Neurotoxin regulation and the temperature stress response in Clostridium botulinum

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http://urn.fi/URN:ISBN:978-951-51-4522-2
Title: Neurotoxin regulation and the temperature stress response in Clostridium botulinum
Author: Mascher, Gerald
Contributor: University of Helsinki, Faculty of Veterinary Sciences, Department of Food Hygiene and Environmental Health
Doctoral Programme in Microbiology and Biotechnology
Publisher: Helsingin yliopisto
Date: 2018-11-23
Language: en
URI: http://urn.fi/URN:ISBN:978-951-51-4522-2
http://hdl.handle.net/10138/260947
Thesis level: Doctoral dissertation (article-based)
Abstract: Clostridium botulinum is a dangerous foodborne pathogen that forms highly resistant endospores and the extremely potent botulinum neurotoxin. Whereas endospores enable the survival and transmission of the organism in many harsh environments, the botulinum neurotoxin blocks neurotransmission and causes the severe and potentially lethal disease botulism in humans and animals. Both traits play an important role in the life of this pathogen and temporally overlap in C. botulinum batch cultures, suggesting common regulation. However, the co-regulation of sporulation and neurotoxin synthesis and the significance of both traits during stress conditions have not been examined in detail. This study focused on the role of the master regulator of sporulation Spo0A in neurotoxin synthesis in Group II C. botulinum type E strains, which lack the well-known neurotoxin gene regulator BotR. Furthermore, the role of the two traits during heat stress in Group I C. botulinum ATCC 3502 was investigated. Group II C. botulinum strains represent the main hazard in minimally-processed anaerobically-packaged foods relying on cold storage, as Group II strains are able to grow and produce toxin at temperatures as low as 3 °C. Cold tolerance mechanisms are scarcely known in psychrotrophic Group II C. botulinum. Studying the mechanisms required for adaption and growth at low temperatures is crucial to counter the safety hazards caused by this dangerous pathogen. The role of a two-component signal transduction system in the cold tolerance of Group II C. botulinum type E was investigated. A better understanding of neurotoxin gene regulation and mechanisms contributing to cold tolerance might enable the development of measures to reduce the risk of botulism outbreaks. The sporulation transcription factor Spo0A was shown to control the initiation of sporulation and neurotoxin synthesis in C. botulinum type E Beluga. The non-sporulating spo0A mutants produced drastically less neurotoxin than the wild-type strain, and in vitro binding assays showed that Spo0A binds to a putative Spo0A-binding box (CTTCGAA) upstream of the neurotoxin gene operon, suggesting the direct activation of neurotoxin synthesis by Spo0A. The sequence and location of the putative Spo0A-binding box is conserved among C. botulinum type E strains, and analysis of spo0A mutations in two more type E strains (K3 and 11/1-1) affirmed the important role of Spo0A in neurotoxin type E synthesis. Spo0A is the first neurotoxin regulator reported in C. botulinum type E strains that lack the neurotoxin gene activating alternative sigma factor BotR. However, co-regulation of sporulation and neurotoxin synthesis is probably not limited to type E strains. Analysis of heat shocked continuously growing C. botulinum type A ATCC 3502 cultures revealed simultaneous downregulation of both traits in response to heat stress, which was affirmed by decreased toxin synthesis and abolished sporulation in batch cultures incubated at 45 °C compared to cultures incubated at 39 °C. This suggests that both traits might be co-regulated in C. botulinum type A, possibly also via Spo0A, which was significantly downregulated after heat shock, whereas the expression of genes encoding the known neurotoxin gene transcription activators BotR and CodY was unaffected or even upregulated during the heat shock response. While heat stress had a negative effect on sporulation and neurotoxin synthesis, the expression of genes related to motility was induced after heat shock. This suggests that motility is the