Project description:Agaricus bisporus is a soil-inhabiting fungus which is cultivated for production of white button mushrooms. A disease of A. bisporus has been previously described with a range of disease symptoms (yield loss, pinning delay, cap distortions and cap browning) which has been given collective name of “Mushroom Virus X” (MVX). The causes of this disease are not clear however prior to this research an association was found between the disease and double-stranded RNA molecules in the mushroom fruitbodies. The experiment was designed to examine causes and host responses of the disease causing the Brown Cap symptom in the cultivated mushroom A. bisporus. This microarray experiment was performed before the Agaricus bisporus genome was sequenced. The gene sequences used to design probes were from known and novel A. bisporus sequences and sequences of transcript fragments identified by Suppression Subtractive Hybridization of non-symptomatic and virus-diseased A. bisporus mushroom fruitbodies. The A. bisporus mushroom fruitbodies were grown on composted wheat straw using commercial cultivation procedures. The gene expression comparison was made of RNA isolated from 32 mushroom fruitbodies (Agaricus bisporus) samples: 20 samples from 5 separate virus-infected commercial mushroom farms with crops displaying the brown symptom (4 replicate samples per farm) and 12 samples from a non-infected crop grown at the University of Warwick. The precise composition of the viral load was the subject of this and future research/papers. Abstract of Manuscript submitted to Applied and Environmental Microbiology: Characterizing the viral agents causing brown cap mushroom disease of Agaricus bisporus by Daniel Eastwood, Julian Green, Helen Grogan, and Kerry Burton (Paper #AEM01093-15). The symptoms of viral infections of fungi range from cryptic to severe but there is little knowledge of the factors involved in this transition of fungal/viral interactions. Brown Cap Mushroom Disease of the cultivated Agaricus bisporus is economically important and represents a model system to describe this transition. Differentially expressed transcript fragments between mushrooms showing the symptoms of Brown Cap Mushroom Disease and control white non-infected mushrooms have been identified and sequenced. Ten of these RNA fragments have been found to be up-regulated over a thousand-fold between diseased and non-diseased tissue but are absent from the Agaricus bisporus genome sequence and hybridise to double-stranded RNA’s extracted from diseased tissue. We hypothesize these transcript fragments are viral and represent components of the disease-causing agent, a bipartite virus with similarities to the family Partitiviridae. The virus fragments were found at two distinct levels within infected mushrooms, at raised levels in infected, non-symptomatic, white coloured mushrooms and much greater levels (3,500-87,000 times greater) in infected mushrooms exhibiting brown colouration. In addition, differential screening revealed 9 up-regulated and 32 down-regulated host Agaricus bisporus transcripts. Chromametric analysis was able to distinguish colour differences between non-infected white mushrooms and white infected mushrooms at an early stage of mushroom growth. This method may be the basis for an ‘on-farm’ disease detection assay. A gene expression comparison was made between diseased mushroom displaying the brown cap symptom with characteristic double-strand RNA profiles (banding pattern on gels) and non-symptomatic virus-free mushrooms. In total RNA was isolated from 32 mushroom fruitbody (Agaricus bisporus) samples: 20 samples from 5 separate virus-infected commercial mushroom farms with crops displaying the brown symptom (4 replicate samples per farm) and 12 samples from a non-infected crop grown at the University of Warwick. Commercially-grown mushrooms are produced in “flushes” at approximately weekly intervals. The samples were collected from commercial farms when symptoms were reported to us but these were from different flushes: Farm1 from the 2nd flush; Farm 2 from the 1st flush; Farm 3 from the 3rd flush; Farm 4 from the 1st flush; and Farm 9 from the 1st flush. To allow for comparisons on the basis of Flush Number, the non-infected mushrooms grown at the University of Warwick were sampled from the first, second and third flushes, 4 mushrooms sampled from each flush.

Project description:The preferential localization of some neoplasms, such as serrated polyps, in specific areas of the intestine suggests that non-genetic factors may be important for their development. To test this hypothesis, we took advantage of transgenic mice that expressed HB-EGF throughout the intestine, but develop serrated polyps only in the cecum. Here we show that a host-specific microbiome was associated with serrated polyps, and that alterations of the microbiota induced by antibiotic treatment or by embryo-transfer rederivation markedly inhibited the formation of serrated polyps in the cecum. Mechanistically, development of serrated polyps was associated with a local decrease in epithelial barrier-function, bacterial invasion, production of antimicrobials, and increased expression of several inflammatory factors such as IL-17, Cxcl2, Tnf-Œ±, and IL-1. Increased number of neutrophils were found within the serrated polyps, and their depletion significantly reduced polyp growth. Together these results indicate that non-genetic factors contribute to the development of serrated polyps and suggest that the development of these intestinal neoplasms in the cecum is driven by the interplay between genetic changes in the host, an inflammatory response, and a host-specific microbiota. SUMMARY: Serrated polyps (SP) are a heterogeneous group of neoplasms found in particular areas of the gut. To define the factors contributing to their specific localization, we analyzed a strain of transgenic mice that carry a genetic alteration throughout the intestinal epithelium, but only develop SP in the cecum. Transcriptome and immunostaining analyses showed increased expression of antimicrobial genes, inflammatory factors, and the presence of bacteria within SP. Alteration of the cecal microbiota by antibiotic treatment or by embryo-transfer rederivation dramatically reduced SP incidence. Microbiome analysis implicated a limited set of bacteria in the development of SP. Together, these results point to a crucial role for the microbiota in the localized development of SP in a genetically susceptible host. We obtained serrated polyp (SP) and surrounding normal (NM) tissue from the ceca of three affected mice (paired design) and assessed expression differences by RNA-Seq.

Project description:Background and aims. The etiopathology of inflammatory bowel diseases is still poorly understood. To date, only few little data are available on the microbiota composition in ulcerative colitis (UC), representing a major subform of inflammatory bowel diseases. Currently, one of the main challenges is to unravel the interactions between genetics and environmental factors in the onset or during the progression and maintenance of the disease. The aim of the present study was to analyse twin pairs discordant for UC for both gut microbiota dysbiosis and host expression profiles at a mucosal level and to get insight into the functional genomic crosstalk between microbiota and mucosal epithelium in vivo. Methods. Biopsies were sampled from the sigmoid colon of both healthy and diseased siblings from UC discordant twin pairs but also from healthy twins. Microbiota profiles were assessed by 16S rDNA libraries while mRNA expression profiles were analysed from the same volunteers using Affymetrix microarrays. Results. UC patients showed a dysbiotic microbiota with lower diversity and more species belonging to Actinobacteria and Proteobacteria phyla. On the contrary, their healthy siblingsM-bM-^@M-^Y microbiota contained more bacteria from the Lachnospiracea and Ruminococcaceae family than did healthy individuals . Sixty-three host transcripts significantly correlated with bacterial genera in healthy individuals whereas only 43 and 32 correlated with bacteria in healthy and UC siblings from discordant pairs, respectively. Several transcripts related to oxidative and immune responses were differentially expressed between unaffected and UC siblings. Conclusion. A loss of crosstalk between gut microbiota and host was highlighted in UC patients. This defect was also striking in healthy siblings from discordant pairs, as was the lower biodiversity within the microbiota. Our results suggest disease-relevant interactions between host transcriptome and microbiota. Moreover, unusual aerobic bacteria were noticed in UC mucosal microbiota, whereas healthy siblings from discordant pairs had higher percentages of potentially beneficialusual commensal bacterial species. Paired samples (twins) were analyzed to obtain data independent of genetic variation

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