Bacillus cereus bakteerin itiöt ja kereulidi-toksiini ruokaperäisen sairauden aiheuttajina

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.Bacillus cereus on gram-positiivinen bakteeri, jolla on kaksi muotoa: i. sauvamainen kasvullinen solu (n. 0,002 mm × 0,004 mm), joka lisääntyy kahtia jakautumalla, ja ii. lepomuoto, pieni (0,001 mm) soikea itiö. Itiöillä on kyky säilyä ympäristössä ravinnotta ja vedettäkin elossa jopa vuosisatoja . Itiöt kestävät jokseenkin kaikkia aineita tai toimenpiteitä, joita käytetään bakteerien tappamiseen: kuumennusta 90°C lämmössä, säteilytystä, desinfiointiaineita ja sekä hapanta (pH ≤ 1) ja emäksistä ( pH ≥ 13) ympäristöä. Itiöiden pinta on rasvahakuinen (hydrofobinen) ja senvuoksi itiöt takertuvat kaikenlaisiin pintoihin, kuten teräkseen, muoveihin ja myös eläviin soluihin. Suotuisissa oloissa B. cereuksen itiöt kuoriutuvat ja kasvavat soluiksi joilla on kyky tuottaa ruokamyrkytyksiä aiheuttavia toksiineja. Osa toksiineista on lämpöherkkiä myrkyllisiä proteiineja joita muodostuu syödyn ruuan saapuessa ruuansulatuskanavaan (ripulitoksiinit). Toinen toksiinityyppi on kuumennusta kestävä peptidi, kereulidi, jota muodostuu valmiissa ruuassa tai sen aineksissa . Kereulidi on kestävä myrkky, jonka tuhoaminen ei onnistu keittämällä tai millään muullakaan ruuankäsittelymenetelmällä. Kereulidipitoisen ruuan nauttiminen aiheuttaa oksennustautia ja vakavan myrkytyksen, joka voi johtaa jopa kuolemaan. Tässä väitöskirjassa tutkittiin kereulidin tuottokykyä B. cereus kannoista, jotka oli kerätty 8 eri valtion alueelta erilaisista ruoka-aineista ja ympäristönäytteistä. 1041 bakteerikantaa kerättiin maaperästä, vesi- ja vesistönäytteistä, ilmasta, eläinten rehuista ja kuivikkeista, ruohosta, lannasta ja tuotantolaitteista. Näistä vain 1,2 % oli kereulidin tuottokykyisiä. Sensijaan niistä 144 kannasta, jotka eristettiin ruuista, 24% tuotti kereulidia. Kereulidin läsnäolon ja määrän mittaamiseen käytettiin kahta eri menetelmää: myrktllisyys nisäkässoluille (siittiötesti) ja nestekromatografia-massaspektrometria analyysiä jolla tunnistettiin kereulidin tyypillinen kemiallinen rakenne. B. cereus on yksi yleisimmistä, ellei yleisin, bakteeri nykyisissä elintarvikkeissa. Useimmista elintarvikkeista löytyy useampaa kuin yhtä B. cereuksen alalajia. 11% B. cereus kannoista, jotka eristettiin satunnaisesti valituista jauhemaisista vauvanruokavalmisteista, osoittautui kereulidin tuottajiksi. Jauhemaiset vauvanruokavalmisteet voivat sisältää 1000 .10 0000 pmy (pesäkettä muodostavaa yksikköä) B. cereus bakteeria / g . Näinollen jokainen kerta-ateria (200 ml, 30 g jauhetta veteen liuotettuna) luultavimmin sisältää myös kereulidin tuottajia. Kun 200 ml :aan steriloitua vauvanruokaa lisättiin > 100 000 pmy kereulidia tuottavaa B. cereus bakteeria ja pullo jätettiin seisomaan huoneenlämpöön 24 h, niin siihen kertyi ruokamyrkytyksen aiheuttava määrä kereulidia (> 0.1 mg). Mielenkiintoista oli, että jos vauvanruokaa laimennettiin vedellä 4 15 kertaisesti, siihen kertyi 10 50 kertaa enemmän kereulidia (g kohti) kuin laimentamattomaan. Alttius kereulidin tuottoon vaihteli valmisteesta toiseen: eniten kereulidimyrkkyä kertyi sellaisiin valmisteisiin, jotka sisälsivät sekä viljaa että maitoa, kun taas puhtaasti maitopohjaisiin valmisteisiin myrkkyä kertyi vähiten. Meijeriteollisuus käyttää järeitä menetelmiä pitääkseen prosessilaitteensa puhtaana, mutta siitä huolimatta löytyy sellaisia B. cereuksen genotyyppejä jotka pystyvät pesiytymään maitosiiloihin. Tässä väitöstyössä havaittiin, että näiden B. cereus alatyyppien itiöt olivat muita kestävämpiä prosessipesuissa käytetyille menetelmille: ne säilyivät pitkään lisääntymiskykyisinä kuumassa 1% natrium hydroksidiliuoksessa (pH > 13), tarttuivat tehokkaasti teräs- ja muihin pintoihin, pystyivät itämään kylmässä, +8°C, ja kasvamaan biofilminä maidossa. Vain harva (1.1%) maitosiilojen B. cereuksista tuotti kereulidia. Tässä väitöstyössä osoitettiin, että kereulidia tuottavat B. cereus kannat kykenivät estämään kereulidia tuottamattomien kantojen kasvun silloin, kun molempia oli samassa ruoka-aineessa. Bakteerin kykyä estää toisen bakteerin kasvua kutsutaan antagonismiksi. Pitkään tiedettiin, että B. cereus antagonoi muiden Bacillus lajien, gram-negatiivien bakteerien ja kasvipatogeenisten sienten kasvua. Tässä väitöstyössä osoitettiin ensi kertaa B. cereus-lajin sisäisen antagonismin, brother-killing (kannibalismi), olemassaolo. Myös jotkut kereulidia tuottamattomat B. cereus kannat olivat antagonistisia lajitovereilleen Tämä osoittaa, että antagonismi ei ollut kereulidin aiheuttamaa. Mielenkiintoinen oli havainto, että antagonistinen alatyyppi oli erittäin yleinen löytö ruokamyrkytystapauksissa. Näyttää siltä, että antagonistinen ominaisuus on virulenssitekijä joka tehostaa erityiseti kereulidia tuottavien B. cereus kantojen kykyä aiheuttaa ihmiselle sairautta

