The role and function of hepatotoxins microcystin and nodularin produced by M.aeruginosa PCC 7806 and N. spumigena NSORlO respectively have yet to be elucidated. The mode of transcriptional regulation of these toxins, incorporating DNA binding proteins, was investigated, as an attempt to further understand the key control mechanisms acting on the toxins. The DNA binding proteins that control nitrogen and iron responsive transcription, NtcA and Fur, were identified from M. aeruginosa PCC7806 and N. spumigena NSOR10. Cloning and over-expression in E. coli was followed by mobility shift assays to determine binding characteristics of NtcA and Fur to the promoters, mcyA/D and ndaA/C, those regions that control the toxin encoding gene clusters in M. aeruginosa PCC 7806 and N. spumigena NSOR10, respectively. The results from these studies suggested a role for iron and nitrogen in the transcriptional control of microcystin and nodularin. biosynthesis. As NtcA and Fur classically act to regulate nitrogen and iron dependent genes, a link may be made to the putative function and control of microcystin and nodularin. By identifying the transcription factors NtcA and Fur in these genera, a greater understanding of the link between nutrient levels in the environment and hepatotoxin production in cyanobacteria may be possible.

As toxinas produzidas por cianobactérias em ecossistemas aquáticos de superfície utilizados para abastecimento público constituem uma preocupação mundial, com casos de intoxicação relatados em diversos países.Sérios problemas de saúde e até mesmo óbito podem ocorrer como consequência dessas intoxicações. Em ambientes de água doce eutrofizados, florações de espécies de Microcystis são frequentemente observadas e, devido à sua ampla distribuição geográfica e capacidade de produzir toxinas, este é um dos gêneros de cianobactérias mais extensivamente estudados. Microcystis spp. são conhecidas pela produção da potente hepatotoxina microcistina. No entanto, um estudo recente com a linhagem M. aeruginosa SPC777 isolada da represa Billings (São Paulo, SP) relatou a sua capacidade de produção simultânea da [L-ser7] microcistina-RR e da neurotoxina saxitoxina (goniautoxinas 1, 2, 3 e 4). Esse foi o primeiro relato de produção de saxitoxina por uma cianobactéria unicelular. Nesse contexto, o presente estudo teve como objetivo identificar e sequenciar os genes envolvidos na biossíntese da microcistina e saxitoxina na linhagem M. aeruginosa SPC777 e reavaliar a produção destas toxinas após vários anos de cultivo em laboratório. Para isso, foi feito o sequenciamento do genoma de M. aeruginosa SPC777 na plataforma SOLiD V3 e a montagem ab initio das leituras foi realizada usando os algoritmos Edena e Velvet. Análises Blast no banco de dados do NCBI foram realizadas na busca de similaridade por sequências dos genes mcy e sxt e de genes que flanqueiam os agrupamentos envolvidos na biossíntese de ambas as toxinas. Além disso, PCR e sequenciamento Sanger foram empregados para auxiliar a busca dos genes de interesse. Os dez genes envolvidos na biossíntese da microcistina (mcyA-J) foram encontrados e anotados a partir do genoma da M. aeruginosa SPC777, assim como os genes dnaN e uma1, que são normalmente encontrados flanqueando o agrupamento gênico da microcistina. O arranjo dos genes mcy no agrupamento seguiu a mesma ordem de outros descritos na literatura, mas foram encontradas diferenças na sequência de nucleotídeos para alguns dos genes. Para saxitoxina, apenas cinco genes entre aqueles diretamente envolvidos na biossíntese desta neurotoxina foram encontrados usando PCR e sequenciamento Sanger. As sequências parciais dos genes sxt apresentaram alta identidade com outros encontrados em cianobactérias tóxicas. Além disso, a tradução dessas sequências em aminoácidos e as funções protéicas e domínios preditos confirmaram sua identidade como genes da sintetase de saxitoxina. Análises químicas em HPLC-MS/MS mostraram a produção de microcistina, com a detecção do íon m/z 1036, que corresponde à microcistina-YM. Entretanto, não foi observada produção de saxitoxinas. Pelo que sabemos, este é o primeiro agrupamento gênico de microcistina sequenciado de uma