• Characterization of the Interaction between Candida albicans and Streptococcus mutans

      Mutawalli, Khalid Hassan; Jabra-Rizk, Mary Ann (2014)
      The oral microbial communities are some of the most complex microbial floras in the human body. Occurrence of oral disease results from disturbance of the equilibrium of this ecosystem. Dental caries or tooth decay is the most common oral disease particularly in children characterized by irreversible destruction of the tooth mediated by demineralization of dental surfaces. These processes are the result of interactions between the various microbial species embedded in the biofilm formed on tooth surface known as dental plaque. These complex interactions between metabolically active microbial species cause fluctuations in pH ultimately resulting in dissolution of the dental hard tissues and formation of carious lesions. The bacterial species Streptococcus mutans has long been considered the etiologic agent of caries, however recent in vitro evidence seem to indicate a role for the fungal species Candida albicans in mediating cariogenic development via its physical and metabolic interactions with S. mutans. However, in depth investigations are required to determine mechanistically precise details of adhesion and signaling under conditions of co-existence. To that end, the goal of this proposal is to characterize the interaction between C. albicans and S. mutans using biologically relevant in vitro model systems. Specifically, we aim to demonstrate that the strong co-adherence of these diverse oral pathogens to each other and to oral surfaces results in the formation of mature biofilms, a pre-requisite for the development of dental caries. Importantly, as design of effective therapies to treat caries has been a challenge, this project also aimed to develop a novel antimicrobial bioadhesive hydrogel formulation for use as oral topical agent with targeted action geared towards blocking microbial adhesion to surfaces and in turn prevention and eradication of biofilms. The accomplishment of the work proposed in this project will provide crucial insights into the potential role of C albicans in the development of dental caries, an area of research that is yet to be explored. The ultimate goal is to contribute to our understanding of the various factors and conditions that play a role in microbial colonization and the progression of colonization to infection. Such crucial information will have important clinical implications as it aids in the identification and the design of novel therapeutic strategies aimed at the prevention and/or treatment of dental caries and oral infections in general. Significantly, the novel findings generated strongly indicate that the presence of C. albicans in the oral environment should be considered as an additional factor in evaluating risks of caries.
    • Clinical and Therapeutic Implications of Biofilm-associated Fungal-Bacterial Interactions: Candida albicans and Staphylococcus aureus

      Kong, Eric Fei; Jabra-Rizk, Mary Ann (2017)
      Biofilm-associated polymicrobial infections, particularly those involving fungi and bacteria, are responsible for significant morbidity and mortality and tend to be challenging to treat. Candida albicans and Staphylococcus aureus are considered leading microbial pathogens primarily due to their ability to form biofilms on indwelling medical devices. However, the impact of mixed species biofilm growth on therapy remains largely understudied. Here, we demonstrate that in mixed biofilms, C. albicans confers S. aureus significantly enhanced tolerance to antimicrobials mediated by impairment of drug diffusion through the biofilm matrix composed of C. albicans secreted fungal cell wall polysaccharides. Further, we demonstrated a key role for the C. albicans secreted quorum sensing molecule farnesol in modulating S. aureus response to therapy via modulation of drug efflux pumps. Importantly, farnesol was also found to inhibit the production of the carotenoid pigment staphyloxanthin, an important virulence factor in S. aureus involved in protection against oxidative stress. The significance of this inhibition was established through demonstration of the enhanced susceptibility of the depigmented S. aureus cells to oxidants and macrophage phagocytic killing. Theoretical computational binding models indicated that the mechanism for pigment inhibition may be due to farnesol competitively inhibiting the binding of farnesyl diphosphate to CrtM, an essential enzyme in the biosynthesis of staphyloxanthin. To begin to explore the implications of this fungal-bacterial interactions in a host, we utilized a clinically relevant subcutaneous catheter mouse model where central venous catheter segments infected in vitro with S. aureus and C. albicans individually or in combination, are implanted subcutaneously in mice. Vancomycin treatment of mice with infected catheters demonstrated that where therapy significantly reduced S. aureus recovery from catheters in mice infected with S. aureus, in co-infected animals, vancomycin had no impact on S. aureus recovery, underscoring the therapeutic implications of mixed biofilm-associated infections. The combined findings from this work provide lacking mechanistic insights into interspecies interactions in biofilm with great clinical relevance. The understanding of the interspecies dynamics of signaling central to the persistence and antimicrobial resistance of polymicrobial infections will greatly aid in overcoming limitations of current therapies and in designing novel therapeutic strategies to target these complex infections. Therefore, future research should focus on designing animal model systems to study the role of secreted quorum sensing molecules in mediating these interactions in vivo, and the impact of these interactions on pathogenesis.
    • Farnesol-Induced Apoptosis in Oral Squamous Carcinoma Cells is Mediated by MRP1 Extrusion and Depletion of Intracellular Glutathione

