens and food contaminants that also cause superficial and subcutaneous infections in humans, such as onychomycosis and keratomycosis.32 They have recently emerged as major opportunistic agents in immunocompromised hosts, especially in patients with hemopathy.33, 34 They are now considered as the third most common fungal genus (after Candida and Aspergillus) isolated from systemic infections in bone marrow transplantation patients.35 F. oxysporum is responsible for about 30% of the human infection caused by this genus.36 Regarding phytopathogenic fungi (Table 2), only PAX 1–3 were tested. At 10, 20 and 40 μg ml−1, PAX 1 and 3 have strong activity against majority of fungi except Botrytis cinerea myc and Piricularia oryzae sp. PAX 2 has similar activity as PAX 1 and 3 against Cladosporium sp., F. culmorum sp. and Phytophthora. Nevertheless, at 10 and 20 μg ml−1, this molecule was less active against other fungi.

Table 1 Chemical structure characteristics of PAX and minimal inhibitory concentration (MIC)a against bacteria and fungi including human pathogens
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Table 2 Antifungal activity of PAX (percent inhibition of fungal growth)
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No cytotoxic activity with doses up to 1 mg ml−1 against CHO cells was measured. Injections of the five PAX into the hemocoels of different species of insects did not result in increased mortality. Moreover, these molecules did not exhibit cytolytic activities against sheep erythrocytes or insect hemocytes. These results show that the PAX had no entomotoxic effects (data not shown).

Chemical structure elucidation of PAXs

Five compounds referred as PAX 1–5 were isolated, purified to homogeneity as a white powder and characterized by mass spectrometry. ESI-MS experiments revealed the molecular weights of different PAXs (PAX 1: 1051 Da, PAX 2: 1079 Da, PAX 3: 1065 Da, PAX 4: 1093 Da). PAXs are soluble in water and alcohols and show an UV λmax of 214 nm (methanol).

Structure elucidation of PAX 3

The acid hydrolysate of PAX 3 yielded seven amino acids, one glycine (Gly) and six lysine (Lys). Its NMR analysis carried out by homo and hetero nuclear experiments is described below.

The NMR data of PAX 3 (1065 Da) were recorded in DMSO-d6 (Table 3). Its 1D spectrum showed eight amide signals spanning the 8.5–7.3 p.p.m. chemical shift area. Among them, one is a triplet (8.18 p.p.m., 5.7 and 5.7 Hz) thus suggesting the presence of a glycine and another displays an unusual doublet of doublet (7.36 p.p.m., 6.5 and 4.0 Hz) indicating that this amide group is bound with a methylene group. The six other amide signals are doublets with a 3JHN-CH coupling constant ranging from 5.2 to 7.1 Hz. The natural abundance 1H-15N HSQC confirmed the presence of these eight amide protons and the absence of a C-terminal amide group (data not shown). In agreement with the acid hydrolysis, the TOCSY experiment allowed us to unambiguously identify a glycine and six lysine residues (Figure 1a). These later being also characterized from the intense signal at 2.74 p.p.m. typical of the CɛH2 protons. Surprisingly, the two-amide signals at 8.38 (doublet) and at 7.36 p.p.m. (doublet of doublet) were found to share the same spin system indicating that the ζ amino group of this lysine was involved in an amide bond to be identified. From the analysis of the DQF-COSY, TOCSY and NOESY data the assignment of the peptidic part was established on the basis of the classical sequential NOEs and is reported in Figure 1. The peptidic sequence was shown to consist of seven residues as following: G1K2K3K4K5K6K7. Unexpectedly, this lysine-rich peptide includes an unusual 5-lysine macrocycle closed by an amide bond between the K7 carboxyl group and the ζK3 amino group. Such a cyclization which gives rise to a 5-residue macrolactame ring was mainly supported by the strong intensity dNN NOE between the K7 and the ζK3 amide protons and by the successive dNN NOEs observed all around the cycle. Owing to the overlap of the K6 and K7 amide signals, the dNN6–7 NOE could not be observed in pure DMSO-d6. The addition of 17% water was enough to separate these two resonances and observe this essential NOE to confirm the cyclic structure giving rise to a 5-residue macrolactame ring. As a result, this cyclization constrains the K3 side chain. This is in agreement with the strong inequivalency observed for the CɛH2 resonances of K3 at 3.45 and 2.77 p.p.m. (Figure 1a).

