Discovery of a Proteolytic Flagellin Family in Diverse Bacterial Phyla That Assembles Enzymatically Active Flagella
This week we profile a recent publication in Nature Communications from Dr. Ulrich Eckhard (pictured) during his time as a postdoctoral fellow in the laboratory of Dr. Chris Overall at the University of British Columbia.
Can you provide a brief overview of the Overall labs research focus?
The Overall Lab at the UBC Life Sciences Institute is focused on understanding the role that specialised enzymes called proteases play in normal processes and in human disease. Proteases cut other proteins in two ways. The first is just degradation to remove old or worn out proteins. Second, and more relevant for cell to cell signalling, is the very precise limited cuts in a protein called “processing”. Notably, precise proteolytic processing can alter protein function such as by turning a proteins function on or off, or even generate new functions.
There are over 500 different proteases in humans and because of their importance in disease, many are important drug targets, especially for arthritis and cancer. It is important to know what proteins are cut by each individual protease to enable more selective drug targeting within a disease pathway, and to identify new pathways that are regulated by proteases. This knowledge can improve the prediction of the effects of inhibiting a protease with a drug in order to minimize detrimental side reactions.
Over the past decade, the Overall Lab has developed new and powerful mass spectrometry-based proteomic techniques, called degradomics, to identify protease substrates on a global scale. For example, we are able to identify all protease substrates and their precise cleavage sites in cells or tissues using one of our methods called TAILS (Terminal Amine Isotopic Labeling of Substrates). Differential labelling allows us to examine up to 10 samples at once, enabling, for example, a comparison of proteolysis over time in a particular patient, or between healthy and diseased samples. As we continue to apply degradomics to study proteolysis in different biological systems, we are realizing how important proteases are in regulating the many processes that are required for life.
Essential for progress is discovering new proteases in biology. To do so the technique known as bioinformatics uses complex computer algorithms to seek new proteases hidden in diverse genomes. Prof. Andrew Doxey, University of Waterloo, is an expert in applied bioinformatics and found the first hints of these hidden proteases in bacteria. Our two labs teamed up together to make the enzyme and characterise this unique and highly unexpected finding that is expected to rapidly advance understanding of bacterial infections and new ways to treat human and livestock diseases.
What is the significance of the findings in this publication?
The bacterial flagellum is a complex multi-component structure including a whip-like appendage, the flagellar filament, which is composed of approx. 20,000 protein units (flagellins) that link up together and form a structure of about 10 micrometers in length. We discovered and characterized a new family of flagellins that encompass a surface exposed enzymatically active protease domain.
Bacteria use this domain by embedding proteolytically active flagellins, which we termed flagellinolysins, in the flagellar filament. There are thousands of these protease domains per flagella, making this one of the largest enzyme structures known. Now, this new type of flagella is capable of digesting proteins in the bacteria’s environment including biofilms and animal or human tissues.
As these flagella sweep the bacteria along, the proteolytic flagella can dissolve the proteinaceous material nearby. This may improve the movement ability of the bacteria to “drill” through its environment or to invade tissues in infection. The protein breakdown products may also be “eaten” to nourish the invading bacteria.
This finding updates the long-held view that bacteria use their flagella mostly for movement, and demonstrates that flagella can also function as gigantic enzymatic complexes. This also improves our understanding of how these organism and related pathogens cause disease. This may suggest new ways to treat or slow down bacterial infections. Furthermore, there may be ways of using these enzymes and/or the flagella-scaffold in biotechnology to specifically degrade things we want to break down, including pathogenic biofilms, which are associated with more than 80 per cent of infections.
What are the next steps for this research?
Our discovery of proteolytically active flagellins (flagellinolysins) opens many new research avenues in biomedical research. TAILS N-terminomics, one of the key proteomic methods developed in the Overall Lab at UBC, will identify substrates both in bacterial biofilms and animal hosts to discover new host-pathogen interactions. Structural studies using X-ray crystallography will reveal atomic details of substrate recognition by the proteases, and will help rational drug design. Finally, gene knock-out studies will be used to explore the evolutionary advantage of proteolytic flagella and the impact on pathogenicity and virulence will be examined.
This research was funded by:
This work was supported by a postdoctoral fellowship from the Michael Smith Foundation of Health Research (MSFHR) to Ulrich Eckhard, an Internal Collaborative Training Award from the UBC Centre for Blood Research to Giada Marino, a Canada Research Chair in Protease Proteomics and Systems Biology (Prof. Christopher Overall), and project grants from the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), as well as with infrastructure grants from the MSFHR and the Canada Foundation for Innovation (CFI).