Streptococcus sanguinis is a gram-positive, non-motile bacterium native to human mouths. It is the primary cause of endocarditis and is also responsible for tooth decay. Two-component systems (TCSs) are commonly found in bacteria. In response to environmental signals, TCSs may regulate the expression of virulence factor genes. Gene co-expression networks are exploratory tools used to analyze system-level gene functionality. A gene co-expression network consists of gene expression profiles represented as nodes and gene connections, which occur if two genes are significantly co-expressed. An adjacency function transforms the similarity matrix containing co-expression similarities into the adjacency matrix containing connection strengths. Gene modules were determined from the connection strengths, and various network connectivity measures were calculated. S. sanguinis gene expression profile data was loaded for 2272 genes and 14 samples with 3 replicates each. The soft thresholding power β=6 was chosen to maximize R2 while maintaining a high mean number of connections. Nine modules were found. Possible meta-modules were found to be: Module 1: Blue & Green, Module 2: Pink, Module 3: Yellow, Brown & Red, Module 4: Black, Module 5: Magenta & Turquoise. The absolute value of module membership was found to be highly positively correlated with intramodular connectivity. Each of the nine modules were examined. Two methods (intramodular connectivity and TOM-based connectivity followed by network mapping) for identifying candidate hub genes were performed. Most modules provided similar results between the two methods. Similar rankings between the two methods can be considered equivalent and both can be used to detect candidate hub genes. Gene ontology information was unavailable to help select a module of interest. This network analysis would help researchers create new research hypotheses and design experiments for validation of candidate hub genes in biologically important modules.

The gram-positive bacterium Streptococcus sanguinis is a member of human indigenous oral microbialflora and has long been recognized as a key player in the bacterial colonization of the mouth. S. sanguinis is also the most common viridians streptococcal species implicated in infective endocarditis. Although many studies have focused on two-component systems in closely related Streptococcus species such as S. mutans, S. pneumoniae and S. gordonii; the mechanism of the response regulator in S. sanguinis is still unknown. The ability of S. sanguinis to adapt and thrive in hostile environments suggests this bacterium is capable of sensing and responding to various environmental stimuli. The present study clearly demonstrates that a number of RR genes, SSA_0204, SSA_0217, SSA_1810, SSA_1794, and SSA_1842, in S. sanguinis are essential to the recognition and response to various environmental stresses. Results from this study also identified genes SSA_0260, SSA_0261, and SSA-0262, involved in acidic tolerance and suppressed by SSA_0204 response regulator.

Streptococcus sanguinis is a gram-positive facultative anaerobe that is indigenous to the oral cavity and a primary colonizer of the oral cavity. It serves as a tether for the attachment of several oral bacteria that colonize the tooth surface, form dental plaque, and cause periodontal disease. Previous experiments with streptococcal strains have suggested that cellular chain morphology of streptococci may influence the competitiveness, susceptibility to phagocytosis, acidurance, and aggregation of the bacterium. The purpose of this study was to systematically determine gene functions that contribute to cellular chain length morphology in the SK36 strain of S. sanguinis. Gene functions for 2048 mutants were elucidated along with Clusters of Orthologous Groups (COG) functions that may be related to or regulate chain formation and morphology. The COG functions with high ratios of genes involved with chain length morphology per number of total non-essential mutant COG functions were in the following order: Cell division and Chromosome Separation, Defense Mechanisms, and Signal Transduction Mechanisms, and Cell Motility and Secretion. Examination of gene annotations of the 326 mutants involved with chain morphology suggests that cellular chain length is dependent on cell wall division and septation, peptidoglycan synthesis, and cell wall mobility. Some of the genes that contribute to chain length properties may be co-regulated which may suggest that chain length phenotypes are a transcriptionally regulated property that further studies may confirm.

