Hassallidins, antifungal glycolipopeptides, are PNAS PLUS widespread among 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 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 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 a 526 bp deletion in the condensation domain strate specificity-conferring nature of the adenylation domains of hasV (Fig. 1A) (16). This deletion would render the gene (SI Appendix, Table S2). The substrate specificity codes of the cluster inactive. No hassallidins were detected from Anabaena first eight modules have an exact or nearly exact match to those sp. 90. We screened 99 Anabaena strains (SI Appendix, Table S3) of adenylation domains with known substrate specificities (SI using PCR primers specific for hasV (SI Appendix,TableS4)

Fig. 1. Biosynthetic pathway of hassallidin. (A) Organization of the has gene cluster in Anabaena sp. SYKE748A showing the location of the deletion in the

has gene cluster of Anabaena sp. 90. (B) Proposed biosynthetic pathway of hassallidins and predicted domain structure of HasV1–2, HasN1–2, HasO1–3,and HasY1–2. Stereochemistries of Thr-2, 3, and 4 are putative. A, adenylation domain; C, condensation domain; Dhfa, C14 to C18 dihydroxy fatty acid; E, epi- merisation domain; M, methylation domain; T, thiolation domain; Te, thioesterase domain.

E1910 | www.pnas.org/cgi/doi/10.1073/pnas.1320913111 Vestola et al. Downloaded by guest on September 23, 2021 to identify other strains that might produce hassallidins. The data) instead of Lys. Second, the formation of D-Thr from L-Thr PNAS PLUS cell extracts from Anabaena cultures were analyzed by liquid requires epimerization of both of its chiral centers, which is not chromatography/mass spectrometry (LC/MS). A group of ions explainable by the enzyme activities encoded in the hassallidin 2 between m/z 1,220 and 1,311 was deduced to represent different NRPS. We defined the stereochemistries of L-Thr and D-allo- 4 aglyconic lipopeptide variants from the known hassallidin A and Thr in Fig. 1 based on the location of epimerase in the HasN1 B with an m/z of 1,220 (Fig. 2 and SI Appendix, Fig. S1). A series module, but still the stereochemistries of the three threonines in of ions with increased m/z values of 132, 162, and 203 and their positions 2, 3, and 4 remain putative. sodium adducts were found together with ions representing NMR analysis of Anabaena sp. SYKE748A hassallidin D con- aglyconic lipopeptide structures (SI Appendix, Fig. S2 A and B). firmed the structures and sequence of subunits 1–10 and identified A mass of 132 Da is equal to a pentose, and 162 Da is equivalent the mono- and disaccharides and their positions, mannose bound to 3-OH of N-MeThr9 and GlcNAc-(1→3)-pentopyranose bound to a hexose residue. In addition, series of ions were detected with 1 1 aglyconic lipopeptide plus 162, 204, 246, 288, and 330 mass units to 3-OH of 2,3-dihydroxy-hexadecanoic acid (Dhh )(Fig.3 (SI Appendix, Fig. S2). The difference between these fragments and SI Appendix, NMR Results, Figs. S3 and S4, and Table S6). of 42 Da equates with an acetyl group, indicating that the hexose Anabaena GenusasaProducerofDiverse Structural Variants of sugar is decorated with 0–4 acetyl groups. Moreover, the mass Hassallidins. Anabaena strains carrying the hassallidin gene clus- difference of 203 Da between ions indicates an acetylated hex- + 2 ter produced hassallidins with various modifications both within osamine residue (161 42). MS analysis showed that these and between the strains. Anabaena sp. SYKE748A produces monosaccharides together with the aglyconic lipopeptide form the 2 at least 40 different structural variants of hassallidin (Fig. 2). native hassallidin (SI Appendix,Fig.S2C and D). MS of the Structurally, all of the newly discovered hassallidins produced by aglyconic lipopeptide variants indicate that the chain length of 1 10 Anabaena contain a peptide ring with eight amino acids, a side dihydroxy fatty acid and amino acid were the origins of vari- chain with one amino acid, 2,3-dihydroxy fatty acid, and two to ation (SI Appendix, Table S5). Fragmentation of the protonated three sugar moieties (Fig. 3 and Table 1). The amino acid aglyconic lipopeptide was conducted, revealing Thr, Tyr, dehy- backbone was otherwise invariable and comparable to hassallidins drobutyrine (Dhb), Gln, Gly, and N-MeThr residues that are in A and B (17, 18), with the exception of the amino acid in position agreement with the hassallidin A and B peptide aglyconic lipo- 10 being either glutamine (hassallidin C) or tyrosine (hassallidin peptide structures (Fig. 3 and SI Appendix, Fig. S1 and Table S5). D) (Fig. 3). The length of the 2,3-dihydroxy fatty acid in posi-

