Lecture 9 - The Human Microbiome Flashcards
Microbiota & microbiome
Microbiota
The microorganisms that typically inhabit a specific environment
Microbiome
the totality of microbes, their genomes and environmental interactions in a defined environment. e.g. the gut of a human, soil sample
Human microbiota – an introduction
In health, internal human tissues are normally free of microbes
In contrast, surface tissues (skin & mucous membranes) are constantly exposed to environmental microorganisms and are readily colonized by diverse microbial (primarily bacterial) species
Also includes viruses, fungi (mostly yeasts), protists
For decades, it was proposed that the human body contains 10x as many bacterial cells as it does human cells
More recent estimates suggest it is more likely to be 1:1 ratio
HMP – Human Microbiome Project
“A strategy to understand the microbial components of the human genetic & metabolic landscape, and how they contribute to normal physiology & predisposition to disease”
The Human Microbiome Project is a worldwide interdisciplinary effort. Primarily utilising the availability of highly parallel DNA sequencers, but also supported by high-throughput mass spectrometers. Together, these technologies enable characterization of whole microbial communities.
Determine whether individuals share a core human microbiome
If all humans have a core microbiome, how is it acquired and transmitted? (methods of delivery/birth, family size, living environment)
How similar are the microbiomes between members of a family, or members of a community, or between different ethnic groups?
Can changes in the human microbiome be correlated with changes in human health?
How stable is the microbiome, & can variation be systematically studied? (does it vary day to day?)
To what extent to factors such as genetics, diet & socio-economic background influence the composition of the microbiome? (twin studies, adopted children)
Does the microbiome contribute directly to human health & disease?
The role of 16S rRNA in the HMP
(no information on metabolics though)
identifies genus
Prokaryotic ribosomes consist of small (30S) and large (50S) subunits; each subunit consisting of a complex of RNA & proteins
16S rRNA is a component of the small subunit
Approximately 1,500 nucleotides long
Contains highly conserved & highly variable regions
The small subunit of ribosomes is responsible for the formation of the initiation complex, performs the decoding of the genetic information, and controls the fidelity of codon-anticodon interactions. The large subunit catalyzes the peptide bond formation and provides the path for the nascent polypeptide chain. The 16S rRNA of the small subunit has many functions. For example, it plays a structural role, acting as a scaffold for the ribonucleoprotein complex. It also plays an important role in stabilizing the correct codon-anticodon pairings.
The fact that the 16S rRNA contains both conserved and variable regions make it very useful for identifying distinguishing between) taxonomic groups. Phylogenetic trees showing the relatedness of different organisms are frequently drawn on the basis of 16S rRNA sequence variation.
16S rDNA sequence analysis of complex samples - PCR
Sequencing of 16S rRNA is well-suited to characterizing the composition of complex microbial populations
Extract DNA from sample (rDNA)
Perform PCR analysis on this DNA using universal 16S rDNA primers (will amplify the 16S rDNA gene from majority/all species present in the sample due to the highly conserved part of the 16S subunit (common to all species))
Sequence this complex mixture, and identify the species present based on the 16S rDNA sequences
Primers anneal to the highly conserved regions but the products will contain the highly variable regions as well
But 16S rRNA is only a feature of prokaryotic ribosomes - using this method we cannot capture any fungi as they have no 16S RNA
Metagenome sequencing for microbiome analysis
Extracting DNA from sample and sequencing everything (no PCR)
Host + microbial DNA
Metagenome sequencing - Essential for defining the microbiome
‘Metagenomics’ is the direct sequencing of DNA recovered from environmental samples
natural environment, host environment etc.
As with 16S rRNA analysis, it allows study of organisms that cannot be easily cultured in the lab – but yields far more information…..
Allows us to identify the repertoire of functions and metabolic pathways that are present within the microbiota
The only way to define all microbial species (prokaryotic, eukaryotic, viral)
The major advantage of metagenomic techniques is that they don’t rely on laboratory cultures of organisms. Surveys based on rRNA sequences taken directly from the environment have suggested that culture-based methods find less than 1% of the bacteria and archaea species in a given sample. This highlights what you might potentially be missing within a sample, if you rely on culture-based methods for its investigation.
