Microbial interactions trigger the production of antibiotics

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ScienceDirect Microbial interactions trigger the production of antibiotics Tina Netzker1, Michal Flak1,2, Mario KC Krespach1,2, Maria C Stroe1,2, Jakob Weber1,2, Volker Schroeckh1 and Axel A Brakhage1,2 Since the discovery of penicillin, antibiotics have been instrumental in treating infectious diseases. However, emerging antibiotic multi-resistance coinciding with a nearly exhausted drug pipeline is a major concern for the future of the therapy of infections. A novel approach for the discovery of antibiotics relies on the analysis of microbial consortia in their ecological context, taking into account the potential natural role of antibiotics. Co-cultivations of microorganisms have been successfully applied for the isolation of unknown secondary metabolites including antibiotics, and, thus, open new avenues to the production of bioactive compounds while at the same time providing insight into the natural function of the produced molecules and the regulation of their formation. Addresses 1 Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI) , Beutenbergstrasse 11a, 07745 Jena, Germany 2 Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany Corresponding author: Brakhage, Axel A ([email protected])

Current Opinion in Microbiology 2018, 45:117–123 This review comes from a themed issue on Antimicrobials Edited by Gilles van Wezel and Gerard Wright

https://doi.org/10.1016/j.mib.2018.04.002 1369-5274/ã 2018 Elsevier Ltd. All rights reserved.

Introduction Microorganisms colonize all habitats on earth. This is possible due to their versatile metabolism, which allows for adapting to ever-changing environmental conditions such as temperature changes, availability of water, or interaction with other microorganisms and predators. In addition, most microorganisms also produce low-molecular-weight compounds, called secondary metabolites (SMs) or natural products (NPs). By contrast to primary metabolites, which are indispensable for basic cellular activity, SMs are generally believed to support the survival of the producing microorganism, by serving as direct www.sciencedirect.com

‘defense weapons’ against other microorganisms, aiding inter-species and intra-species communication or by conferring an advantage in nutrient acquisition [1,2]. The genes for the biosynthesis of SMs are largely encoded in clusters. The majority of these clusters encode a central polyketide synthase (PKS) or non-ribosomal peptide synthetase (NRPS) gene [3]. SMs often possess valuable therapeutic properties and have been used as antibiotics, anti-tumor agents, and cholesterol-lowering compounds [2]. Of particular value are antibiotics used to treat bacterial and fungal infections. However, bacterial antibiotic resistance has become an increasing threat, due to overuse and misuse of therapeutics, coupled with a lack of new drugs, a phenomenon known as the antibiotic innovation gap [4]. The ‘antibiotic resistance crisis’ is a worsening problem [5], requiring political intervention, underlined by the WHO [6] and G20 statements [7], and the renewed interest in scientific discovery of novel antibiotics. In the early years of antibiotic discovery, scientists exploited microbial monocultures to find new antimicrobial compounds. Additionally, SMs have been chemically modified to create semi-synthetic antibiotics. Another strategy was based on de novo synthesis of compounds and their testing for desired properties. These approaches were highly successful, yielding all ‘key access antibiotics’ on the WHO Model List of Essential Medicines [8]. For example, the Nobel Prize winning antibiotics penicillin and streptomycin are produced in monocultures. Nevertheless, screening for novel compounds in the established SM producers such as actinomycetes or filamentous fungi has led to a high rediscovery rate of known compounds, a challenging problem, which could be diminished by further development of dereplication methods. Since the 1960s, only six new antibiotic classes were introduced into clinical practice [9]. Today, the increasing number of sequenced microbial genomes has uncovered the huge biosynthetic potential of microorganisms. However, there is a lack in identification and understanding the triggers activating the production of these hidden secondary metabolites [10]. Consequently, more targeted methods must be established to find novel antibiotics and unique chemistry. As shown by the increasing number of publications within the last years, cultivation of two or more species together (co-cultivation) has become a promising way to identify novel antimicrobial compounds [11–14]. To further optimise Current Opinion in Microbiology 2018, 45:117–123

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this approach, substantial technological advances have been made such as the development of microfluidic cell encapsulation, iChip or a co-cultivation chamber. This review discusses recent trends in co-cultivation with regards to identifying the underlying molecular mechanisms, discovering novel metabolites, and further developing enabling technologies.