The acid hydrolysis of Anabaena hassallidin C and D for the tion 1 varied, containing either 14, 15, 16, or 18 carbon atoms. MICROBIOLOGY determination of amino acid chirality confirmed the presence Hassallidins from Anabaena possessed a previously unidentified of L-Thr, D-Thr, D-allo-Thr, D-Tyr, D-Gln, Gly, N-MeThr, L-Gln pattern of glycosylation. The sugar variants of hassallidins dis- (hassallidin C), and L-Tyr (hassallidin D). This amino acid content is covered were disaccharide N-acetylhexosamine–pentose (M1–M2; fully compatible with the prediction given by the hassallidin gene 203 Da–132 Da), mono or dipentose (M1 and M2), deoxyhexose cluster (Fig. 1 and SI Appendix, Table S2), with two exceptions. (M1 and/or M2; 146 Da), and acetylhexose possessing dif- In position 10, there is either L-Gln or L-Tyr (Tyr from NMR ferent degrees of acetylation with 0–4 acetyl groups attached (M1

Fig. 2. Total ion current and extracted ion chromatograms (TICCs and EICs) showing all of the hassallidins and their aglyconic core variants produced by a single strain of Anabaena sp. SYKE748A. The arrows indicate the retention times of different hassallidin variants, which are grouped into eight categories: A to H. Numbers 0–4 above the arrows represent the acetylation degree of the sugar (acetyl) hexose in hassallidin variants. The multiplier of the EIC signal is in parentheses.

Vestola et al. PNAS | Published online April 17, 2014 | E1911 Downloaded by guest on September 23, 2021 Fig. 3. The structure of hassallidins A–D. Dhfa, dihydroxy fatty acid, dihydroxy tetradecanoic acid in hassallidin A and B, and dihydroxy hexadecanoic acid (Dhh) in hassallidin C and D. Dhb, dehydrobutyric acid.

and M3; 162, 204, 246, 288, or 330 Da) (Fig. 3 and Table 1). The lyzed the cell extract from two C. raciborskii strains by LC/MS main variants produced by Anabaena sp. SYKE748A and many and discovered that C. raciborskii strains CS-505 and ATC 9502 other Anabaena strains were hassallidin C and hassallidin D (Fig. 3). both produce hassallidins (Tables 1 and 2 and SI Appendix, Table We screened 99 Anabaena strains for the presence of hasV (SI S7). The hassallidins produced by C. raciborskii strains contained Appendix, Table S3), and we amplified two other NRPS genes, a C18 dihydroxy fatty acid side chain and two sugars, a pentose hasO and hasN, from the Anabaena strains that contained hasV. (M2) and a hexose (M3). The amino acid in position 10 in strain We were able to identify the hassallidin genes from 23 Anabaena CS-505 was threonine according to the MS2 analysis of the strains and to show that 20 of these strains also produce has- aglyconic lipopeptide unit (SI Appendix, Table S7) fitting to the sallidins (Table 2). No hassallidin production was detected in the predicted amino acid based on substrate specificity (SI Appendix, remaining three strains despite the presence of the hassallidin Table S2). This prompted us to screen other heterocyst-forming genes. Among these three inactive strains is the present-day cyanobacterial genera for the ability to produce hassallidins. By culture of Anabaena sp. 90 that, due to a 526-bp deletion in hasV, screening 104 cyanobacterial strains (SI Appendix,TableS3) from no longer produces hassallidin (16). Using PCR primers specific different heterocyst-forming genera other than Anabaena for for hasV, we found that the deletion occurred between 2003 and hasN and analyzing the cell extracts by LC/MS, we identified 2006; Anabaena sp. 90 was isolated in 1986 (SI Appendix, Fig. has genes and hassallidins from an Aphanizomenon strain, two S5). Using 10-y-old freeze-dried material, we were able to show Nostoc strains, and a Tolypothrix strain but only has genes from that Anabaena sp. 90 produced hassallidins before the deletion two other Tolypothrix strains (Table 2). However, there was less event. In addition, we found that a culture of Anabaena sp. structural variation in hassallidins identified from strains of 90M3, an apdA mutant of Anabaena sp. 90 constructed in 1999 Cylindrospermopsis, Nostoc, Aphanizomenon,andTolypothrix than (13), produces hassallidins (SI Appendix, Fig. S5). Anabaena (Table 1 and SI Appendix, Table S7). To find the phylogenetic relationship between hassallidin- Diversity of Cyanobacterial Hassallidin Producers. Bioinformatic producing strains, we constructed a 16S rRNA-based phyloge- analysis of the Anabaena sp. SYKE748A hassallidin gene clusters netic tree (Fig. 4). The producing strains were evenly distributed strongly suggests that Cylindrospermopsis raciborskii CS-505 also throughout the Nostocales order (subsection IV), comprising encodes a cryptic hassallidin gene cluster, as it showed significant five separate clusters. resemblance with 65–72% identity between HasN, HasO, HasV, and HasY and 55–84% identity between some of the additional Antifungal Activity. The minimum inhibitory concentration (MIC) ORFs (SI Appendix, Table S1). To test this possibility, we ana- of hassallidin D was ≤2.8 μg/mL (1.5 μM) against all Candida

