Growth temperature variation and heat stress response of Clostridium botulinum

Clostridium botulinum, the causative agent of botulism in humans and animals, is frequently exposed to stressful environments during its growth in food or colonization of a host body. The wide genetic diversity of the strains of this foodborne pathogen has been thoroughly studied using different molecular biological methods; however, it is still largely unknown how this diversity reflects in the ability of different C. botulinum strains to tolerate environmental stresses. In contrast to cold tolerance, which has been the focus of intensive research in recent years, the molecular mechanisms C. botulinum utilizes in response to heat shock and during adaptation to high temperature stress are poorly understood. The aims of this study were to investigate the strain variation of Group I and II C. botulinum with regard to growth at low, high, and optimal temperature; the roles of hrcA, the negative regulator of Class I heat shock genes (HSG) and dnaK, a molecular chaperone coding Class I HSG, in the response of the Group I C. botulinum strain ATCC 3502 to heat and other environmental stresses; and the molecular mechanisms this strain employs in response to acute and prolonged heat stress. The maximum and minimum growth temperatures of 23 Group I and 24 Group II C. botulinum strains were studied. Further, maximum growth rates of the Group I strains at 20, 37, and 42°C and of the Group II strains at 10, 30, 37, and 42°C were determined. Within their groups, the C. botulinum strains showed significant variation in growth-limiting temperatures and their capability to grow at extreme temperature, especially at high temperature. Largest strain variation was found for Group I within type B and for Group II within type E strains, which further showed more mesophilic growth tendencies than the other Group II strains. However, the genetic background of the selected C. botulinum strains reflected only weakly in their growth characteristics. Group I strains showed larger physiological variation despite being genetically more closely related than Group II. A number of strains of both groups showed faster growth at temperatures above than at their commonly assumed optimal growth temperatures of 30°C for Group II and 37°C for Group I strains. In addition, they possessed higher maximum growth temperatures than the average of the studied strains. These strains can be expected to have higher than assumed optimal growth temperatures and pronounced high temperature stress tolerance. Good correlation was detected between maximum growth temperatures and growth rates at high temperature, although not for all strains. Therefore direct prediction from one studied growth trait to the other was impossible. These findings need to be taken into account when estimating the safety of food products with regard to C. botulinum by risk assessment and challenge studies. The role of Class I HSGs in C. botulinum Group I strain ATCC 3502 was studied by quantitative real-time reverse transcription PCR and insertional inactivation of the Class I HSGs hrcA and dnaK. During exponential and transitional growth, Class I HSGs were constantly expressed followed by down-regulation in the stationary phase. Exposure of mid-exponentially growing culture to heat shock led to strong, transient Class I HSG up-regulation. Inactivation of hrcA resulted in over-expression of all Class I HSGs, which confirmed its role as negative regulator of Class I HSGs in C. botulinum. Both inactivation mutants showed impaired high temperature tolerance as indicated by reduced growth rates at 45°C, a reduced maximum growth temperature, and increased log-reduction after exposure to lethal temperature. The growth of the dnaK mutant was more strongly affected than that of the hrcA mutant, emphasizing the importance of the molecular chaperone DnaK for C. botulinum. Reduced growth rates were evident for both mutants under optimal conditions and heat stress, but also under low pH, and high saline concentration. This suggests a probable role for Class I HSG in cross protection of C. botulinum against other environmental stresses. C. botulinum ATCC 3502 was grown in continuous culture and exposed to heat shock followed by prolonged high temperature stress at 45°C. Changes in the global gene expression pattern induced by heat stress were investigated using DNA microarray hybridization. Class I and III HSGs, as well as members of the SOS regulon, were employed in response to acute heat stress. High temperature led to suppression of the botulinum neurotoxin coding botA and the associated non-toxic protein-coding genes. During adaptation and in the heat-adapted culture, motility- and chemotaxis-related genes were found to be up-regulated, whereas sporulation related genes were suppressed. Thus, increase in motility appeared to be the long-term high-temperature stress-response mechanism preferred to sporulation. Prophage