preferred choice when facing elevated temperatures, probably to search for environments with less harmful temperatures. In order to grow and produce neurotoxin at cold temperatures, bacteria have to sense low temperatures and adjust their metabolism and structure for efficient growth in cold environments. We identified the first two-component signal transduction system (TCS) induced during the cold-shock response and needed for efficient growth at low temperatures in psychrotrophic C. botulinum type E. Expression of the TCS genes clo3403 (encoding a histidine kinase for sensing) and clo3404 (encoding a DNA regulator for responding) was increased after cold shock and prolonged compared to the expression pattern observed at the optimal growth temperature, suggesting that the TCS CLO3403/CLO3404 is needed for cold adaptation. Furthermore, inactivation of the TCS genes clo3403 and clo3404 resulted in impaired growth with significantly reduced maximum growth rates at low temperatures but not at the optimum temperature compared to wild-type growth. The important role of the TCS CLO3403/CLO3404 for cold tolerance in C. botulinum type E was confirmed by successful complementation of the mutations. In summary, this study demonstrated that sporulation and neurotoxin synthesis are co-regulated via the master regulator of sporulation Spo0A in C. botulinum type E and that heat stress has a negative effect on both traits in C. botulinum ATCC 3502, which also suggests common regulation in type A strains. Sporulation-dependent neurotoxin synthesis might play a central role in the life of this dangerous pathogen and represents a key intervention point for control. Finally, we identified a TCS (CLO3403/CLO3404) important for cold adaptation in psychrotrophic C. botulinum type E, which represents a major hazard in anaerobically-packaged chilled foods.Clostridium botulinum on vaarallinen ruokamyrkytysbakteeri, joka muodostaa kestäviä itiöitä ja tuottaa voimakkainta luonnollista myrkkyä, botulinumneurotoksiinia. Itiöiden avulla bakteeri selviytyy epäsuotuisissa olosuhteissa ja päätyy elintarvikkeisiin tai elimistöön. Neurotoksiini nieltynä tai elimistössä muodostuessaan estää hermoimpulssin aiheuttaen ihmisille ja eläimille henkeä uhkaavan velttohalvauksen, botulismin. C. botulinumia esiintyy yleisesti elintarvikkeiden raaka-aineissa ja lämmönkestävät itiöt selviävät useimmista nykyaikaisista elintarvikkeiden prosesseista. Osa C. botulinum -kannoista kasvaa ja tuottaa toksiinia jopa jääkaappilämpötiloissa. Nämä kannat muodostavat merkittävän terveysriskin pakatuissa kylmäsäilytetyissä elintarvikkeissa. Itiömuodostus ja toksiinituotanto ovat keskeisiä elementtejä C. botulinumin biologiassa ja epidemiologiassa. Mekanismit, joilla bakteeri säätelee itiömuodostusta ja toksiinituotantoa, tunnetaan huonosti. On myös epäselvää, miten erilaiset ympäristöolosuhteet vaikuttavat C. botulinumin itiöitymiseen ja toksiinituotantoon ja miten bakteeri sopeutuu erilaisiin olosuhteisiin esimerkiksi elintarvikkeiden valmistuksen ja säilytyksen aikana. Väitöskirjatyössä osoitettiin, että itiöivillä bakteereilla itiöitymisen pääsäätelijänä yleisesti tunnettu Spo0A-proteiini säätelee myös C. botulinumin neurotoksiinituotantoa. Spo0A on ensimmäinen neurotoksiinituotannon säätelytekijä, joka on kuvattu E-tyypin toksiinia tuottavilla C. botulinum –kannoilla. Näitä kantoja esiintyy yleisesti Suomen vesistöissä ja niiden kaloissa, ja ne aiheuttavat botulismiriskiä ihmiselle tyhjiöpakattujen lämminsavukalatuotteiden välityksellä. Lisäksi väitöskirjatyössä löydettiin kaksoiskomponenttijärjestelmiin kuuluva säätelytekijäpari, jonka avulla bakteeri todennäköisesti aistii ympäristön lämpötilaa ja sopeutuu kasvamaan ja tuottamaan toksiinia ja itiöitä kylmässä. Korkeaan lämpötilaan bakteerin havaittiin sopeutuvan aktivoimalla liikkumista edesauttavia tekijöitä ja vähentämällä itiöitymistä ja neurotoksiinituotantoa. C. botulinumin kasvua, sopeutumista ja toksiinituotantoa säätelevien solutason mekanismien ja ympäristötekijöiden selvittäminen avaa uusia mahdollisuuksia hallita bakteerin aiheuttamia terveysriskejä elintarvikkeissa ja elimistössä.
Subject: Food Hygiene
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