Table 1. The structural variation of hassallidins in strains of cyanobacteria from Anabaena, Nostoc, Aphanizomenon, Tolypothrix, and Cylindrospermopsis genera

A–M, 1,220, 1,234, 1,248, 1,276, 1,255, 1,269, 1,283, 1,311, 1,249, 1,232, 1,241, 1,282, and 1,298 m/z. Variation in aglyconic lipopeptides (A–M) can be explained 10 by the hydrocarbon chain length (Cn,14–18; U, unknown) of the dihydroxy fatty acid in position 1 and by the amino acid in position 10 (Aa ;Q,glutamine;X, unknown amino acid; Y, tyrosine). Monosaccharides M1, M2, and M3 of hassallidins are N-acetylhexosamine (purple triangle), pentose (green inverted triangle), deoxyhexose (blue diamond), and hexose (red circle), which contains 0–4 acetyl (Ac) groups. Monosaccharide positions have been deduced based on the identified positions in hassallidins A, B, and D. Black squares indicate presence. Strain numbers are as in Table 2. Detailed variation is shown in SI Appendix,TableS7.

E1912 | www.pnas.org/cgi/doi/10.1073/pnas.1320913111 Vestola et al. Downloaded by guest on September 23, 2021 Table 2. Detected (LC/MS) hassallidin production of cyanobacterial strains and results of PCR amplification of has NRPS genes PNAS PLUS indicating the presence of the has gene cluster No. Strain code Origin, location Year has NRPS genes Hassallidins LC/MS

Anabaena sp. 1 90, y 2009 Lake Vesijärvi, Finland 1986 ■□ 2 90, y 1998 (freeze dried) Lake Vesijärvi, Finland 1986 ■■ 390M3(apdA mutant) Lake Vesijärvi, Finland 1986 ■■ 4 299A Lake Vesijärvi, Finland 1992 ■□ 5 299B Lake Vesijärvi, Finland 1992 ■■ 6 258 Lake Tuusulanjärvi, Finland 1990 ■■ 7 SYKE748A Lake Tuusulanjärvi, Finland 1999 ■■ 8 SYKE763A Lake Tuusulanjärvi, Finland 1999 ■■ 9 0TU33S16 Lake Tuusulanjärvi, Finland 2000 ■■ 10 0TU43S8 Lake Tuusulanjärvi, Finland 2000 ■■ 11 1TU33S8 Lake Tuusulanjärvi, Finland 2001 ■■ 12 1TU35S12 Lake Tuusulanjärvi, Finland 2001 ■■ 13 1TU44S9 Lake Tuusulanjärvi, Finland 2001 ■■ 14 1TU44S16 Lake Tuusulanjärvi, Finland 2001 ■■ 15 SYKE971/6 Lake Kotojärvi, Finland 1999 ■■ 16 PH256 Lake Knud, Denmark 1994 ■□ 17 NIVA-CYA 269/2 Lake Frøylandsvatnet, Norway 1990 ■■ 18 NIVA-CYA 269/6 Lake Frøylandsvatnet, Norway 1990 ■■ 19 XPORK5C Porkkala Cape, the Baltic Sea coast, Finland 1999 ■■ 20 XSPORK7B Porkkala Cape, the Baltic Sea coast, Finland 1999 ■■ 21 XSPORK36B Porkkala Cape, the Baltic Sea coast, Finland 1999 ■■ 22 XSPORK14D Porkkala Cape, the Baltic Sea coast, Finland 1999 ■■ ■■ 23 BECID19 The Gulf of Finland, Vuosaari, Finland 2001 MICROBIOLOGY Cylindrospermopsis raciborscii 24 ATC-9502 Lake Balaton, Hungary 1994 ■■ 25 CS-505 Freshwater, Solomon Dam, Australia 1996 ■■ Aphanizomenon gracile 26 Heaney/Camb 1986 140 1/1 Freshwater, Lough Neagh, Ireland 1986 ■■ Nostoc sp. 27 159 Lake Haukkajärvi, Finland 1986 ■■ 28 113.5 Lichen associated ? ■■ Tolypothrix sp. 29 PCC 9009 Watkins Glen State Park, New York, United States ? ■■ 30 PCC 7504 Aquarium, Stockholm, Sweden 1972 ■□ 31 PCC 7101 Borneo, soil 1950 ■□

