Induced production of cytochalasans in co-culture of marine fungus Aspergillus flavipes and actinomycete Streptomyces sp.

Abstarct Secondary metabolites profiles of co-culture of Aspergillus flavipes and Streptomyces sp. that isolated from the same habitat showed an induced production of a series of cytochalasans (five aspochalasins and rosellichalasin, determined by MS and NMR analysis). These cytochalasans were found to be produced by A. flavipes in LC–MS comparison analysis, and biological activity assays revealed that they were able to cause cytotoxic effects against Streptomyces sp. within a wide range of concentrations without causing any effect to the producer A. flavipes, which favoured the producer in competition. Further induction mechanism study applying membrane-separated culture and morphology study with scanning electron microscopy (SEM) suggested that the successful induction of active secondary metabolites required microbial physical contact.


Introduction
Microbial secondary metabolites were thought to be produced because of the selectional advantages they confer on the producing organisms (Stone & Williams 1992), such as inhibiting or killing other microorganisms (antibiotics), toxicity against hosts for colonisation (Duke & Dayan 2011) and regulating intraspecies populations (quorum sensing) (Gonzalez & Keshavan 2006). Several papers in natural products research area have reported the ABSTARCT Secondary metabolites profiles of co-culture of Aspergillus flavipes and Streptomyces sp. that isolated from the same habitat showed an induced production of a series of cytochalasans (five aspochalasins and rosellichalasin, determined by MS and NMR analysis). These cytochalasans were found to be produced by A. flavipes in LC-MS comparison analysis, and biological activity assays revealed that they were able to cause cytotoxic effects against Streptomyces sp. within a wide range of concentrations without causing any effect to the producer A. flavipes, which favoured the producer in competition. Further induction mechanism study applying membrane-separated culture and morphology study with scanning electron microscopy (SEM) suggested that the successful induction of active secondary metabolites required microbial physical contact. induced production of secondary metabolites when one microbe was cocultivated with other microbes (Scherlach et al. 2013;Teles et al. 2013;Li et al. 2014;Yu et al. 2015), and we gained some insights of the interactions between bacteria and fungi for secondary metabolites production recently (Schroeckh et al. 2009;Nützmann et al. 2011), but the complex of interactions and the fundamental mechanisms of how and why these compounds were induced still need more investigations.
Aspergillus flavipes belongs to a genus that was a major contributor of fungal origin secondary metabolites. This fungus existed in various environments and owned very abundant secondary metabolites genes. It had been reported to produce different kinds of secondary metabolites, such as peptide deformylase inhibitors flavimycins (Kwon et al. 2012), cytotoxic cerebroside analogues (Jiang et al. 2004), cytotoxic aspochalasins (Zhou et al. 2004;Lin et al. 2009;Liu et al. 2014), antimicrobial butyrolactones (Bai et al. 2014) and cyclopeptides . A. flavipes's ability to produce varieties of secondary metabolites means it was a good candidate for studying the role of secondary metabolites in microbial interactions.
In this work, fungus A. flavipes and actinomycete Streptomyces sp. which isolated from the same marine sediment, were applied to microbial co-culture and showed an induced production of secondary metabolites, we further identified these compounds, and investigated why and how these compounds were produced.

Cocultivation of A. flavipes with Streptomyces sp. for the production of cytochalasans
Secondary metabolites of cell-free medium of the co-culture and monoculture were analysed using hPLC. The co-culture (Figure 1(c)) displayed many newly produced metabolites compared to the Streptomyces sp. (Figure 1(a)) and A. flavipes (Figure 1(b)) monocultures. The induced compounds (compounds 1-6) were isolated and identified (supplementary material) as rosellichalasin (1) (Xiao et al. 2013), aspochalasin E (2) (Naruse et al. 1993), aspochalasin P (3) (Lin et al. 2009), aspochalasin h (4) (Tomikawa et al. 2002), aspochalasin M (5) (Naruse et al. 1993), and 19,20-dihydro-aspochalasin D (6) (Tomikawa et al. 2001) (Figure 1(d)). LC-ToF-MS analysis was carried out to determine the producer of these compounds. The culture medium of A. flavipes monoculture displayed m/z peaks of rosellichalasin (1) Figure S1), but the culture medium of Streptomyces sp. monoculture did not, which unambiguously supported that all six cytochalasans were produced by A. flavipes. These data suggest that when A. flavipes encountered Streptomyces sp., it elevated the ambient concentration of cytochalasans, which were produced at a very low production rate in monoculture and could not be detected by hPLC analysis.