      Intapa, Chaidan; Jabra-Rizk, Mary Ann (2013)
      Farnesol is a key intermediate in the sterol biosynthesis pathway in eukaryotic cells that has exhibited significant anti-cancer and antimicrobial activity. We have shown that farnesol triggers apoptosis in oral squamous carcinoma cells (OSCCs) and in the fungal pathogen Candida albicans via a classical apoptotic process. However, the exact mechanism of farnesol cytotoxicity in eukaryotic cells has not been fully elucidated. In the cell, hydrophobic xenobiotics conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. This process results in the formation of glutathione S-conjugates which act as substrates for export by ATP-binding cassette transporters (ABC transporter) and are extruded from the cell. This study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with multidrug resistance-associated protein 1 (MRP1) - mediated extrusion of glutathione-farnesol conjugates and oxidized glutathione results in total glutathione depletion, oxidative stress and ultimately cell death. The combined findings demonstrated that farnesol exposure resulted in significant decrease in intracellular glutathione levels concomitant with intracellular Reactive oxygen species (ROS) accumulation and decrease in cell viability. However, addition of exogenous glutathione maintained intracellular levels and enhanced cell viability. Furthermore, gene and protein expression studies demonstrated significant up-regulation of MRP1 in cells treated with farnesol. However, MRP1 blocking and monoclonal antibody specific inhibition of MRP1 enhanced cell tolerance to farnesol. This is the first study describing the involvement of MRP1-mediated glutathione efflux as a mechanism for farnesol-induced apoptosis in OSCCs. Understanding of the mechanisms underlying farnesol-cytotoxicity in eukaryotic cells may lead to the development of this redox-cycling agent as an alternative chemotherapeutic target.
    • Impaired Histatin-5 Levels and Salivary Antimicrobial Activity against C. albicans in HIV Infected Individuals

      Al Thunayyan, Awdah Anwar; Jabra-Rizk, Mary Ann (2013)
      HIV-infected individuals constitute a population highly susceptible to opportunistic infections particularly oral candidiasis (OC) caused by the most pathogenic human fungal species Candida albicans. The susceptibility to OC is enhanced under reduced CD4+ T-cells however local host defenses may also play a key role. Host-produced salivary antimicrobial peptides are considered to be an important part of the host innate immune system involved in protection of the oral cavity against colonization and infection by microbial species. Histatin-5 (Hst-5) specifically has exhibited potent anti-candidal properties in vitro. However, its importance in protecting the oral mucosa against candidal colonization and importantly, its contribution to the observed enhanced susceptibility of HIV+ individuals to candidiasis has not been previously investigated most likely due to the lack of feasible and sensitive methods for measuring salivary Hst-5 concentrations. To that end, the goal of this study was to develop a novel immunoassay to accurately measure and analyze salivary Hst-5 levels within the context of HIV infection and oral candidal colonization in order to validate the hypothesis that salivary Hst-5 levels are compromised in HIV+ individuals. The results from these studies demonstrated that salivary Hst-5 levels are significantly (60%) decreased in HIV+ individuals compared to healthy subjects concomitant with enhanced candidal prevalence. The findings generated from this project provided new insights into oral innate immune defense mechanisms and the enhanced susceptibility of HIV+ individuals to oral candidiasis.
    • In Vitro and in Vivo Characterization of Candida albicans and Streptococcus mutans Interactions

