Thesis advisor: Babak Momeni / Rothia mucilaginosa is a gram-positive bacterium that is found in the human oral microbiota. Under normal circumstances, it exhibits a commensal relationship with humans. However, in immunocompromised patients and those with medical implants, these bacteria can pose a serious health risk. In this thesis I outline experiments performed to reveal more about the normal role of R. mucilaginosa within the oral microbiota in the hopes of better understanding its transition to virulence. R. mucilaginosa was grown alongside several of its major co-inhabitants in the oral microbiota in pairwise fashion on solid growth media to search for signs of interspecies interactions. After initial screening, those pairs that showed consistent patterns of interaction were selected for further analysis via fluorescence microscopy. A consistent interaction pattern was confirmed between R. mucilaginosa and Streptococcus gordonii. Interactions were also noted between Corynebacterium durum and R. mucilaginosa as well as between Corynebacterium durum and Neisseria elongata. / Thesis (BS) — Boston College, 2018. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Scholar of the College. / Discipline: Biology.

INTRODUCTION: Celiac disease is a T-cell mediated-inflammatory disorder of the small intestine precipitated by gluten ingestion. Gluten can be effectively degraded by Rothia aeria, a natural resident oral microbe. Rothia bacteria can potentially be developed as a first probiotic for celiac disease. Aims: To select the optimal culture conditions for R. aeria enzyme expression in vitro and determine the ratio of R. aeria to gluten to achieve digestion within 2 hr incubation, which is the residence time of foods in the stomach/upper intestines. MATERIALS AND METHODS: R. aeria were cultivated in Brain Heart Infusion (BHI) with different strength (4%, 20% and 100%), and chemical defined media M9 supplemented with different carbon sources (glucose, succinate, glycerol, or casein). The effect of incubation time and temperature (28°C and 37°C) on the enzyme expression of the bacteria grown in BHI was also assessed. The enzyme activities of R. aeria (standardized with OD 0.6) were measured with the para-nitroanilide-derivatized substrate. Gliadin degradation was investigated by incubating a fixed gliadin concentration (250 µg/ml) with different amount of R. aeria cell (OD620 1.0, 0.5, 0.25), and analyzed by SDS-PAGE. RESULTS: The OD620 of 24 hr R. aeria culture in 4%, 20%, and 100% BHI were 0.240, 0.932, and 2.033, respectively. No bacterial growth was observed in M9 broth +2% glucose, succinate, or glycerol, but grew well in M9 broth +2% casein with OD620 19.54. R. aeria exhibited highest activity when grown in 100% BHI (0.22AU/min) and lowest in 4% BHI (0.1AU/min). Enzyme activities in M9+2% casein were low (0.035AU/min), not in proportional to the OD of R. aeria culture. The specific enzyme activities of R. aeria in 100% BHI was high in log phase (9 to 12 hr or 12 to 18 hr), but the yield of total activity was less than that of in stationary phase (20 to 44 hr). The activity of the cells grown at 37°C was higher than at 28°C. R. aeria suspension with an OD620 of 1.0 exhibited rapid degradation of gliadins at 250ug/ml. CONCLUSIONS: Full strength BHI broth and 20 to 44 hr cultivation at 37°C are considered as optimal cultivation condition to obtain a R. aeria cell culture with high enzyme activity. Starvation condition do not enhance enzyme expression.

