Chemically labeled toxins or bioactive peptides show a heterogeneous intracellular distribution and low spatial overlap with autofluorescence in bloom-forming cyanobacteria

Abstract

Harmful algal blooms formed by colony-forming cyanobacteria deteriorate water resources by producing cyanotoxins, which frequently occur at high intracellular concentrations. We aimed to localize toxic microcystins (Mcs) and bioactive anabaenopeptins (Aps) at the subcellular level under noninvasive conditions. Since both metabolites are synthesized nonribosomally, the relaxed specificity of key enzymes catalyzing substrate activation allowed chemical labeling through a standard coppercatalyzed click chemistry reaction. the genera Planktothrix and Microcystis specifically incorporated unnatural amino acids such as N-propargyloxy-carbonyl-L-lysine or O-propargyl-L-tyrosine, resulting in modified AP or MC peptides carrying the incorporated alkyne moiety. The labeled cells were quantitatively differentiated from the unlabeled control cells. MCs and APs occurred intracellularly as distinct entities showing a cell-wide distribution but a lowered spatial overlap with natural autofluorescence. Using the immunofluorescence technique, colocalization with markers of individual organelles was utilized to relate the distribution of labeled Mcs to cellular compartments, e.g., using RbcL and FtsZ (cytosol) and PsbA (thylakoids). The colocalization correlation coefficients calculated pairwise between organelles and autofluorescence were highly positive as opposed to the relatively low positive indices derived from labeled MCs. The lower correlation coefficients imply that only a portion of the labeled Mc molecules were related spatially to the organelles in the cell.

High Levels of Anabaenopeptins Detected in a Cyanobacteria Bloom from N.E. Spanish Sau-Susqueda-El Pasteral Reservoirs System by LC–HRMS

“…The present results agree with recent studies in which analysis of cyanotoxins and CPs in surface waters reveals more than MC [ 31 , 32 , 35 , 41 , 42 , 51 , 65 , 67 , 68 ]. However, these cyanopeptides have received little attention and there are few toxicity and health risk studies about APs and OCA Y.…”

“…Therefore, the analysis confirms the end of bloom episode and the quality of the water already allowed its use without any risk associated with algae. The present results agree with recent studies in which analysis of cyanotoxins and CPs in surface waters reveals more than MC [31,32,35,41,42,51,65,67,68]. However, these cyanopeptides have received little attention and there are few toxicity and health risk studies about APs and OCA Y.…”

“…Tandem mass spectrometry (MS/MS) has been widely adopted for the routine quantitative analyses of target cyanotoxins in water samples using the very sensitive multiple reaction monitoring (MRM) mode of triple quadrupole (QqQ) mass spectrometers [23,[25][26][27][28][29][30][31]. For structural characterization and quantitation purposes, 3D ion trap (IT) [32][33][34][35][36][37], linear ion trap (LIT) [38], time-of-flight (TOF) [39,40], Q-TOF [41][42][43][44][45] and Orbitrap mass spectrometers [26,[46][47][48][49][50][51][52] were employed. High mass accuracy of TOF and Orbitrap instruments is beneficial for LC-MS and LC-MS/MS analyses of cyanotoxins [24].…”

“…To our knowledge, such high concentrations of cyanobacterial peptides (CPs) have never been reported before. Finally, it is important to highlight that effective consumer protection requires efficient detection of the whole spectrum of cyanobacterial toxin congeners, especially, considering that different bioactive metabolites are often detected simultaneously [31,32,35,38,41,42,51]. The HRMS approach has been demonstrated to be particularly suitable for the detection and structural confirmation of other CPs, more than MCs, which are not commonly monitored.…”

Toxic compounds produced by cyanobacteria belonging to several species of the order Nostocales: A review

“…including Nostoc, strains ASN-M (Nowruzi et al, 2012), PCC73102 (Guljamow et al, 2017;Rouhiainen et al, 2010), and CENA543 (Kurmayer et al, 2020;Shishido et al, 2017 (Harms et al, 2016;Murakami et al, 2000), protein phosphatases (Sano, Usui, Ueda, Osada, & Kaya, 2001) and/or serine proteases (trypsin, chymotrypsin) (Mazur-Marzec et al, 2018).…”