A current trend — co-cultivation of microorganisms Microorganisms never live isolated in the environment but are constantly in close proximity to other organisms exchanging and sharing chemical signals, such as SMs. However, for most SMs, their ecological role remains obscure [2]. A long-lasting hypothesis was that they are used as weapons for competition with other microorganisms [15], while other studies showed that low concentrations of SMs can modulate gene expression, cell physiology, and act as elicitors to induce silent SM gene clusters in the confronted organisms, suggesting a role as signalling molecules [16–18] (Figure 1a). Recent studies on the interactions of various Streptomyces spp. with each other showed that, depending on neighbouring strains and nutrient availability, their antibiotic production was increased and that of the competitors was repressed [19,20]. This finding supports the classical hypothesis about the role of SMs as weapons against other microorganisms. Another study, however, supports the hypothesis that SMs play a crucial role as chemical communication molecules when fusaric acid from the fungus Fusarium oxysporum promotes the colonization of its hyphae by the bacterium Pseudomonas fluorescens [21]. The given examples are consistent with the idea, that SMs elicit both positive and negative effects on the target organisms. Both findings further fuel the increasing interest in co-cultivation experiments not only as an approach to identify novel compounds, but also to understand the underlying molecular mechanisms and the role of SMs in nature. Co-cultivation of microorganisms aims at simulating ecological interactions not present in microbial monocultures. Therefore, novel cultivation methods, as well as analytical methods are required that will be discussed below. A number of novel SMs have been already isolated from co-cultivated microorganisms [11,12]. An early example is the mixed culture of the fungus Aspergillus nidulans and the bacterium Streptomyces rapamycinicus that led to the activation of a silent fungal gene cluster and, as a consequence, the production of the archetypal polyketide orsellinic acid [22] (Figure 1). The same streptomycete species was also able to activate a silent gene cluster in A. fumigatus, which led to the discovery of the fumicyclines [23]. Co-culturing of Micromonospora sp. with Rhodococcus sp. on a microscale platform resulted in the identification of keyicin, an antibiotic selectively active against gram-positive bacteria [24]. The new macrolide Current Opinion in Microbiology 2018, 45:117–123

antibiotic called berkeleylactone was identified by co-cultivation of Penicillium fuscum and Penicillium camembertii/clavigerum [25]. Berkeleylactones show an unprecedented mode of antibacterial activity, since they do not stall the ribosome or inhibit protein biosynthesis, as comparable macrolide antibiotics do. Both keyicin and berkeleylactones underline the value of studying terrestrial or marine-derived microbial interactions to identify new potential anti-infective compounds with new mode of actions. Another potent source for unknown SMs is the human microbiota. Lactocillin [26], a ribosomally synthesised thiopeptide, as well as the non-ribosomal peptide lugdunin [27], are produced by human commensals. Both compounds prevent the growth of neighbouring pathogens, indicating their involvement in structuring of the human microbiome, as well as their potential as antibiotics. Bipartite microbial cultivations provide a fascinating source for the identification of new compounds with biological activity. However, to simulate natural ecological conditions, multiple species interactions are essential (Figure 1b). This requires the formation of synthetic microbial consortia with strains shown to interact with each other. For the investigation of such multi-species consortia, appropriate cultivation methods and analytical tools need to be developed (see below). One of the currently best-studied multi-species systems is the symbiosis between fungus-growing ants and associated actinomycetes, which evolved 50–60 million years ago. Attine ants cultivate the basidiomycete Leucoagaricus gonglyophorus by providing nutrients, and, in return, the fungus serves as their main food source [28]. The ants protect the fungus by grooming and weeding, production of antimicrobials, and distribution of SMs produced by the associated Pseudonocardia strains [29]. Several compounds inhibiting parasitic fungi such as Escovopsis spp. have been discovered, among them the well-known antifungal compounds antimycin [30] and candicin [31], or a new nystatin derivative [32]. Investigation of produced SMs by ant-associated bacteria also led to the new antifungals dentigerumycin [33] and selvamicin [34]. These compounds not only inhibit the growth of parasitic fungi but also suppress the growth of closely related species. Recently, it was shown that Pseudonocardia sp. involved in the interaction produced the plasmid-encoded rebeccamycin analogue, which probably provides defence against closely related bacteria [35]. Ants are not the only insects utilizing symbiotic actinomycetes to protect their fungal cultivar. Similarly, termites cultivating distinct fungi are also colonized by SM-producing actinomycetes. For example, Beemelmanns et al. isolated the antibacterial macrotermycins A–D from Amycolatopsis sp. [36]. These examples illustrate the benefit of multipartner interactions over monocultures or co-cultures for the mining of valuable NPs. More of these natural systems need to be studied not only to discover interesting compounds, but also to acquire a profound knowledge about the www.sciencedirect.com

