Hassallidins, Antifungal Glycolipopeptides, Are Widespread
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Hassallidins, antifungal glycolipopeptides, are PNAS PLUS widespread among cyanobacteria and are the end-product of a nonribosomal pathway Johanna Vestolaa, Tania K. Shishidoa, Jouni Jokelaa, David P. Fewera, Olli Aitiob, Perttu Permib, Matti Wahlstena, Hao Wanga, Leo Rouhiainena, and Kaarina Sivonena,1 aDepartment of Food and Environmental Sciences, Viikki Biocenter 1, University of Helsinki, FI-00014, Helsinki, Finland; and bProgram in Structural Biology and Biophysics, Institute of Biotechnology/Nuclear Magnetic Resonance Laboratory, University of Helsinki, FI-00014, Helsinki, Finland Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved March 25, 2014 (received for review November 7, 2013) Cyanobacteria produce a wide variety of cyclic peptides, including their products are unknown (4, 12). The mining of microbial the widespread hepatotoxins microcystins and nodularins. Another genomes provides a useful approach for natural product dis- class of peptides, cyclic glycosylated lipopeptides called hassalli- covery. The initial genome annotation of the toxic bloom-forming dins, show antifungal activity. Previously, two hassallidins (A and B) cyanobacterium Anabaena sp. 90 identified gene clusters for ana- were reported from an epilithic cyanobacterium Hassallia sp. baenopeptins, anabaenopeptilides, and microcystins (13–15). Sur- and found to be active against opportunistic human pathogenic prisingly, a fourth cryptic inactive gene cluster was found and fungi. Bioinformatic analysis of the Anabaena sp. 90 genome iden- proposed to be responsible for the biosynthesis of hassallidins (16). tified a 59-kb cryptic inactive nonribosomal peptide synthetase In the present study, we report the detailed characterization of the gene cluster proposed to be responsible for hassallidin biosynthe- gene cluster from the genome of Anabaena sp. SYKE748A and sis. Here we describe the hassallidin biosynthetic pathway from show that it is responsible for the production of numerous novel Anabaena sp. SYKE748A, as well as the large chemical variation glycosylated lipopeptides. These compounds resemble antifungal and common occurrence of hassallidins in filamentous cyanobacte- hassallidins A and B, which were isolated from an epilithic cyano- ria. Analysis demonstrated that 20 strains of the genus Anabaena bacterium Hassallia sp. (17, 18). A large number of new hassallidin MICROBIOLOGY carry hassallidin synthetase genes and produce a multitude of variants was detected, showing that the ability to produce these hassallidin variants that exhibit activity against Candida albicans. compounds is widespread in heterocyst-forming cyanobacteria. The compounds discovered here were distinct from previously – reported hassallidins A and B. The IC50 of hassallidin D was 0.29 Results 1.0 μMagainstCandida strains. A large variation in amino acids, The Hassallidin Biosynthetic Gene Cluster. The assembly and anno- sugars, their degree of acetylation, and fatty acid side chain length tation of the Anabaena sp. 90 genome revealed a cryptic non- was detected. In addition, hassallidins were detected in other cyanobacteria including Aphanizomenon, Cylindrospermopsis ribosomal peptide gene cluster, coding for a previously unknown raciborskii, Nostoc,andTolypothrix. These compounds may protect natural product that was proposed to be hassallidin (16). To some of the most important bloom-forming and globally distributed identify the hassallidin gene cluster from an active producer cyanobacteria against attacks by parasitic fungi. of hassallidins, we obtained a draft genome of Anabaena sp. SYKE748A. The identified hassallidin gene cluster was located nonribosomal peptide synthesis | natural product discovery | on three contigs and had a sequence similarity greater than genome mining | secondary metabolites | bioactive peptide Significance yanobacteria are known for their propensity to form toxic Cblooms that have caused the deaths of wild and domestic animals New antifungal compounds are needed due to an increasing all over the world (1). However, they are also a rich source of natural incidence of invasive fungal infections and resistance to many products that exhibit antimicrobial, anticancer, and immunosup- currently used drugs. Here we show that cyanobacteria are pressive activities that can be exploited in drug development (2, 3). a rich source of antifungal compounds such as glycosylated These secondary metabolites have versatile and often highly complex lipopeptides, called hassallidins, which are commonly produced chemical structures with diverse biosynthetic origins. The majority by filamentous nitrogen-fixing cyanobacteria. A diverse group of bioactive compounds