Highly Toxic Microcystis aeruginosa Strain, Isolated from Sao Paulo—Brazil, Produce Hepatotoxins and Paralytic Shellfish Poison Neurotoxins

Abstract

While evaluating several laboratory-cultured cyanobacteria strains for the presence of paralytic shellfish poison neurotoxins, the hydrophilic extract of Microcystis aeruginosa strain SPC777—isolated from Billings’s reservoir, Sa˜o Paulo, Brazil—was found to exhibit lethal neurotoxic effect in mouse bioassay. The in vivo test showed symptoms that unambiguously were those produced by PSP. In order to identify the presence of neurotoxins, cells were lyophilized, and the extracts were analyzed by HPLC–FLD and HPLC–MS. HPLC–FLD analysis revealed four main Gonyautoxins: GTX4(47.6%), GTX2(29.5%), GTX1(21.9%), and GTX3(1.0%). HPLC– MS analysis, on other hand, confirmed both epimers, with positive Zwitterions M? 395.9 m/z for GTX3/GTX2 and M? 411 m/z for GTX4/GTX1 epimers. The hepatotoxins (Microcystins) were also evaluated by ELISA and HPLC–MS analyses. Positive immunoreaction was observed by ELISA assay. Alongside, the HPLC–MS analyses revealed the presence of [L-ser7] MCYST-RR. The N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA was chosen as the target sequence to detect the presence of the mcy gene cluster. PCR amplification of the NMT domain, using the genomic DNA of the SPC777 strain and the MSF/MSR primer set, resulted in the expected 1,369 bp product. The phylogenetic analyses grouped the NMT sequence with the NMT sequences of other known Microcystis with high bootstrap support. The taxonomical position of M. aeruginosa SPC777 was confirmed by a detailed morphological description and a phylogenetic analysis of 16S rRNA gene sequence. Therefore, co-production of PSP neurotoxins and microcystins by an isolated M. aeruginosa strain is hereby reported for the first time.