genes, including regulatory genes, were activated by high temperature and might therefore contribute to the high temperature tolerance of C. botulinum strain ATCC 3502. Further, remodeling of parts of the protein metabolism and changes in carbohydrate metabolism were observed.Clostridium botulinum on vakavan taudin aiheuttaja sekä ihmisille että eläimille. Sen kasvuun vaikuttavat useat tekijät elintarvikkeiden valmistuksen ja säilytyksen aikana. Tämän elintarvikkeiden välityksellä leviävän taudinaiheuttajan laajaa geneettistä monimuotoisuutta on tutkittu monilla molekyylibiologisilla menetelmillä. Silti ei vielä suurelta osin tiedetä, miten tämä monimuotoisuus vaikuttaa C. botulinum -kannoilla ympäristön aiheuttaman stressin sietokykyyn. Verrattuna kylmänsietokykyyn, jota tutkimusryhmämme on laajasti tutkinut viime vuosina, tiedetään hyvin vähän siitä, miten C. botulinumin käyttämät molekyylitason mekanismit reagoivat lämpösokkiin sekä sopeutuvat korkean lämpötilan aiheuttamaan stressiin. Tässä tutkimuksessa selvitettiin ryhmien I ja II C.botulinum -kantojen välisiä kasvueroja alhaisessa, korkeassa sekä optimaalisessa lämpötilassa. Luokan I lämpösokkigeenien ilmentymiseen vaikuttavien geenien hrcA (lämpösokkigeenien negatiivinen säätelijä) sekä dnaK (lämpösokkigeeni, joka osallistuu solun sokkivasteessa tuottaman kaitsijaproteiinin koodaamiseen) rooleja tutkittiin ryhmän I C.botulinum -kannan ATCC 3502 vasteessa lämpöön ja muuhun ympäristön aiheuttamaan stressiin. Lisäksi selvitettiin molekyylitason mekanismeja joita kyseinen kanta käyttää altistuessaan äkilliselle ja pitkittyneelle lämpöstressille. Tutkimuksessa tarkasteltiin 23 ryhmän I ja 24 ryhmän II C. botulinum -kantaa. Niiden maksimi- ja minimikasvulämpötilat määritettiin sekä tarkasteltiin kasvunopeuksia eri lämpötiloissa. Ryhmien sisällä havaittiin merkittävää vaihtelua kasvua rajoittavissa lämpötiloissa sekä kyvyssä kasvaa äärimmäisissä lämpötiloissa, erityisesti korkeassa lämpötilassa. Ryhmän I kantojen välillä ilmeni suurempaa fysiologista vaihtelua huolimatta siitä, että ne ovat geneettisesti läheisempiä keskenään kuin ryhmän II kannat. Useat tutkituista ryhmien I ja II kannoista kasvoivat nopeammin yleisesti oletettua optimaalista kasvulämpötilaa korkeammissa lämpötiloissa. Lisäksi näiden kantojen maksimikasvulämpötila oli keskimääräistä korkeampi kuin muilla tutkituilla kannoilla. Voidaan olettaa, että näillä C. botulinum -kannoilla on merkittävä lämpöstressinsietokyky, sekä niiden todellinen maksimikasvulämpötila on korkeampi kuin on tähän mennessä luultu. Kantojen väliset fysiologiset vaihtelut tulee ottaa huomioon elintarvikkeiden turvallisuutta ja C. botulinum -riskiä arvioitaessa. Lämpösokkigeenien roolia ryhmän I C. botulinum ATCC 3502 -kannalla tutkittiin kvantitatiivisella polymeraasiketjureaktiolla sekä inaktivoimalla bakteerin genomista lämpösokkigeenit hrcA ja dnaK. Lämpösokkigeenit olivat aktiivisina nopeasti lisääntyvän kasvun aikana kunnes niiden ilmentyminen hiljeni kasvun tasaannuttua. Kun nopean kasvun vaiheessa oleva bakteerikasvusto altistettiin lämpösokille, havaittiin lämpösokkigeenien voimakas, mutta lyhytaikainen ilmentyminen. C. botulinumilla hrcA -geenin toimimattomuus lisäsi merkittävästi muiden luokan I lämpösokkigeenien aktiivisuutta, vahvistaen näin kyseisen geenin roolia lämpösokkigeenien negatiivisena säätelijänä. Sekä hrcA- että dnaK geenien toiminnan hiljentäminen heikensi tutkitun kannan lämmönsietokykyä selvästi, mikä näkyi hidastuneena kasvuna 45 °C:ssa, alentuneena maksimikasvulämpötilana, sekä korkeassa lämpötilassa nopeutuneena solujen tuhoutumisena. dnaK -geenin toimimattomuus heikensi kasvua voimakkaasti viitaten kyseisen geenin koodaaman kaitsijaproteiinin keskeiseen rooliin C. botulinum -bakteerilla. Geenien toiminnan inaktivoinnin todettiin lisäksi alentavan kasvunopeutta happamassa ympäristössä sekä korkeassa suolapitoisuudessa. Havainto antaa viitteitä lämpösokkigeenien mahdollisesta roolista myös muissa kuin lämpötilaan liittyvissä ympäristöstresseissä. C. botulinum ATCC 3502 -kannan jatkuvassa kasvatuksessa geenien ilmentymisen havaittiin muuttuvan kun kasvusto altistettiin lämpösokille sekä sitä seuraavalle kasvulämpötilan nostolle 45 °C:een. Näitä geeni-ilmentymisen muutoksia tutkittiin DNA-mikrosirumenetelmällä. Lämpöstressin todettiin aktivoivan luokan I ja III lämpösokkigeenejä, sekä geenejä jotka osallistuvat solun DNA-vaurioiden korjaamiseen. Korkea lämpötila vaimensi myös botulinumneurotoksiinia koodaavan botA -geenin sekä siihen liittyvien muiden geenien ilmentymistä. Lämpösopeutumisen aikana ja sen saavuttamisen jälkeen havaittiin liikkuvuuteen ja ympäristön kemiallisten ärsykkeiden aistimiseen liittyvien geenien ilmentymisen voimistuneen. Samalla bakteerin lepomuotojen eli itiöiden muodostumiseen liittyvien geenien ilmentyminen heikentyi. Lämpötilan todettiin aktivoivan myös eri säätelygeenejä, joiden arvellaan parantavan C. botulinum ATCC 3502 -kannan lämmönsietokykyä