linhagem de Microcystis isolada da América do Sul,além de serem os primeiros genes sxt descritos em uma cianobactéria unicelular. Este estudo propiciou novos conhecimentos sobre a origem dos genes mcy e sxt e contribuiu para uma melhor compreensão da evolução destas toxinas / The toxins produced by cyanobacteria in surface aquatic ecosystems used for public supply constitute a worldwide concern, with poisoning cases reported in several countries. Serious health problems and even death can occur as a consequence of these poisonings. In eutrophic freshwater environments, blooms of Microcystis species are often observed and, due to its wide geographic distribution and ability to produce toxins, this is one of the most extensively studied cyanobacterial genera. Microcystis spp. are known for the production of the potent hepatotoxin microcystin. Nonetheless, a recent study with the strain M. aeruginosa SPC777 isolated from Billings reservoir (São Paulo, SP) reported its ability for simultaneous production of [L-ser7] microcystin-RR and the neurotoxin saxitoxin (gonyautoxins 1, 2, 3 and 4). This was the first report of saxitoxin production by a unicellular cyanobacterium. In this context, the present study aimed at the identification and sequencing of the genes involved in the biosynthesis of microcystin and saxitoxin in the strain M. aeruginosa SPC777 and re-evaluation ofthe production of these toxins after several years of cultivation in laboratory. For this, whole genome sequencing of M. aeruginosa SPC777 was done in the platform SOLiD V3 and ab initio assembly of the reads was performed using the algorithms Edena and Velvet. Blast analyses in the NCBI database were performed in the searchfor similarity to mcy and sxt gene sequences and to genes flanking the clusters involvedin the biosynthesis of both toxins. Furthermore, PCR and Sanger sequencing were employed to help the search for the genes of interest. The ten genes involved in microcystin biosynthesis (mcyA-J) were found and annotated from the genome of M. aeruginosa SPC777, as well as the genes dnaN and uma1, which are usually found flanking the microcystin gene cluster. The arrangement of mcy genes in the cluster has followed the same order than others described in literature, but differences were found in the sequence of nucleotides for some of the genes. For saxitoxin, only five genes among those directly involved in the biosynthesis of this neurotoxin were found using PCR and Sanger sequencing. The partial sxt gene sequences have shown high identities to others found in toxic cyanobacteria. Additionally, their translation into amino acids and the predicted protein functions and domains confirmed their identity as saxitoxin synthetase genes.HPLC-MS/MS chemical analyses have shown the production of microcystin, with the detection of the ion m/z1036, which corresponds to the microcystin-YM. Nevertheless, saxitoxin production was not observed. As far as we know, this is the first microcystin gene cluster sequenced from a Microcystis strain isolated from South America and it is also the first time that sxt genes are described in a unicellular cyanobacterium. This study has brought new insightson the origin of the mcy and sxt genes and contributed to a better understanding of the evolution of these toxins

As toxinas produzidas por cianobactérias em ecossistemas aquáticos de superfície utilizados para abastecimento público constituem uma preocupação mundial, com casos de intoxicação relatados em diversos países.Sérios problemas de saúde e até mesmo óbito podem ocorrer como consequência dessas intoxicações. Em ambientes de água doce eutrofizados, florações de espécies de Microcystis são frequentemente observadas e, devido à sua ampla distribuição geográfica e capacidade de produzir toxinas, este é um dos gêneros de cianobactérias mais extensivamente estudados. Microcystis spp. são conhecidas pela produção da potente hepatotoxina microcistina. No entanto, um estudo recente com a linhagem M. aeruginosa SPC777 isolada da represa Billings (São Paulo, SP) relatou a sua capacidade de produção simultânea da [L-ser7] microcistina-RR e da neurotoxina saxitoxina (goniautoxinas 1, 2, 3 e 4). Esse foi