The bacterium Streptococcus sanguinis is a primary member of the human oral microflora and also has been recognized as a key player in the bacterial colonization of the mouth. It is considered the most common viridians streptococcal species implicated in infective endocarditis. In all kingdoms of life, ATP binding cassette (ABC) transporters are essential to many cellular functions. Sequencing of the SK36 genome provided the opportunity to study ABC transporter mutants and their relationship with acidity of the oral environment. Despite numerous studies that have focused on carbohydrate uptake systems in closely related streptococcal species such as S. mutans, S. pneumonia and S. pyogenes, the mechanism of the response of these ABC transporters to acidic conditions in S. sanguinis is still unknown. The capability of S. sanguinis to adapt in these harsh environments suggests this bacterium is capable of responding to various environmental stimuli. The purpose of this study was to examine ABC mutants to identify functions that contribute to acid tolerance in S. sanguinis. This study demonstrates that two acid-sensitive mutant genes, SSA_1507 and SSA_1508, identify genes involved in acid tolerance. The two mutants grew on different sugars and none of them showed a defect in sugar utilization at acid pH. We couldn’t recognize any significant differences in sugar uptake for the two acid sensitive mutants or in mutants of their neighboring genes. Thus, the observed acid sensitivity is not due to a failure to take up any of the common sugars tested. The cytoplasmic pH of S. sanguinis was studied with the fluorescent pH indicator (BCECF) and SK36 was observed to have a wider pH range than either of the two acid-sensitive mutants SSA_1507 or SSA_1508. In these two mutants, intracellular pH was not as well maintained. At all pH values tested, the mutants displayed a lower intracellular pH than the wild type. These observations indicate that the cell membrane of these two mutants is unable to protect the interior components from adverse effects of higher pH values and lower pH values, and prove that these two mutant genes SSA_1507 and SSA_1508 are unable to grow in lower pH values. These results support a role for these ABC transporters in proton pump or export and indicate that the mutants are less able to pump out protons.

Streptococcus sanguinis is an important component of the dental plaque biofilm and is believed to play a beneficial role in the oral cavity. S. sanguinis is also a leading cause of infective endocarditis (IE), a potentially lethal infection of the cardiac valves. S. sanguinis possesses genetic competence, the ability to acquire exogenous DNA into its genome. In the well characterized system of S. pneumoniae, genetic competence requires a quorum sensing system encoded by the early comCDE genes, as well as late genes controlled by the alternate sigma factor, ComX. Previous studies in other streptococcal species have suggested functions for the com regulon apart from DNA uptake. Here we characterized functions of the S. sanguinis com regulon genes in genetic competence, IE virulence, and biofilm formation. Our findings indicated that the early regulatory genes and those under the control of ComX in S. sanguinis play similar roles in genetic competence as their orthologs in other competent streptococci; however the sequence and mechanism of processing of the quorum sensing signal, competence-stimulating peptide, CSP, were determined to be unique. Using a rabbit endocarditis model, we determined that the comCDE and comX genes were not required for virulence, bacteremia, or pathology under a variety of infection conditions. In contrast, examination of biofilms by microscopy and crystal violet staining indicated that S. sanguinis CSP enhanced biofilm formation in a comDE-dependent manner. Deletion of the early com gene SSA_0195 eliminated this effect, while expression of the gene from an inducible promoter increased biofilm formation in the absence of CSP. Deletion of the comX gene resulted in biofilms with increased staining, cell death, and profoundly altered structure. Treatment with DNase I reduced biofilm formation in a com-independent manner. Taken together, these results suggest that expression of SSA_0195 is both necessary and sufficient for CSP-dependent biofilm enhancement, and that the late gene activator, ComX, is required to maintain normal biofilm architecture. Our findings suggest the com regulon of S. sanguinis may be an important determinant of competitiveness in the mouth, where native CSP production may occur at levels sufficient to influence biofilm formation.

