Your body is a wonderland. A wonderland teeming with trillions of bacteria, that is. But it’s not as horrifying as it might sound. In fact, there’s mounting evidence that many aspects of our health are closely intertwined with the composition and hardiness of our microscopic compatriots, though exactly how is still mostly unclear.

Now, researchers at the Stanford University School of Medicine have discovered that these microbial hitchhikers — collectively known as the human microbiome — are churning out tens of thousands of proteins so small that they’ve gone unnoticed in previous studies. The proteins belong to more than 4,000 new biological families predicted to be involved in, among other processes, the warfare waged among different bacterial strains as they vie for primacy in coveted biological niches, the cell-to-cell communication between microbes and their unwitting hosts, and the critical day-to-day housekeeping duties that keep the bacteria happy and healthy.

Because they are so small — fewer than 50 amino acids in length — it’s likely the proteins fold into unique shapes that represent previously unidentified biological building blocks. If the shapes and functions of these proteins can be recreated in the lab, they could help researchers advance scientific understanding of how the microbiome affects human health and pave the way for new drug discovery.

A paper describing the research findings was published Aug. 8 in Cell. Ami Bhatt, MD, PhD, assistant professor of medicine and of genetics, is the senior author. Postdoctoral scholar Hila Sberro, PhD, is the lead author.

‘A clear blind spot’

“It’s critically important to understand the interface between human cells and the microbiome,” Bhatt said. “How do they communicate? How do strains of bacteria protect themselves from other strains? These functions are likely to be found in very small proteins, which may be more likely than larger proteins to be secreted outside the cell.”

But the proteins’ miniscule size had made it difficult to identify and study them using traditional methods.

“We’ve been more likely to make an error than to guess correctly when trying to predict which bacterial DNA sequences contain these very small genes,” Bhatt said. “So until now, we’ve systematically ignored their existence. It’s been a clear blind spot.”

The fact that she found thousands of new protein families definitely surprised us all.

It might be intimidating for the uninitiated to think too deeply about the vast numbers of bacteria that live on and in each of us. They account for far more cells in and on the human body than actual human cells do. Yet these tiny passengers are rarely malicious. Instead, they help with our digestion, supplement our diet and generally keep us running at our peak. But in many cases, it’s been difficult to pick apart the molecular minutiae behind this partnership.

Bhatt and her colleagues wondered if answers might be found in the small proteins they knew were likely to wiggle through the nets cast by other studies focusing on the microbiome. Small proteins, they reasoned, are more likely than their larger cousins to slip through the cell membrane to ferry messages — or threats — to neighboring host or bacterial cells. But how to identify and study these tiny Houdinis?

“The bacterial genome is like a book with long strings of letters, only some of which encode the information necessary to make proteins,” Bhatt said. “Traditionally, we identify the presence of protein-coding genes within this book by searching for combinations of letters that indicate the ‘start’ and ‘stop’ signals that sandwich genes. This works well for larger proteins. But the smaller the protein, the more likely that this technique yields large numbers of false positives that muddy the results.”

A big surprise

To tackle the problem, Sberro decided to compare potential small-protein-coding genes among many different microbes and samples. Those that were identified repeatedly in several species and samples were more likely to be true positives, she thought. When she applied the analysis to large data sets, Sberro found not the hundreds of genes she and Bhatt had expected, but tens of thousands. The proteins predicted to be encoded by the genes could be sorted into more than 4,000 related groups, or families, likely to be involved in key biological processes such as intercellular communication and warfare, as well as maintenance tasks necessary to keep the bacteria healthy.

“Honestly, we didn’t know what to expect,” Bhatt said. “We didn’t have any intuition about this. The fact that she found thousands of new protein families definitely surprised us all.”

The researchers confirmed the genes encoded true proteins by showing they are transcribed into RNA and shuttled to the ribosome for translation — key steps in the protein-making pathway in all organisms. They are now working with collaborators to learn more about the proteins’ functions and to identify those that might be important to the bacteria fighting for space in our teeming intestinal carpet, for example. Such proteins might serve as new antibiotics or drugs for human use, they believe.

“Small proteins can be synthesized rapidly and could be used by the bacteria as biological switches to toggle between functional states or to trigger specific reactions in other cells,” Bhatt said. “They are also easier to study and manipulate than larger proteins, which could facilitate drug development. We anticipate this to be a valuable new area of biology for study.”

Bhatt is a member of Stanford Bio-X, the Stanford Cancer Institute, the Stanford Maternal & Child Health Research Institute and a fellow of Stanford ChEM-H.

Other Stanford co-authors are graduate student Brayon Fremin; postdoctoral scholars Soumaya Zlitni, PhD, and Fredrik Edfors, PhD; and Michael Snyder, PhD, professor and chair of genetics.

Researchers from One Codex, the Joint Genome Institute of the Department of Energy, the Alexander Fleming Biomedical Sciences Research Center in Greece, and Lawrence Berkeley National Laboratory also contributed to the study.

The study was supported by the National Institutes of Health (grants HG000044, K08CA184420, P30CA124435, and 1Ro1AT010123201), the PhRMA Foundation, the U.S. Department of Energy and a Damon Runyon Clinical Investigator Award.

Stanford’s departments of Medicine and of Genetics also supported the work.