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Evolutionary Stability of Antibiotic Protection in a Defensive Symbiosis

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Evolutionary Stability of Antibiotic Protection in a Defensive Symbiosis Evolutionary stability of antibiotic protection in a defensive symbiosis Tobias Engla,b,1, Johannes Kroissa,c, Marco Kaid,2, Taras Y. Nechitayloa, Ales Svatosd, and Martin Kaltenpotha,b,1 aInsect Symbiosis Research Group, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; bEvolutionary Ecology, Institute of Organismic and Molecular Evolution, Johannes Gutenberg University, 55128 Mainz, Germany; cThermo Fisher Scientific GmbH, 63303 Dreieich, Germany; and dResearch Group Mass Spectrometry/Proteomics, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved January 18, 2018 (received for review November 15, 2017) The increasing resistance of human pathogens severely limits the However, the presence of these compounds in situ and their efficacy of antibiotics in medicine, yet many animals, including relevance for the protective activity of the insects’ nutritional solitary beewolf wasps, successfully engage in defensive alliances resources have received little attention (32). By contrast, HPLC– with antibiotic-producing bacteria for millions of years. Here, we high-resolution MS, NMR, and imaging MS were successfully report on the in situ production of 49 derivatives belonging to used for in situ characterization of the chemical mediators in three antibiotic compound classes (45 piericidin derivatives, 3 another defensive symbiosis involving solitary beewolf wasps and streptochlorin derivatives, and nigericin) by the symbionts of 25 bee- antibiotic-producing Streptomyces bacteria (19, 21, 34). wolf host species and subspecies, spanning 68 million years of This specialized protective symbiosis occurs in the clade of evolution. Despite a high degree of qualitative stability in the antibiotic Philanthini digger wasps that are associated with host-specific mixture, we found consistent quantitative differences between species strains of the actinobacterium Streptomyces philanthi (35). and across geographic localities, presumably reflecting adaptations to Beewolf females harbor the symbiotic bacteria in antennal res- combat local pathogen communities. Antimicrobial bioassays with the ervoirs (36) and secrete them into their brood cells, where they three main components and in silico predictions based on the structure are transferred to the larval cocoon and protect the developing and specificity in polyketide synthase domains of the piericidin larva against opportunistic mold fungi from the environment biosynthesis gene cluster yield insights into the mechanistic basis and (37). In the European beewolf, Philanthus triangulum, this de- ecoevolutionary implications of producing a complex mixture of antimicrobial compounds in a natural setting. fensive activity is achieved through the symbiont-mediated pro- duction of a mixture of antimicrobial compounds, including defensive symbiosis | protective mutualism | antibiotic resistance | streptochlorin and at least eight different piericidins on the co- Philanthus | Streptomyces philanthi coon (34). Despite the opportunity for environmental acquisition of nonspecific bacteria and phylogenetic evidence for occasional horizontal transfer of symbionts between host species, the symbiosis ince the discovery of penicillin in 1928 by Alexander Fleming, has remained specific since its origin about 68 Mya, involving the use of antibiotics for the treatment of infectious diseases S monophyletic clades of wasps and bacteria, respectively (35). has revolutionized human medicine (1). However, the past 60 y have seen a dramatic increase in antibiotic-resistant pathogens, rendering many of the previously potent antibiotics effectively Significance useless (2–5). The rapid evolution of resistance through muta- tion (6) and the spread of resistance genes by horizontal gene Insights from natural applications of antibiotics are important transfer (7–9) raise the question of how antibiotics can maintain to gain a deeper understanding of the evolutionary processes their efficiency in a natural context over long evolutionary that underlie the maintenance of an antibiotic defense and timescales. Unfortunately, our knowledge about the ecology and prevent the rise and spread of antibiotic resistance. Using evolution of antibiotics remains severely limited. While they can 25 species and subspecies of beewolf digger wasps that en- Streptomyces be used as chemical weapons under certain symbiotic or free- gage in a defensive symbiosis with bacteria, we living conditions (10–12), in other situations they seem to serve tracked evolutionary changes in the antibiotic cocktail that ’ as signaling molecules that affect gene expression in con- or protects