The Lilliputian Colonies in Our Machine (Part 2) – Our Microbiome

The story of Gulliver's Travels happened in four parts at four locations: Lilliput, Brobdingnag, Laputa and the land of the Houyhnhnms. I wrote about Eukaryotic cells and a special organelle, the exosome, in Part 1 of this series. Prokaryotic cells, like bacteria, on the other hand, don't have nuclear membranes, chromosomes or organelles. Fungi are eukaryotes. However, they are both small, nimble and wily, and have existed almost the entirety of our living planet. This is a very brief introduction to the microbiome -- bacteria, archaea, protists, fungi and viruses. Recent estimates have the microbiome numbers in us humans at about 10x the number of cells within us – a total of about 350 trillion! Therefore, one could ask, who is regulating whom?

In mid-April, I had the privilege of being on a microbiome bioinformatics panel discussion at the Translational Microbiome conference in Boston. Two of the doyens in microbiome research, Rita Colwell and Jack Gilbert presented fascinating keynotes there (both have backgrounds in the marine microbiome). A shout-out to Dr. Gilbert, a co-author of the recent book "Dirt is Good".

I will try and cobble this little tale of the microbiome in four very short stories, along with a few tidbits I learned at the conference... of course, we cannot do full justice to all the families, species, stories and beautiful pleomorphism of these "origin of life" creatures. We cannot start the journey without its beginning…

"…so let's collapse the planet's entire history into a single calendar year... it is 31st December just before the stroke of midnight...humans have existed for these 30 minutes or fewer. Before October, almost every living thing on the planet consisted of single cells." Ed Young from “I Contain Multitudes”, HarperCollins US 2016, ISBN 9780062368621

The origin story:

Did bacteria come from space? Hitch a ride on a meteor or a comet? The jury is still out -- I am a believer in the RNA- and Lipid-first hypothesis (the egg came first!). The chart below looks to tie the origin story timeline together:

Another view of this timeline is the “family tree of life” charted below by Leonard Eisenberg which so elegantly shows what is summarized by Ed Young in his book "I Contain Multitudes": "…so let's collapse the planet's entire history into a single calendar year... it is 31st December just before the stroke of midnight...humans have existed for these 30 minutes or fewer. Before October, almost every living thing on the planet consisted of single cells."

The deep ocean of very early earth may be the oldest proof-of-life with the recent discovery 3.77 billion year old fossils which may contain archea or bacteria. A new tree of life has recently been represented based on genome sequences which includes 92 named bacterial phyla, 26 archaeal phyla and all five of the eukaryotic supergroups.

The four anatomical and ontological locations in our little tale:

1: That Strep in our throat and on our skin; guide-RNAs, tracrRNA and CRISPR.

The science press is agog over CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), for good reasons – a summary of the functions of CRISPR are succinctly depicted below:

Image Source: Sander JD & Joung JK, "CRISPR-Cas systems for editing, regulating and targeting genomes", Nature Biotechnology 32, 347–355 (2014)

The origin of CRISPR is a more interesting story: a 20-year history from Haloferax mediterranei, an archaeal microbe with extreme salt tolerance to Streptococcus pyogenes that uses the trans-activating CRISPR RNA (tracrRNA) for adaptive immunity. S pyogenes is the strep throat and skin bacteria. Perhaps a look at how guide RNA affects rRNA and microRNA across bacterial species could create a precision platform? Biomimicry is, after all, the sincerest form of flattery to our single-celled ancestors!

2: Mycome-Land: the fungi near Seattle; mold versus bacteria

The norths of our trees are mossy and there are firs and ferns aplenty – I live in Greater Seattle and my wonderful city has grown on me like fungi, which I am... It is not surprising that the largest (and perhaps oldest living) organism in the world is a fungus, found in my neck of the woods. Yeast is the most used and most studied fungus among humans. The term “mycome” was coined by another doyen of the microbiome, Mahmoud Ghannoum. Alexander Fleming was an accidental mycologist whose discovery of penicillin as the “world’s first antibiotic” with the mold on the orange rind in a messy lab is a well-worn story in many a science class. The real story is more nuanced with other key players: Dr. Howard Florey, a professor of pathology at Oxford and Dr Ernst Chain a biochemist there. It took more than 12 years and 2000 liters of mold cultured fluid to make enough pure penicillin to treat sepsis in one person – Albert Alexander, an Oxford cop, in 1940. I cannot ignore mycobacteria when mentioning the mycome – especially tuberculosis (TB).

Continuing on the origin story theme, I would be remiss not to mention one of the most innovative and creative Design of Experiment (DoE) methods from the Kushony Lab at Harvard Medical School called “The Evolution of Bacteria.” See for yourself why antimicrobial resistance is the largest global threat looming around the corner that we are not paying much attention to:

3: Gods and rivers; pond-scum and phages: the enemy of my enemy is my future?

