Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Background:Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17.Methods:Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18.Results:The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 μg), goat (HU50 = 13.2 ± 0.3 μg), rabbit (HU50 = 34.7 ± 0.5 μg), and human (HU50 = 25.6 ± 0.6 μg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 μg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction...(AU)

Postpartum uterine infection and endometritis in dairy cattle

After parturition, uterine involution, regeneration of the endometrium, return of ovarian cyclic activity, and the control of pathogenic bacteria in the uterus is required before cows are likely to conceive again. However, pathogenic bacteria often cause uterine disease in modern dairy cattle, leading to decreased productivity and reduced fertility. This review aims to provide an overview of postpartum uterine infection and disease in dairy cattle. Metritis and endometritis are the main postpartum clinical conditions; although, subclinical endometritis is an emerging issue. Postpartum uterine disease is associated with the isolation of Escherichia coli, Trueperella pyogenes, and anaerobic pathogenic bacteria. Sensing of bacteria or their pathogen-associated molecules, such as lipopolysaccharide, by the innate immune system generates inflammatory responses. Endometrial inflammation includes increased expression of complement, calgranulins, interleukins and acute phase proteins, as well as the chemotaxis of neutrophils and macrophages to the site of infection. Uterine disease is also characterised by tissue damage, including endometrial cytolysis caused by the cholesterol-dependent cytolysin, pyolysin. The responses to pathogens are energetically expensive, and depletion of the key cellular nutrients, glucose or glutamine, impairs inflammatory responses by endometrial tissues. For sustainable intensification of the dairy industry over the next 50 years, it is vital to understand why high-milk-yield cows are so susceptible to uterine pathology and develop new ways to prevent uterine disease.

Postpartum uterine infection and endometritis in dairy cattle

After parturition, uterine involution, regeneration of the endometrium, return of ovarian cyclic activity, and the control of pathogenic bacteria in the uterus is required before cows are likely to conceive again. However, pathogenic bacteria often cause uterine disease in modern dairy cattle, leading to decreased productivity and reduced fertility. This review aims to provide an overview of postpartum uterine infection and disease in dairy cattle. Metritis and endometritis are the main postpartum clinical conditions; although, subclinical endometritis is an emerging issue. Postpartum uterine disease is associated with the isolation of Escherichia coli, Trueperella pyogenes, and anaerobic pathogenic bacteria. Sensing of bacteria or their pathogen-associated molecules, such as lipopolysaccharide, by the innate immune system generates inflammatory responses. Endometrial inflammation includes increased expression of complement, calgranulins, interleukins and acute phase proteins, as well as the chemotaxis of neutrophils and macrophages to the site of infection. Uterine disease is also characterised by tissue damage, including endometrial cytolysis caused by the cholesterol-dependent cytolysin, pyolysin. The responses to pathogens are energetically expensive, and depletion of the key cellular nutrients, glucose or glutamine, impairs inflammatory responses by endometrial tissues. For sustainable intensification of the dairy industry over the next 50 years, it is vital to understand why high-milk-yield cows are so susceptible to uterine pathology and develop new ways to prevent uterine disease.(AU)

Identification of two novel cytolysins from the hydrozoan Olindias sambaquiensis (Cnidaria)

Although the hydrozoan Olindias sambaquiensis is the most common jellyfish associated with human envenomation in southeastern and southern Brazil, information about the composition of its venom is rare. Thus, the present study aimed to analyze pharmacological aspects of O. sambaquiensis venom as well as clinical manifestations observed in affected patients. Crude protein extracts were prepared from the tentacles of animals; peptides and proteins were sequenced and submitted to circular dichroism spectroscopy. Creatine kinase, cytotoxicity and hemolytic activity were evaluated by specific methods.(AU)

Identification of two novel cytolysins from the hydrozoan Olindias sambaquiensis (Cnidaria)

Although the hydrozoan Olindias sambaquiensis is the most common jellyfish associated with human envenomation in southeastern and southern Brazil, information about the composition of its venom is rare. Thus, the present study aimed to analyze pharmacological aspects of O. sambaquiensis venom as well as clinical manifestations observed in affected patients. Crude protein extracts were prepared from the tentacles of animals; peptides and proteins were sequenced and submitted to circular dichroism spectroscopy. Creatine kinase, cytotoxicity and hemolytic activity were evaluated by specific methods.

Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil

The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.(AU)

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms

Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.(AU)

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms

Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.(AU)

Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

Pore-forming cytolysins of 19 kDa from sea anemones present a remarkable cytolytic property. In the present work, a purified 19-kDa cytolysin was obtained from the sea anemone Heteractis magnifica. The purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mM sodium phosphate buffer (pH 7.4), followed by anion exchange chromatography in DEAE-Sepharose® column (GE Healthcare, Sweden) and gel filtration chromatography using Sephadex® G-50 matrix (GE Healthcare, Sweden). The active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. The final active fraction showed a prominent protein band of molecular mass of 19 kDa when analyzed by SDS-PAGE.(AU)

Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

It is well established that sea anemones comprise a rich source of cytolytic toxins. The present study reports the isolation and characterization of a cytolysin obtained from the sea anemone Heteractis magnifica collected in the Andaman Islands of the Indian Ocean. The crude extract was screened for hemolytic activity by a blood agar plate method and a 6-mm zone of clearance was observed after incubation. The hemolytic property of the crude extract, tested by the microtiter plate method, revealed positive results at concentrations as low as 120 ng/mL. Furthermore, it was favored by alkaline pH and was stable up to 60°C. On the other hand, the hemolytic effect was abolished by the addition of human serum. Purification steps involved ammonium sulfate precipitation and subsequent desalting by dialysis, followed by anion- and cation-exchange chromatographies. The purified fractions displayed the presence of a 19-kDa cytolysin when analyzed by SDS-PAGE. The conserved region of the cytolysin (with 303 bp) was amplified by RT-PCR and was sequenced. The sequence showed maximum homology (97 percent) with the already reported cytolysins from other sea anemone species.(AU)

Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

Pore-forming cytolysins of 19 kDa from sea anemones present a remarkable cytolytic property. In the present work, a purified 19-kDa cytolysin was obtained from the sea anemone Heteractis magnifica. The purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mM sodium phosphate buffer (pH 7.4), followed by anion exchange chromatography in DEAE-Sepharose® column (GE Healthcare, Sweden) and gel filtration chromatography using Sephadex® G-50 matrix (GE Healthcare, Sweden). The active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. The final active fraction showed a prominent protein band of molecular mass of 19 kDa when analyzed by SDS-PAGE.(AU)

Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

It is well established that sea anemones comprise a rich source of cytolytic toxins. The present study reports the isolation and characterization of a cytolysin obtained from the sea anemone Heteractis magnifica collected in the Andaman Islands of the Indian Ocean. The crude extract was screened for hemolytic activity by a blood agar plate method and a 6-mm zone of clearance was observed after incubation. The hemolytic property of the crude extract, tested by the microtiter plate method, revealed positive results at concentrations as low as 120 ng/mL. Furthermore, it was favored by alkaline pH and was stable up to 60ºC. On the other hand, the hemolytic effect was abolished by the addition of human serum. Purification steps involved ammonium sulfate precipitation and subsequent desalting by dialysis, followed by anion- and cation-exchange chromatographies. The purified fractions displayed the presence of a 19-kDa cytolysin when analyzed by SDS-PAGE. The conserved region of the cytolysin (with 303 bp) was amplified by RT-PCR and was sequenced. The sequence showed maximum homology (97 percent) with the already reported cytolysins from other sea anemone species.(AU)

Hemolytic toxin from the soft coral Sarcophyton trocheliophorum: isolation and physiological characterization

The unifying characteristic of cnidarians is the production of protein and polypeptide toxins. The present study describes the identification of a hemolytic toxin from the soft coral Sarcophyton trocheliophorum. The crude extract was highly cytotoxic (EC50 = 50 ng/mL) against human erythrocytes. It was also tested for hemolytic activity by the blood agar plate method, resulting in a hemolytic halo of 12 mm with 50 µg of protein. The stability of the venom under different physiological conditions was analyzed. The venom hemolytic activity was augmented by alkaline and neutral pH whereas it was reduced in acidic pH. The activity was stable up to 60º C. The hemolytic activity was completely abolished by the addition of serum and reduced significantly during frequent freezing-thawing cycles. Toxin purification was performed by ammonium sulfate precipitation and subsequently desalted by dialysis against 10 mM sodium phosphate buffer (pH 7.2), followed by anion exchange chromatography on DEAE cellulose column and gel filtration chromatography using Sephadex G-50 matrix. The purified active fractions possessed a prominent protein of approximately 45 kDa, as revealed by SDS-PAGE.(AU)

Hemolytic toxin from the soft coral Sarcophyton trocheliophorum: isolation and physiological characterization