      Khoury, Zaid; Jabra-Rizk, Mary Ann; 0000-0001-9596-3560 (2019)
      The oral cavity is a complex environment harboring diverse microbial species that often co-exist within biofilms formed on oral surfaces. Within a biofilm, inter species interactions can be synergistic in that the presence of one organism generates a niche for another enhancing colonization. Among these species are the opportunistic fungal pathogen Candida albicans and the bacterial species Streptococcus mutans, the causative microorganisms strongly linked to the development of oral candidiasis and dental caries, respectively. Recent studies have reported the enhanced prevalence of C. albicans in children with early childhood caries indicating that this fungal-bacterial interaction may have clinical implications. In this study, we aimed to elucidate and characterize this interaction between these diverse species. Specifically, we designed in vitro and in vivo studies to validate the hypothesis that the presence of C. albicans in the oral cavity augments S. mutans colonization, potentially mediating dental caries development. Using various C. albicans mutant strains and a GFP-tagged S. mutans, metabolic viability and fluorescent biofilm assays were performed to assess S. mutans recovery from mixed biofilms and to elucidate the mechanisms of interactions. Additionally, to visualize the architecture of formed biofilms, confocal scanning laser fluorescent and electron scanning microscopy were used. Importantly, a clinically relevant mouse model of oral co-infection was developed to demonstrate C. albicans mediated enhanced S. mutans colonization in a host. The findings demonstrated significantly higher recovery of S. mutans from biofilms with C. albicans in vitro. Images revealed a high bacterial affinity to C. albicans, and secreted fungal cell wall polysaccharides were identified as the key factor mediating biofilm formation, particularly mannans. Importantly, analyses of harvested tissue demonstrated significantly higher S. mutans recovery from teeth of co-infected mice compared to mice infected only with S. mutans. Collectively, the findings strongly indicate that the presence of C. albicans in the oral environment may impact the development of dental caries and should be considered as a factor in evaluating the risk of caries. Results obtained in this thesis will support future studies using animal models of dental caries to further characterize this relationship in a closely related model in our laboratory.
    • Interactions Between Candida albicans and Streptococcus mutans in the Oral Cavity

      Al Saud, Hadeel; Jabra-Rizk, Mary Ann (2017)
      The oral microbial communities are some of the most complex microbial floras in the human body. Occurrence of oral disease results from disturbance of the equilibrium of this ecosystem. Dental caries or tooth decay is the most common oral disease particularly in children characterized by irreversible destruction of the tooth structure. These processes are the result of interactions between the various microbial species embedded in the biofilm formed on tooth surface known as dental plaque. The bacterial species Streptococcus mutans has long been considered the etiologic agent of caries, however our in vitro findings demonstrated that S. mutans avidly adheres to C. albicans forming mixed biofilms on abiotic surfaces. As mucosal biofilms are implicated in a wide variety of infections, it is imperative that we understand the implications of the interactions between colonizing microbial species, as they co-exist on host tissue surfaces. To that end, in this project, we developed a clinically relevant mouse model of oral co-infection to investigate the interactions between C. albicans and S. mutans in a host. In this model, experimental animals were orally infected with C. albicans then they were exposed to S. mutans through drinking water, whereas control animals received S. mutans. Four days post infection with C. albicans, animals were euthanized and tongues and teeth were harvested and assessed for microbial burden as well as tissue histopathology analysis. The combined findings from the study demonstrated that in the presence of C. albicans, a significantly higher numbers of S. mutans were recovered from tongues and teeth of mice compared to mice infected with only S. mutans. Histopathology of tongue sections revealed massive invasion of tissue by the invasive C. albicans hyphae along with accumulation of large amounts of S. mutans. In contrast, in mice with S. mutans alone, minimal amount of S. mutans was seen adhering to the tongues. The combined findings from this study indicate a potential role for C. albicans in mediating cariogenic development via its physical interactions with S. mutans. Therefore, future studies should focus on developing novel therapeutic strategies geared towards prevention of adhesion receptors to block adhesive microbial reactions.
    • Protection of the Oral Mucosa by Salivary Histatin-5 against Candidiasis in a Murine Model of Oral Infection

      Al Shaye, Sara Yousef; Jabra-Rizk, Mary Ann (2011)
      The oral cavity is a primary target for opportunistic infections, particularly oral candidiasis caused by Candida albicans. A commensal fungus commonly colonizing mucosal surfaces, under conditions of immune dysfunction, C. albicans can become a pathogen causing recurrent infections. Yet, the role of host oral innate immunity in the development of candidiasis has not been fully elucidated. Specifically, the host salivary antimicrobial peptide histatin-5 (Hst-5) has been proposed to play a protective role in the oral cavity against C. albicans. However, investigations demonstrating its efficacy on oral tissue are lacking. To this end, in this study, an ex vivo and in vivo murine model of oral infection were developed. Viable C. albicans counts and histopathological analyses demonstrated a significant protective effect for Hst-5 on mouse oral tissue against C. albicans. More importantly, host saliva exerted a comparable anticandidal effect. However, this effect was neutralized upon treatment of saliva with proteases and C. albicans, previously shown to degrade Hst-5, indicating that Hst-5 is likely the salivary component responsible for the observed protection. Combined, the findings from this study demonstrate for the first time the efficacy of salivary Hst-5 in protecting host oral tissue against C. albicans infection, thereby affirming the therapeutic potential of this natural host peptide.
    • Streptococcus mutans Bacterial Adherence on Lithium Disilicate Porcelain Specimens

