The cyanobacterial hepatotoxin microcystin binds to proteins and increases the fitness of microcystis under oxidative stress conditions

Abstract

Microcystins are cyanobacterial toxins that represent a serious threat to drinking water and recreational lakes worldwide. Here, we show that microcystin fulfils an important function within cells of its natural producer Microcystis. The microcystin deficient mutant ΔmcyB showed significant changes in the accumulation of proteins, including several enzymes of the Calvin cycle, phycobiliproteins and two NADPH-dependent reductases. We have discovered that microcystin binds to a number of these proteins in vivo and that the binding is strongly enhanced under high light and oxidative stress conditions. The nature of this binding was studied using extracts of a microcystin-deficient mutant in vitro. The data obtained provided clear evidence for a covalent interaction of the toxin with cysteine residues of proteins. A detailed investigation of one of the binding partners, the large subunit of RubisCO showed a lower susceptibility to proteases in the presence of microcystin in the wild type. Finally, the mutant defective in microcystin production exhibited a clearly increased sensitivity under high light conditions and after hydrogen peroxide treatment. Taken together, our data suggest a protein-modulating role for microcystin within the producing cell, which represents a new addition to the catalogue of functions that have been discussed for microbial secondary metabolites.

Figure 1. Proteomic comparison of Microcystis aeruginosa PCC 7806 wild type and microcystin-deficient ΔmcyB mutant.

Proteome of soluble fraction of Microcystis aeruginosa PCC 7806 wild type (WT, blue colouring) and microcystin-deficient ΔmcyB mutant (orange colouring) after exposition to high light of 70 µmol photons m⁻²s⁻¹ for 2 hours. Gels were obtained after separation of soluble protein extracts (400 µg) by 2-DE (first dimension: IEF pH range 4–7 linear, second dimension: SDS-PAGE applying 12.5% acrylamide) and Coomassie-staining. Representative well resolved gels of WT and ΔmcyB were grouped, respectively, warped and fused to one gel via Delta2D version 4.0 (DECODON, Greifswald, Germany). Differential protein spots and selected non-differential protein spots analyzed for standardization are indicated with arrows. Highly abundant phycobiliproteins (PB), associated linker proteins (PBL) as well as proteins of more than three isoforms are encircled. Protein identities are given in Table 1 and Table S1.

Figure 2. Immunoblot analyses of soluble Microcystis protein extracts using a microcystin-specific antibody.

A) Protein extracts of the ΔmcyH, ΔmcyB mutants and wild type. B) Protein extracts of ΔmcyB mutant prior and after addition of microcystin-LR and DTNB pre-treatment (left). Protein extracts of ΔmcyB mutant with addition of microcystin-LR or cysteamin-linked microcystin-LR MC* (right). C) Modified microcystin variant with a cysteamin group attached to the N-methyldehydroalanine moiety.

Figure 3. Comparative proteomic analysis of phycobiliproteins.

A) Mass shift of phycobiliproteins (PB) in Microcystis aeruginosa PCC 7806 wild type (blue colouring) compared to microcystin-deficient ΔmcyB mutant (orange colouring) as seen by 2DE-analysis. Proteins with differential mass in wild type and mutant are encircled. B) Corresponding immunoblot analysis using the microcystin-specific antibody.

Figure 4. Impact of light and oxidative stress on microcystin binding to proteins.

A) Immunoblot analyses of soluble Microcystis protein extracts with an anti-microcystin antibody after high light treatment of 700 µmol photons m⁻² s⁻¹ for up to 4 hours. The irradiation time of the individual samples is indicated. The sample 4′h was irradiated with high light for three hours and subsequently transferred to low light for one hour. B) Immunoblot analysis of a low light adapted culture (C) that was treated for 4 hours with 700 µmol photons m⁻² s⁻¹ high light (HL), 10 µM H₂O₂ or in parallel grown under low light in the absence of iron (-Fe). Immunoblot analysis of a low light adapted culture of the ΔmcyB mutant and a culture treated for three hours with high light (700 µmol photons m⁻² s⁻¹) after microcystin addition.

Figure 5. Quantitative analysis of RbcL.

A) Immunoblot analysis of in vitro expressed and affinity purified RbcL protein using a RbcL-specific antibody and a microcystin-specific antibody, respectively. Microcystin was added as indicated. B) Representative Coomassie-stained gel pictures (upper panel) and immunoblot analyses (lower panel) with a RbcL-specific antibody of soluble protein extracts of M. aeruginosa PCC 7806 wild type (left) and the ΔmcyB mutant (right) after high light treatment of 700 µmol photons m⁻² s⁻¹ for up to four hours. The irradiation time of the individual samples is indicated. The sample 4′h was irradiated with high light for three hours and subsequently transferred to low light for one hour.

Figure 6. Protein degradation assay.

A) Coomassie gel picture showing M. aeruginosa PCC7806 wild type and ΔmcyB cell extracts treated for up to 3 hours with 0.2 µg/mL subtilisin. B) Immunoblot analysis using a specific RbcL antibody. C) Quantification of RbcL signal intensity from three independent subtilisin assays for M. aeruginosa PCC7806 wild type and ΔmcyB mutant. * The starting points were adjusted to 100% D) Control treatment without addition of subtilisin for three hours. E) Control digestion of BSA with subtilisin in presence or absence of microcystin.

Figure 7. Photographs of M. aeruginosa PCC 7806 wild type (WT) and ΔmcyB mutant.

A)grown on 6-well plates for five days under low light (30 µmol photons m⁻² s⁻¹) or high light (300 µmol photons m⁻² s⁻¹) conditions, B)grown on 6-well plates for five days under low light conditions with increasing concentrations of H₂O₂ supplemented to the medium.