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Figure 1

(a) effects activation

Microbial community

signal molecules/ stimuli

silent gene cluster

Microbial community

(b)

Monoculture

Co-culture

Complex culture

(c)

Monocultures

Co-culture Current Opinion in Microbiology

Production of SMs by microorganisms in monoculture, co-culture or by complex consortia leading to activation of silent gene clusters and production of potentially novel SMs. (a) Microorganisms excrete a plethora of NPs that act as chemical mediators like antibiotics or potential inductors of silent gene clusters in neighbouring species. The thereby produced compounds might affect other microorganisms. (b) Microbial monocultures lead to the production of a number of compounds that can be increased by co-cultivation. It is likely that the full capacity of NP formation can only be exploited in complex microbial consortia. (c) Microscope images of fungal (left) and bacterial (right) monocultures and in cocultivation on solid agar (left) and in liquid culture (right).

regulation underlying their production. We envision that by understanding the underlying principles of microbial communication, it will be possible to predict which microorganisms ‘talk to each other’.

New cultivation methods New cultivation methods are needed for isolation of unknown species, such that ideally favour the growth of slow-growing species. As 99% of all microorganisms still appear to be ‘unculturable’, meaning they do not grow under standard laboratory conditions, strategies have to be developed to explore their underexploited www.sciencedirect.com

potential. Successful examples are the diffusion chamber [37] and the iChip device [38], consisting of hundreds of miniaturized diffusion chambers, used to grow ‘unculturable’ marine and terrestrial microorganisms. Both devices follow the vision of transferring the natural environment to the lab bench without impeding growth, nutrient factors, or signals generated by surrounding organisms, restricting only movement. The use of iChip and diffusion chamber led to a 40–50% growth recovery, compared to only 1% in standard Petri dishes [38,39], and to the identification of several new environmental isolates like Psychrobacter sp. [40], Nocardia sp. [41], or Eleftheria Current Opinion in Microbiology 2018, 45:117–123

