Combining biochemistry to dentistry : from in vitro Candida glabrata observations to an in vivo clinical lingonberry application

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Title: Combining biochemistry to dentistry : from in vitro Candida glabrata observations to an in vivo clinical lingonberry application
Author: Pärnänen, Pirjo
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences, Molecular and Integrative Biosciences, The Department of Biochemistry and Biotechniques
Doctoral Programme in Integrative Life Science
Publisher: Helsingin yliopisto
Date: 2020-06-16
Thesis level: Doctoral dissertation (article-based)
Abstract: Our studies focused on using Candida glabrata (C. glabrata) as a model organism to isolate and investigate the role of C. glabrata cell wall proteases as host protein- degrading virulence factors and the inhibition of their action. The cell wall proteins of microbes are in the frontline of first contact with the host cells in oral mucosa. C. glabrata is the second most prominent Candida yeast, and it is commonly found in the normal oral microbial flora causing opportunistic yeast infections, particularly in hospitalized patients. It is considered innately azole- resistant and treatment is more difficult compared to the typical Candida albicans (C. albicans) infections. Azoles are the most commonly used antifungal agents used in candidosis. There is an urgent need of development of topical antimicrobial agents, and wild berries such as lingonberry, have been increasingly studied. Lingonberries are known to have antioxidant, anti-inflammatory, antimicrobial and anticancerous properties and are considered beneficial to health. To this background we studied in vitro and in vivo the effects of a patented, fermented lingonberry juice (Lingora®, from now on abbreviated as FLJ). It was specially developed to be used as a mouthwash on C. glabrata and other typical microbes of the oral flora related to yeast infections and caries. Our primary goal was to isolate, identify and characterize C. glabrata cell wall proteases with biochemical methods: enzymatic treatment of C. glabrata cells, MDPF-zymography, SDS-PAGE, 2D-PAGE and LC-MS/MS. These methods may be used to isolate and identify novel Candida cell wall proteases enabling their further characterization and inhibition studies. Further in vitro studies were conducted on the effect of FLJ on intracellular protein expression of C. glabrata with the 2D-DIGE method. The proteins were identified by LC-MS/MS. The inhibition of proliferation and invasion of two aggressive oral tongue squamous cell carcinoma (OTSCC) lines (HSC-3, SCC-25) with FLJ and curcumin were measured in vitro by colorimetric ELISA and three- dimensional Myogel spheroid assay. Finally, we conducted a clinical pilot study including oral examinations, microbial cultivations and measurements of active MMP-8 concentrations using PerioSafe® point-of-care test. FLJ was used as a mouthwash to see if it has also in vivo effects on three microbes of the oral microbiota. From the C. glabrata cell wall we identified a novel, uncharacterized 25 kDa serine protease, Cwp1.2., with an estimated pI of 7.6 and gelatinolytic activity. This activity was inhibited by PMSF, a known serine protease inhibitor. Certain C. glabrata intracellular protein expressions related to glycolysis, oxidative phosphorylation, oxidative stress and biofilm formation were significantly diminished after treatment with FLJ. These proteins include e.g. heat shock protein and redoxin, which are expressed by C. glabrata when predisposed to stress. Downregulation of these proteins causes C. glabrata cells to be more vulnerable to environmental stress and may cause lower virulence. FLJ showed to inhibit proliferation and invasion of two aggressive OTSCC cell lines similar to curcumin. FLJ is safe, has no known interactions with medications and could be studied to be used as an adjunctive therapy in management