A Whole New (Microbial) World

The microbiome is a diverse collection of bacteria closely intertwined with all aspects of human health. Over recent years, efforts to shift microbiome research from descriptive to mechanistic have revealed remarkable genetic diversity. However, the challenge of linking gene to phenotype is a complex one, especially because current annotation techniques disregard an entire class of genes. Known as small open reading frames (sORFs), these genes encode potentially significant proteins – and that’s why Ami Bhatt and her team at Stanford University wanted to uncover their diverse functions (1).

“Small proteins are easy to miss for two reasons,” says Bhatt, an Assistant Professor of Medicine and Genetics. “Most bioinformatic algorithms do not annotate proteins made up of less than 30–50 amino acids. This is because start and stop codons are often closely located – and annotating them without additional filters would produce a lot of false positives. Also, biochemical approaches to finding proteins usually involve physical dialysis or size-based separation, which are optimized for proteins of a regular size.” These challenges leave many small proteins undetected, which could therefore hinder our understanding of microbial communication.

Yet it is these elusive proteins’ small size that makes them so interesting. “Because they can be transcribed and translated quickly, they could be used by bacteria as biological switches to toggle between functional states or to trigger specific reactions in other cells,” Bhatt says. The proteins may also serve regulatory functions and play a crucial role in larger cellular structures.

In an attempt to unmask the tiny proteins, the team compared potentially relevant genes among different microbes to isolate those that appeared repeatedly (and were therefore more likely to be true positives). Although they expected to find hundreds of genes, the search uncovered tens of thousands. The proteins encoded by these genes are believed to play a role in important biological processes, such as intercellular communication and warfare. “The most exciting part was realizing that, despite using these microbial genome datasets for years, we’ve never taken note of this entire class of proteins that was right under our noses.”

The next steps will be to recreate the shapes and functions of the newly discovered proteins in the lab. “The proteins that aren’t secondarily modified might be relatively easy to use in heterologous expression or even synthesize directly. And that will help us to determine exactly which sORFs are transcribed and translated, and understand what regulates their expression,” explains Bhatt. “We also want to discover which microbial proteins bind to host-associated signaling receptors.” Ultimately, the newly discovered proteins could advance our understanding of how the microbiome affects human health and open the door for new tests and treatments.