Table 3 NMR data of PAX 3 (dimethyl sulfoxide, 32 °C) and of PAX 4 (dimethyl sulfoxide, 27 °C)
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Figure 1
figure 1

1H NMR data illustrating the assignment of the peptidic moiety. (a) Part of the TOCSY showing the different spin systems (DMSO, 305 K). ζK3 is for the zeta amide proton of K3. The side chain spin system of K3 observed both from the amide and the ζK3 amide proton is boxed. Due to the length of the side chain the ɛCH2 cross-peaks are of weak intensity from the amide chemical shift and not observed in this plot level. (b) Part of the NOESY showing the ‘sequential’ NOE between the αCH2 of the fatty acid and the amide proton of G1 labeled G1/CH2 (DMSO-d6, 305 K, 250 ms of mixing time). (c) Part of the NOESY showing the dNN NOEs (DMSO-d6 with 17% of water, 285 K, 250 ms of mixing time). The strong intensity NOE between HN K7 and ζHN K3 amide protons (7/ζ3) characterizes the cyclization. Notice that the dNN6–7 was not observed in pure DMSO due to the overlap of the K6 and K7 amide signals (part a).

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Clearly, several remaining resonances in the 1H spectrum do not belong to the peptidic part. In particular, the doublet at 0.84 p.p.m. and the multiplet at 1.49 p.p.m. belong to an isopropyl group. By using both homonuclear and heteronuclear data, starting from the sole methyl signal of the spectrum, the (CH3)2-CH-(CH2)3- spin system was unambiguously characterized (Figure 2). This is also the case for the proton resonance at 3.81 p.p.m. that belongs to a CH group whose 13C signal is at 67.58 p.p.m.. These chemical shifts are typical for an alcohol or ether function indicating the presence of an -CH(OH)- or -CH(OR)- group. In addition, from this alcohol or ether group the CO-CH2-CH(O)-(CH2)3- spin system was clearly characterized (Figure 2). Interestingly, these two well-identified spin systems share an identical intense cross-peak at (1H) 1.24/(13C) 28.86 p.p.m. corresponding to several -CH2- groups, suggesting that the two spin systems could be linked together by an aliphatic chain. Thus, the acyl fragment would be a 3-hydroxy fatty acid. Owing to the overlap of several methylene groups the acyl chain length was deduced from the molecular weight as being an iso-15:0 3-hydroxy fatty acid. Moreover, the ‘sequential’ NOE between the C2H2 (2.23 p.p.m.) of the 3-hydroxy fatty acid and the amide proton of G1 unambiguously characterized the amide bond link between the fatty acid and the peptidic fragments. Finally, the PAX 3 structure was fully corroborated by MS-MS fragmentation (Figure 3).

Figure 2
figure 2

Part of the 1H-13C HSQC-TOCSY showing the spin system of the iso-15:0 3-hydroxy fatty acid. The two partial spin systems identified from the carboxyl (large rectangles) and from the methyl (small rectangles with labels in italics) groups are boxed. They share the same CH2 cross-peak (twofold labeled CH2*) corresponding to the overlap of several equivalent methylene groups at 1.24/28.86 p.p.m. located between the two extremities of the fatty acid. The length of the acyl chain was deduced from the molecular weight obtained by mass spectrometry.

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Figure 3
figure 3

Key fragmentations of the 1066 [M+H]+ ions of PAX 3.

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Altogether, these data suggest the presence of the iso-15:0 3-hydroxy fatty acid linked to the cyclopeptidic part at the G1 residue to yield a lysine-rich cyclolipopeptide with a 5-residue macrolactame ring (Figure 4). The configurations of the α-carbons of the amino acids as well as that of the C3 of the iso-15:0 3 hydroxy acid were not determined.

Figure 4
figure 4

Chemical structure of PAX 3.

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The PAX 1, PAX 2 and PAX 4, 1H NMR spectra in DMSO (dimethyl sulfoxide) were very similar to that of PAX 3. The two main changes were easily observed and characterized by the combination of 1H NMR and ESI/MS data. The first one with regard to the peptidic part with the K2R mutation, whereas the second one with regard to the length of the fatty acid. The PAX 2 (1079 Da) and PAX 4 (1093 Da) chemical structures consist of the K2R mutation (Tables 1 and 3) with the iso-14:0 and the iso-15:0 3-hydroxy acid, respectively. In contrast, PAX 1 (1051 Da) displays a K2 residue and the iso-14:0 3-hydroxy acid.

Although the 1H NMR spectrum of PAX 5 could not be obtained, its molecular weight identical to PAX 2 (1079 Da) suggested that it could be its normal isomer. However, when compared with PAX 3, the +14 Da delta mass could be explained either by one methylene group extension of the 3-hydroxy fatty acid to yield a 16:0 homolog or by a simultaneous presence of a K2R mutation (+28 Da) and a 14:0 3-hydroxy fatty acid (−14 Da). To choose between these two hypothesis, more material should be isolated to record the PAX 5 NMR spectrum.

