69 research outputs found
Bacillus cereus bakteerin itiöt ja kereulidi-toksiini ruokaperÀisen 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 ÂșC, radiation, disinfectants and extreme alkaline (â„ 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 ÂșC. Fourth, spores capable of germinating at 8 ÂșC 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 à 0,004 mm), joka lisÀÀntyy kahtia jakautumalla, ja ii. lepomuoto, pieni (0,001 mm) soikea itiö. ItiöillÀ on kyky sÀilyÀ ympÀristössÀ ravinnotta ja vedettÀkin elossa jopa vuosisatoja . Itiöt kestÀvÀt jokseenkin kaikkia aineita tai toimenpiteitÀ, joita kÀytetÀÀn bakteerien tappamiseen: kuumennusta 90°C lÀmmössÀ, sÀteilytystÀ, desinfiointiaineita ja sekÀ hapanta (pH †1) ja emÀksistÀ ( pH ℠13) ympÀristöÀ. Itiöiden pinta on rasvahakuinen (hydrofobinen) ja senvuoksi itiöt takertuvat kaikenlaisiin pintoihin, kuten terÀkseen, muoveihin ja myös elÀviin soluihin.
Suotuisissa oloissa B. cereuksen itiöt kuoriutuvat ja kasvavat soluiksi joilla on kyky tuottaa ruokamyrkytyksiÀ aiheuttavia toksiineja. Osa toksiineista on lÀmpöherkkiÀ myrkyllisiÀ proteiineja joita muodostuu syödyn ruuan saapuessa ruuansulatuskanavaan (ripulitoksiinit). Toinen toksiinityyppi on kuumennusta kestÀvÀ peptidi, kereulidi, jota muodostuu valmiissa ruuassa tai sen aineksissa . Kereulidi on kestÀvÀ myrkky, jonka tuhoaminen ei onnistu keittÀmÀllÀ tai millÀÀn muullakaan ruuankÀsittelymenetelmÀllÀ. Kereulidipitoisen ruuan nauttiminen aiheuttaa oksennustautia ja vakavan myrkytyksen, joka voi johtaa jopa kuolemaan.
TÀssÀ vÀitöskirjassa tutkittiin kereulidin tuottokykyÀ B. cereus kannoista, jotka oli kerÀtty 8 eri valtion alueelta erilaisista ruoka-aineista ja ympÀristönÀytteistÀ. 1041 bakteerikantaa kerÀttiin maaperÀstÀ, vesi- ja vesistönÀytteistÀ, ilmasta, elÀinten rehuista ja kuivikkeista, ruohosta, lannasta ja tuotantolaitteista. NÀistÀ vain 1,2 % oli kereulidin tuottokykyisiÀ. Sensijaan niistÀ 144 kannasta, jotka eristettiin ruuista, 24% tuotti kereulidia. Kereulidin lÀsnÀolon ja mÀÀrÀn mittaamiseen kÀytettiin kahta eri menetelmÀÀ: myrktllisyys nisÀkÀssoluille (siittiötesti) ja nestekromatografia-massaspektrometria analyysiÀ jolla tunnistettiin kereulidin tyypillinen kemiallinen rakenne.