INTRODUCTION: Foods enter the oral cavity and mix with saliva. Some foods are not well tolerated, for instance, gluten proteins in individuals suffering from celiac disease (CD). Celiac disease is a chronic immune-mediated inflammation of the duodenum, triggered by gliadin component of gluten contained in wheat, barley and rye. In previous studies we showed that oral Rothia bacteria can degrade gliadin in vitro. The objective of this study was to gain more insights into the role of Rothia bacteria and subtilisin-A enzyme on gliadin digestion in vivo, with the ultimate goal to find new therapeutic options for CD. MATERIALS AND METHODS: Part I: Rothia bacterial proteins were analyzed for enzyme activity and subjected to LC-ESI-MS/MS. For in vivo, mice chow was prepared with and without R. aeria. Gliadin epitope abolishment was assessed in the mice stomach contents (n=9/group) by ELISA. Part II: Subtilisin-A was dissolved in various solutions, temperatures and incubation time to assess enzyme activity by using enzyme substrate (Suc-AAPF-pNA). Part III: PEGylation of subtilisin-A (Sub-A) was performed by mixing Sub-A with methoxy-polyethylene glycol (mPEG) and further encapsulated by polylactic-glycolic acid (PLGA). The activity of the modified enzyme to detoxify the immunogenic gliadin epitopes was evaluated at pH3.0. RESULTS: Part I: R. aeria gliadin-degrading enzyme was found to be a member of the subtilisin family. In vivo, gliadins immunogenic epitopes were reduced by 32.6%. Part II: Sub-A dissolved at pH1.5 showed a band of 27kDa, while it only showed bands below 10kDa when dissolved at pH7.0, suggesting auto-proteolysis. The enzyme activity was completely lost at temperatures exceeding 60°C and also reduced 4-fold after 6hr incubation at 37°C. Part III: PEGylation protected Sub-A from autolysis. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gliadin immunogenic epitopes were decreased by 60% in the stomach of the mice fed with chow containing Sub-A-mPEG-PLGA. CONCLUSION: The results provide proof for the contribution of oral Rothia bacteria to gliadin digestion and pharmaceutical modification can protect Sub-A from auto-digestion as well as from acidic insults, thus rendering the usefulness of coated subtilisins as a digestive aid for gluten degradation. / 2019-01-13T00:00:00Z

Rothia mucilaginosa (Rm) is a gram-positive bacterium residing in the oral cavity. Recent studies in our laboratory have shown that this microorganism is able to cleave gluten, including immunogenic domains implicated in celiac disease. This can be beneficial to patients with celiac disease because exploitation of Rm can provide a novel mode of treatment. The enzymes responsible for this cleavage are as yet unknown. The purpose of this study was to optimize the transformation efficiencies of Rm cells through electroporation, with the ultimate goal to create knock-out mutants for enzyme activity. We have determined various aspects of Rm cells relevant for this project: (1) the growth curve characteristics of Rm; (2) the presence of endogenous restriction enzyme activities; and (3) the conditions facilitating Rm electroporation by varying electroporation voltages. Furthermore, electroporation and transformation of the plasmid pUC18 was conducted in Escherichia coli. The growth curve of Rm cells in BHI growth medium incubated at 37°C while shaking showed a doubling time of approximately 3 hours in the logarithmic growth phase. Using a cell sonicate of Rm cells incubated with Lambda DNA and four different restriction enzyme buffers, we found that there were no apparent endogenous restriction enzyme activities detectable. For the electroporation experiments, we used previously published protocols for the bacterium Staphylococcus aureus, as a standard condition to electroporate Rm cells. Those studies have shown that changing electrical parameters during the electroporation would yield a high efficiency rate of gram-positive bacterial transformation (Lofblom et. al., 2006; Metzler et. al., 1992). Therefore in our study, we increased the field strengths (kV*cm-1) to electroporate Rm cells. Rm cells could not be successfully transformed, and we observed that field strengths exceeding 18 kV*cm-1 destroyed Rm cells. On the other hand, the transformation of E. coli with pUC18 was successful. Our studies have laid the groundwork for investigating the transformation of Rm cells, and future studies can use the results obtained to further investigate ways to optimize transformation of Rm cells for potential utility in celiac patients.

Tesis por compendio / [ES] Se ha estimado que obtenemos más de las tres cuartas partes del nitrato que ingerimos de la fruta y la verdura. Los vegetales ricos en nitratos incluyen verduras de hoja verde y ciertos tubérculos (p. ej., remolachas y rábanos). Las glándulas salivales concentran activamente el nitrato plasmático, lo que da lugar a concentraciones elevadas de nitrato en la saliva (5 a 8 mM) después de una comida rica en nitratos. El nitrato es un factor ecológico que puede inducir cambios rápidos en la estructura y función de las comunidades polimicrobianas. Sin embargo, los efectos sobre la microbiota oral no se han estudiado en detalle, mientras que un número limitado de estudios previos a esta tesis indican que es probable que el nitrato sea beneficioso para la salud bucal. El objetivo de esta tesis es, por tanto, estudiar los cambios