Metagenomics of the Human Microbiome Tyler Kosowan MMIC 7050 October 3rd, 2013

Overview Introduction Environmental Acquisition Dysbiosis and Pathogenesis Human Microbiome Project Metagenomics Future Prospects Summary

Introduction Microbiome - to the environmental population of microorganisms that exist in and on the bodies of humans including the genes they contain Microbiota- the separate microbial taxa located at the different areas of the body

Introduction History - Antonie van Leewenhoek, 1680s, compared the microbiota of his own oral and fecal samples - At the time he was able to record obvious differences in microbes between the two bodily habitats

Introduction Symbiotic human microbiota cell count to date: - 1012 to 103 microbial cells harbored by each person Human body cell count: - 3.72 × 1013 cells

Introduction The human body is able to make acceptions to fighting the natural microflora It still must be able to distinguish foreign pathogens that don’t belong - in some cases the difference can be at the species level or lower A disruption of this balance may lead to illness

Environmental Acquisition Microbes colonize us from birth. - initially opportunistic microbes colonize different areas of our bodies - other microbes eventually out compete these initial microbes to establish our adult microbiome.

Environmental Acquisition Various sites of differing microbiomes around the human body. The numbers show the quantity of microorganisms per gram-weight of homogenized tissue, collected fluid, or per square centimeter of tissue surface Davis, C., 1996, Medical Microbiology 4th Edition, University of Texas Medical Branch at Galveston, Galveston, Texas; Chapter 6

Environmental Acquisition Varying conditions of sites around the body include: - temperature, pH, and redox potential as well as metabolic concerns such as water, oxygen, and nutrient levels

Environmental Acquisition Example - Neonates: ● Initially the neonatal gut is colonized by gram-positive bacteria such as bifidobacteria and lactobacilli if the infant is breast-fed ● This bacterial population is displaced over time by a gram-negative flora like Enterobacteriaceae when the infant is bottle-fed

Environmental Acquisition Diet and microbiome changes - the stable, adult microbiome may change from introduction of genes - it has been observed that bacteria in the gut can pick up genes through horizontal gene transfer ● Example, antibiotics in chicken feed was found in bacteria in the chicken’s gut.

Dysbiosis and Pathogenesis Dysbiosis is when there is a shift from the natural order of the microflora at a region of the human body. In the gut the natural microflora have an influence on the immune system

Dysbiosis and Pathogenesis Cryptopatch diagram showing structure consisting of lymphoid tissue inducer cells and dendritic cells Littman, D., and Pamer, E., 2011, Role of the Commensal Microbiota in Normal and Pathogenic Host Immune Responses, Cell Host and Microbe, 10(4):311-323

Dysbiosis and Pathogenesis Certain types of bacteria in the lamina propria of the gut are able to influence the collection of Treg and Th17 cells at areas in the gut. - seen at both the large and small intestines Gnotobiotic mice lacking these bacteria presented a lack of those T-cells in the gut - addition of a gram-positive, anaerobic, segmented, filamentous bacterium resorted the collection of T-cells to the gut

Dysbiosis and Pathogenesis Methods of the natural microflora to outcompete invading microorganisms in the gut Davis, C., 1996, Medical Microbiology 4th Edition, University of Texas Medical Branch at Galveston, Galveston, Texas; Chapter 6

Dysbiosis and Pathogenesis An example of pathogenic dysbiosis is with the bacterium Clostridium difficile - it takes advantage of a decrease in gut microflora ● Usually because the patient is taking broad spectrum antibiotics or is immunocomprimised - the bacteria adhere to the exposed intestinal epithelium using rybosylated actin filaments and begin replicating

Dysbiosis and Pathogenesis Rates of short hospital stay discharges of patients with Clostridium difficile listed as any diagnosis, by age in America from 1996 to 2003 McDonald, L., Owings, M., and Jernigan, D., 2006, Clostridium difficile Infection in Patients Discharged from US Short-stay Hospitals, 1996–2003, Emerging Infectious Diseases, 12(3): 409-415

Human Microbiome Project First discussed in 2005 Ongoing project - pilot study has completed Goal was to show it is possible to characterize the human microbiome sufficiently to enable study of the human microbiome variability Worked to standardize data resources and technological improvements

Human Microbiome Project Scheme for studying the ‘‘normal’’ human microbiome in the Human Microbiome Project. Both 16sRNA sequences and whole-genome shotgun sequences (WGS) were combined to give an overall look at the different microflora at different sites of the human body The NIH HMP Working Group, 2009, The NIH Human Microbiome Project, Genome Research, 19:2317–232

Human Microbiome Project Pilot study - 250 “normal” volunteers - 5 major sites sampled from ● mouth, skin, gastrointestinal tract, vagina (if applicable to subject), and nasal cavity - DNA extracted from samples ● used for 16S rRNA PCR and whole-genome shotgun library construction

