Toxin Production and Immunoassay Development. 1. Palytoxin

Том 36(2020) №3 стр. 82-89; DOI 10.21519/0234-2758-2020-36-3-82-89А.В. Гаева1*, О.В. Громова1, О.С. Дуракова1, С.В. Генералов1, Л.Ф. Ливанова1, О.А. Волох1 Определение специфической активности компонентов холерной химической вакцины с использованием культуры клеток 1ФКУЗ «Российский научно-исследовательский противочумный институт «Микроб»» Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека, Саратов 410005 *[email protected] Поступила - 2019-11-26; После доработки - 2020-03-16; Принята к публикации - 2020-05-15 Список литературы Описаны методы определения динамики продукции токсинов штаммом Vibrio cholerae 569B при глубинном культивировании в биореакторе и антигенной активности специфической фракции холерогена-анатоксина по анатоксинсвязыванию с использованием клеточных культур. Показана высокая степень соответствия результатов, полученных методами, применяемыми для контроля этапов производства холерной химической вакцины и рассмотренными в данной работе. Отмечено, что применение клеточной линии СНО-К1 наиболее перспективно для замены биомоделей на промежуточных этапах контроля активных компонентов холерной химической вакцины. Разработанный методический подход впервые предлагается использовать на этапах производства холерной бивалентной химической вакцины. культура клеток, Vibrio cholerae, холерная химическая вакцина, контроль производства, холера. Vol 36(2020) N 3 p. 82-89; DOI 10.21519/0234-2758-2020-36-3-82-89A.V. Gaeva1*, O.V. Gromova1, O.S. Durakova1, S.V. Generalov1, L.F. Livanova1, O.A. Volokh1 Determination of Specific Activity of Cholera Chemical Vaccine Components using Cell Culture 1Russian Research Anti-Plague Institute «Microbe» of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Saratov, 410005 *[email protected] Received - 26.11.2019; Accepted - 15.05.2020 References The methods has been described to determine the dynamics of toxin production by the Vibrio cholerae 569B strain during submerged cultivation in bioreactor and of the antigenic activity of specific choleragen anatoxin fraction by anatoxin binding levels using cell cultures. High degree of consistency was observed between the results obtained via the method under consideration and those obtained via control methods at different stages of cholera chemical vaccine production. It was shown that the CHO-K1 cell line is the most promising substitute for biomodels at the intermediate stages of control of active cholera chemical vaccine components. The developed methodological approach was first proposed for use at the stages of cholera chemical bivalent vaccine manufacturing. cell culture, Vibrio cholerae, cholera chemical vaccine, production control, cholera.

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Toxin Production in Soybean (Glycine max L.) Plants with Charcoal Rot Disease and by Macrophomina phaseolina, the Fungus that Causes the Disease

Toxins ◽

10.3390/toxins11110645 ◽

2019 ◽

Vol 11 (11) ◽

pp. 645 ◽

Cited By ~ 8

Author(s):

Hamed K. Abbas ◽

Nacer Bellaloui ◽

Cesare Accinelli ◽

James R. Smith ◽

W. Thomas Shier

Keyword(s):

Culture Media ◽

Leaf Disc ◽

Toxin Production ◽

Macrophomina Phaseolina ◽

Economic Losses ◽

Limit Of Quantification ◽

Charcoal Rot ◽

Wide Range ◽

Soybean Plants ◽

Rot Disease

Charcoal rot disease, caused by the fungus Macrophomina phaseolina, results in major economic losses in soybean production in southern USA. M. phaseolina has been proposed to use the toxin (-)-botryodiplodin in its root infection mechanism to create a necrotic zone in root tissue through which fungal hyphae can readily enter the plant. The majority (51.4%) of M. phaseolina isolates from plants with charcoal rot disease produced a wide range of (-)-botryodiplodin concentrations in a culture medium (0.14–6.11 µg/mL), 37.8% produced traces below the limit of quantification (0.01 µg/mL), and 10.8% produced no detectable (-)-botryodiplodin. Some culture media with traces or no (-)-botryodiplodin were nevertheless strongly phytotoxic in soybean leaf disc cultures, consistent with the production of another unidentified toxin(s). Widely ranging (-)-botryodiplodin levels (traces to 3.14 µg/g) were also observed in the roots, but not in the aerial parts, of soybean plants naturally infected with charcoal rot disease. This is the first report of (-)-botryodiplodin in plant tissues naturally infected with charcoal rot disease. No phaseolinone was detected in M. phaseolina culture media or naturally infected soybean tissues. These results are consistent with (-)-botryodiplodin playing a role in the pathology of some, but not all, M. phaseolina isolates from soybeans with charcoal rot disease in southern USA.