B. cereus on yksi yleisimmistÀ, ellei yleisin, bakteeri nykyisissÀ elintarvikkeissa. Useimmista elintarvikkeista löytyy useampaa kuin yhtÀ B. cereuksen alalajia. 11% B. cereus kannoista, jotka eristettiin satunnaisesti valituista jauhemaisista vauvanruokavalmisteista, osoittautui kereulidin tuottajiksi. Jauhemaiset vauvanruokavalmisteet voivat sisÀltÀÀ 1000 .10 0000 pmy (pesÀkettÀ muodostavaa yksikköÀ) B. cereus bakteeria / g . NÀinollen jokainen kerta-ateria (200 ml, 30 g jauhetta veteen liuotettuna) luultavimmin sisÀltÀÀ myös kereulidin tuottajia. Kun 200 ml :aan steriloitua vauvanruokaa lisÀttiin > 100 000 pmy kereulidia tuottavaa B. cereus bakteeria ja pullo jÀtettiin seisomaan huoneenlÀmpöön 24 h, niin siihen kertyi ruokamyrkytyksen aiheuttava mÀÀrÀ kereulidia (> 0.1 mg). Mielenkiintoista oli, ettÀ jos vauvanruokaa laimennettiin vedellÀ 4 15 kertaisesti, siihen kertyi 10 50 kertaa enemmÀn kereulidia (g kohti) kuin laimentamattomaan. Alttius kereulidin tuottoon vaihteli valmisteesta toiseen: eniten kereulidimyrkkyÀ kertyi sellaisiin valmisteisiin, jotka sisÀlsivÀt sekÀ viljaa ettÀ maitoa, kun taas puhtaasti maitopohjaisiin valmisteisiin myrkkyÀ kertyi vÀhiten.
Meijeriteollisuus kÀyttÀÀ jÀreitÀ menetelmiÀ pitÀÀkseen prosessilaitteensa puhtaana, mutta siitÀ huolimatta löytyy sellaisia B. cereuksen genotyyppejÀ jotka pystyvÀt pesiytymÀÀn maitosiiloihin. TÀssÀ vÀitöstyössÀ havaittiin, ettÀ nÀiden B. cereus alatyyppien itiöt olivat muita kestÀvÀmpiÀ prosessipesuissa kÀytetyille menetelmille: ne sÀilyivÀt pitkÀÀn lisÀÀntymiskykyisinÀ kuumassa 1% natrium hydroksidiliuoksessa (pH > 13), tarttuivat tehokkaasti terÀs- ja muihin pintoihin, pystyivÀt itÀmÀÀn kylmÀssÀ, +8°C, ja kasvamaan biofilminÀ maidossa. Vain harva (1.1%) maitosiilojen B. cereuksista tuotti kereulidia.
TÀssÀ vÀitöstyössÀ osoitettiin, ettÀ kereulidia tuottavat B. cereus kannat kykenivÀt estÀmÀÀn kereulidia tuottamattomien kantojen kasvun silloin, kun molempia oli samassa ruoka-aineessa. Bakteerin kykyÀ estÀÀ toisen bakteerin kasvua kutsutaan antagonismiksi. PitkÀÀn tiedettiin, ettÀ B. cereus antagonoi muiden Bacillus lajien, gram-negatiivien bakteerien ja kasvipatogeenisten sienten kasvua. TÀssÀ vÀitöstyössÀ osoitettiin ensi kertaa B. cereus-lajin sisÀisen antagonismin, brother-killing (kannibalismi), olemassaolo. Myös jotkut kereulidia tuottamattomat B. cereus kannat olivat antagonistisia lajitovereilleen TÀmÀ osoittaa, ettÀ antagonismi ei ollut kereulidin aiheuttamaa. Mielenkiintoinen oli havainto, ettÀ antagonistinen alatyyppi oli erittÀin yleinen löytö ruokamyrkytystapauksissa. NÀyttÀÀ siltÀ, ettÀ antagonistinen ominaisuus on virulenssitekijÀ joka tehostaa erityiseti kereulidia tuottavien B. cereus kantojen kykyÀ aiheuttaa ihmiselle sairautta
CC2D1A regulates human intellectual and social function as well as NF-ÎșB 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 ÎșB (NF-ÎșB). Cc2d1a gain and loss of function both increase activation of NF-ÎșB, revealing a critical role of Cc2d1a in homeostatic control of intracellular signaling. Cc2d1a knockdown in neurons reduces dendritic complexity and increases NF-ÎșB activity, and the effects of Cc2d1a depletion can be rescued by inhibiting NF-ÎșB 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 1928ĂÂ 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
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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
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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
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 âmutation loadâ 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
The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies
Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology
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