The gut microbiota in human health & disease
Characteristics of the gut microbiota have been implicated in a variety of human conditions:
Obesity Childhood-onset asthma Inflammatory bowel disease (colitis) Colorectal carcinomas Cardiovascular disease Multiple sclerosis
e.g. Gut-brain connections
Gut microbiota & obesity – studies in mouse model
Mouse model of obesity (mutation in leptin gene)
ob/ob mice are obese; +/+ and ob/+ mice are lean
Intestinal contents were recovered from mice, DNA isolated, and PCR performed using universal 16S rDNA primers
Subsequent sequencing of PCR products to define the composition of the microbial community
Leptin is a hormone that plays a key role in regulating energy intake and expenditure, including appetite and metabolism.
Obesity was associated with a shift in the relative abundance of taxa present
50% reduction in Bacteroidetes
Significant increase in Firmicutes
A similar shift in the relative abundance of Bacteroidetes & Firmicutes was observed in obese humans (and going on a diet restored the balance back to the ‘lean’ model of Bacteriodetes vs Firmicutes)
Cause or Consequence?
Is the shift in the relative abundance of Bacteroidetes-Firmicutes the cause of weight gain, or a consequence of consuming specific dietary components that alters their abundance?
Microbiota-mediated ‘transmission’ of obesity
“Conventionally-raised” (CONV-R) mice fed various different diets before being sacrificed
The microbiota from these different sacrificed mice was transplanted into mice that had been raised in a germ-free environment
Assessed weight gain in these recipient mice, as well as monitoring microbiota composition etc.
An “obese microbiome” promoted weight gain when transplanted to a germ-free mouse, compared to comparable transplantation of a “lean microbiome”
Shifting microbiota alters metabolic potential
Metagenomics (whole gut DNA sequencing) of the microbiota to profile the microbiome:
The shift in abundance observed within the microbiota of obese mice alters the metabolic potential of the microbiota
Increase in genes encoding enzymes involved in the breakdown of dietary polysaccharides
A lower amount of energy remained in the faeces of obese mice compared to lean counterparts
Increased capacity of the obese microbiome for energy extraction
Helicobacter pylori
Gram-negative, highly motile, spiral-shaped bacterium
Colonizes the non-acid-secreting mucosa of the stomach and the upper intestinal tract
Associated with gastritis, ulcers & gastric cancers
Prevalence of colonization ranges from 20% in developed countries to more than 90% in the developing world
Approx. 10% will develop peptic ulcers, 1-3% gastric cancer & 0.1% MALT lymphoma
Helicobacter pylori & peptic ulcers
H. pylori is invasive, and colonizes the non-acid producing surfaces of the gastric mucosa
The gastric mucus layer protects H. pylori from stomach acid
A combination of H. pylori products and host responses cause inflammation, tissue destruction & ulceration
Such peptic ulcers traditionally treated with long-term antacids, only with partial success (typically relapsing within 1 year)
Discovery of association with H. pylori revolutionised treatment and led to awarding of Nobel Prize in 2005
Ulcers now effectively treated with a 14-day course of antibiotics
Helicobacter pylori & carcinogenesis
Some H. pylori isolates carry the cag pathogenicity island (cag PAI) (pathogenicity islands are part of the genome that encodes factors associated with pathogenicity)
The cag PAI encodes the CagA protein (Cytotoxin-associated gene A) and the Cag type IV secretion system (SM lectures). Hollow needle injects effectors into the host cell
Patients harbouring cag+ strains are at heightened risk of gastric cancer
CagA is injected into host cell by the type IV secretion system, whereupon it interacts with numerous host cell proteins. In doing so, it activates downstream signalling pathways and enhances the proliferative ability of the gastric epithelial cells.
Helicobacter pylori & gastric MALT lymphomas
H. pylori can also be associated with gastric MALT lymphomas (blood cancer)
MALT, mucosa-associated lymphoid tissue; part of the normal response to infection at mucosal sites
The inflammation associated with chronic H. pylori infection/colonisation can be associated with development of gastric MALT lymphoma
Constant stimulation of lymphocytes recruited to the infection can promote mutations in the lymphocytes, resulting in lymphomas (uncontrolled lymphocyte proliferation)