o primeiro relato de produção de saxitoxina por uma cianobactéria unicelular. Nesse contexto, o presente estudo teve como objetivo identificar e sequenciar os genes envolvidos na biossíntese da microcistina e saxitoxina na linhagem M. aeruginosa SPC777 e reavaliar a produção destas toxinas após vários anos de cultivo em laboratório. Para isso, foi feito o sequenciamento do genoma de M. aeruginosa SPC777 na plataforma SOLiD V3 e a montagem ab initio das leituras foi realizada usando os algoritmos Edena e Velvet. Análises Blast no banco de dados do NCBI foram realizadas na busca de similaridade por sequências dos genes mcy e sxt e de genes que flanqueiam os agrupamentos envolvidos na biossíntese de ambas as toxinas. Além disso, PCR e sequenciamento Sanger foram empregados para auxiliar a busca dos genes de interesse. Os dez genes envolvidos na biossíntese da microcistina (mcyA-J) foram encontrados e anotados a partir do genoma da M. aeruginosa SPC777, assim como os genes dnaN e uma1, que são normalmente encontrados flanqueando o agrupamento gênico da microcistina. O arranjo dos genes mcy no agrupamento seguiu a mesma ordem de outros descritos na literatura, mas foram encontradas diferenças na sequência de nucleotídeos para alguns dos genes. Para saxitoxina, apenas cinco genes entre aqueles diretamente envolvidos na biossíntese desta neurotoxina foram encontrados usando PCR e sequenciamento Sanger. As sequências parciais dos genes sxt apresentaram alta identidade com outros encontrados em cianobactérias tóxicas. Além disso, a tradução dessas sequências em aminoácidos e as funções protéicas e domínios preditos confirmaram sua identidade como genes da sintetase de saxitoxina. Análises químicas em HPLC-MS/MS mostraram a produção de microcistina, com a detecção do íon m/z 1036, que corresponde à microcistina-YM. Entretanto, não foi observada produção de saxitoxinas. Pelo que sabemos, este é o primeiro agrupamento gênico de microcistina sequenciado de uma linhagem de Microcystis isolada da América do Sul,além de serem os primeiros genes sxt descritos em uma cianobactéria unicelular. Este estudo propiciou novos conhecimentos sobre a origem dos genes mcy e sxt e contribuiu para uma melhor compreensão da evolução destas toxinas / The toxins produced by cyanobacteria in surface aquatic ecosystems used for public supply constitute a worldwide concern, with poisoning cases reported in several countries. Serious health problems and even death can occur as a consequence of these poisonings. In eutrophic freshwater environments, blooms of Microcystis species are often observed and, due to its wide geographic distribution and ability to produce toxins, this is one of the most extensively studied cyanobacterial genera. Microcystis spp. are known for the production of the potent hepatotoxin microcystin. Nonetheless, a recent study with the strain M. aeruginosa SPC777 isolated from Billings reservoir (São Paulo, SP) reported its ability for simultaneous production of [L-ser7] microcystin-RR and the neurotoxin saxitoxin (gonyautoxins 1, 2, 3 and 4). This was the first report of saxitoxin production by a unicellular cyanobacterium. In this context, the present study aimed at the identification and sequencing of the genes involved in the biosynthesis of microcystin and saxitoxin in the strain M. aeruginosa SPC777 and re-evaluation ofthe production of these toxins after several years of cultivation in laboratory. For this, whole genome sequencing of M. aeruginosa SPC777 was done in the platform SOLiD V3 and ab initio assembly of the reads was performed using the algorithms Edena and Velvet. Blast analyses in the NCBI database were performed in the searchfor similarity to mcy and sxt gene sequences and to genes flanking the clusters involvedin the biosynthesis of both toxins. Furthermore, PCR and Sanger sequencing were employed to help the search for the genes of interest. The ten genes involved in microcystin biosynthesis (mcyA-J) were found and annotated from the genome of M. aeruginosa SPC777, as well as the genes dnaN and uma1, which are usually found flanking the microcystin gene cluster. The arrangement of mcy genes in the cluster has followed the same order