of OTSCC. The clinical mouthwash pilot study with FLJ results showed statistically significant reduction in Candida and S. mutans counts. Our in vitro studies also indicate growth inhibitory effect on the most common periodontitis- related bacteria. Bleeding on probing (BOP), visible plaque index (VPI) and trend of active matrix metalloprotease-8 (aMMP-8) values were also reduced during the FLJ mouthwash period. Lactobacilli counts increased during the mouthwash period. Although lactobacilli are thought to be related to caries the clinical parameters and clinical outcome indicate a balancing effect on the oral microbial flora from a dysbiotic to a symbiotic direction. This diminished microbial related inflammatory burden should be studied further in context with broader positive general health effects. The results show several beneficial aspects of FLJ in the oral environment. The methodology used in these studies might be applicable to other oral microbes in developing novel antimicrobial agents related to cell wall proteases of Candida. Combined in vitro and in vivo studies showed effects of FLJ on C. glabrata intracellular proteins, host cell derived proteins including anti-inflammatory effects, tongue carcinoma cells and oral microbiota.Suun paikallishoitona käytettäviä aineita on rajatusti ja nykytutkimus on laajentunut luonnonmarjojen tutkimiseen. Marjojen katsotaan olevan osa terveellistä ruokavaliota. Puolukalla tiedetään olevan antioksidatiivisia, anti-inflammatorisia, antimikrobiaalisia ja tiettyjä syöpiä ehkäiseviä ominaisuuksia, mutta sen vaikutuksia suuhun ei ole tutkittu. Suukäyttöön soveltuvaksi puolukkamehusta poistettiin patentoidulla menetelmällä valtaosa luontaisista sokereista fermentoimalla (Lingora®). Sen vaikutuksia tutkittiin sekä laboratoriokokeilla että kliinisesti suun hiivasienitulehduksissa esiintyvien Candida- lajien ja karieksessa esiintyvien mikrobien (Streptococcus mutans, laktobasillit) kasvua ehkäisevänä aineena. Lisäksi laboratoriokokeessa tutkittiin fermentoidun puolukkamehun vaikutusta kahden aggressiivisen kielisyöpäsolulinjan jakaantumiseen ja leviämiseen. Candida glabrata (C. glabrata) on toiseksi yleisin suun mikrobiomissa esiintyvä Candida- hiivasienilaji, joka aiheuttaa vaikeita tulehduksia varsinkin sairaalapotilailla. Mikrobien soluseinä on kontaktissa suun limakalvon soluihin ja yksi tutkimusten tavoite olikin käyttää C. glabrataa malliorganismina ja tunnistaa uusia C. glabratan soluseinän proteaaseja sekä tutkia fermentoidun puolukkamehun vaikutusta C. glabratan solunsisäisten proteiinien määrään, joilla saattaa olla merkitystä sen taudinaiheuttamiskyvylle. C. glabratan soluseinästä löydettiin uusi proteaasi, jota tutkimalla on mahdollista kehittää uusia hiivasienilääkkeitä. Tutkimuksissa käytettyjä menetelmiä voi soveltaa proteaasien tutkimukseen muillakin mikrobeilla uusien antimikrobiaalisten aineiden kehittämisessä. C. glabratan solunsisäiseen aineenvaihduntaan ja stressinsietoon liittyvien proteiinien määrät vähenivät fermentoidulla puolukkamehulla. Tällä on hiivasienten kasvua sekä mahdollisesti taudinaiheuttamiskykyä heikentävä vaikutus. Laboratoriokokeissa fermentoitu puolukkamehu esti myös merkittävästi kielisyöpäsolujen kasvua ja leviämistä. Kliinisessä kokeessa fermentoitu puolukkamehu esti merkittävästi Candida- hiivasienten ja Streptococcus mutans- bakteerien kasvua. Laktobasillien määrä lisääntyi, jonka katsotaan olevan hyödyllistä yleisterveydelle. Ienverenvuoto (BOP) ja näkyvän plakin määrä (VPI) vähenivät myös ja aMMP-8 määrässä näkyi laskeva trendi. Jatkotutkimukset ovat meneillään. Fermentoitu puolukkamehu on turvallinen, eikä sillä ole tunnettuja merkittäviä lääkeaineinteraktioita. Nämä laboratorio- ja kliinisen kokeen tulokset osoittavat sillä olevan useita hyödyllisiä vaikutuksia ja sitä voidaan käyttää suun kotihoidon tukena.
Subject: faculty of Biological and Environmental Sciences
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