Heat shock and prolonged heat stress attenuate neurotoxin and sporulation gene expression in group I Clostridium botulinum strain ATCC 3502

Foodborne pathogenic bacteria are exposed to a number of environmental stresses during food processing, storage, and preparation, and in the human body. In order to improve the safety of food, the understanding of molecular stress response mechanisms foodborne pathogens employ is essential. Many response mechanisms that are activated during heat shock may cross-protect bacteria against other environmental stresses. To better understand the molecular mechanisms Clostridium botulinum, the causative agent of botulism, utilizes during acute heat stress and during adaptation to stressfully high temperature, the C. botulinum Group I strain ATCC 3502 was grown in continuous culture at 39 degrees C and exposed to heat shock at 45 degrees C, followed by prolonged heat stress at 45 degrees C to allow adaptation of the culture to the high temperature. Growth in continuous culture was performed to exclude secondary growth phase effects or other environmental impacts on bacterial gene transcription. Changes in global gene expression profiles were studied using DNA microarray hybridization. During acute heat stress, Class I and III heat shock genes as well as members of the SOS regulon were activated. The neurotoxin gene botA and genes encoding the neurotoxin-associated proteins were suppressed throughout the study. Prolonged heat stress led to suppression of the sporulation machinery whereas genes related to chemotaxis and motility were activated. Induced expression of a large proportion of prophage genes was detected, suggesting an important role of acquired genes in the stress resistance of C. botulinum. Finally, changes in the expression of a large number of genes related to carbohydrate and amino acid metabolism indicated remodeling of the cellular metabolism.Peer reviewe

Genomic and Phenotypic Characterization of Clostridium botulinum Isolates from an Infant Botulism Case Suggests Adaptation Signatures to the Gut

