“If you understand the principles of howbiofilms assemble and cause diseases, we can learn how to prevent or disassemblethem effectively.” —DR. MICHEL KOO

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OF KOREAN DESCENT, Dr. Hyun (Michel)Koo was born in Germany, raised in Brazil,and has worked in the United States for adecade and a half. As a result, he has masteredEnglish, Korean, and Portuguese. But thattally does not include the fact that he is alsofluent in the complementary but distinct languages of biochemistry, microbiology,and clinical dentistry.

Now a professor in the Department ofOrthodontics at Penn Dental Medicine, Dr.Koo’s comfort in many realms has led him toclinically relevant discoveries in unexpectedplaces, from the Amazon rainforest, to exoticfruits and plants, to humble beehives andcranberries.

The driving force linking these diversediscoveries is Dr. Koo’s desire to understandand find treatments against disease-causingbiofilms, the sticky mix of microbes, glue-likepolymers and other materials that affixesitself to many surfaces. His focus is on thebiofilm known as plaque that accumulateson the tooth’s surface, leading to breakdownof enamel and onset of the disease dentalcaries, commonly called tooth decay or cavities.

DR. MICHEL KOO EMPLOYING UNIQUE APPROACHESTO TARGET BIOFILMS AND TOOTH DECAY

UNRAVELINGBIOFILMSOPPOSITE: Hyun (Michel) Koo, DDS, PhD, joined PennDental Medicine last September as Professor, Departmentof Orthodontics. In his lab, Dr. Koo focuses on under-standing how pathogenic biofilms cause oral infectiousdiseases, particularly dental caries (or tooth decay). Heuses advanced molecular and imaging technologies tounveil the principles of biofilm assembly on tooth surfaces.At the same time, he is developing new therapeuticapproaches to target the biofilm at its core—the extra-cellular matrix-scaffold—using naturally-occurring andsynthetic agents.

“Our main purpose is to find out howmicrobes build up biofilms and cause diseasesand then hopefully find a better way to preventthem,” he says.

A UNIQUE APPROACHDr. Koo’s interest in research emerged as adental student in Brazil. While most studentswent home for summer vacation, he spentthe breaks in the lab doing research thatstoked his interest in biochemistry andmicrobiology. After graduating, he began to

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UNRAVELINGBIOFILMSpractice dentistry but maintained a ferventcuriosity that eventually drove him back tothe classroom. But instead of seeking furtherdental-specific training, he entered a master’sprogram in food science and biochemistry atthe State University of Campinas (UNICAMP),Brazil—the first dental student ever to do so.

“I thought, the oral cavity is the firstpoint of entry for everything we consume,”he says. “I was excited to learn from the fas-cinating world of food science if there wasanything to be discovered about preventingoral disease or promoting oral health.”

Working with a mentor (Dr. Yong Park),Dr. Koo began a search for new substancesthat could provide oral health benefits usingbiotechnology in the form of microbialenzymes and plant-food chemistry.

“Sugar is the arch criminal of dentalcaries as it fuels harmful bacteria to build upplaque and make acids that dissolve teeth,”says Dr. Koo. “We wanted to find specificmicrobes in nature that could use this sugarto produce new types of sugar molecules thathave similar sweetening properties but couldn’tbe metabolized in the oral cavity by bacteria.”

To find these novel sugar-metabolizingmicrobes, Dr. Koo collected samples in dozensof different ecosystems.

“We looked in places from the cleanest,purest ecosystems like the Amazon rainforestto rotting food and insects in São Pauloopen markets,” he says.

He amassed an array of more than 3,000samples and identified a few new microbesthat could produce alternative sweeteningyet non-cariogenic and non-caloric sugars;some of which are now being developed bythe food industry.

A pull toward unlikely sources for dis-covery continued in another project Dr. Koodeveloped, this time looking at honeybeehives. Bees seal and protect their hives witha substance called propolis, which they pro-duce from resins they collect as they visit a variety of plants. Dr. Koo and colleaguesfound that a number of small molecules isolated from propolis have anti-biofilm

BIOFILM ARCHITECTURE IN 3DDr. Koo continued his scientific inquiries inBrazil while earning a Ph.D. in oral biology(Dr. Jaime Cury, mentor) at FOP-UNICAMPin a joint program with the University ofRochester, where he subsequently joined thefaculty. “At Rochester, I had an incredibleopportunity for academic growth, particularlyworking with Dr. William Bowen, a world-authority in dental caries research,” saysKoo. While continuing to look for toothdecay therapies, he also delved more deeplyinto understanding how pathogenic bacteria,such as S. mutans, assemble plaque biofilms.

Using advanced imaging technologythat allows him to reconstruct a three-dimen-sional picture of a biofilm, Dr. Koo has foundthat the bacteria are able to cluster tightlytogether because they are enmeshed in amatrix comprised of glue-like polymermolecules and other extracellular materials.

properties. By targeting an enzyme producedby the main culprit behind dental caries, the bacterium Streptococcus mutans, theresearchers demonstrated that propolis-derivedcompounds could inhibit the formation ofbiofilms and the development of dentalcaries in rodents.

