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06 - February 12, 2013.docx

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February 12, 2013 – The microbiome in immune system development, function and disease Diversity of genes of microbiome >>>>> human genome Large scale projects to map human microbiomes – Metagenomics – without prior culture – these organisms discussed today are difficult to culture, does not like to live as single organisms Probiotic – do not colonize humans; come largely from cows and non-humans; cultured in labs; not part of humans, go through us Prebiotics – foodstuffs – promote growth of beneficial bacteria – Commensal bacteria – bacteria that are part of us; million genes – display mutually beneficial interactions with us – critical for nutrition, metabolism, etc – bacteria in intestines that without whom, we cannot digest food; they make vitamins – critical!! High through put sequencing – method of choice when confronted with the diversity 16S ribosomal gene – all bacteria have this gene Moutains and valleys of diversity – make sets of oligonucleotide primers based on conserved regions of gene, PCR amplify, make libraries of amplicons; these amplicons include more variable regions where the sequences used to distinguish – DNA Isolate DNA Barcodes and adaptors – isolate DNA from fifty samples, second round of PCR to add barcodes to distinguish hem from each other, and have them sequenced all together – this increases efficiency – use computorial approach to individualize the fifty samples Quantitative – can determine frequency of given group of bacteira in A and B? Map of diversity in human microbiome – sequence microbiota from different sources from internal and external body Take 16S data from large samples and try to organize by making sense of bacteria – taxonomic group of bacteria contained in samples Phylogenetic map of microbial diversity – many different parts of body evaluated – height of bars displays relative abundance of groups of bacteria Include opportunistic pathogens – ex. Surveying lungs of cystic fibrosis patients vs controls – Survey health and disease states of bacteria Microbiome diversity – other sets of algorithms to try to affiliate variable parts of sequences with KNOWN bacteria, which we have good sequences (this does not include majority of bacteria in gut) OTU- operational taxonomic unit – unknown whether strain or species – operational definition, no external meaning Sequences are related to each other in phylogenetic tree – representation of any of OTU grouping is collated in table – more biological replicates, the better the statistical analysis – generate massive OTU tables What is the diversity of the flora? New approach – shotgun metagenome sequencing – take all the genomic DNA From the sample of gut or fecal matter – blast apart into tiny fragments, put tags on ends, and high throhgput sequence them – when reassemble the sequences – try to relate them to all bacteria genes in databases – for sample 1 through 4, what are the frequency of – get back hundreds of millions of sequences – computational – compare samples to each other regarding frequency of different genes in them – No reference genome – What factors?? – how do they correlate with overall composition of bacteria – Principle component analysis – statistical method – take complex data set and find structure inside of it EX. Enterotype – change in microbial diversity in gut as function of age on left Red – young; blue – old Signatures can be found that distinguish the groups On the right – Malawi and Central America – more similarity than USA Order out of seeming chaos Left side – regular sequencing using 16S – diversity in OUT, taxonomy Right side – using shotgun sequencing – similarity Many different combinations ofbacteria that can confer optimal function in given environment – shown in this comparison – do not get hung up on Diversity – how and when people acquire gut microbiota – Correlating changes in intestinal microbiome with milestones in life – lots of variation of proportion of major phyla over time – but later in life, Environmental factors affecting human microbiota – Seniors have far less stability in gut flora – more likely subject to infection by enteric pathogens Gut epithelial barrier In inner lumen of colon – 10^12 g bacteria One cell thick layer of epitheial that separtes that density to essentially zero Need to avoid innate immune sensing of bacteria – compromise the one-cell thick barrier and deliver into systemic immune system bacteria that will disturb metabolic Immune system on the zero side of bacteria barrier/epithelium Stratification – luminal side to cellular side – in lumen of small intestine and large intestine Small intestine –w here vast majority of nutrients absorbed, need access to metabolic products and nutrients produced by commensal bacteria – Large intestine – density of bacteira very high – outer mucus layer that is essential to keeping bacteria- rich regions segregated from inner layer, which is more bacteria-poor Mucus – mixture of differnet molecules – produced by goblet cells – secrete mucin-rich proteins, important for repairing tears in gut – these cells turnover at RAPID rate – Goblet cells also receiving input from bacteria products – production of mucins regulated by bacteria products there – a number of bacteria groups called peptidoglycan – stimulate secretion of mucus – short chain fatty acids and low molecular fatty acids (butarate) that cause increase in release of mucins – two way interaction to produce stratification – feedback loop Mouse expts – one mucin-producing enzyme, Muc2 – knockout – increased translocation of commensal bacterial into systemic circulation, reduced integrity of lining of epithelium – The mucus layer also – segmented filamentous bacteria line epithelial interfaces – biofilims help exclude pathogens – ecology problem, competing for space – high fitness of commensal bacteria helps exclude pathogenic bacteria competing the niches – Stratification II – epitheial cells that line the gut make series of specialized antimicrobial peptides – paneth cells – make antimicrobial peptides called alpha defensins – related to defensins that amphibians have on skin – Inner mucus layer is kill zone – concentration of a number of differnet host secreted factors – closer to the wall, heavier the armament becomes Antimicrobial lectin – Generation of secreted IgA – IgA is commensal cell specific – ferried across epitheial surface on specialized immunoglobulin receptor – Microbe-specific IgA – present in high concentration in barriers – contribute to limiting bacterial penetration Some bacteria do get throug
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