Phages (more accurately, bacteriophages) are viruses that infect bacteria. They are found everywhere bacteria are found... soil, the earth's crust, oceans, rivers and, of course, pond scum. From another “great flood” origin story: the Ganges (Ganga) is the holiest river in India, whose origin in Hindu mythology is from the locks of Lord Shiva (who is called “Gangadhara” or literally Ganga-holder). Torrential rainfall is threatening to cleave the earth. Lord Shiva’s hair was the “via media” to tame the Ganges before it descended to earth. Is this a reference to the stellar origin of bacteria (followed by viruses and phages)?

Image source: “Lord Shiva the Gangadhara” painting, National Museum, New Delhi – Jaipur, Rajasthan style, 1800

It was in the waters of the Ganges that Ernest Hanbury Hankin reported the first "bacteriocide" (a bacteriophage) in 1896 in the Annals of the Pasteur Institute. After penicillin stole the thunder of phages in 1920 – the mantle of the first antibiotic – their use declined drastically; the only countries that still used it in the 20th century were Russia, Georgia, Poland and Cuba.

In the past decade, there has been a steady resurgence of Phage Therapy. The last resort after most antimicrobials have failed have so far been primarily copper and silver. A more recent story of pond-scum and phages comes from a Connecticut ophthalmologist, a Pseudomonas aeruginosa infection from his aortic mesh in the hospital, an enterprising physician and virologist, and our hero – a pond-scum phage from the OMKO1 virus. We need more interest and funding from the usual suspects to accelerate the use of phages.

4: The Kingdom of Monera...and the denizens in our gut

Monera was a phylogenetic kingdom for all prokaryotes. The first example of a denizen of Monera in our gut is the bacteria Vibrio cholerae, which was one of Rita Colwell’s conserved microbiome keynote stories on the microbiome studies from the National Institute of Cholera and Enteric Disease (NICED) in Kolkatta, India (along the river Ganges):

• The microbiome of healthy Indian descent is much different than that of healthy Western European descent
• The Indian microbiome encodes for higher rates of antimicrobial resistance than the Human Microbiome Project (HMP) samples
• The Indian microbiome has a predominance of carbohydrate metabolism genes

As from the summary above, pathogenicity genes in bacteria are mobile, the bioinformatics needed to understand this gets complicated quickly, especially across geographies.

There are many examples of gut bacteria influencing health and disease

• Breast milk and its influence on the gut microbiome of the growing infant
• A specific bacteria, Prevotella copri, found in the gut about 75% of patients with Rheumatoid Arthritis (RA)
• Cardiovascular disease (CVD) risk and its relation to the gut microbiome via metabolic syndrome, Type 2 Diabetes (T2D) and obesity.
• Clostridium difficle or C difficle colitis (a disease of the digestive tract) is a serious side effect of antibiotic therapy and hospital acquired infection (HAI). Fecal Microbiota Transplantation (FMT) has been shown to be an accepted therapy regimen.
•  The “microbiota-gut-brain-axis.”

Epilogue: Bioinformatics and Infrastructures

We are entering the realm of what the past US Defense Secretary Rumsfeld termed as the "unknown unknowns" with the rising popularity of microbiome research.

Listed below is the informatics influenced by microbiome databases, methods, integration and standards:

• 16S rRNA, Whole Genome Sequencing and Proteomic markers for microbiomics
• Single-cell genome sequencing
• The Human Microbiome Project (HMP)
• Mock Microbiome Communities from NIST
• Epidemiology and Precision Population Health
• Integrative imaging with Pathology and Radiology
• International Metagenomics and Microbiome Standards Alliance (IMMSA)

This systems thinking for a microbiome informatics pipeline is summarized in the diagram below:

Image Source: Agiar-Pulido V, et al, "Metagenomics, Metatranscriptomics, and Metabolomics Approaches for Microbiome Analysis", Evolutionary Bioinformatics 2016:12(S1) 5–16 doi: 10.4137/EBO.S36436.

The microbiome is rapidly gaining traction as a biomarker for drug development, a drug delivery and therapeutic platform. a manufacturing platform using synthetic yeast, phage-based antimicrobial system and the gut microbiome as multiple biomarker (microRNA in the microbiome is stable) and therapeutic pathways. This will strain the information technology resources (compute, storage and data movement) that is already under stress with Genomics, Radiology and Pathology. Let us ensure that we build a stable and useful infrastructure to study the building blocks of us humans that is as old as life on earth!

## ?2017 Sanjay Joshi