Tesis Doctorado
Transport of saxitoxin (stx) and its analogues in filamentous freshwater cyanobacteria
Autor
Soto-Liebe, Katia Leandra
Institución
Resumen
Paralytic shellfish poisoning toxins (PSTs) are a group of naturally occurring neurotoxicalkaloids (saxitoxin and analogues) produced among several genera of freshwatercyanobacteria and marine dinoflagellates. The functional role of PSTs in the ecology andevolution of toxigenic cyanobacteria and dinoflagellates remains unknown, though subject to continual speculation, regarding chemical defense and/or ion transport and regulatory interactions. Determining whether these toxins are exported to the environment and knowing the mechanisms used in this process, can allow us to understand the role that PSTs play in the environment.The sxt cluster is responsible for the production of saxitoxin (STX) and its derivatives.In cyanobacteria, the sxt cluster has a core of 15 genes which have been conserved, both in sequence and synteny, among 5 clusters described to date. This gene cluster encodes MATE (multidrug and toxic compound extrusion) transporter family proteins: SxtF and SxtM,candidates for toxin export. This protein family, represented by NorM in the bacterium Vibrio parahaemolyticus, confers resistance to multiple cationic toxic agents through Na+/drug antiporters.The aim of this project was to identify whether PSTs can be exported by SxtF andSxtM in cyanobacteria, evaluating the role of sodium in this process, and to determine thesub-cellular location of both proteins. The accurate identification of the PSTs profiles in cyanobacteria was a primary task in order to identify the function of the genes present in the cluster. Presently, it has not been possible to perform mutagenesis experiments or heterologous expression of this kind of cluster. Cylindrospermopsis raciborskii T3, a toxigenic freshwater cyanobacterium, has been used as a model to study the physiology and elucidate the biosynthetic pathway of PSTs.Nevertheless; there are several inconsistencies and contradictions in the published toxin profiles for T3. An initial report claimed that this strain produced STX and gonyautoxins 2 and 3 (GTX2/3), whereas a subsequent study arose the profile: STX and the N-sulfocarbamoyl derivatives C1/2. When the T3 strain was used to identify the sequence of the sxt cluster, thetoxin profile was considered to comprise STX, NEO, dcSTX and B1.Our analysis of the complete genome of another PST-producing cyanobacterium,Raphidiopsis brookii D9, closely related to C. raciborskii T3, as well as the PST profile in both strains by liquid chromatography coupled to mass spectrometry (LC-MS/MS), has resolved many issues regarding the correlation between biosynthetic pathways, putative genes and the T3 toxin profile. Our reassessment of the PST profile of T3 by LC-MS/MS confirmed production of only STX, NEO and dcNEO. We analyzed SxtF/M sequences of eight PST-producing cyanobacteria. Phylogeneticanalyses of SxtF/M showed a high conservation of SxtF (100% identity) in theCylindrospermopsis-Raphidiopsis clade, suggesting conserved substrate affinity by this transporter. Two domains involved in Na+ and drug recognition from NorM proteins (MATEfamily) of V. parahaemolyticus and V. cholerae are present in SxtF/M. The Na+ recognition domain was conserved in both SxtF/M, indicating that Na+ can maintain the role as a cation anti-transporter. Although the putative consensus motif for toxin binding differed between SxtF and SxtM, implying differential substrate binding, by protein modeling and docking analysis, we found that there is no marked affinity between the recognition domain and aspecific PST analogue. This agrees with our results of PST export in R. brookii D9, where we observed that the response to Na+ incubation was similar to different analogues. It was not possible to establish a correlation between presence of sxtF and/or sxtM and a specific PST profile.Based upon the importance of monovalent cations (such as sodium) in themaintenance of cellular homeostasis and ion channel function, we examined the effect ofhigh extracellular concentrations of Na+, K+ and Li+ on growth, cellular integrity, toxinproduction and release to the external medium in R. brookii D9 (GTX2/3 and STX producing).We observed toxin export in response to high (17 mM) NaCl and KCl concentrations in thegrowth medium that was not initially related to osmotic stress effects, compared to theosmolyte mannitol. Addition of exogenous PSTs with the same compositional profile as theone produced by R. brookii D9 was able to partially mitigate the effect of high Na+ (17 mM).Finally, bioinformatic analysis shows that at least six proteins encoded by sxt genescould be located or anchored to the membrane (SxtA, SxtD, SxtF, SxtJ, SxtM and SxtU). Weconfirmed by immunolocalization the presence of SxtA, enzyme responsible for the earlystages of STX synthesis, SxtF and SxtM, associated with cytoplasmic and thylakoidsmembranes in R. brookii D9 and C. raciborskii MVCC14. We propose that Sxt proteins could form a “complex” anchored to the membrane. Posterior studies are necessary to determinewhy these proteins are also located in thylakoids membranes. These results reassert the hypothesis regarding the involvement of Na+ and SxtF/M in toxin recognition, and therefore in toxin export. PFCHA-Becas Doctor en Ciencias Mención Genética Molecular y Microbiología 233p. PFCHA-Becas TERMINADA