Bacteria form biofilms by attaching themselves to the host and to each other by sticky hair-like filaments called pili. When biofilms accumulate, periodontitis follows. Now, researchers in Japan have determined the structure of pili and how they assemble, which could lead to strategies for fighting gum disease.
“Pili are vital for both the survival of the bacteria and the creation of the biofilms,” said Dr. Satoshi Shibata, first author and staff scientist at the Okinawa Institute of Science and Technology Graduate University (OIST). “By taking a close look at these pili, our research has provided insights into how we can prevent biofilms from forming.”
Previous studies by the researchers revealed how most members of the Bacteroidiaclass of bacteria such as Porphyromonas gingivalis, which causes periodontitis, have unique Type V pili. But until now, the structure and assembly process of these pili were unknown.
“Besides periodontal pathogens, Type V pili are seen in major colon bacteria such as Bacteroidesand Prevotellaspecies and their Type V pili may contribute to formation of colon microbiota,” said professor Koji Nakayama of Nagasaki University.
Pili themselves are made up of smaller protein units called pilins. In the case of P gingivalis, most of these are FimA pilins. Although pilins are only linked by weak interactions, they can assemble into very stable pili. The first step in determining how this assembly occurred was to take a close look at the structure of individual pilins.
“Detailed structural information of FimA is very important because pathogenicity of P gingivalisstrains is closely related with the FimA subtypes,” said professor Katsumi Imada of Osaka University, who crystalized FimA pilins and revealed their unassembled state at atomic resolution.
“Based on findings from earlier experiments by the Nakayama group, we theorized that these pilins assembled themselves via a mechanism of protease-mediated strand exchange,” said Shibata. “So, our next experiment took a close look at fully assembled pili using cryo-electron microscopy.”
Associate professor Mikio Shoji of the Nakayama group and Shibata prepared a genetically engineered version of the FimA pilins, which successfully assembled into pili, after a protease (a protein that cuts other proteins) was added.
Shibata then collected thousands of images on the high-end cryo-electron microscope at OIST and processed the data on the university’s “Sango” supercomputer, resulting in a complete three-dimensional atomic model of the assembled pilus structure.
“When we added the protease, the pilins started to assemble into elongated pili like train cars connecting to form a train,” said associate professor Matthias Wolf, who leads OIST’s Molecular Cryo-Electron Microscopy Unit. “This happened because the protease cut a retaining loop and released a protein strand, known as the donor strand, which triggered the assembly to begin.”
Once released, the donor strand flipped out of the pilin and inserted itself into a neighboring pilins groove, connecting the two pilins. The researchers then took a closer look at the amino acid composition at the end of the donor strand and found that it played a critical role in the assembly mechanism.
Using biochemistry, crystallography, and cryo-electron microscopy, the researchers mutated the protein, which prevented the pili from forming and proved how these key amino acids contribute to pilin polymerization.
Ultimately, the researchers said, this study is a step toward new antibacterial drugs not just for the diseases caused by P gingivalisbut also for those caused by any bacteria with Type V pili.
“We’re now trying to create an inhibitor that prevents pili from assembling,” said Shibata. “This structure serves as a target to create new drugs, which are desperately needed to counter increasing antibiotic resistance. Finding novel antimicrobial compounds is a critical advantage in fighting these pathogens.”
The study, “Structure of Polymerized Type V Pilin Reveals Assembly Mechanism Involving Protease-Mediated Strand Exchange,” was published by Nature Microbiology.
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