“Starting with food science and com-pounds found in nature, we now have a betterunderstanding of oral diseases, and the basisof products that could prevent those diseases,”he says.

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“If you understand the principles of howbiofilms assemble and cause diseases, we canlearn how to prevent or disassemble themeffectively,” Dr. Koo says.

Studying these microbial communitiesenmeshed in a polymeric matrix could pavethe way for relevant findings beyond themouth—ranging from barnacles on a ship hullto plaque on heart valves and medical devices—as biofilms are often associated with manydiseases in humans as well as industrial andnaval issues.

“We always have the idea in mind ofhow our research in the oral cavity might havebroader applicability,” Dr. Koo says.

ABOVE: (top) Dr. Koo’s research has shown thatmolecules found in cranberries, called proanthocyanidins,could disrupt the assembly of the biofilm matrix and beused to develop new therapies against cariogenic biofilms.(bottom) Prospective anti-biofilm agents are initiallytested in the laboratory using biofilms formed onhydroxyapatite (a tooth enamel-like material).

OPPOSITE: Biofilm structure is highly complex. Bacterial‘islets’ or microcolonies (green) are enmeshed and sur-rounded by an intricate extracellular ‘scaffold,’ known asmatrix (red), which is comprised of polysaccharides andother materials.

“Biofilms are highly organized microbialcommunities, forming a structure almostlike a tissue,” says Dr. Koo. “We show howthe ‘scaffolding’ of the matrix creates ahighly compartmentalized architecture, whilemaking the biofilms very sticky on the surface.”

In 2012, Dr. Koo and colleagues publisheda paper in PLoS Pathogens demonstratingthat not only does the matrix provide a scaffold and make the biofilm gooey andsticky, but it also helps to create acidicmicroenvironments. By devising a novelthree-dimensional pH-mapping technique,Dr. Koo showed highly localized, matrixdelineated acidic compartments throughoutthe biofilm. These niches of low pH affects

everything from the balance of microorgan-isms that can thrive inside of them, to thebehavior of S. mutans, to the severity of damageto tooth enamel that results in cavities.

“Our data offer new avenues for furtherelucidation of how cell-matrix interactionsgovern the formation of pathogenic biofilms,while introducing new tools and methods for biofilm research,” Dr. Koo says.

Dr. Koo’s probing of the matrix structureand biofilm microenvironment has led tonew ways of thinking about how to preventor treat plaque build-up on the teeth. Insteadof solely targeting the bacteria themselves,Dr. Koo and his team are looking for ways to degrade or stop matrix production. Thisstrategy has the added benefit of avoidingtreatment with antibiotics, which some bacteria can eventually learn to evade.

DEVELOPING NOVEL THERAPIESMuch like his previous explorations in theAmazon and other ecosystems, Dr. Koo hascontinued to look in some unlikely placesfor new anti-biofilm compounds to target thematrix. His investigations have revealedpotentially useful compounds in cranberriesand in the waste product from the wine-makingindustry—the leftover grape skin, pulp andseeds called pomace. Dr. Koo’s research hasshown that molecules found in cranberriescalled proanthocyanidins can reduce thesynthesis of polysaccharides in the matrix.

In parallel, Dr. Koo has methodicallyexamined the bioactivity of each of the food-derived compounds discovered in his lab to help design an effective therapy againstcariogenic biofilms. He has looked at how com-binations of various molecules could synergize,enhancing the overall therapeutic effect.

In one line of research, Dr. Koo and colleagues examined the possibility of pairingthe proven standby, fluoride, with newly discovered anti-biofilm agents. The rationalewas simple yet promising: Fluoride helps toprevent mineral loss and rebuild tooth mineralduring acid attack, but has limited effectsagainst bacteria and biofilm formation.

“We thought that including agents thatimpair acid production, a terpenoid, andbiofilm matrix build-up, a flavonoid, couldcomplement and enhance the effectivenessof fluoride,” Dr. Koo says.

He discovered that the therapeutic effectof this combination therapy was superior tothat of the potent antimicrobial chlorhexidineas well as fluoride, effectively reducing thedevelopment of caries disease in an animalmodel. Recently, Dr. Koo and colleagues published a paper in Antimicrobial Agentsand Chemotherapy revealing the mechanismsby which these food-derived compounds,together with fluoride, disrupt the assemblyof the biofilm matrix, and enhance the overallanti-caries activity.

“Our main purpose is tofind out how microbes build up biofilms andcause diseases and thenhopefully find a better way to prevent them.” —DR. MICHEL KOO

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DISCOVERING A CROSS-KINGDOMPARTNERSHIP This knowledge of biofilm architecture andassembly extends to an important publichealth concern. Dr. Koo has a special interestin a disease that affects children early in life,called early childhood caries. It involves ahighly destructive and painful form of toothdecay that affects toddlers, particularly thosefrom backgrounds of poverty.