Filled square, positive result; unfilled square, negative result. See also SI Appendix, Table S5.

strains tested (Fig. 5). The half maximum inhibitory concentration (ATCC 11006) with the tested amount of cell extract. The cell IC50 was 0.55 (0.29), 1.53 (0.82), and 1.86 (1.00) μg/mL (μM) extracts of Anabaena sp. SYKE748A and XSPORK36B strains for Candida albicans HAMBI 261 (ATCC 11006), C. albicans showed weak activity against Aspergillus flavus HAMBI 829, HAMBI 484 (ATCC 10231), and Candida krusei HAMBI 486 whereas extracts from other tested strains did not inhibit this (ATCC 6258), respectively (Fig. 5). Interestingly, the MIC of the fungus (SI Appendix, Fig. S7). linear form of hassallidin D was found to be 36 μg/mL (20 μM) against C. albicans HAMBI 261 (ATCC 11006). The disk diffu- Discussion sion bioassay using 10 μg of hassallidin D shows inhibition of A multitude of previously unidentified hassallidin variants are Cryptococcus albidus HAMBI 264 (ATCC 10666) and Filobasi- produced among heterocystous cyanobacteria. They exhibit anti- diella neoformans (ATCC 10226), respectively, with inhibition fungal activity against important opportunistic pathogenic fungi. zones of 17 and 11 mm. No inhibition of Aspergillus strains by Particularly Anabaena strains proved to be a good source of hassallidin D was observed. Antifungal activity of cell extracts hassallidins, with multiple different variants produced simulta- containing 10 μg of hassallidin D was tested against C. albicans neously (Fig. 2 and Table 1). The newly discovered hassallidin HAMBI 261 (ATCC 11006) using the disk diffusion method. variants possessed unusually diverse structures distinct from the The cell extracts of Anabaena strains 90M3, SYKE748A, previously found hassallidins A and B (17, 18), due to the co- XSPORK7B, XSPORK36B, Nostoc sp. 159, and C. raciborskii occurrence of variable lengths of the dihydroxy fatty acid chain ATC 9502 inhibited the growth of C. albicans HAMBI 261 (constituting 14, 15, 16, or 18 carbons) and complex glycosylation (ATCC 11006) (SI Appendix, Fig. S6). Nostoc sp. 159 extract had patterns with three variable monosaccharide moieties comprised the largest inhibition zone of all of the strains tested (SI Ap- of five optional monosaccharides: N-acetylhexosamine (M1), pendix, Table S8). However, Anabaena sp. SYKE748A proved to pentose (M1 and M2), hexose (M1 and M3), deoxyhexose (M1 produce the highest amount of hassallidins. Anabaena sp. 90, or M2), or acetylhexose possessing different degrees of acetylation Tolypothrix sp. PCC 7415 and PCC 7504 strains, C. raciborskii CS- (M3) (Table 1). The dihydroxy fatty acid side chain of hassallidins A 505, and Aphanizomenon gracile Heaney/Camb 1986 140 1/1 did and B contains 14 carbons decorated with mannose (17), and not exhibit antifungal activity against C. albicans HAMBI 261 hassallidin B also has an additional rhamnose (18). The peptide

Vestola et al. PNAS | Published online April 17, 2014 | E1913 Downloaded by guest on September 23, 2021 Fig. 4. Evolutionary history of cyanobacteria-producing hassallidin based on 16S rRNA genes. Phylogenetic tree was inferred using MEGA 5, indicating in the nodes of the bootstrap values above 50% for maximum parsimony, maximum likelihood, and neighbor-joining bootstrap replicates. Strains detected to produce hassallidins are highlighted. An asterisk indicates two strains that have the hasN gene but no hassallidins were detected by LC/MS due to deletion in the hassallidin gene cluster in Anabaena sp. 90 and possible mutation in Tolypothrix sp. PCC 7504.