The ability of bacteria to assimilate free DNA from the environment is known as competence. Though many studies have focused on competence regulation in Streptococcus pneumoniae and Streptococcus gordonii, Streptococcus sanguinis has yet to be examined. Physiological characterization of competence in S. sanguinis strain SK36 and its comC mutant, JFP41, led to the genome-wide transcriptional analysis of cells induced to competence via addition of competence-stimulating peptide (CSP). A total of 128 genes were induced at least 2-fold, 74 of which were classified as either “early” or “late” based on their induction patterns. Expression patterns were verified using qRT-PCR. This study identified genes not up-regulated in S. pneumoniae or S. gordonii and lays the foundation for bioinformatic studies to identify conserved binding sites upstream from CSP-regulated genes. These results also shed light on the possible existence and identity of expected CSP exporters in S. sanguinis, which have so far eluded discovery.

VERAS, I. N. Análise do perfil morfológico de candida tropicalis durante a formação do biofilme sob influência de metabólitos extracelulares de bactérias do gênero Streptococcus. 2014. 77 f. Dissertação ( Mestrado em Biotecnologia) - Campus de Sobral, Universidade Federal do Ceará, Sobral, 2014. / Submitted by Djeanne Costa (djeannecosta@gmail.com) on 2016-06-29T14:58:00Z No. of bitstreams: 1 2014_dis_insveras.pdf: 2042523 bytes, checksum: adf18ca2b4b9df61f42a9d6cff04ce95 (MD5) / Approved for entry into archive by Djeanne Costa (djeannecosta@gmail.com) on 2016-06-29T14:58:25Z (GMT) No. of bitstreams: 1 2014_dis_insveras.pdf: 2042523 bytes, checksum: adf18ca2b4b9df61f42a9d6cff04ce95 (MD5) / Made available in DSpace on 2016-06-29T14:58:25Z (GMT). No. of bitstreams: 1 2014_dis_insveras.pdf: 2042523 bytes, checksum: adf18ca2b4b9df61f42a9d6cff04ce95 (MD5) Previous issue date: 2014 / Bacteria and fungi are found together in a multitude of environments, and particularly in the form of biofilm adherent species which interact through various signaling mechanisms. In the oral cavity, Candida species coexist with numerous bacterial species, and evidence suggests that bacteria can modulate biofilm formation as well as induce the formation of hyphae. Thus, to characterize such interactions are essential to the understanding of pathogenesis of diseases and possibly the discovery of new therapeutic strategies. In this sense, the main objective of this study was to evaluate, in vitro, the mechanisms involved in the interaction between bacteria Streptococcus oralis, Streptococcus sanguinis, and Streptococcus parasanguinis yeast Candida tropicalis. In the study the effect of extracellular metabolites present in the supernatant of C. tropicalis (SCT) on the pre-formed biofilm (6H) S. oralis, S. sanguinis and S. parasanguinis was analyzed. The effect of extracellular metabolites of S. oralis (SSO), S. sanguinis (SSS) and S. parasanguinis (SSP) on planktonic growth and biofilm / Bactérias e fungos são encontrados juntos em uma infinidade de ambientes e, particularmente, na forma de biofilme, onde as espécies aderentes interagem através de diversos mecanismos de sinalização. Na cavidade oral, espécies de Candida coexistem com inúmeras espécies bacterianas, e evidências sugerem que bactérias podem modular a formação de biofilme, bem como induzir a formação de hifas e pseudo-hifas. Assim, caracterizar tais interações é essencial para o entendimento da patogênese das doenças e, possivelmente, a descoberta de novas estratégias terapêuticas. Nesse sentido, o objetivo principal deste trabalho foi avaliar, in vitro, os mecanismos envolvidos na interação entre as bactérias Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis e a levedura Candida tropicalis. No estudo foi analisado o efeito dos metabólitos extracelulares presentes no sobrenadante de C. tropicalis (SCT) sobre o biofilme pré-formado (6h) de S. oralis, S. sanguinis e S. parasanguinis. Também foi avaliado o efeito dos metabólitos extracelulares de S. oralis (SSO), S. sanguinis (SSS) e S. parasanguinis (SSP) sobre o crescimento planctônico, formação de biofilme e capacidade de filamentação de C. tropicalis, utilizando apenas o sobrenadante da cultura de cada um dos estreptococos em diferentes concentrações (100, 50 e 25%). Além disso, foi analisado o efeito dos metabólitos extracelulares dos estreptococos sobre o biofilme pré-formado (6h) de C. tropicalis. Para verificar o efeito dos metabólitos extracelulares foram utilizados dois métodos: o método turbidimétrico que se baseia na leitura da densidade óptica (OD) das suspensões celulares e a coloração cristal violeta (CV) que permite a quantificação indireta da formação de biofilme através da coloração com cristal violeta. Em seguida, foram examinadas as características dos biofilmes, formados por 24 horas, através da análise por microscopia óptica comum. Os resultados referentes ao biofilme foram submetidos ao ANOVA com pós-teste Bonferroni, com diferença estatística de p<0,01. Nossos resultados sugerem que substâncias solúveis produzidas por S. oralis, S. sanguinis e S. parasanguinis induzem a formação de hifas de C. tropicalis sem interferir no crescimento planctônico. Além de diminuir drasticamente o desenvolvimento do biofilme dessa levedura quando em contato com SSS e SSP. Tal fato reforça a ideia de que existe grande heterogeneidade dentro de biofilmes polimicrobianos, especialmente entre leveduras e bactérias. E que o resultado dessa interação depende das condições as quais estes micro-organismos serão submetidos.

Objetivo: Evaluar el efecto inhibitorio de la terapia fotodinámica (TPD) con Diodo Emisor de Luz (LED) azul sobre Streptococcus mutans y Streptococcus sanguinis en presencia y ausencia de riboflavina (E - 101). Materiales y métodos: Se realizaron cuatro tratamientos en presencia y ausencia de la exposición de luz LED azul y riboflavina al 0.5% sobre Streptococcus mutans y Streptococcus sanguinis. Las bacterias fueron cultivadas en medio BHI y la unidad de medida utilizada fue las unidades formadoras de colonias (UFC/ml). Resultados: La fotoactivación con luz LED azul a 40 segundos no tuvo efecto inhibitorio sobre S. mutans y S. sanguinis. Sin embargo, al realizar la terapia fotodinámica en presencia de riboflavina, se observó que el crecimiento bacteriano fue menor (p<0.05). Asimismo, se identificó que la viabilidad bacteriana de S. sanguinis es menor que la de S. mutans, con un 40% y 66% respectivamente. Conclusiones: Se concluye que la riboflavina tiene un efecto inhibitorio significativo sobre la viabilidad bacteriana de S. mutans y S. sanguinis. / Objective: To evaluate the inhibitory effect of photodynamic therapy (TPD) with blue Light Emitting Diode (LED) on Streptococcus mutans and Streptococcus sanguinis in presence and absence of riboflavin (E-101). Materials and methods: Four treatments were performed in presence and absence of blue LED and riboflavin (0.5%) exposure on Streptococcus mutans and Streptococcus sanguinis. The bacteria were grown in BHI medium and the unit of measurement used was the colony forming units (CFU / ml). Results: Photoactivation with blue LED light at 40 seconds had no inhibitory effect on S. mutans and S. sanguinis. However, when performing photodynamic therapy in presence of riboflavin, it was observed that bacterial growth was lower (p <0.05). Likewise, it was identified that bacterial viability of S. sanguinis is lower than S. mutans, with 40% and 66% respectively. Conclusions: It is concluded that riboflavin has a significant inhibitory effect on the bacterial viability of S. mutans and S. sanguinis. / Tesis