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Ecophysiological Aspects and sxt Genes Expression Underlying Induced Chemical Defense in STX-Producing Raphidiopsis raciborskii (Cyanobacteria) against the Zooplankter Daphnia gessneri

Toxins ◽

10.3390/toxins13060406 ◽

2021 ◽

Vol 13 (6) ◽

pp. 406

Author(s):

Mauro C. P. Vilar ◽

Thiago F. C. P. Rodrigues ◽

Luan O. Silva ◽

Ana Beatriz F. Pacheco ◽

Aloysio S. Ferrão-Filho ◽

...

Keyword(s):

Induced Defense ◽

Toxin Production ◽

Grazing Pressure ◽

Chemical Defenses ◽

Widespread Occurrence ◽

Genes Expression ◽

Freshwater Bodies ◽

Predator Response ◽

Aquatic Food ◽

Shed Light

Cyanobacteria stand out among phytoplankton when they form massive blooms and produce toxins. Because cyanotoxin genes date to the origin of metazoans, the hypothesis that cyanotoxins function as a defense against herbivory is still debated. Although their primary cellular function might vary, these metabolites could have evolved as an anti-predator response. Here we evaluated the physiological and molecular responses of a saxitoxin-producing Raphidiopsis raciborskii to infochemicals released by the grazer Daphnia gessneri. Induced chemical defenses were evidenced in R. raciborskii as a significant increase in the transcription level of sxt genes, followed by an increase in saxitoxin content when exposed to predator cues. Moreover, cyanobacterial growth decreased, and no significant effects on photosynthesis or morphology were observed. Overall, the induced defense response was accompanied by a trade-off between toxin production and growth. These results shed light on the mechanisms underlying zooplankton–cyanobacteria interactions in aquatic food webs. The widespread occurrence of the cyanobacterium R. raciborskii in freshwater bodies has been attributed to its phenotypic plasticity. Assessing the potential of this species to thrive over interaction filters such as zooplankton grazing pressure can enhance our understanding of its adaptive success.

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Hunting the eagle killer: A cyanobacterial neurotoxin causes vacuolar myelinopathy

Science ◽

10.1126/science.aax9050 ◽

2021 ◽

Vol 371 (6536) ◽

pp. eaax9050

Author(s):

Steffen Breinlinger ◽

Tabitha J. Phillips ◽

Brigette N. Haram ◽

Jan Mareš ◽

José A. Martínez Yerena ◽

...

Keyword(s):

United States ◽

Aquatic Vegetation ◽

Southeastern United States ◽

Hydrilla Verticillata ◽

Toxin Production ◽

The United States ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Bald Eagles ◽

Cyanobacterial Species

Vacuolar myelinopathy is a fatal neurological disease that was initially discovered during a mysterious mass mortality of bald eagles in Arkansas in the United States. The cause of this wildlife disease has eluded scientists for decades while its occurrence has continued to spread throughout freshwater reservoirs in the southeastern United States. Recent studies have demonstrated that vacuolar myelinopathy is induced by consumption of the epiphytic cyanobacterial species Aetokthonos hydrillicola growing on aquatic vegetation, primarily the invasive Hydrilla verticillata. Here, we describe the identification, biosynthetic gene cluster, and biological activity of aetokthonotoxin, a pentabrominated biindole alkaloid that is produced by the cyanobacterium A. hydrillicola. We identify this cyanobacterial neurotoxin as the causal agent of vacuolar myelinopathy and discuss environmental factors—especially bromide availability—that promote toxin production.