backbone of the newly discovered hassallidins is identical to was measured to be ≤2.8 μg/mL (1.5 μM). Previously, MIC val- hassallidins A and B, except that the amino acid in position 10 ues of 4.8 μg/mL for hassallidins A and B against C. albicans have was either glutamine (hassallidin C), tyrosine (hassallidin D), or in been reported (28). Interestingly, the linear form of hassallidin D one case threonine (Fig. 3 and Table 1). Dihydroxy fatty acid side has an MIC of ≤36 μg/mL (20 μM), showing the importance of chain compounds such as cormycin A with antifugal bioactivity ring structure for bioactivity. Nevertheless, this result shows that has been previously reported from Pseudomonas corrugata,but hassallidin is also slightly active in its linear form. Many other the family of those compounds does not contain any sugars (19). bioactive compounds lose their bioactivity when the ring struc- In a majority of glycosylated natural products, the sugar moieties ture is opened (e.g., refs. 29, 30). The structural differences be- belong to the 6-deoxyhexose family (20). The most exceptional tween the hassallidin variants produced might influence their feature of hassallidin produced by Anabaena is the presence of bioactivity. For example, the sugar moieties of many glycosylated an unusual acetylhexose with an acetylation degree ranging from compounds play a significant or even fundamental role in their 0 to 4. Acetylated sugar structures have been previously found bioactivity (31). The number of sugars has also been reported only in a few natural products (21–23) and never in natural prod- to influence the activity. The antitumor activity of landomycin ucts produced by cyanobacteria to date, and overall they are rare increases with the length of the deoxysugar chain, although its among secondary metabolites produced by microbes. mode of action is yet not fully understood (32). On the other hand, All tested Anabaena strains inhibited the growth of C. albicans, with the exception of Anabaena sp. 90 wild type, which, due to the additional sugar group of hassallidin B was not reported to a deletion, no longer produces hassallidins (16). A few natural have an influence on its antifungal activity, although no aglycon products produced by cyanobacteria have been previously de- form was examined (28). The function of the acetyl groups of scribed to exhibit antifungal activity—for example, cryptophycin acetylhexose in hassallidin is unknown, but for antitumor com- (24, 25), tanikolide (26), and nostofungicidine (27). These com- pound chromomycin A3, the acetylation of sugars was essential for pounds and hassallidins share a lactone structure and a long hy- its biological activity (31). Previous study indicates that hassallidin drocarbon side chain, 5-hydroxy-5-hydroxymethylpalmitic acid in A affects the integrity of the plasma membrane (33). This mecha- tanikolide, 3-amino-6-hydroxystearic acid in nostofungicidine, and nism of action differs from the inhibition of the cell wall synthesis by 2,3-dihydroxymyristic, 2,3-dihydroxypalmitic or 2,3-dihydroxystearic echinocandins, as caspofungin (33). The incidence of invasive fungal acid in hassallidins. Both could be essential elements for the infections in humans has been increasing, and the need for new antifungal activity. The MIC value for hassallidin D isolated selective antifungals is growing due to the toxicity, low efficacy, from Anabaena sp. SYKE748A against C. albicans and C. krusei and drug resistance of many currently used drugs (34).

