Antitumor Anthraquinones from an Easter Island Sea Anemone: Animal or Bacterial Origin?

Abstract

The presence of two known anthraquinones, Lupinacidin A and Galvaquinone B, which have antitumor activity, has been identified in the sea anemone (Gyractis sesere) from Easter Island. So far, these anthraquinones have been characterized from terrestrial and marine Actinobacteria only. In order to identify the anthraquinones producer, we isolated Actinobacteria associated with the sea anemone and obtained representatives of seven actinobacterial genera. Studies of cultures of these bacteria by HPLC, NMR, and HRLCMS analyses showed that the producer of Lupinacidin A and Galvaquinone B indeed was one of the isolated Actinobacteria. The producer strain, SN26_14.1, was identified as a representative of the genus Verrucosispora. Genome analysis supported the biosynthetic potential to the production of these compounds by this strain. This study adds Verrucosispora as a new genus to the anthraquinone producers, in addition to well-known species of Streptomyces and Micromonospora. By a cultivation-based approach, the responsibility of symbionts of a marine invertebrate for the production of complex natural products found within the animal's extracts could be demonstrated. This finding re-opens the debate about the producers of secondary metabolites in sea animals. Finally, it provides valuable information about the chemistry of bacteria harbored in the geographically-isolated and almost unstudied, Easter Island.

Keywords: Actinobacteria; Easter Island; anthraquinones; chromatography; marine invertebrates; sea anemone; spectroscopy; symbionts.

Figure 1

Identified molecules from the Easter Island sea anemone Gyractis sesere.

Figure 2

Chemical analysis of the crude extract of the sea anemone Gyractis sesere. (A) ¹H NMR spectra of the crude extract of marine anemone Gyractis sesere acquired in CDCl₃, 600 MHz. Highlighted in the zoomed area are the frequencies of characteristic resonances originating from hydroxyl exchangeable protons in vicinity to ketogroups. (B) UV chromatogram (254 nm) of the crude extract of the sea anemone Gyractis sesere highlighting the specific peaks for ■RT: 24.3 min, and ▲: RT: 25.2 min. (C) High resolution mass for ■ (m/z [M + H]⁺ 341.1378) and (D) high resolution mass for ▲ (2) (m/z [M + H]⁺ 369.3510). *RT: Retention Time.

Figure 3

Genera and number of Actinobacteria species strains isolated from the sea anemone Gyractis sesere.

Figure 4

HPLC chromatograms of the crude extracts of the sea anemone Gyractis sesere, its respective actinobacterial isolates, and the purified anthraquinones, Lupinacidin A (1) and Galvaquinone B (2). Approximate retention times of Lupinacidin A (1) and Galvaquinone B (2) are highlighted by boxes.

Figure 5

Phylogenetic tree based on 16S rRNA gene sequence of Verrucosispora sp. SN26_14.1. The tree was calculated using a neighbor-joining statistical method and Jukes–Cantor model. • Red dots highlight Lupinacidin A (1) and Galvaquinone B producers (2).

Figure 6

Biosynthetic gene cluster of anthraquinones producers. Rsd: Streptomyces olivaceus SCSIO T05 gene cluster [28], Rsl: Streptomyces bottropensis (Streptomyces. sp. Gc C4/4) gene cluster [29], Vex: Verrucosispora sp. SN26_14.1. C1: aromatase, K₁: acyl carrier protein, K₂: ketosynthase (beta), K₃: ketosynthase (alpha), A: acyl transferase, K₄: 3-oxoacyl-ACP synthase III, T₁: ABC-transporter (substrate binding), T₂: ABC-transporter (ATP binding), T₃: ABC-transporter trans-membrane, O₁: luciferase-like monooxygenase, O₂: flavin reductase, P: phosphotransferase, R₁: SARP family regulator, C₂: second ring cyclase, O₃: 3-oxoacyl-ACP reductase, O₄: anthrone monooxygenase, O₅: NADH: flavin oxidoreductase, C₃: cyclase, R₂: SARP regulatory protein, R₃: LAL-family regulator, O₆: luciferase-like monooxygenase, R₄: MarR family transcriptional regulator, T₄: drug resistance transporter, O₇: putative NADPH quinone reductase, O₈: putative NADPH: quinone oxidoreductase, O₉: FAD-dependent oxidoreductase, O₁₀: C9-keto reductase, H: amidohydrolase, -3: unknown function, -2: major facilitator superfamily protein, -1: Transcriptional regulatory protein, 1: cupin, 2: citrate/H⁺ symporter, 3: transcriptional regulator.