Notice that in cyclolipopeptides there are two types of cyclizations involving the carboxylic group, one involving an amide bond and another an ester bond leading to a macrolactame or a macrolactone ring, respectively.37

Conclusion

This report describes the production, the purification and the characterization of a new antimicrobial family from X. nematophila. Their significant antifungal activity and their lack of cytotoxicity and entomotoxicity increase the potential interest of these molecules for vegetal or human health application.

Methods

Producing organism

Xenorhabdus nematophila F1 (Ecologie Microbienne des Insectes et Interactions Hôtes-Pathogène collection) was grown on Luria-Bertani medium (LB, composed of bactotryptone 10 g l−1, yeast extract 5 g l−1 and NaCl 10 g l−1) for liquid culture and on LB-agar for solid cultures. The phase status (I or II) of this strain was determined by culturing on NBTA (Nutrient agar (Difco, Detroit, MI, USA) 31 g l−1, bromothymol blue 25 mg l−1 and 2, 3, 5-triphenyl tetrazolium chloride 1% 40 mg l−1) and measuring antibacterial activity against M. luteus. Xenorhabdus exhibits two colony forms or variants when cultured in vitro. Modifications of the outer membrane induce differential adsorption of dyes by variants. Phase I variants absorb dyes and are blue on NBTA plates, whereas phase II colonies are red. Phases I and II of strains are indicated as suffixes (/1 and /2, respectively) attached to strain designations. This strain was maintained at 15 °C on NBTA medium.

Bacterial strains and antimicrobial agents

The following reference strains were used for the evaluation of antimicrobial activity: P. aeruginosa CIP 76.110, E. coli CIP 76.24, Proteus vulgaris CIP 58.60, S. aureus CIP 76.25, S. epidermidis CIP 68.21, Bacillus cereus ATCC 14579, Streptococcus pneumoniae CIP 103.566, M. luteus CIP 53.45 and C. albicans CIP 48.72 and clinical isolates (strains are indicated as suffixes H and a number) obtained from patients with infection at the University Hospital of Nîmes. Phytopathogenic fungi were obtained from Rhobio (Lyon, France). Vancomycin and polymyxin (Sigma-Aldrich, St Louis, MO, USA) were provided as standard powders by the manufacturers.

Antibacterial susceptibility testing methods

The minimal inhibitory concentration (MIC) was defined as the lowest antibiotic concentration, which yielded no visible growth. MIC was determined as recommended by the Clinical and Laboratory Standards Institute.38 Antibiotics were tested at final concentrations (prepared from serial twofold dilutions) ranging from 100 to 0.78 mg l−1. The test medium was Mueller–Hinton broth, and the inoculum was 5 × 105 CFU ml−1. The inoculated microplates were incubated at 37 °C under shaking for 18 h before reading.

Antifungal susceptibility testing methods

The PAX activities were tested against the human pathogen F. oxysporum and against different phytopathogenic fungi by the M38-A microdilution method39 with RPMI 1640 medium as recommended in the Clinical and Laboratory Standards Institute M23-A document.40 The NCCLS M27-A2 broth microdilution method was used when C. albicans was tested.41

Fungal inoculi were prepared from 7-day cultures grown on potato dextrose agar and adjusted spectrophotometrically to optical densities that ranged from 0.09 to 0.17 and diluted (1:50) in RPMI 1640 broth. The density of the inoculum suspension of C. albicans isolate was adjusted to a density of a 0.5 McFarland standard and diluted 1:1000 in RPMI 1640 broth.41 Microdilution trays (96 U-bottom shaped) containing 100 μl antifungal dilutions were added with 100 μl PAX solutions (final concentration ranging from 100 to 0.78 mg l−1 for F. oxysporum and C. albicans; 10, 20 and 40 μg ml−1 for other fungi). After inoculation of the trays, all microdilution trays were incubated at 30 °C in ambient air. As described in the M38-A method,39 MICs for F. oxysporum were determined by visual examination at 48 h. MICs were defined as the lowest drug concentration that showed absence of growth or complete growth inhibition (100%). MICs for C. albicans were determined at 48 h and corresponded to 100% of growth inhibition.41 Regarding phytopathogenic fungi, the culture absorbance was measured at 600 nm, 5 days after the beginning of the experiments. The activity results correspond to a percentage of growth inhibition ((1−Abs600 nm culture with PAX)/Abs600 nm culture without PAX).

Cytotoxicity test

Chinese Hamster Ovary (CHO) cells were grown in RPMI medium supplemented with 5% (v/v) fetal calf serum. The cells were incubated for 24 h at 37 °C in the absence of serum and in the presence of PAX (final concentration ranging from 1000 to 7.8 mg l−1).