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terrae [42]. The latter two microorganisms produce the novel antibiotics neocitramicin I & II [41] and teixobactin [42], respectively. These findings render the diffusion chamber and the iChip interesting tools to grow uncultured microorganisms and support the hypothesis that an environmental signal exchange can aid growth and induce the production of SMs. A promising cultivation method for multispecies analysis is based on droplet microfluidics, which meets the necessary requirements [43–45]. Furthermore, microfluidics enables the study of one-to-one interactions between microorganisms in a defined and controlled manner. For example, communication via quorum sensing was induced in an encapsulated LasR-producing Escherichia coli strain by diffusion of the autoinducer N-acyl homoserine lactone (AHL) from adjacent droplets [46]. This E. coli strain was also able to sense other AHL-producing bacteria in neighbouring droplets [46]. For the investigation of multispecies interactions (10 species), suitable imaging and labelling techniques are needed to distinguish between different organisms and to follow interactions spatially and spatiotemporally. Besides the successful establishment of ideal growth conditions for a multispecies cultivation system, a more challenging issue would be the establishment of conditions for a stable NP production by the microbial consortium along with real-time monitoring. Another major draw-back lies in the fact that most microbial NPs remain hidden in the global microbiome to which ‘unculturable’ microorganisms contribute to a major extent. Culture-independent NP discovery based on meta-genome sequencing, identification of biosynthetic gene clusters and their expression in appropriate host cells allows to tackle this problem at least in part. It led to the discovery of the malacidins, a distinct class of antibiotics that are commonly encoded in soil microbiomes but have never been reported in culture-based NP discovery efforts. The malacidins are active against multidrug-resistant pathogens, sterilize methicillin-resistant Staphylococcus aureus skin infections in an animal wound model and do not select for resistance under laboratory conditions [47].

Methods and perspectives for the analysis of bioactive compounds With the advent of genome mining, it has become apparent that the number of SM gene clusters far exceeds the number of compounds discovered [2,10]. Nevertheless, the largest proportion of research addressing bioprospecting microbes for antibiotics has so far focused on monocultures, often based on the ‘one strain many compounds’ (OSMAC) strategy [48,49]. Axenic culture extracts were screened using either bioactivity-guided or structureguided fractionation to discover and identify novel Current Opinion in Microbiology 2018, 45:117–123

chemistry [50]. These workflows traditionally involve a separation technique (thin-layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC), capillary electrophoresis) and a detection technique (mass spectrometry (MS), nuclear magnetic resonance (NMR), UV–Vis/IR spectroscopy) (reviewed in [51]). Although these techniques remain a gold standard in NP discovery, a growing tendency towards a less targeted, more holistic approach can be noticed [52]. This includes the application of modern imaging and analysis methods, with higher resolution, sensitivity and throughput options [53], as well as devices allowing for online data collection, from simple plate readers to proprietary systems such as the BioLector [54]. Although mixed cultures represent a source of untapped chemical diversity, finding suitable culture conditions and sample analysis techniques brings about a spectrum of challenges. The analysis of a multispecies experiment usually falls into one of the following three categories: (i) the metabolite(s) production can be easily visualized and/or monitored with simple methods such as TLC or high pressure liquid chromatography (HPLC) (summarized in [55]); (ii) the metabolites are spatially confined, for instance localized in a confrontation zone, which can be detected by imaging techniques such as Matrix-Assisted Laser Desorption/Ionization (MALDI) imaging mass spectrometry (IMS) [56,57] or (iii) the production is not obvious, falls under detection threshold level, and requires a generalized, untargeted secondary metabolomics approach coupled with data mining [51]. The state of the art of metabolomics of SMs is in sharp contrast with that of primary metabolites, for which robust networks and large databases have been developed (e.g. [58]). Comparative MS-based metabolomics has only recently been used for prediction of the activity of SMs and provides an avenue to compound discovery independent of abundance [59]. Since the ‘golden age’ of NP discovery, MS, coupled with chromatographic methods for complexity reduction, has been at the forefront of the discovery of SMs as a relatively inexpensive and sensitive methodology. In the field of MS, incremental advances and technological innovation have led to a number of devices suited for a range of applications, depending on the needed sensitivity, resolution, throughput, or sample amount requirements. Although the variation in the MS devices remains low [60], the coupled ionization modalities are becoming more and more specialized, allowing for in vivo, space-resolved compound discovery and monitoring. Over the past two decades, MS imaging (MSI) applied to microbiology has been enabled by MALDI, representing an approximated half of all microbial MSI publications [52]. Boya et al. [61] used MALDI–MS/MS to visualize antagonistic interactions between ant-derived streptomycetes and the fungal pathogen Escovopsis sp. and identified the antibiotics actinomycins D, X2, and www.sciencedirect.com