In early life, the immature human gut microbiota is prone to colonization by pathogens that are usually outcompeted by mature microbiota in the adult gut. Colonization and neurotoxin production by a vegetative Clostridium botulinum culture in the gut of an infant can lead to flaccid paralysis, resulting in a clinical outcome known as infant botulism, a potentially life-threatening condition. Beside host factors, little is known of the ecology, colonization, and adaptation of C. botulinum to the gut environment. In our previous report, an infant with intestinal botulism was shown to be colonized by neurotoxigenic C. botulinum culture for 7 months. In an effort to gain ecological and evolutionary insights into this unusually long gut colonization by C. botulinum, we analyzed and compared the genomes of C. botulinum isolates recovered from the infant feces during the course of intoxication and isolates from the infant household dust. A number of observed mutations and genomic alterations pinpointed at phenotypic traits that may have promoted colonization and adaptation to the gut environment and to the host. These traits include motility, quorum-sensing, sporulation, and carbohydrate metabolism. We provide novel perspectives and suggest a tentative model of the pathogenesis of C. botulinum in infant botulism. IMPORTANCE While the clinical aspects of infant botulism and the mode of action of BoNT have been thoroughly investigated, little is known on the pathogenesis and adaptive mechanisms of C. botulinum in the gut. Here, we provide for the first time a comprehensive view on the genomic dynamics and plasticity of C. botulinum over time in a case of infant botulism. The genomic and phenotypic analysis of C. botulinum isolates collected during the disease course offers an unprecedented view of C. botulinum ecology, evolution, and pathogenesis and may be instrumental in developing novel strategies for prevention and treatment of toxicoinfectious botulism. While the clinical aspects of infant botulism and the mode of action of BoNT have been thoroughly investigated, little is known on the pathogenesis and adaptive mechanisms of C. botulinum in the gut. Here, we provide for the first time a comprehensive view on the genomic dynamics and plasticity of C. botulinum over time in a case of infant botulism.Peer reviewe

Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid

Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.Peer reviewe

The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC) of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies). For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs). The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans.Peer reviewe

Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.Peer reviewe

Crystal structures of OrfX1, OrfX2, and the OrfX1–OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A–G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1–OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.Peer reviewe

Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy

Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials

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Puu- ja hirsitaloteollisuusyritysten markkina-alue-, tuote- ja asiakasvalintoihin perustuvat kokonaisstrategiat

TutkimusartikkeliPuu- ja hirsitaloteollisuus on heterogeeninen toimiala, jolla toimii kooltaan, tuotannoltaan ja omistusrakenteeltaan monenlaisia yrityksiä. Tämä aiheuttaa toimialan kehittämiselle haasteita, mutta tarjoaa samanaikaisesti myös mahdollisuuksia edistää puu- ja hirsitaloyritysten liiketoimintaa ja niissä tapahtuvaa oppimista. §§ Toimialakohtaisten ja alueellisten kehittämishankkeiden on kritisoitu vastaavan huonosti erityyppisten puu- ja hirsitalovalmistajien tarpeita. Alan yritysten tekemien strategiavalintojen nykyistä syvällisempi tunteminen helpottaisi kehittämistoimenpiteiden nykyistä parempaa kohdentamista. Tutkimuksen tavoitteena oli löytää yritysryhmiä, joihin kuuluu tuote- ja palveluvalikoimiltaan, markkina-alueiltaan ja asiakassegmenteiltään keskenään samankaltaisia puu- ja hirsitalovalmistajia. §§ Tutkimustulosten mukaan puu- ja hirsitaloteollisuudessa toimii tekemiensä strategisten valintojen näkökulmasta keskenään samankaltaisia yrityksiä, joissa on havaittavissa ryhmittymistä tuote- ja palvelukokonaisuuksien lisäksi tiettyihin markkina-alueisiin ja asiakkaisiin. Tuloksissa löydettiin seuraavat markkina-alue-, tuote- ja asiakasvalinnoista koostuvat kokonaisstrategiat: tuotannollinen erikoistuminen toimien useissa markkina- ja asiakassegmenteissä, tuotannollinen erikoistuminen toimien kapeissa markkina- ja asiakassegmenteissä sekä tuotannollinen laaja-alaisuus toimien kapeissa markkina- ja asiakassegmenteissä. §§ Yritysyhteistyö, osaamisen kehittäminen ja tiedonkulun edistäminen ovat alan kilpailukyvyn kehittämiselle kriittisiä tekijöitä. Tulevaisuudessa olisi tärkeää tutkia erityyppisiä strategisia linjauk–sia toteuttavien yritysten kykyä hyödyntää tietoa omassa liiketoiminnassaan, kartoittaa niiden tietotarpeita ja selvittää erityyppisille yrityksille soveltuvia tiedonvälityskanavia