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sxtA4+ and sxtA4- Genotypes Occur Together within Natural Pyrodinium bahamense Sub-Populations from the Western Atlantic

Microorganisms ◽

10.3390/microorganisms9061128 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1128

Author(s):

Kathleen Cusick ◽

Gabriel Duran

Keyword(s):

Molecular Mechanisms ◽

Harmful Algal Bloom ◽

Single Cells ◽

Natural Populations ◽

Indian River Lagoon ◽

Toxin Production ◽

Genotype Frequency ◽

Rrna Gene ◽

Western Atlantic ◽

Pyrodinium Bahamense

Saxitoxin (STX) is a secondary metabolite and potent neurotoxin produced by several genera of harmful algal bloom (HAB) marine dinoflagellates. The basis for variability in STX production within natural bloom populations is undefined as both toxic and non-toxic strains (of the same species) have been isolated from the same geographic locations. Pyrodinium bahamense is a STX-producing bioluminescent dinoflagellate that blooms along the east coast of Florida as well as the bioluminescent bays in Puerto Rico (PR), though no toxicity reports exist for PR populations. The core genes in the dinoflagellate STX biosynthetic pathway have been identified, and the sxtA4 gene is essential for toxin production. Using sxtA4 as a molecular proxy for the genetic capacity of STX production, we examined sxtA4+ and sxtA4- genotype frequency at the single cell level in P. bahamense populations from different locations in the Indian River Lagoon (IRL), FL, and Mosquito Bay (MB), a bioluminescent bay in PR. Multiplex PCR was performed on individual cells with Pyrodinium-specific primers targeting the 18S rRNA gene and sxtA4. The results reveal that within discrete natural populations of P. bahamense, both sxtA4+ and sxtA4- genotypes occur, and the sxtA4+ genotype dominates. In the IRL, the frequency of the sxtA4+ genotype ranged from ca. 80–100%. In MB, sxtA4+ genotype frequency ranged from ca 40–66%. To assess the extent of sxtA4 variation within individual cells, sxtA4 amplicons from single cells representative of the different sampling sites were cloned and sequenced. Overall, two variants were consistently obtained, one of which is likely a pseudogene based on alignment with cDNA sequences. These are the first data demonstrating the existence of both genotypes in natural P. bahamense sub-populations, as well as sxtA4 presence in P. bahamense from PR. These results provide insights on underlying genetic factors influencing the potential for toxin variability among natural sub-populations of HAB species and highlight the need to study the genetic diversity within HAB sub-populations at a fine level in order to identify the molecular mechanisms driving HAB evolution.

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Spo0A Suppresses sin Locus Expression in Clostridioides difficile

mSphere ◽

10.1128/msphere.00963-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Babita Adhikari Dhungel ◽

Revathi Govind

Keyword(s):

Diarrheal Disease ◽

Toxin Production ◽

The United States ◽

Upstream Region ◽

Pcr Analysis ◽

Bacterial Cells ◽

Master Regulator ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR′ (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI. However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile. IMPORTANCE Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

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Effect of Salt Level and Nitrite on Toxin Production by Clostridium botulinum Type E Spores in Smoked Great Lakes Whitefish1

Journal of Food Protection ◽

10.4315/0362-028x-50.3.212 ◽

1987 ◽

Vol 50 (3) ◽

pp. 212-217 ◽

Cited By ~ 9

Author(s):

S. L. CUPPETT ◽

J. I. GRAY ◽

J. J. PESTKA ◽

A. M. BOOREN ◽

J. F. PRICE ◽

...

Keyword(s):

Great Lakes ◽

Water Activity ◽

Salt Concentration ◽

Clostridium Botulinum ◽

Toxin Production ◽

Water Phase ◽

Salt Level ◽

Botulinum Type ◽

Clostridium Botulinum Type E ◽

Average Water

The effect of salt level and nitrite on botulinal safety of smoked whitefish was investigated. An average water-phase (wp) salt concentration of 4.4% inhibited outgrowth of Clostridium botulinum type E spores (103 spores/g) for over 35 d in temperature-abused (27°C) smoked whitefish. Incorporation of nitrite (220 mg/kg) during brining to the smoked salted (4.4%, wp) whitefish inhibited toxin production for 56 d at 27°C. An average salt concentration of 6.2% (wp), with or without nitrite, totally inhibited toxin production for the duration of the study (83 d). The effect of pH and water activity in temperature-abused smoked whitefish as a means of controlling toxin production by C. botulinum type E spores was evaluated.

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