The induced cytochalasans were cytotoxic to Streptomyces sp.
In the followed bioactivity study, all six cytochalasans exhibited strong toxicity against Streptomyces sp. (Figure 1(e)), as the inhibiting rate ((oD control − oD test )/oD control × 100%) was 50-80% at concentrations of 2-16 μg/mL, and most of these cytochalasans showed an inhibiting rate of 60% even at concentration of 2 μg/mL, but had no effect on the fungus A. flavipes at the same concentration. This result indicates that the cytochalasans helped A. flavipes to compete with Streptomyces.
Cytochalasans are fungal metabolites that are structurally characterised by the presence of a reduced isoindone nucleus fused to a macrocyclic ring (Thomas 1991). Since the finding of the first cytochalasan, more than 100 related structures have been isolated from various fungal sources. almost all bioactivity studies of cytochalasans, including the cytochalasans isolated in this work, have focused on their toxicity against human cancer cell lines (Zhou et al. 2004;Lin et al. 2009;Xiao et al. 2013), as cytochalasans have been well known for their abilities to bind to actin filaments and block polymerisation and the elongation of actin (Scherlach et al. 2010). Some aspochalasins have shown antimicrobial activity (Betina et al. 1972;Flashner et al. 1982) but few in number. In the current study, it was found that when the cytochalasan producer A. flavipes was co-cultivated with actinomycete Streptomyces sp., its production of cytochalasans was greatly stimulated, and these cytochalasans could inhibit the growth of its competitor Streptomyces sp. to a great extent from 2 to 32 μg/mL (Figure 1(e)), which was an important experimental support for the potential ecological role of cytochalasans.

Induced production of cytochalasans required physical contact
The production of cytochalasans by A. flavipes was greatly stimulated when it was co-cultivated with Streptomyces sp. To explore the mechanism of stimulation, a series of experiments was carried out. We first cultured A. flavipes in Streptomyces sp. spent medium, and with heat-inactivated Streptomyces sp. mycelium, the hPLC analysis results (Figure  2(a)) showed no production of cytochalasans, thereby excluding the possibility of small molecules and proteins produced by Streptomyces sp. mono-culture. We also co-cultivated A. flavipes and Streptomyces sp. separately (Figure 2(b)), using dialysis membrane (8-14 k MWCo) and microfiltration membrane (0.22 μm), to test the signal small molecules and proteins produced only in co-culture, the hPLC analysis results again showed no induction of cytochalasans. So we assumed that the induced production of cytochalasans was activated through physical interaction. The cross-section SEM images of mycelium balls in co-culture (Figure 2(c)) showed that the whole mycelia of Streptomyces sp. were tightly packed by mycelia of A. flavipes, and in the area where the Streptomyces sp. was tangled with A. flavipes, many Streptomyces sp. mycelia were stuck to the A. flavipes (Figure 2(d)). It was reported previously that the physical interaction between actinomycete and fungus could lead to specific metabolic response (Schroeckh et al. 2009), therefore, we conclude that the induction of cytochalasans was probably through physical interaction.

Bacterial strains and culture methods
The strains used in this study were Streptomyces sp. CGMCC4.7185 (16S rDNa Gene Bank accession number KJ729120) and A. flavipes CGMCC 3.15449 (ITS Gene Bank accession number KP760851), isolated from marine sediments of the Nanji Islands (China, 27 o 42′N，121 o 08′E), both strains have been deposited at the China General Microbiological Culture Collection Center (CGMCC) with open accession. The microbes were cultured in a medium composed of 5 g yeast extract, 5 g glycerol and 1 L 75% seawater (ph 7.5) in 500-mL flasks (containing 200 mL medium) under 180 rpm at 28 °C. To produce the co-culture, 5 mL of microbial seed broth (A. flavipes and Streptomyces sp. in a ratio of 1:4 (v/v)) were added to the 200 mL culture medium and cocultivated for eight days.

Secondary metabolites isolation and structures determination
about 60 L co-culture medium was extracted with equal volume of ethyl acetate twice. The resulting ethyl acetate part was fractioned by silica gel column chromatography using Ch 2 Cl 2 -Meoh gradient (100-90%) of increasing polarity. Fractions displayed the induced peaks in hPLC analysis were further purified by preparative hPLC (Beijing Chuangxintongheng LC3000 Semi-preparation Gradient hPLC System equipped with Sepax amethyst C-18 (5 μm, 21.2 × 250 mm) column) to afford relative cytochalasans. The structures were determined by LC/ToF/MS analysis and nuclear magnetic resonance (Bruker ascend TM 600) spectrometer analysis. Spectral data were supplied in supplementary material.