CC2D1A regulates human intellectual and social function as well as NF-κB signaling homeostasis.

Autism spectrum disorder (ASD) and intellectual disability (ID) are often comorbid, but the extent to which they share common genetic causes remains controversial. Here, we present two autosomal-recessive founder mutations in the CC2D1A gene causing fully penetrant cognitive phenotypes, including mild-to-severe ID, ASD, as well as seizures, suggesting shared developmental mechanisms. CC2D1A regulates multiple intracellular signaling pathways, and we found its strongest effect to be on the transcription factor nuclear factor κB (NF-κB). Cc2d1a gain and loss of function both increase activation of NF-κB, revealing a critical role of Cc2d1a in homeostatic control of intracellular signaling. Cc2d1a knockdown in neurons reduces dendritic complexity and increases NF-κB activity, and the effects of Cc2d1a depletion can be rescued by inhibiting NF-κB activity. Homeostatic regulation of neuronal signaling pathways provides a mechanism whereby common founder mutations could manifest diverse symptoms in different patients

Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group.

One hundred representative strains of Bacillus cereus were selected from a total collection of 372 B. cereus strains using two typing methods (RAPD and FT-IR) to investigate if emetic toxin-producing hazardous B. cereus strains possess characteristic growth and heat resistance profiles. The strains were classified into three groups: emetic toxin (cereulide)-producing strains (n=17), strains connected to diarrheal foodborne outbreaks (n=40) and food-environment strains (n=43), these latter not producing the emetic toxin. Our study revealed a shift in growth limits towards higher temperatures for the emetic strains, regardless of their origin. None of the emetic toxin-producing strains were able to grow below 10 degrees Celsius. In contrast, 11% (9 food-environment strains) out of the 83 non-emetic toxin-producing strains were able to grow at 4 degrees Celsius and 49% at 7 degrees Celsius (28 diarrheal and 13 food-environment strains). non-emetic toxin-producing strains. All emetic toxin-producing strains were able to grow at 48 degrees Celsius, but only 39% (16 diarrheal and 16 food-environment strains) of the non-emetic toxin-producing strains grew at this temperature. Spores from the emetic toxin-producing strains showed, on average, a higher heat resistance at 90 degrees Celsius and a lower germination, particularly at 7 degrees Celsius, than spores from the other strains. No difference between the three groups in their growth kinetics at 24 degrees Celsius, 37 degrees Celsius, and pH 5.0, 7.0, and 8.0 was observed. Our survey shows that emetic toxin-producing strains of B. cereus have distinct characteristics, which could have important implication for the risk assessment of the emetic type of B. cereus caused food poisoning. For instance, emetic strains still represent a special risk in heat-processed foods or preheated foods that are kept warm (in restaurants and cafeterias), but should not pose a risk in refrigerated foods