E1914 | www.pnas.org/cgi/doi/10.1073/pnas.1320913111 Vestola et al. Downloaded by guest on September 23, 2021 that C. raciborskii produces hassallidins. This has gene cluster PNAS PLUS (SI Appendix,TableS1) carries four NRPS genes that code for nine modules, which is in accordance with the number of amino acids in hassallidin. -producing C. raciborskii has been reported to be the cause of the outbreak of hep- atoenteritis in 1979, in Palm Island, Australia, where 148 people were poisoned due to a contamination of a drinking water reservoir (43–45). Interestingly, when tested in mice, pure cylindrospermop- sin damaged only the liver, but the cylindrospermopsin-containing cell extract caused injuries also to the kidneys, adrenal glands, lungs, and intestines. It has been suggested that the cell lysates might have contained an unknown compound in addition to cylindrospermopsin (45). Considering that hassallidins have also shown cytotoxicity against eukaryotic human acute T-cell leukemia with IC50 0.2 μg/mL (28), they may have contributed to the additional symptoms. Fig. 5. MIC and half maximum inhibitory concentration (IC50) of hassallidin In addition to Anabaena and Cylindrospermopsis, we discov- DagainstC. albicans HAMBI 261 (ATCC 11006), C. albicans HAMBI 484 (ATCC ered that hassallidin production ability is even further dispersed 10231), and C. krusei HAMBI 486 (ATCC 6258). among cyanobacteria. Some of the strains from genera Nostoc, Aphanizomenon,andTolypothrix also produced hassallidins (Table The structural characterization, genetic organization, and 2). However, no hassallidin production of two other strains of predicted substrate specificities of adenylation domains attest Tolypothrix (PCC 7101 and PCC 7504) containing hasN was to the involvement of the gene cluster from the draft genome of detected. The phylogenetic tree showed that the hassallidin- cyanobacterium Anabaena sp. SYKE748A in hassallidin bio- producing strains fell into five separate clusters (Fig. 4). More- synthesis (Fig. 1). We verified this by the discovery of the natural over, the strains producing hassallidins are widely dispersed to mutation during the genome sequencing process and loss of various different habitats, including freshwater, brackish water, hassallidin production by the Anabaena sp. 90 (Fig. 1 and Table and lichen association, in distant geographic areas (Table 2 and 2) (16). The complex structure of hassallidins indicates that in SI Appendix, Table S3). Little is known about the importance of MICROBIOLOGY addition to the four key NRPS proteins, several putative tailoring secondary metabolites for the producer strains, but they might enzymes are required to complete the biosynthesis. Four pro- be a way to combat rival organisms or parasites. It is known that teins—HasD, HasQ, HasT, and HasX—exhibit conserved motifs some genera of cyanobacteria are susceptible to infections caused toward glycosyltransferase with fairly high similarities (SI Ap- by parasitic chytrid fungi (46, 47). It has been shown that oli- pendix, Table S1) and are proposed to be involved in the glyco- gopeptides produced by Planktothrix cyanobacteria disturb the sylation of hassallidins. In most known cases, the number of infection caused by the fungi Rhizophydiales (46) and the wild glycosyltransferases corresponds to the number of sugars at- type harboring an entire oligopeptide repertoire is more resistant tached (35). Some glycosyltransferases are described to work to fungal infection than the oligopeptide-minus mutants (47). It iteratively, catalyzing the attachment of more than one sugar to has also been reported that the presence of Anabaena spiroides in the aglycon (36). However, the has gene cluster seems to encode the water reduces the number of Hyphomycetes fungi (48). Due one additional glycosyltransferase, whose function remains unclear. to their antifungal activity, it is possible that hassallidins could be In addition, a predicted acyltransferase, HasR, was found to be a means of defense against fungal infections or play a role as an encoded in the has gene cluster. Acyl/acetyltransferases have been allelochemical. Although hassallidins show promising antifungal activity, little is known about their effect on other organisms. shown to be responsible for the acetylation of the sugar moieties in an antifungal glycolipid flocculosin (23). An antitumor polyketide Materials and Methods chromomycin A contains two O-acetylated sugars, but only one 3 Cyanobacterial Cultures. Strains of Anabaena, Nostoc, Cylindrospermopsis, acyltransferase is found in the biosynthetic gene cluster, which is Tolypothrix, and Aphanizomenon were grown in Z8 medium either with or proposed to catalyze both sugar O-acetylation reactions (21). Thus, without nitrogen (49). Strains were cultivated in 40 mL culture at 17–25 °C − it is possible that HasR could be a candidate for the transfer of one under continuous illumination of 3–15 μmol·m 2·s1. Anabaena sp. SYKE748A or more acetyl groups required to decorate the hassallidin sugars. was mass cultivated in 5 × 2.7 L batches with aeration of filter-sterilized The acylation of hassallidins most likely includes the action of the pressurized air. After cultivation, the clear medium was decanted away, and the rest of the culture was freeze dried. additional C domain in the first HasV1 module. The atypical N-terminal C domain has been reported in lipopeptide biosynthetic gene clusters (37), and thus we postulate that it is catalyzing the Sequencing of the Hassallidin Gene Cluster. DNA extraction from the Anabaena sp. N-acylation of the first threonine in the peptide chain and repre- SYKE748A strain was made using the phenol-chloroform method (15, 16). Ultra high-coverage sequence data were produced by Illumina MiSeq. The genome sents the first step in the assembly of hassallidin. Based on the se- was assembled using Newbler (version 2.8), after adapter removal and short quence similarities, lipidation might involve the action of proteins read filtering. The hassallidin gene cluster was located on three contigs, and HasG, HasH, and HasL to work in concert with the first C domain. the gaps were closed by PCR and Sanger sequencing to produce the final intact How Dhb is enzymatically formed from threonine is generally un- 59-kb gene cluster. known. However, this modification has been detected in cyano- bacteria in nodularin and biosyntheses (38, 39). Identification of the Hassallidin Gene Cluster. The gene cluster from strain C. raciborskii has previously been reported to produce only SYKE748A was annotated according to that in Anabaena sp. 90 (16), due to two different types of toxins: tricyclic alkaloidic hepatotoxin their extremely high sequence similarities (99.8%). The putative roles of the cylindrospermopsin and neurotoxic tetrahydropurine has gene cluster encoded proteins were assigned by sequence similarity using (40, 41). The genome of C. raciborskii CS-505 contains a cryptic the National Center for Biotechnology Information (NCBI) BLASTp and Inter- ProScan Sequence searches (48). An in silico approach was used for prediction NRPS gene cluster in addition to the NRPS/PKS hybrid gene of domain organization (50). The substrate specificity conferring code of cluster responsible for the production of cylindrospermopsin (41, adenylation domains in NRPSs (51) was performed using NRPSpredictor soft- 42). The significant similarity between the amino acid sequences ware (52, 53). Identification of the hassallidin gene cluster in C. raciborskii was of the has gene cluster and the genes in the genome of C. based on the genome of strain CS-505 (41) (NCBI database accession no. raciborskii CS-505 (SI Appendix,TableS1) led us to discover NZ_ACYA01000025, www.ncbi.nlm.nih.gov/nuccore/NZ_ACYA01000025).