than others described in literature, but differences were found in the sequence of nucleotides for some of the genes. For saxitoxin, only five genes among those directly involved in the biosynthesis of this neurotoxin were found using PCR and Sanger sequencing. The partial sxt gene sequences have shown high identities to others found in toxic cyanobacteria. Additionally, their translation into amino acids and the predicted protein functions and domains confirmed their identity as saxitoxin synthetase genes.HPLC-MS/MS chemical analyses have shown the production of microcystin, with the detection of the ion m/z1036, which corresponds to the microcystin-YM. Nevertheless, saxitoxin production was not observed. As far as we know, this is the first microcystin gene cluster sequenced from a Microcystis strain isolated from South America and it is also the first time that sxt genes are described in a unicellular cyanobacterium. This study has brought new insightson the origin of the mcy and sxt genes and contributed to a better understanding of the evolution of these toxins

Student Number : 9902006N - MSc(Med) Dissertation - School of Medicine - Faculty of Health Sciences / Hepatocellular carcinoma (HCC) is the most common primary malignant tumour of the liver. Aflatoxin B1 (AFB1) is a potent hepatocarcinogen, and dietary iron overload has been shown to contribute to HCC development in black africans. Both are well studied hepatotoxins. The aim of this study was to use a Wistar rat model over a 12 month period to investigate synergy and the extent thereof between AFB1 ingestion and dietary iron overload. 25ug/day of AFB1, reconstituted in DMSO, was administered by gavaging the animals, over a period of 10 days with a 2 day interval in between. The chow diet was supplemented with 0.75% (w/w) ferrocene iron. Experimental subjects were divided into 4 groups. Group 1 was fed the normal chow diet. Group 2 was fed 0.75% (w/w) ferrocene iron alone. Group 3 was gavaged 250μg AFB1 alone. Group 4 was fed the 0.75% (w/w) ferrocene iron and gavaged 250μg AFB1. A number of assays were conducted to investigate synergy. Colorimetric assays were used to measure serum iron, total-iron binding capacity, ALT, AST, GGT, nitrite production, lipid peroxidation and hydroxyproline concentrations. ELISA’s were used to determine ferritin, 8-isoprostane and 8-hydroxyguanosine concentrations. Nontransferrin bound iron was measured using an HPLC method. A chemiluminescent assay was used to measure superoxide anion production. Cytokines were measured using a suspension array system. Mutagenicity was assessed using the Ames mutagenicity assay using salmonella typhimirium strains TA97, TA98, TA100 and TA102. Iron profiling indicated that iron overloading occurred with the ingestion of the ferrocene diet. Biomarkers of oxidative stress, as illustrated by the measurement of 8-hydroxyguanosine and lipid peroxidation, showed additive synergistic effects between the two carcinogens. The anti-inflammatory interleukin-10 was shown to be markedly elevated with the co-administration of the two carcinogens, indicating the elevated inflammatory processes. Additive synergistic effects were noted in terms of the liver disease marker ALT. The salmonella typhimirium strain TA102 used in the Ames mutagenicity test showed increased colony counts with respect to the coadministration of carcinogens (P<0.05), although no synergistic effect was noted. In a few of the presented parameters, the AFB1 group was not significantly different to the control group, although significant differences between the Fe group and the Fe + AFB1 groups were noted. The implication of which is that the presence of AFB1 is increasing the activity of Fe as a carcinogen, thereby acting as a co-carcinogen. Examples of such parameters illustrating this are presented in the results section including serum ALT, serum nitrite, liver and serum lipid peroxidation, liver and serum 8-hydroxyguanosine, some of the mutagenicity assays, and interleukin-10. The conclusion of this study suggests that AFB1 acts as a co-carcinogen in the presence of iron overloading, implying that a synergistic relationship between these two toxins exists.