ARL3 mutations cause Joubert syndrome by disrupting ciliary protein composition

Joubert syndrome (JBTS) is a genetically heterogeneous autosomal recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTSrelated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997-109106128 (hg 19)), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP Ribosylation Factor-Like GTPase 3, ARL3, is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localisation in cilia. We propose that ARL3 provides a potential hub in the network of encoded ciliopathy genes, whereby perturbation of ARL3 results in the mislocalisation of multiple ciliary proteins due to abnormal displacement of lipidated protein cargo

FBXO32, encoding a member of the SCF complex, is mutated in dilated cardiomyopathy

Ubiquitination defect in cells expressing mutant FBXO32. a Co-immunopricipitation analysis. HEK293 cells were transfected with the indicated plasmids and immunoblot analysis was performed from total cell lysates using a specific anti-ubiquitin antibody. FBXO32 expression is shown as well as GAPDH. The blot is representative of three independent experiments. b Immunoblot analysis of the ubiquitination in cardiomyocytes. Cells were transfected with the Flag-FBXO32-WT or Flag-FBXO32-Mutant and whole cell extracts were analyzed by immunoblotting using the indicated antibodies. (TIF 1928 kb

A founder CEP120 mutation in Jeune asphyxiating thoracic dystrophy expands the role of centriolar proteins in skeletal ciliopathies

Jeune asphyxiating thoracic dystrophy (JATD) is a skeletal dysplasia characterized by a small thoracic cage and a range of skeletal and extra-skeletal anomalies. JATD is genetically heterogeneous with at least nine genes identified, all encoding ciliary proteins, hence the classification of JATD as a skeletal ciliopathy. Consistent with the observation that the heterogeneous molecular basis of JATD has not been fully determined yet, we have identified two consanguineous Saudi families segregating JATD who share a single identical ancestral homozygous haplotype among the affected members. Whole-exome sequencing revealed a single novel variant within the disease haplotype in CEP120, which encodes a core centriolar protein. Subsequent targeted sequencing of CEP120 in Saudi and European JATD cohorts identified two additional families with the same missense mutation. Combining the four families in linkage analysis confirmed a significant genome-wide linkage signal at the CEP120 locus. This missense change alters a highly conserved amino acid within CEP120 (p.Ala199Pro). In addition, we show marked reduction of cilia and abnormal number of centrioles in fibroblasts from one affected individual. Inhibition of the CEP120 ortholog in zebrafish produced pleiotropic phenotypes characteristic of cilia defects including abnormal body curvature, hydrocephalus, otolith defects and abnormal renal, head and craniofacial development. We also demonstrate that in CEP120 morphants, cilia are shortened in the neural tube and disorganized in the pronephros. These results are consistent with aberrant CEP120 being implicated in the pathogenesis of JATD and expand the role of centriolar proteins in skeletal ciliopathie

Mutations in phospholipase C eta-1 ( PLCH1 ) are associated with holoprosencephaly

Funder: NIHR Cambridge Biomedical Research centreBackground: Holoprosencephaly is a spectrum of developmental disorder of the embryonic forebrain in which there is failed or incomplete separation of the prosencephalon into two cerebral hemispheres. To date, dominant mutations in sonic hedgehog (SHH) pathway genes are the predominant Mendelian causes, and have marked interfamilial and intrafamilial phenotypical variabilities. Methods: We describe two families in which offspring had holoprosencephaly spectrum and homozygous predicted-deleterious variants in phospholipase C eta-1 (PLCH1). Immunocytochemistry was used to examine the expression pattern of PLCH1 in human embryos. We used SHH as a marker of developmental stage and of early embryonic anatomy. Results: In the first family, two siblings had congenital hydrocephalus, significant developmental delay and a monoventricle or fused thalami with a homozygous PLCH1 c.2065C>T, p.(Arg689*) variant. In the second family, two siblings had alobar holoprosencephaly and cyclopia with a homozygous PLCH1 c.4235delA, p.(Cys1079ValfsTer16) variant. All parents were healthy carriers, with no holoprosencephaly spectrum features. We found that the subcellular localisation of PLCH1 is cytoplasmic, but the p.(Cys1079ValfsTer16) variant was predominantly nuclear. Human embryo immunohistochemistry showed PLCH1 to be expressed in the notorcord, developing spinal cord (in a ventral to dorsal gradient), dorsal root ganglia, cerebellum and dermatomyosome, all tissues producing or responding to SHH. Furthermore, the embryonic subcellular localisation of PLCH1 was exclusively cytoplasmic, supporting protein mislocalisation contributing to the pathogenicity of the p.(Cys1079ValfsTer16) variant. Conclusion: Our data support the contention that PLCH1 has a role in prenatal mammalian neurodevelopment, and deleterious variants cause a clinically variable holoprosencephaly spectrum phenotype