Vestola et al. PNAS | Published online April 17, 2014 | E1915 Downloaded by guest on September 23, 2021 DNA Isolation, PCR Amplification, and Sequencing. Genomic DNA from the freeze dried. Purification yielded 2.4 mg of native hassallidin D for testing cyanobacterial strains was extracted using either DNeasy Plant Mini Kit the antifungal activity. Hassallidin D purification for the NMR and bioassay (Qiagen Gmbh) or E.Z.N.A SP Plant DNA Mini Kit (Omega Bio-Tek Inc.). PCR (linear hassallidin D) is described in SI Appendix. Amino acid analysis of amplifications of DNA from Anabaena were performed in iCycler (Bio-Rad) hassallidin C and D was made using the Marfey method as previously de- using primers hasV-fw and hasV-rev, hasN-fw and hasN-rev, and hasO-fw scribed in ref. 56. and hasO-rev, designed to amplify parts from hasV, hasN, and hasO (SI Appendix, Table S4). PCR conditions are described in SI Appendix. The PCR NMR Spectroscopy. All NMR spectra were collected using a Varian Unity products were sequenced by Macrogen, Inc. INOVA 600 MHz NMR spectrometer at 40 °C in DMSO-d6. Detailed analysis is Primers AF and AR (SI Appendix, Table S4) were used in PCR to screen presented in SI Appendix. cyanobacteria from other genera for the gene hasN. PCR conditions are described in SI Appendix. PCR products were cloned into the pCR2.1-TOPO Antifungal Assay. Microdilution assay was used to investigate the hassallidin D cloning vector (TOPO-TA Cloning-Kit, Invitrogen) and transformed to TOP10 MIC and the half maximum inhibitory concentration (IC )ofC. albicans and Escherichia coli cells (Invitrogen). Colonies were tested by PCR, and plasmids 50 C. krusei. A stock solution of hassallidin D with a concentration of 2.24 mg/mL containing inserts of the right size were purified using QiaprepSpin Mini- in DMSO was used for a serial dilution assay. Twofold dilutions from the prep Kit (Qiagen). The sequencing was performed by ABI PRISM BigDye stock solution in DMSO were prepared, and this serial dilution ranged from Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Internal primers 2.24 to 0.0044 mg/mL. The final concentration in the well was from 22.4 to μ (1194Rc, 16S545F, 16S979F, 16S1092R, and 359F; SI Appendix, Table S4) were 0.04 g/mL. Two microliters of each dilution containing hassallidin D in μ used to sequence 16S rRNA gene fragments. The contigs were assembled DMSO was used per well containing 98 L of RPMI-1640 medium, in tripli- using Phred/Phrap/Consed (Philip Green), considering bases with quality ≥20. cates (R6504, with L-glutamine and without NaHCO3; Sigma). Two microliters The 16S rRNA phylogenetic tree was constructed in Molecular Evolutionary of DMSO was used as the negative control. Potato dextrose agar me- Genetic Analysis (MEGA) 5 (54) using maximum likelihood (K2+G+I model), dium plates containing C. albicans HAMBI 261 (ATCC 11006), C. albicans neighbor-joining (MCL+G model), and maximum parsimony (closest neigh- HAMBI 484 (ATCC 10231), and C. krusei HAMBI 486 (ATCC 6258) grown at 35 °C bor interchange on random trees) methods. The 16S rRNA partial gene for 24 h were used to prepare the inoculum. The test inoculum was prepared sequences data have been submitted to the GenBank database. as described in the guidelines of the Clinical and Laboratory Standards In- stitute document M27-A3 (57). We added 100 μL of the test inoculum per Extraction, Chemical Analyses, Purification, and Amino Acid Analysis of well, allowing a final concentration of 0.5 × 103 to 2.5 × 103 cells/mL. The Hassallidin D. Extraction and chemical analyses were performed as de- MIC value was the lowest concentration in which no growth of the organism