      Vo, Diane; Masri, Radi, 1975-; Jabra-Rizk, Mary Ann (2015)
      Streptococcus mutans as it pertains to dental and oral health is significant for its role as the primary etiologic factor of caries. While primary caries results from initiation of lesions in virgin tooth structure, secondary caries is a significant contributing factor to the replacement of dental restorations. Caries formation is directly related to plaque accumulation, which is mediated by bacteria adhesion to intraoral surfaces. In the case of the restored tooth, bacteria must adhere to the restorative material, particularly along margins in order to cause recurrent pathology. A material that has recently come into much favor is lithium disilicate, a glass based system with fillers in a homogenous glass. Lithium disilicate restorations can be either (1) pressed or (2) milled to fabricate inlays, onlays, veneers or single unit crowns. These restorations can be full-contour, or may be cut back and subsequently modified with (3) veneering fluorapatite, or (4) glazed. With respect to bacterial adhesion to restorative surfaces, the overwhelming factor is surface roughness. The threshold for this effect has previously been found to be 0.2 µm Ra value, above which there was a positive correlation between surface roughness and plaque retention. Specimens were fabricated for each of the four preparation types per manufacturer's recommendations and incubated with S. mutans UA159 wild-type. Biofilms adherent to specimens were then sonicated, redispersed, and plated for quantification. Results were tested with an analysis of variance (ANOVA). Significant differences that were found were further analyzed by Tukey's Honestly Significant Difference (HSD) test. Pearson's r was also be used to evaluate the relationship between surface roughness and biofilm accumulation. A p-value of ≤ 0.05 was considered significant. Surface roughness, as quantified by Ra values, indicated that Press and CAD groups were not significantly different from one another, but were significantly lower than that of ZirPress/Ceram, which was lower than surface roughness of the Ceram Glaze group (F = 513.898, p ≤ 0.0005). Similarly, CFUs/ml for the CAD and Press groups were significantly lower than the ZirPress/Ceram group, which were also significantly lower than those of the Ceram Glaze group (F = 201.721, p ≤ 0.0005). A strong positive association was also seen between surface roughness and biofilm accumulation (r = .95). Many factors, such as caries risk, presence of other restorations, and individual patient hygiene, influence whether these differences in surface roughness and biofilm accumulation become clinically relevant to the formation of caries. The present study has demonstrated that different preparations vary in their surface roughness and biofilm accumulation measurements, and that these differences in surface quality are associated with bacterial adherence.
    • Therapeutic Evaluation of a Novel Topical Antimicrobial Formulation against Candida-Associated Denture Stomatitis in an Experimental Rat Model

      Sultan, Ahmed; Jabra-Rizk, Mary Ann; 0000-0001-5286-4562 (2019)
      Candida-associated denture stomatitis (DS), caused by the fungal species Candida albicans, is the most common manifestation of oral candidiasis and is prevalent in up to 70% of denture wearers. DS tends to be a persistent and recurrent oral condition as a consequence of the ability of C. albicans to adhere to denture material and invade associated palatal tissue. There are currently no effective therapeutic strategies targeting DS, and despite antifungal therapy, infection is often re-established after treatment ceases. Therefore, it has become crucial to identify novel therapeutic approaches. Antimicrobial peptides have attracted significant attention as candidates for drug development due to their potent antimicrobial and anti-inflammatory properties, lack of toxicity and lack of development of drug resistance. Specifically, histatin-5 (Hst-5), naturally produced and secreted by host salivary glands, has demonstrated potent antifungal activity, including against strains resistant to traditional antifungals. However, our laboratory has previously demonstrated vulnerability for Hst-5 to proteolysis by C. albicans secreted proteolytic enzymes at specific amino acid residues. Therefore, to generate a resistant derivative of Hst-5, we engineered a variant (K11R-K17R) with substitutions in the amino acid residues at the cleavage sites. The new peptide proved to be more stable, and unlike the native Hst-5, resistant to proteolysis by C. albicans proteases. Importantly, for clinical application, we designed a polymer-based bioadhesive hydrogel as a delivery system for the peptide and developed a therapeutic formulation specifically designed for oral topical application. The potency of the new formulation in inhibiting C. albicans adherence and biofilm formation on denture acrylic material was demonstrated in vitro indicating a potential clinical applicability against DS. To that end, using 3D digital design and printing technology, we engineered and fabricated a universal intraoral device that was successfully used in the animals to develop clinical disease mimicking DS as in humans. Using the novel animal model, we established the clinical utility of the formulation for the prevention of biofilm formation on denture device and DS development. Importantly, in addition to DS, the formulation can also be used for treatment of other forms of candidiasis as well as serve in augmenting host natural immune defenses.