Mutations in phospholipase C eta-1 ( PLCH1 ) are associated with holoprosencephaly

Funder: NIHR Cambridge Biomedical Research centreBackground: Holoprosencephaly is a spectrum of developmental disorder of the embryonic forebrain in which there is failed or incomplete separation of the prosencephalon into two cerebral hemispheres. To date, dominant mutations in sonic hedgehog (SHH) pathway genes are the predominant Mendelian causes, and have marked interfamilial and intrafamilial phenotypical variabilities. Methods: We describe two families in which offspring had holoprosencephaly spectrum and homozygous predicted-deleterious variants in phospholipase C eta-1 (PLCH1). Immunocytochemistry was used to examine the expression pattern of PLCH1 in human embryos. We used SHH as a marker of developmental stage and of early embryonic anatomy. Results: In the first family, two siblings had congenital hydrocephalus, significant developmental delay and a monoventricle or fused thalami with a homozygous PLCH1 c.2065C>T, p.(Arg689*) variant. In the second family, two siblings had alobar holoprosencephaly and cyclopia with a homozygous PLCH1 c.4235delA, p.(Cys1079ValfsTer16) variant. All parents were healthy carriers, with no holoprosencephaly spectrum features. We found that the subcellular localisation of PLCH1 is cytoplasmic, but the p.(Cys1079ValfsTer16) variant was predominantly nuclear. Human embryo immunohistochemistry showed PLCH1 to be expressed in the notorcord, developing spinal cord (in a ventral to dorsal gradient), dorsal root ganglia, cerebellum and dermatomyosome, all tissues producing or responding to SHH. Furthermore, the embryonic subcellular localisation of PLCH1 was exclusively cytoplasmic, supporting protein mislocalisation contributing to the pathogenicity of the p.(Cys1079ValfsTer16) variant. Conclusion: Our data support the contention that PLCH1 has a role in prenatal mammalian neurodevelopment, and deleterious variants cause a clinically variable holoprosencephaly spectrum phenotype

ARMC9 and TOGARAM1 define a Joubert syndrome-associated protein module that regulates axonemal post-translational modifications and cilium stability

Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy, characterized by a pathognomonic hindbrain malformation. All known JBTS-genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we use the recently identified JBTS-associated protein ARMC9 in tandem-affinity purification and yeast two-hybrid screens to identify a novel ciliary module composed of ARMC9-TOGARAM1-CCDC66-CEP104- CSPP1. TOGARAM1-variants cause JBTS and disrupt its interaction with ARMC9. Using a combination of protein interaction analyses and characterization of patient-derived fibroblasts, CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrate that dysfunction of ARMC9 or TOGARAM1 results in short cilia with decreased axonemal acetylation and glutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in both ARMC9 and TOGARAM1 patient cells lines suggest a role for this new JBTS-associated protein complex in ciliary stability

Characterizing the morbid genome of ciliopathies

Background Ciliopathies are clinically diverse disorders of the primary cilium. Remarkable progress has been made in understanding the molecular basis of these genetically heterogeneous conditions; however, our knowledge of their morbid genome, pleiotropy, and variable expressivity remains incomplete. Results We applied genomic approaches on a large patient cohort of 371 affected individuals from 265 families, with phenotypes that span the entire ciliopathy spectrum. Likely causal mutations in previously described ciliopathy genes were identified in 85% (225/265) of the families, adding 32 novel alleles. Consistent with a fully penetrant model for these genes, we found no significant difference in their “mutation load” beyond the causal variants between our ciliopathy cohort and a control non-ciliopathy cohort. Genomic analysis of our cohort further identified mutations in a novel morbid gene TXNDC15, encoding a thiol isomerase, based on independent loss of function mutations in individuals with a consistent ciliopathy phenotype (Meckel-Gruber syndrome) and a functional effect of its deficiency on ciliary signaling. Our study also highlighted seven novel candidate genes (TRAPPC3, EXOC3L2, FAM98C, C17orf61, LRRCC1, NEK4, and CELSR2) some of which have established links to ciliogenesis. Finally, we show that the morbid genome of ciliopathies encompasses many founder mutations, the combined carrier frequency of which accounts for a high disease burden in the study population. Conclusions Our study increases our understanding of the morbid genome of ciliopathies. We also provide the strongest evidence, to date, in support of the classical Mendelian inheritance of Bardet-Biedl syndrome and other ciliopathies