Human Microbiome Project 16S PCR fragments were sequenced with a 454 Pyrosequencer Whole-genome libraries were sequenced using paired-end illumina shotgun sequencing removal of human sequences, poor quality sequences, and repeat sequences Similar sequences at each sample site were binned and placed into operational taxonomic units

Human Microbiome Project Observable patterns of α and β diversity at sites - α diversity, the amount of different sequences from one sample site - β diversity, the difference in sequence diversity between sites at one major sample site Example, saliva sampling sites had very high alpha diversity of operational taxonomic units (OTUs), but expressed one of the lowest beta diversities

Human Microbiome Project No microbial taxa found at every major sampling site Some bacteria considered pathogenic were found but at very low numbers.

Metagenomics A technique that can show data about an organism that would otherwise be uncultivable - most bacteria part of our natural microflora are uncultivable Many fields of science can benefit from the knowledge attained through these methods

Metagenomics The DNA extracted for 16S rRNA PCR amplification interacts with designed primers - primers don’t need to be highly specific as 16S is a highly conserved gene - some primers may need to be designed for special cases PCR amplimers from this are known as “Polonies”

Metagenomics Reads from the 454 sequencer were aligned using highly stringent parameters to construct contigs denoting one bacteria per alignment set Similar contigs were binned together as operational taxonomic units (OTUs) Short reads are harder to align - newer software able to deal with this ● ex) Velvet, ALLPATHS, and Euler-SR

Metagenomics Whole-genome shotgun libraries can give us a closer look at each bacteria found Software can look for potential ORFs from the segments of the genomes submitted - ex) Motif EXtraction can identify putative enzymes by looking for peptide fragments and then comparing to known enzymes to consider possible funtion

Future Prospects Many questions still remain - What is the difference between a competitive and mutual inter-microbial reaction? More data is required for statistical significance - Repeat studies need to be done Other areas of meta-analysis need to be focused on to see the whole picture - metatransciptomics and metaproteomics Higher throughput and efficient software will need to be designed to cross-analyse all meta-data.

Summary Our body has many environmental niches that microbes colonize, some being helpful to us When the natural state of a microbial niche is disrupted the host can become sick The Human Microbiome Project was created to further our knowledge of the dynamics going on in our own bodies Metagenomics was a method utilized in the Human Microbiome Project that lead to a fast and efficient method of data mining for the vast variety of bacteria at major body site - no culturing required - found bacteria that currently are considered uncultivable

References Bianconi, E. et al., 2013, An Estimation of the Number of Cells in the Human Body, Annals of Human Biology, doi:10.3109/03014460.2013.807878 Davis, C., 1996, Medical Microbiology 4th Edition, University of Texas Medical Branch at Galveston, Galveston, Texas; Chapter 6 Gevers, D., Pop, M., Schloss. P.D., and Huttenhower, C., 2012, Bioinformatics for the Human Microbiome Project, PLoS Computational Biology, 8(11): e1002779. doi:10.1371/journal.pcbi.1002779 Heinlen, L. and Ballard, J., 2010, Clostridium difficile Infection, American Journal of the Medical Sciences, 340(3): 247–252 Littman, D., and Pamer, E., 2011, Role of the Commensal Microbiota in Normal and Pathogenic Host Immune Responses, Cell Host and Microbe, 10(4):311-323 McDonald, L., Owings, M., and Jernigan, D., 2006, Clostridium difficile Infection in Patients Discharged from US Short-stay Hospitals, 1996–2003, Emerging Infectious Diseases, 12(3): 409-415 Netherwood, T. et al., 1999, Gene Transfer in the Gastrointestinal Tract, Applied and Environmental Microbiology, 65(11): 5139-5141 Peterson, J. et al., 2009, The NIH Human Microbiome Project, Genome Research, 19(12): 2317–2323 The Human Microbiome Project Consortium, 2012, Structure, Function and Diversity of the Healthy Human Microbiome, Nature, 486: 207-214 The NIH HMP Working Group, 2009, The NIH Human Microbiome Project, Genome Research, 19:2317–232 Ursell, L., 2012, Defining the Human microbiome, Nutrition Reviews, 70(1):S38–S44 Wen, L. et al., 2008, Innate Immunity and Intestinal Microbiota in the Development of Type 1 Diabetes, Nature, 455(7216):1109-1113 Wooley, J. and Ye, Y., 2009, Metagenomics: Facts and Artifacts, and Computational Challenges, Journal of Computer Science and Technology, 25(1): 71-81 Wooley, J., Godzik, A., and Friedberg, I., 2010, A Primer on Metagenomics, Plos Computational Biology, 6(2): 1-13