“It’s a costly and terrible disease andvery psychologically damaging to kids becausethey can’t even smile,” he says. “The toothdecay can become so severe that treatmentoften requires surgery in the operating room.”

Some of Dr. Koo’s latest work, to bepublished in the May issue of Infection andImmunity, aims to identify the factors thatmake these infections so virulent in children,with an eye toward preventing or treatingthe disease.

Though researchers had long known S. mutans was a primary culprit in the disease,Dr. Koo and collaborators, as well as otherscientists, probed dental plaque from childrenwith the disease and almost always foundthe fungus Candida albicans together withhigh levels of S. mutans. The discoverypiqued Dr. Koo’s interest, because althoughC. albicans sticks to the cheek and tongue, it was not believed to be a common resident in dental plaque formed on teeth.

“We were puzzled,” Dr. Koo says. “Candidausually does not associate with S. mutans,nor does it colonize teeth very effectively.”

The investigators discovered that anenzyme secreted by S. mutans, which the

bacterium uses to produce extracellularpolysaccharides from sugar, also binds toand enables Candida to produce a glue-likepolymer in the presence of sugar. Candidathen uses this same polymer to adhere toteeth and to bind S. mutans, two abilities itotherwise lacks.

“The combination of the two organismsled to a greatly enhanced production of thebiofilm matrix,” Dr. Koo says, “drastically boost-ing the ability of the bacterium and the fungusto colonize the teeth, increasing the bulk of thebiofilms and the density of the infection.”

And because of the biofilm’s compart-ments of low pH, this accumulation led togreater levels of acid next to the teeth that candissolve enamel, leading to cavity formation.The investigators showed that infection byS. mutans and C. albicans together doubledthe number of cavities and boosted theirseverity several fold in rats.

“It is an intriguing interaction where afungus is converted into a fierce stimulatorof cariogenic biofilm formation,” says Dr. Koo

“Our data will certainly open the way totest agents to prevent this disease,” Dr. Koosays, “and, even more intriguing, the possibilityof preventing children from acquiring thisinfection.”

NEW HORIZONSThe novelty and merit of Dr. Koo’s work hasbeen recognized through several awards,including the IADR Distinguished ScientistAward and IADR/GSK Innovation in OralCare Award, as well as funding from theNational Institutes of Health, the U.S.

Department of Agriculture, and industry.Having just come aboard Penn DentalMedicine’s faculty last September, he sayshe is honored to be part of what he sees as“a world-class winning team for research,”with ample opportunities for cross-disciplinarycollaboration within the School and across thecampus to make new discoveries and to bringthem into clinical use. For example, Dr. Koo isstarting to collaborate with Dr. Henry Daniell,Professor, Departments of Biochemistry andPathology, on a project to investigate thepotential of using his antimicrobial peptidesto control cariogenic biofilms.

Dr. Koo’s recent sabbatical leave in Dr. Ken Yamada’s lab (a leading scientist incell/matrix biology) at NIDCR/NIH broughtnew ideas to study biofilms. He’s particularlyinterested in taking advantage of the nano-technology and engineering expertise atPenn, perhaps utilizing the new Singh Centerfor Nanotechnology, located just at the otherend of Penn’s campus from Penn DentalMedicine. He plans to build on previous work,in which he has explored strategies for drugdelivery using nanoparticles.

“One of the major challenges of topically-delivered compounds is rapid clearance ofthe agents in the mouth before they have timeto exert their full therapeutic effect,” he notes.

By engineering low-cost and high-adhesive nano-carriers, the therapeutics can be retained for longer periods of time,increasing drug efficacy. He hopes thesetechnologies will lead to development ofmore-effective therapies against biofilm-associated oral diseases.

At Penn Dental Medicine, Dr. Koo seeshimself able to fully realize the potential ofthese and other clinically relevant technologies.

“There are so many ways different disci-plines can help us, from biomedical sciencesto nanotechnology to engineering approachesand Penn’s philosophy is centered in promotingthe integration of knowledge,” he says. “I’m inthe right environment with all the necessarysupport to further advance our mission ofconducting innovative research and developingnew therapies to make a difference.”

—By Katherine Unger Baillie

UNRAVELINGBIOFILMS

“Our data offer new avenuesfor elucidation of how cell-matrix interactions govern the formation of pathogenicbiofilms, while introducingnew tools for biofilm research.”—DR. MICHEL KOO

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TOP: Members of the Koo Lab, left to right: Jinzhi He, Dr. Yuan Liu,Dr. Michel Koo, Yong Li, Dr. Lizeng Gao, Dr. Geelsu Hwang, Dr. Dongyeop Kim.

ABOVE: The 3D architecture of a fungal-bacterial biofilm.

RIGHT: This close-up image of the biofilm illustrates the spatialrelationship between Candida albicans cells and a Streptococcusmutans microcolony. Yeast (red) and hyphal forms of C. albicans(blue) are found associated in the periphery of the microcolonystructure (green); specific areas where fungal cells are associatedwith the microcolony are highlighted in orange.