The cytotoxicity was measured using the cell cytotoxicity Kit I (Roche Applied Sciences, Meylan, France).

Insect toxicity tests

The common cutworm, Spodoptera littoralis, was reared on an artificial diet42 at 24 °C, and the wax moth, Galleria mellonella, was reared on pollen and wax at 28 °C. A locust, Locusta migratoria, was reared on grass at 28 °C. Eggs of the tobacco hornworm, Manduca sexta, were obtained from Monika Stengl (University of Regensburg, Regensburg, Germany). M. sexta larvae were reared on an artificial diet43 at 27 °C with light-dark cycles consisting of 16 h of light and 8 h of darkness. Fifth-instar larvae of each insect species were selected and surface sterilized with 70% (v/v) ethanol before intrahemocoelic injection. The larvae were divided into groups of 12 larva, and each larva was injected with 10 μl of one of the purified PAX, corresponding to a dose of 0.1 μg per insect, or with phosphate-buffered saline. The treated larvae were incubated individually for up to 96 h, and then the number of dead insects was recorded.

A liquid hemolysis assay with sheep erythrocytes44 was used to determine hemolytic activity of purified PAX. Cytolytic assays were performed with insect hemocytes by collecting hemolymph samples from S. littoralis larvae in an anticoagulant buffer.45 Hemocytes were centrifuged, rinsed in phosphate-buffered saline to remove plasmatic factors, and resuspended in the same buffer (2 × 104 hemocytes ml−1). The suspensions (10 μl) were each mixed with 10 μl of a purified PAX, corresponding to a 0.1-μg dose, deposited on a slide, and incubated for 20 min at 28 °C. Hemocytes with phosphate-buffered saline were used as a control. Cell lysis was observed with a light microscope and was recorded.

NMR and MS analysis

The NMR samples were prepared from the lyophylized lipopeptide. They were disolved in DMSO-d6 to yield 1.0–1.5 mM solution. Chemical shifts are expressed with respect to the DMSO-d6 residual signal set at 2.50 and 39.5 p.p.m. for 1H and 13C spectra, respectively. All NMR experiments were carried out on a Bruker Avance 600 spectrometer (Bruker Analytik GmbH, Rheinstetten, Germany) equipped with a triple resonance cryoprobe, and spectra were recorded at temperatures ranging from 295 to 310 K. Double-quantum filtered-COSY (DQF-COSY),46, 47 z-filtered total-correlated spectroscopy (z-TOCSY)48, 49 and NOESY50 spectra were acquired in the phase-sensitive mode, using the States-TPPI method.51 We obtained z-TOCSY spectra with a mixing time of 90 ms and NOESY spectra with mixing times of 150 and 250 ms, respectively. The 1H-15N HSQC, 1H-13C HSQC, 1H-13C HMBC, 1H-13C HSQC-TOCSY experiments52, 53, 54 were carried out with the same sample. All data were processed with XWINNMR software (Bruker Analytik GmbH). The assignment of the peptidic part was achieved using the general strategy described by Wüthrich.55 Owing to the close proximity of the K6 and K7 amide signals in DMSO-d6, the dNN6–7 NOE could not be observed. Thus, two other data sets were recorded with 17 and 33% of water at several temperatures ranging from 285 to 310 K. In these conditions, these two amide signals were enough separated to observe the dNN6–7 NOE.

The assignment of the non-peptidic part was obtained from the analysis of the homo and heteronuclear data. Owing to the overlap of 1H resonances as well as the 13C resonances of the central methylene groups of the fatty acid, giving rise to an unique HSQC cross-peak, the length of the fatty acid chain was calculated to be in agreement with the molecular weight measured by mass spectrometry. For PAX 1, 2 and 4, only 1H-NMR data were recorded.

LC-MS was first performed to obtain the m/z value of the protonated PAX derivatives. ESI-LC-MS data were obtained in the positive mode on a Waters alliance LC-MS system (Waters ZQ mass detector, Waters photodiode array detector 2696, Waters alliance HPLC systems 2790). The HPLC column used was a C18 column (Waters; X-terra RP18; 5 μm; 4.6 × 250 mm) maintained at 35 °C. Solvents were (A) water +0.1% TFA (B) acetonitrile +0.1% TFA and the flow rate was 1 ml min−1. The mobile phase composition was 80% A from 0 to 5 min, ramped to 80% B at 35 min. Samples were dissolved in solvent A (100 μl). Sample injection volume was 10 μl. UV-visible detection was by absorbance at 200–600 nm. Solvent flow to the MS was diverted to waste for the first 5 min to minimize salt build-up. PAX 3 MS-MS fragmentation data were obtained on a Waters Micromass Q-T of micro mass spectrometer.