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. 2019 Sep 9;11(9):521.
doi: 10.3390/toxins11090521.

First Report of Microcystis Strains Producing MC-FR and -WR Toxins in Japan

Tsuyoshi Ikehara  1 , Kyoko Kuniyoshi  2 , Haruyo Yamaguchi  3 , Yuuhiko Tanabe  4 , Tomoharu Sano  5 , Masahiro Yoshimoto  6 , Naomasa Oshiro  7 , Shihoko Nakashima  8 , Mina Yasumoto-Hirose  9
Affiliations

First Report of Microcystis Strains Producing MC-FR and -WR Toxins in Japan

Tsuyoshi Ikehara et al. Toxins (Basel). .

Abstract

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography-mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) inhibition assay. The representative MC, MC-LR, and related cyanobacterial toxins strongly inhibit PP2A activity and can therefore be assayed by measuring the extent of PP2A inhibition. However, these methods require reference toxin standards for the quantification and identification of known MCs. To obtain various MC-producing cyanobacterial strains, we surveyed and collected MC-producing cyanobacteria from environmental sources of water in Okinawa, Japan. Using a dual assay (LC-MS analysis and PP2A inhibition assay), we identified and isolated Microcystis strains producing five MC variants (MC-LR, -RR, -LA, -FR and -WR). Approximately 4 mg of MC-WR and -FR toxins were purified from the laboratory culture of the Microcystis isolate NIES-4344. Pure MC-WR and -FR variants were prepared for future use as toxin standards in LC-MS analysis. Phylogenetic analysis based on ftsZ revealed that the NIES-4344 strain belongs to the identified groups in Microcystis aeruginosa. This is the first report of Microcystis strains producing mainly MC-WR and -FR toxins in Japan.

Keywords: Cyanobacteria; Microcystis; environmental water; liquid chromatography–mass spectrometry; microcystins; protein phosphatase 2A inhibition assay.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Preparation of test solutions for PP2A inhibition assay and LC-MS analysis.
Figure 2
Figure 2
Chromatograms of nine standard MCs obtained via LC-MS analysis.
Figure 3
Figure 3
Chromatograms showing the composition of MCs isolated from crude extracts prepared from water samples containing cyanobacteria at six sites. (a) Site No. 1; (b) Site No. 7; (c) Site No. 8; (d) Site No. 10; (e) Site No. 11; (f) Site No. 12.
Figure 4
Figure 4
Chromatograms showing the composition of MCs of cyanobacterial strains isolated from different sampling sites. (a) Site No. 1; (b,c) Site No. 7; (d) Site No. 12.
Figure 5
Figure 5
1H NMR spectra of purified MC variants. (a) MC-WR; (b) MC-FR.
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