The unifying characteristic of cnidarians is the production of protein and polypeptide toxins. The present study describes the identification of a hemolytic toxin from the soft coral Sarcophyton trocheliophorum. The crude extract was highly cytotoxic (EC50 = 50 ng/mL) against human erythrocytes. It was also tested for hemolytic activity by the blood agar plate method, resulting in a hemolytic halo of 12 mm with 50 µg of protein. The stability of the venom under different physiological conditions was analyzed. The venom hemolytic activity was augmented by alkaline and neutral pH whereas it was reduced in acidic pH. The activity was stable up to 60º C. The hemolytic activity was completely abolished by the addition of serum and reduced significantly during frequent freezing-thawing cycles. Toxin purification was performed by ammonium sulfate precipitation and subsequently desalted by dialysis against 10 mM sodium phosphate buffer (pH 7.2), followed by anion exchange chromatography on DEAE cellulose column and gel filtration chromatography using Sephadex G-50 matrix. The purified active fractions possessed a prominent protein of approximately 45 kDa, as revealed by SDS-PAGE.(AU)

Análise genotípica e filogenética com base nos genes do pilus tipo IV de Moraxella bovis e da citotoxina de M. bovis, Moraxella bovoculi e Moraxella ovis

Historicamente, acreditava-se que a ceratoconjuntivite infecciosa bovina (CIB) estáva sob competência exclusiva de Moraxella bovis. Contudo, outras espécies do Gênero Moraxella também vêm sendo estudadas quanto à participação na patogenia da CIB, como Moraxella ovis e principalmente Moraxella bovoculi. Esta tese descreve análises filogenéticas e de dados genotípicos com base nos genes codificadores dos pili tipo IV dos tipos Q e I (TfpQ/I) de M. bovis e da citotoxina de M. bovis (MbxA), M. bovoculi (MbvA) e M. ovis (MovA). A diferenciação entre M. bovis, M. bovoculi e M. ovis foi previamente realizada por PCR (região intergenica 16S-23S) conforme protocolo estabelecido na literatura. Após, é descrita uma análise molecular com base na região 3' dos genes tfpQ/I (compreendendo subdomínio 1-C N-terminal e o domínio C-terminal) de 16 isolados de campo e cinco cepas vacinais de M. bovis, provenientes da América do Sul. Todas as 47 sequencias do gene tfp tipo Q e tipo I analisadas resultaram em 31 alelos designados de 1 até 31. A reconstrução filogenética resultou em uma distinção dos 31 alelos em onze grupos (designados de A até J e Epp). A análise da sequencia de aminoácidos (aa) deduzidos da região C-terminal mostrou níveis de similaridade entre 67 e 100% dentro dos grupos, enquanto a análise da região D (subunidade C-terminal) resultou níveis de similaridade entre 60 e 100%. Além disso, um estudo filogenético com base na região 3' dos genes da citotoxina foi realizado para investigar a relação genética entre os isolados de M. bovis (n = 17), M. bovoculi (n = 11) e M. ovis (n = 7) e cepas de referência. A reconstrução filogenética permitiu a diferenciação entre as espécies, sendo que os isolados mais antigos de M. bovoculi permaneceram em ramo mais próximos aos isolados de M. bovis. O nível de similaridade de aminoácidos entre as sequencias de MbxA ficou em 99.9% de média, enquanto entre as sequencias de MbvA e MovA foi respectivamente de 98.8% e 99.3%. A similaridade entre MbvA e MovA foi de 96.6%, enquanto MbxA em relação a MbvA e MovA foi de 77.6%. Assim, é possível concluir que o gene tfp pode ser adequado para inferir distinção entre os isolados de M. bovis originários da América do Sul, enquanto o gene codificador da citotoxina é adequado para classificação filogenéticas dos isolados de M. bovis, M. bovoculi e M. ovis, e, talvez para a compreensão das relações evolutivas entre e dentro de cada espécie.