reported from cyanobacteria are cyclic or of hassallidins and their complex nonribosomal biosynthesis linear peptides that can be heavily modified, including derivatization were characterized in detail. Hassallidins and their previously such as epimerization, glycosylation, acylation, formylation, methyl- unidentified biosynthetic enzymes offer new material for drug ation, halogenation, or sulphation (4). Many of these peptides are development. In addition, these compounds may have an made nonribosomally with peptide cores consisting of an array of ecological role in protecting cyanobacteria from parasitic fungi. proteinogenic or nonproteinogenic amino acids as well as com- ponents of polyketide origin. They are assembled on large enzyme Author contributions: J.V., T.K.S., J.J., D.P.F., L.R., and K.S. designed research; J.V., T.K.S., J.J., D.P.F., O.A., M.W., H.W., and L.R. performed research; P.P. and K.S. contributed new complexes by a thiotemplate mechanism in which the non- reagents/analytic tools; J.V., T.K.S., J.J., D.P.F., O.A., M.W., H.W., and L.R. analyzed data; ribosomal peptide synthetases (NRPSs) act simultaneously as and J.V., T.K.S., J.J., D.P.F., P.P., H.W., L.R., and K.S. wrote the paper. template and biosynthetic machinery (5, 6). NRPSs are organized The authors declare no conflict of interest. into modules, each of which is responsible for one amino acid This article is a PNAS Direct Submission. – activation and peptide bond formation (5, 7 9). The selectivity Freely available online through the PNAS open access option. of NRPS adenylation domains combined with NRPS catalytic Data deposition: The sequence reported in this paper has been deposited in the GenBank domain organization that generally follows the colinearity princi- database (accession nos. KJ502174, KF631395, KF631396, KF631397, KF631398,and ple (7, 10) offers a means to predict the amino acid building blocks KF631399.). and thus the putative peptide structure. 1To whom correspondence should be addressed. E-mail: [email protected]. Microbial genomes are replete with NRPS gene clusters (11, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 12). However, the majority of these gene clusters are cryptic and 1073/pnas.1320913111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1320913111 PNAS | Published online April 17, 2014 | E1909–E1917 Downloaded by guest on September 23, 2021 99.8% to that of Anabaena sp. 90. The hassallidin synthetase Appendix, Table S2). The last HasY2 module shows lower iden- (has) gene clusters from Anabaena sp. 90 and SYKE748A are tity to known adenylation domains. bidirectional and span 59 kb of genomic DNA (Fig. 1A). They Sequence comparison with database proteins suggests the in- consist of 26 ORFs, designated as hasA–hasZ, that are flanked by volvement of four glycosyltransferases (HasD, HasQ, HasT, and two transposase genes (SI Appendix, Table S1). The cluster HasX), which are predicted to catalyze the addition of sugars to encodes four multidomain NRPS proteins, HasN, HasO, HasV, hassallidin (Fig. 1 and SI Appendix, Table S1). The putative and HasY, with sizes of 345 kDa, 412 kDa, 244 kDa, and 323 acyltransferase HasR in the gene cluster could have a role in kDa, respectively. The NRPSs contain nine modules, which are acetylation reactions (SI Appendix, Table S1). Moreover, the responsible for the activation and incorporation of nine amino predicted gene products of hasG, hasH, and hasL show similarity acids into the growing polypeptide chain (Fig. 1B). Each of the to enzymes involved in fatty acid synthesis. HasG exhibits con- NRPS modules bears a condensation (C), adenylation (A), and served domains typical of the acyl-protein synthetase superfam- thiolation (T) domain. In addition, modules HasN1, HasN2, and ily, including synthetase and ligase, whereas the deduced function HasO2 each contain an epimerization (E) domain suggesting for HasH is acyl carrier protein, proposing a role in transfer of that the end-product would contain three D-amino acids. The the fatty acid of hassallidin. The putative tailoring enzyme HasY1 module encodes an N-methyltransferase (NMT) domain, HasL shows high similarity to 3-oxoacyl-(acyl-carrier-protein) indicating the presence of an N-methylated amino acid in the reductase, also suggesting a role in lipid side chain biosynthesis end-product. The HasY2 module bears a C-terminal thioesterase (SI Appendix,TableS1). domain that catalyzes the cyclization and release of the peptide backbone by forming an ester bond between amino acids in- Structural Elucidation of Hassallidins. The genome assembly of corporated by modules HasV2 and HasY2. Predictions of the Anabaena sp. 90 revealed that the putative hassallidin gene amino acid backbone of the peptide were made using the sub- cluster contained