the wasps larval offspring against mold fungi. Our heterospecific microorganisms (13–19). results yield insights into the mechanistic basis as well as the Two of the main challenges associated with studying the ecological and evolutionary implications of producing a com- ecology of antibiotics are (i) the difficulty of monitoring complex plex cocktail of antimicrobial compounds in a symbiotic setting. microbial interactions in nature (20) and (ii) the detection and Author contributions: T.E., J.K., and M. Kaltenpoth designed research; T.E., J.K., T.Y.N., quantification of antibiotic production in situ (21). A solution for and M. Kaltenpoth performed research; J.K. and M. Kai contributed new reagents/ the first issue is to consider associations that involve only a analytic tools; T.E., J.K., T.Y.N., A.S., and M. Kaltenpoth analyzed data; and T.E. and limited number of interacting organisms. In particular, defensive M. Kaltenpoth wrote the paper. symbioses between insects and antibiotic-producing symbionts The authors declare no conflict of interest. represent attractive model systems, since a limited number of This article is a PNAS Direct Submission. partners have interacted over millions of years to ensure pro- Published under the PNAS license. tection against coevolving or opportunistic pathogens (11, 12, Data deposition: The data reported in this paper have been deposited in GenBank, https:// 20). A prime example is the tripartite symbiosis of leafcutter www.ncbi.nlm.nih.gov/genbank (accession nos. KX098584, KU759552–KU759556,and – ants, their nutritional fungus gardens, and Actinobacteria that KU759557 KU759562). The MS data reported in this paper have been deposited in the – Dryad Digital Repository (doi:10.5061/dryad.6907h). protect the gardens from coevolved parasitic microfungi (22 26) 1 – To whom correspondence may be addressed. Email: [email protected] or mkaltenpoth@ as well as the ants themselves against entomopathogens (27 29). uni-mainz.de. One major limitation in the study of leafcutter ants and many 2Present address: Department of Biochemistry, Institute for Biological Sciences, University other defensive symbioses has been the focus on bioactive mi- of Rostock, 18059 Rostock, Germany. crobial metabolites produced in vitro, with several antibiotic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. compounds characterized from Pseudonocardia and Streptomyces 1073/pnas.1719797115/-/DCSupplemental. isolates that are associated with the leafcutter ants (25, 30–33). Published online February 14, 2018. E2020–E2029 | PNAS | vol. 115 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1719797115 Downloaded by guest on September 24, 2021 Considering the high selection pressure exerted by antagonistic (ESI)-MS/MS (data available from the Dryad Digital Repository: PNAS PLUS soil microbes as well as the specificity and long coevolutionary doi:10.5061/dryad.6907h). history in the beewolf–Streptomyces symbiosis, it seems sur- Comparisons of the accurate molecular masses of all detected prising that the symbiont-produced antibiotic mixture has pro- substances with 2,583 Streptomyces-produced bioactive sub- vided an efficient defense against pathogens over the past stances in Antibase 2005 (38) revealed actinopyrone, Mer- 68 My. A2026, and traces of nigericin in addition to the previously Here, we addressed three key questions for understanding reported compounds piericidin and streptochlorin. Automated as the evolutionary dynamics of the multicomponent antimicro- well as manual searches yielded additional predicted derivatives of bial defense in beewolf wasps and their ecological relevance. these five core structures. While streptochlorin and eight pier- (i) How did the symbiont-produced antibiotic mixture change icidins exhibiting different modifications had been described over evolutionary timescales and to what extent do the pat- previously as symbiont-produced substances from cocoons of the European beewolf (34), pimprinine (=SF2583B) and its de- terns reflect phylogenetic constraints and ecological adapta- rivative, several Mer-A2026 and actinopyrone derivatives, and tions? (ii) How is the diversity of antimicrobial compounds nigericin had only been known from free-living Streptomyces.The generated on the molecular level? (iii) Can synergistic or an- ’ pimprinines represent chlorine-free streptochlorin analogs, and tagonistic interactions of compounds present in the beewolves Mer-A2026 and the actinopyrones are structurally closely related antibiotic mixture affect their activity against potential to the piericidins, exhibiting a pyridine ring that lacks a methoxy antagonists? group and a pyranone instead of the pyridine ring, respectively. A scan of the MS/MS fragmentation patterns for the pyridine/ Results pyranone fragment yielded
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