microbiológicos inducidos por nitratos e identificar posibles mecanismos de homeostasis generados por este compuesto, con el fin de determinar si el nitrato puede considerarse un prebiótico para la salud bucal. Un segundo objetivo fue aislar cepas reductoras de nitrato y probar su potencial probiótico in vitro. En el capítulo 1, se realizó un estudio in vitro para testar el efecto del nitrato 6,5 mM en comunidades orales cultivadas a partir de la saliva de 12 individuos sanos. En el capítulo 2, se obtuvieron 53 aislados de bacterias reductoras de nitrato y se probó el efecto de seis candidatos a probióticos en comunidades orales sanas cultivadas a partir de saliva de diferentes donantes con o sin nitrato 6,5 mM. En el capítulo 3, se estudió el efecto de un extracto de remolacha rico en nitrato sobre la acidificación oral después de un enjuague con azúcar en 24 individuos sin caries activas. Se tomaron sobrenadantes (capítulos 1 y 2) o muestras de saliva (capítulo 3) para mediciones de nitrato, nitrito, amonio, lactato y pH. Además, la composición bacteriana de la biopelícula in vitro y del pellet salivar se determinó usando secuenciación Illumina del rRNA 16S y/o qPCR del género nitratorreductor Rothia. Los datos demuestran que el nitrato estimula el crecimiento de los géneros beneficiosos Rothia y Neisseria en nuestro modelo in vitro, mientras que potencialmente disminuye las bacterias asociadas a la caries, la halitosis y la enfermedad periodontal. Además, los datos in vitro e in vivo presentados en esta tesis indican que el nitrato puede limitar o prevenir caídas de pH cuando los azúcares son fermentados por la microbiota oral, un mecanismo de resiliencia que podría ser estimulado por el consumo de extractos vegetales ricos en nitratos. Los principales mecanismos de amortiguación del pH por parte del nitrato son el uso de acido láctico durante la desnitrificación (observado tanto in vivo como in vitro) y durante la reducción de nitrito a amonio, así como la producción potencial de amoníaco (observado in vitro). En esta tesis, los efectos del nitrato se observaron después de períodos cortos, es decir, después de 5-9 h de incubación in vitro y 1-4 horas después de la ingesta del suplemento de nitrato in vivo. Los estudios futuros deberían centrarse en los efectos longitudinales de la ingesta diaria de nitratos. En el capítulo 2, se aislaron bacterias reductoras de nitrato pertenecientes a los géneros Rothia y Actinomyces. Una selección de aislados de Rothia aumentó el uso de lactato y la capacidad de reducción de nitratos de las comunidades bucales, lo que potencialmente beneficiaría la salud dental y la salud sistémica, respectivamente. Los datos in vitro e in vivo presentados en esta tesis sugieren que el nitrato puede modular la microbiota oral en aspectos que son beneficiosas para el huésped y, por lo tanto, podría considerarse una sustancia prebiótica para la microbiota oral. Además, los aislados reductores de nitratos pueden estimular los efectos beneficiosos del metabolismo del nitrato, sobre todo en personas con bajos niveles de estas bacterias. / [CA] S'ha estimat que obtenim més de les tres quartes parts del nitrat que ingerim de la fruita i la verdura. Els vegetals rics en nitrats inclouen verdures de fulla verda i uns certs tubercles (p. ex., remolatxes i raves). Les glàndules salivals concentren activament el nitrat plasmàtic, la qual cosa dona lloc a concentracions elevades de nitrat a la saliva (5 a 8 mm) després d'un menjar ric en nitrats. El nitrat és un factor ecològic que pot induir canvis ràpids en l'estructura i funció de les comunitats polimicrobianes. No obstant això, els efectes sobre la microbiota oral no s'han estudiat detalladament, mentre que un nombre limitat d'estudis previs a aquesta tesi indiquen que és probable que el nitrat siga beneficiós per a la salut bucal. L'objectiu d'aquesta tesi és, per tant, estudiar els canvis microbiològics induïts per nitrats i identificar possibles mecanismes d'homeòstasi generats per aquest compost, amb la finalitat de determinar si el nitrat pot considerar-se un prebiòtic per a la salut bucal. Un segon objectiu va ser aïllar soques reductores de nitrat i provar el seu potencial probiòtic in vitro. En el capítol 1, es va realitzar un estudi in vitro per a testar l'efecte del nitrat 6,5 mm en comunitats orals cultivades a partir de la saliva de 12 individus sans. En el capítol 2, es van obtindre 53 aïllats de bacteris reductors de nitrat i es va provar l'efecte de sis candidats a probiòtics en comunitats orals sanes cultivades a partir de saliva de diferents donants amb o sense nitrat 6,5 mm. En el capítol 3, es va estudiar l'efecte d'un extracte de remolatxa ric en nitrat sobre l'acidificació oral després d'un glopeig amb sucre en 24 individus sense càries actives. Es van prendre sobrenadants (capítols 1 i 2) o mostres de saliva (capítol 3) per a mesuraments de nitrat, nitrit, amoni, lactat i pH. A més, la composició bacteriana de la biopel·lícula in vitro i del pèl·let salivar es va determinar usant seqüenciació Illumina del RNAr 16S i/o qPCR del gènere nitratorreductor Rothia. Les dades demostren que el nitrat estimula el creixement dels gèneres beneficiosos Rothia i Neisseria en el nostre model in vitro, mentre que potencialment disminueix els bacteris associats a la càries, l'halitosi i la malaltia periodontal. A més a més, les dades in vitro i in vivo presentades en aquesta