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X0b, elaiophylin, and efomycins A and G. Although destructive to the sample, it facilitated a robust, spatially resolved imaging of a range of metabolites involved in microbial interactions. Matrix-free Laser Desorption/Ionization (LDI) techniques have substituted matrix overlaying with depositions of metals or nanocomposites and are particularly useful in analyses of chemical mixtures containing compounds with endogenous UV chromophores [62,63]. MALDI and LDI approaches are routinely coupled to time-of-flight high resolution mass spectrometry (TOF-HRMS) modalities with tandem MS, providing a sufficient amount of data for compound identification and/or dereplication [64]. The need for less invasive methods of microbial visualization, allowing for topography inherent to solid-phase cultures has led to the development of desorption electrospray ionization (DESI) [65]. This invention has landmarked the advent of ambient MS techniques, enabling the observation of samples in their native environments. Methods evolved on the platform of ambient ionization have been successfully employed in microbiology laboratories and led to the identification of multiple NPs (reviewed in [66]). NanoDESI, the latest improvement on DESI, has been shown as a suitable tool for analysing mixed biofilm cultures [67]. Circumventing the visualization step and substituting with heat mapping, Sica and coworkers developed a non-invasive method that couples a droplet-liquid microjunction-surface sampling probe (droplet-LMJ-SSP) with a standard UHLPC-PDA-HRMS setup. This system enabled the investigation of fungal SM profiles in cultures grown directly on an agar plate, as well as their effects on other fungi in co-cultivation [68,69]. Such analysis yielded several new peptaibol derivatives [70] and has been used to monitor secondary metabolite interaction in a co-cultivation of Xylaria cubensis and Penicillium sp. [69]. It is conceivable that this approach could be used more widely in chemical characterization of diverse microbial co-cultures. Considering the wealth of spectroscopic and spectrometric data being produced, increasing the rates of antibiotic discovery without rediscovering already known compounds is of paramount importance. An increased use of public repositories of reference data for microbial metabolites, such as the GNPS or MassBank, will greatly alleviate the dereplication problem and accelerate the discovery of novel microbial products [71]. Furthermore, untargeted secondary metabolomic applications, akin to their primary metabolomic counterparts, have recently emerged as specialized tools for compound discovery and dereplication, using MS2 spectral networking analysis or fragmentation trees [50,72]. The crucial step is the analysis of the resulting amount of data in order to identify differentially produced metabolites. The data can be subjected to either a simple differential analysis coupled with traditional statistical approaches (partial least square (PLS) or orthogonal projection to latent structure (OPLS)) [56], more innovative approaches such as www.sciencedirect.com

POCHEMON [73], or to an advanced chemometric analysis [51,74]. Combined with the abundance of nucleic acid sequence data, this functional -omics approach falls in line with a general integrative trend in NP research.

Conclusion and perspective The discussed examples illustrate the value of studying multispecies interactions for the identification of new antimicrobial compounds and understanding of general principles of microbial communication. Challenges to be overcome include the development of culture techniques allowing multiple microbial strains to live in a steady state. Deeper understanding of the rational basis of microbial communication based on SMs, will lead to the possibility to predict microbial interaction partners in silico based on their genetic information. This will also help to unravel the ecological role secondary metabolites play (Figure 1). The development and improvement in analytical and imaging techniques (e.g. Raman spectroscopy [75]), along with their combination, will allow for obtaining information on compounds and interaction partners online in situ.

Conflict of interest statement Nothing declared.

Acknowledgements We thank Sandor Nietzsche (Electron Microscopy Center, Jena University Hospital) for providing scanning electron microscopic photographs. Research in the authors’ laboratory was supported by the Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Center 1127 ChemBioSys, the DFG-funded excellence graduate school Jena School for Microbial Communication and the German Federal Ministry of Education and Research-funded project DrugBioTune in the frame of Infectcontrol2020.

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