Historically, infectious bovine keratoconjunctivitis (IBK) was believed to be under the exclusive competence of Moraxella bovis. However, the roles of other species of Genus Moraxella are also being considered in the pathogenesis of IBK, such as Moraxella ovis and particularly Moraxella bovoculi. This thesis describes phylogenetic and genotypic analysis based on the genes encoding type IV pili Q- and I-type (TfpQ/I) of M. bovis and cytotoxin of M. bovis (MbxA), M. bovoculi (MbvA) and M. ovis (MovA). The distinction between M. bovis, M. bovoculi and M. ovis was previously performed by PCR (16S-23S intergenic region) according to the protocol established in the literature. Then, there is described a molecular analysis based on the 3' region of genes tfpQ/I (including 1-C N-terminal subdomain and C-terminal domain) of 16 field strains and five vaccine strains of M. bovis from South America. All 47 sequences of tfp Q- and I-type genes analyzed resulted in 31 alleles designated 1 to 31. The phylogenetic reconstruction resulted in a distinction of 31 alleles in eleven groups (designated A through J and Epp). The analysis of the deduced amino acid sequence (aa) of the C-terminal region showed similarity levels between 67 and 100% within the groups, while the analysis of the D region (C-terminal subunit) resulted in levels of similarity between 60 and 100%. In addition, a phylogenetic analysis based on the 3' region of the cytotoxin gene was performed to investigate the genetic relationship among M. bovis (n = 17), M. bovoculi (n = 11) and M. ovis (n = 7) strains and reference strains. Phylogenetic reconstruction allowed the differentiation among species, and the older M. bovoculi strains remained in branch closer to M. bovis strains. The amino acid similarity level among the MbxA sequences stood at an average of 99.9%, while among the MbvA and MovA sequences the similarity was respectively 98.8% and 99.3%. The similarity between MbvA and MovA was 96.6%, while MbxA for MbvA and MovA was 77.6%. Thus, it is possible to conclude that the tfp gene may be inferred suitable for differentiate among M. bovis strains, while the cytotoxin-encoding gene is suitable for phylogenetic classification of M. bovis, M. bovoculi and M. ovis strains and perhaps for understanding the evolutionary relationships among three species

LEUKOTOXIC ACTIVITY OF ACTINOBACILLUS ACTINOMYCETEMCOMITANS ISOLATED FROM HUMAN AND NON-HUMAN PRIMATES

Actinobacillus actinomycetemcomitans is a clinically relevant periodontopathogenic Gram-negative coccobacillus that produces a leukotoxin of the RTX cytolysin family. In this study, we evaluated the leukotoxic activity of A. actinomycetemcomitans strains isolated from human and marmosets by Trypan blue exclusion and by the chemiluminescence assays. Among eight A. actinomycetemcomitans human strains studied, two (P2.17 and P8.12) were classified as high leukotoxin producers and among eight marmoset strains, one (M22.11) showed high leukotoxin production, as determined by Trypan blue exclusion assay. The reference strains ATCC 29523 and FDC Y4 respectively behaved like moderate and low producers. The chemiluminescence assay was used to evaluate the leukotoxic activity of M22.11 and P2.17 strains submitted to different growth conditions. Leukotoxic activity was detected on cells at the logarithmic phase and was similar under anaerobic and microaerophilic growth conditions. It was greatly reduced when cells were grown at glucose concentrations lower or higher than 0.75% (0.25% and 1.5%) in thioglycolate medium. Leukotoxin production mainly by the M22.11 strain was low in BHI broth, whereas production in TSB medium showed a similar level as in thioglycolate broth medium. Sodium bicarbonate at 10 mM did not affect leukotoxin production.

Actinobacillus actinomycetemcomitans é um cocobacilo Gram negativo, periodontopatógeno clinicamente importante, que produz uma leucotoxina pertencente à família das citolisinas RTX. Neste estudo, avaliou-se a atividade leucotóxica de amostras de A. actinomycetemcomitans isoladas de seres humanos e de calitriquídeos pelos métodos de exclusão de azul de Tripan e quimioluminescência. Duas (P2.17 e P8.12) entre oito amostras de A. actinomycetemcomitans isoladas de seres humanos, e uma (M22.11) entre 8 amostras isoladas de sagüis se apresentaram como altamente produtoras de leucotoxina, como determinado pelo teste de exclusão de azul de Tripan. As amostras de referências de A. actinomycetemcomitans ATCC 29523 e FDC Y4 se comportaram como média e baixa produtoras de leucotoxina, respectivamente. O teste de quimioluminescência foi usado para avaliar a atividade leucotóxica das amostras M22.11 e P2.17 submetidas a diferentes condições de crescimento. A atividade leucotóxica foi detectada em células durante a fase logarítmica e foi similar sob crescimento em anaerobiose e microaerofilia. A atividade leucotóxica foi muito reduzida quando as células foram crescidas em concentrações de glicose mais baixa e mais alta que 0,75% (0,25% e 1,5%), em meio tioglicolato. A produção de leucotoxina, especialmente pela amostra M22.11, foi mais baixa em caldo BHI, enquanto em meio TSB a produção foi em nível similar a aquele em meio tioglicolato. Bicarbonato de sódio 10mM não afetou a produção de leucotoxina.