tesi indiquen que el nitrat pot limitar o previndre caigudes de pH quan els sucres són fermentats per la microbiota oral, un mecanisme de resiliència que podria ser estimulat pel consum d'extractes vegetals rics en nitrats. Els principals mecanismes d'amortiment del pH per part del nitrat són l'ús de àcid làctic durant la desnitrificació (observat tant in vivo com in vitro) i durant la reducció de nitrit a amoni, així com la producció potencial d'amoníac (observat in vitro). En aquesta tesi, els efectes del nitrat es van observar després de períodes curts, és a dir, després de 5-9 h d'incubació in vitro i 1-4 hores després de la ingesta del suplement de nitrat in vivo. Els estudis futurs haurien de centrar-se en els efectes longitudinals de la ingesta diària de nitrats. En aquesta tesi es van aïllar bacteris reductors de nitrat pertanyents als gèneres Rothia i Actinomyces. Una selecció d'aïllats de Rothia va augmentar l'ús de lactat i la capacitat de reducció de nitrats de les comunitats bucals, la qual cosa potencialment beneficiaria la salut dental i la salut sistèmica, respectivament. Les dades in vitro i in vivo presentats en aquesta tesi suggereixen que el nitrat pot modular la microbiota oral en aspectes que són beneficiosos per a l'hoste i, per tant, podria considerar-se una substància prebiòtica per a la microbiota oral. A més, els aïllats reductors de nitrats poden estimular els efectes beneficiosos del metabolisme del nitrat, sobretot en persones amb baixos nivells d'aquests bacteris. El nitrat i els bacteris reductors de nitrat són, per tant, components prometedors per a futurs productes de salut oral. / [EN] It has been estimated that we obtain over three quarters of dietary nitrate from vegetables and fruits. Nitrate-rich vegetable types include leafy greens and certain root vegetables (e.g., beetroots and radishes). The salivary glands actively concentrate plasma nitrate, leading to high salivary nitrate concentrations (5-8 mM) after a nitrate-rich meal. Nitrate is an ecological factor that can induce rapid changes in structure and function of polymicrobial communities. However, the effects on the oral microbiota have not been clarified, whilst a limited number of previous studies did indicate that nitrate is likely to be beneficial for oral health. The aim of this thesis was therefore to study nitrate-induced microbiome changes and identify potential mechanisms for nitrate-induced homeostasis, in order to determine if nitrate can be considered a prebiotic compound for oral health. A second aim was to isolate nitrate-reducing isolates and test their probiotic potential in vitro. In chapter 1, an in vitro study was set up testing the effect of 6.5 mM nitrate on oral communities grown from saliva of 12 healthy individuals. In chapter 2, fifty-three nitrate-reducing isolates were obtained and the effect of six probiotic candidates was tested on healthy oral communities grown from saliva of different donors with or without 6.5 mM nitrate. In chapter 3, the effects of nitrate-rich beetroot extracts on oral acidification after sugar rinsing was tested in 24 individuals without active caries. Supernatants (chapters 1 and 2) or saliva samples (chapter 3) were taken for nitrate, nitrite, ammonium, lactate and pH measurements. Additionally, the bacterial composition of in vitro biofilms and salivary pellets were determined using 16S rRNA gene Illumina sequencing and/or qPCR of the nitrate-reducing genus Rothia. We showed that nitrate stimulates the growth of the beneficial genera Rothia and Neisseria in our in vitro model, while potentially decreasing caries-, halitosis- and periodontal disease-associated bacteria. Additionally, the in vitro and in vivo data presented in this thesis indicate that nitrate can limit or prevent pH drops when sugars are fermented by the oral microbiota - a mechanism of resilience that could be stimulated by the consumption of nitrate-rich vegetable extracts. The main pH buffering mechanisms of nitrate were lactic acid usage during denitrification (observed both in vivo and in vitro) and during the reduction of nitrite to ammonium, as well as the potential production of ammonia (observed in vitro). In this thesis, the effects of nitrate were observed after short periods, i.e., after 5-9 h incubation in vitro and/or after 1-4 hours after nitrate supplement intake in vivo. Future studies should focus on the longitudinal effects of daily nitrate intake. In chapter 2, nitrate-reducing species belonging to the genera Rothia and Actinomyces were isolated. A selection of Rothia isolates increased lactate usage and nitrate reduction capacities of oral communities, potentially benefitting dental health and systemic health, respectively. The in vitro and in vivo data presented in the current thesis suggest that nitrate can modulate the oral microbiota in ways that are beneficial for the host and could thus be considered a prebiotic substance for the oral microbiota. Additionally, nitrate-reducing isolates can stimulate certain beneficial effects of nitrate metabolism. Nitrate and nitrate-reducing bacteria are thus promising components for future oral care products to prevent or treat oral diseases and this should be further investigated. / Rosier, BT. (2022). Nitrate as a Prebiotic and Nitrate-Reducing Bacteria as Probiotics for Oral Health [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/181578 / TESIS / Compendio