scribed in ref. 55 with the following exceptions: Luna C18 (2) column (100 × was observed. The IC50 was calculated by linear interpolation of the concen-

4.6 mm) was eluted with a 30 min gradient from 10% to 100% of acetoni- trations using the following formula: IC50 = (50% – LowI%)/(HighI% – LowI%) ×

trile in 0.1% aqueous formic acid. The LC/MS parameters were optimized (HighC – LowC) + LowC,inwhichCisconcentrationandI%isinhibitionper- with hassallidin ion m/z 1,862 with a negative mode of polarity as follows: centage calculated with the formula I% = 100 – [(ODsample – ODmedium)/

capillary voltage, 3,650 V; capillary offset value, 250 V; skimmer potential, (ODcontrol – ODmedium)] × 100. LowI% is the inhibition percentage corre-

58.0 V; trap drive value, 112.0. Commercial hassallidins A and B (Alexis spondent to the value directly below the MIC, and HighI% is the one cor- Biochemicals, Ezno Life Science Inc.) were used as references. respondent to the MIC. In addition, hassallidin D was found to linearize For bioactivity tests, biomass of Anabaena sp. SYKE748A (2.7 g) was di- during the storage, and this form was used in a microdilution assay. The × vided into two 50 mL plastic tubes. We added 2 35 mL of acetonitrile/ assay was performed as described in ref. 28. Twofold dilutions with a hassallidin dimethyl sulfoxide (1:1) solution, and suspensions were treated for 30 s at concentration between 4.6 and 38 μg/mL were dissolved in 50% (vol/vol) 18,000 rpm with a Silent Crusher homogenizer at an ambient temperature. aqueous methanol. Negative controls containing 50% methanol and me- Suspensions were centrifugated at 10,000 × g for 5 min, and supernatants dium alone and positive controls containing 10 μg of nystatin were tested. were pooled. Acetonitrile was evaporated with a rotary evaporator at 20 °C, The MIC was considered the minimum concentration of hassallidin that and the residue was diluted 20 times with water. The solution was divided strongly inhibited the growth of C. albicans HAMBI 261 (ATCC 11006). The into four and then passed through four primed (first methanol, then 5% bioactivity assay performed using crude extract of different tested strains are (vol/vol) aqueous methanol) solid phase extraction cartridges (Strata C18-E, described in SI Appendix. 5g/20mL,55μm, 70 Å; Phenomenex Inc.). Cartridges were eluted with 50 mL of 90% (vol/vol) aqueous acetonitrile, which was then removed with a rotary evaporator from the effluent. The residue was freeze dried and dissolved to ACKNOWLEDGMENTS. We thank Lyudmila Saari for maintaining and han- dling the cyanobacteria strains and the anonymous reviewers and the editor HPLC eluent. Solution was injected into a semipreparative Luna C8 (2) col- × μ – for their constructive feedback and help. This work was supported by Grants umn (10 150 mm, 5 m, 100 Å; Phenomenex Inc.) in 1 2 mL batches. The 118637 and 258827 from the Academy of Finland (to K.S.). T.K.S. was partially column was eluted isocratically with acetonitrile/0.1% ammoniumacetate funded by the Helsinki Graduate Program in Biotechnology and Molecular (40/60) at an ambient temperature. Acetonitrile was removed with a nitro- Biology, the São Paulo Research Foundation (2009/13455-0), the Centre for gen stream from the collected hassallidin fractions, and the residue was International Mobility (TM-09-6506), and the Finnish Cultural Foundation.

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