The human mouth teems with millions of enamel-eroding, gum-inflaming microbes.
One of these, Porphyromonas gingivalis, is a bacterial homesteader that stakes a claim deep within the spaces between teeth and gums. It’s also the leading cause of tooth loss — secreting proteins that destroy the soft tissues and bone that support teeth to cause periodontal disease.
Now scientists have identified the thousands of proteins the bacterium produces, shedding light on how it interacts with healthy cells in order to thrive, according to dental researchers from the University of Florida and the University of Washington. They describe their findings in the current issue of the journal Proteomics.
“Determining which proteins are expressed in greater levels in the mouth has allowed us to gain clues as to how P. gingivalis might be causing disease, and what we might be able to do with drugs or vaccines to prevent it,” said Richard Lamont, Ph.D., a professor of oral biology at UF’s College of Dentistry and study investigator.
The National Institute of Dental and Craniofacial Research estimates 80 percent of adult Americans have some form of periodontal disease, their symptoms ranging from mild gum irritation to complete tooth loss.
People with periodontal disease also are at increased risk of stroke and heart attack, and the disease makes it difficult to control blood sugar levels in people with diabetes. If that’s not bad enough, pregnant women with periodontal disease are seven times more likely to deliver low-birth-weight, preterm babies. Proteins are important to study because they are the foundation of the cellular structure of every living organism, Lamont said. They carry on the day-to-day biology of life, going about their business as enzymes and antibodies. They can also cause disease.
“The genes themselves are only important in that they encode the proteins,” Lamont said. “It’s the proteins that are most responsible for disease, and in most cases it’s proteins that are vaccine and drug targets.”
The scientists have been trying to understand how P. gingivalis interacts with healthy oral tissues to cause such devastation. In this study, they used cutting-edge molecular research techniques to map all the proteins — known as the proteome — produced by P. gingivalis. Ultimately, the researchers were able to fill hundreds of gaps in the organism’s sequence of roughly 2,000 proteins.
“The approach used in this study is very exciting,” said Hansel Fletcher, Ph.D., an associate professor of microbiology and molecular genetics at Loma Linda University in Loma Linda, Calif. “For the first time, we are able to see that the more than 200 so-called ‘hypothetical’ proteins in P. gingivalis are expressed and have specific functions.”
Until now, scientists had identified less than 2 percent of the pathogen’s proteins and had to guess at what other proteins might be present in the proteome based on similarities to other known proteins, said Fletcher.
“This study has done two things to advance that,” Lamont said. “We’ve identified the complete protein complement of the organism, and we’ve looked at how those proteins are expressed when the organism is in an environment that closely mimics an oral situation.”
To do this, Lamont and his colleagues compared the proteins secreted by P. gingivalis when grown in a medium containing human gum cell proteins with the proteins produced by the bacteria when grown in a neutral medium. Bacterial proteins from the two conditions were separated using a new technique called Multidimensional Protein Identification Technology, or MudPIT.
Once separated, mass spectrometry was used to measure each protein’s mass and charge, identifiers as unique to proteins as the whorls of fingerprints are to people.
The spectrometry measurements were fed into a computer database to create a computational model of the P. gingivalis proteome, resulting in a surprising find.
“Some of the proteins we previously thought were important when they were expressed in the lab proved not to be when the organism is in an environment that mimics the oral cavity,” Lamont said.
To put it simply, the behavior, or protein expression, of the organism when it’s at work in the human mouth is very different from its behavior when it’s vacationing in a Petri dish.
“An organism growing in a lab isn’t causing disease,” Lamont said. “It’s an organism that’s in your gums, your lungs, your heart valves, your arteries causing disease.”
The next step will be to expose P. gingivalis to other oral pathogens to determine what interactions may exist that contribute to infection, he said.
“This study is important in that we now have an understanding of the protein expression on a global scale for this pathogen,” Lamont said. “Now we need to see how it adapts to various situations present in the mouth to cause disease.”
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