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / BactÃrias e fungos sÃo encontrados juntos em uma infinidade de ambientes e, particularmente, na forma de biofilme, onde as espÃcies aderentes interagem atravÃs de diversos mecanismos de sinalizaÃÃo. Na cavidade oral, espÃcies de Candida coexistem com inÃmeras espÃcies bacterianas, e evidÃncias sugerem que bactÃrias podem modular a formaÃÃo de biofilme, bem como induzir a formaÃÃo de hifas e pseudo-hifas. Assim, caracterizar tais interaÃÃes à essencial para o entendimento da patogÃnese das doenÃas e, possivelmente, a descoberta de novas estratÃgias terapÃuticas. Nesse sentido, o objetivo principal deste trabalho foi avaliar, in vitro, os mecanismos envolvidos na interaÃÃo entre as bactÃrias Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis e a levedura Candida tropicalis. No estudo foi analisado o efeito dos metabÃlitos extracelulares presentes no sobrenadante de C. tropicalis (SCT) sobre o biofilme prÃ-formado (6h) de S. oralis, S. sanguinis e S. parasanguinis. TambÃm foi avaliado o efeito dos metabÃlitos extracelulares de S. oralis (SSO), S. sanguinis (SSS) e S. parasanguinis (SSP) sobre o crescimento planctÃnico, formaÃÃo de biofilme e capacidade de filamentaÃÃo de C. tropicalis, utilizando apenas o sobrenadante da cultura de cada um dos estreptococos em diferentes concentraÃÃes (100, 50 e 25%). AlÃm disso, foi analisado o efeito dos metabÃlitos extracelulares dos estreptococos sobre o biofilme prÃ-formado (6h) de C. tropicalis. Para verificar o efeito dos metabÃlitos extracelulares foram utilizados dois mÃtodos: o mÃtodo turbidimÃtrico que se baseia na leitura da densidade Ãptica (OD) das suspensÃes celulares e a coloraÃÃo cristal violeta (CV) que permite a quantificaÃÃo indireta da formaÃÃo de biofilme atravÃs da coloraÃÃo com cristal violeta. Em seguida, foram examinadas as caracterÃsticas dos biofilmes, formados por 24 horas, atravÃs da anÃlise por microscopia Ãptica comum. Os resultados referentes ao biofilme foram submetidos ao ANOVA com pÃs-teste Bonferroni, com diferenÃa estatÃstica de p<0,01. Nossos resultados sugerem que substÃncias solÃveis produzidas por S. oralis, S. sanguinis e S. parasanguinis induzem a formaÃÃo de hifas de C. tropicalis sem interferir no crescimento planctÃnico. AlÃm de diminuir drasticamente o desenvolvimento do biofilme dessa levedura quando em contato com SSS e SSP. Tal fato reforÃa a ideia de que existe grande heterogeneidade dentro de biofilmes polimicrobianos, especialmente entre leveduras e bactÃrias. E que o resultado dessa interaÃÃo depende das condiÃÃes as quais estes micro-organismos serÃo submetidos. / Bacteria and fungi are found together in a multitude of environments, and particularly in the form of biofilm adherent species which interact through various signaling mechanisms. In the oral cavity, Candida species coexist with numerous bacterial species, and evidence suggests that bacteria can modulate biofilm formation as well as induce the formation of hyphae. Thus, to characterize such interactions are essential to the understanding of pathogenesis of diseases and possibly the discovery of new therapeutic strategies. In this sense, the main objective of this study was to evaluate, in vitro, the mechanisms involved in the interaction between bacteria Streptococcus oralis, Streptococcus sanguinis, and Streptococcus parasanguinis yeast Candida tropicalis. In the study the effect of extracellular metabolites present in the supernatant of C. tropicalis (SCT) on the pre-formed biofilm (6H) S. oralis, S. sanguinis and S. parasanguinis was analyzed. The effect of extracellular metabolites of S. oralis (SSO), S. sanguinis (SSS) and S. parasanguinis (SSP) on planktonic growth and biofilm formation capacity filamentation of C. tropicalis was also evaluated using only the supernatant culturing each of streptococci in different concentrations (100, 50 and 25%). Furthermore, the effect of extracellular metabolites of streptococci on the pre-formed biofilm (6h) of C. tropicalis were analyzed. To verify the effect of extracellular metabolites two methods were used: The turbidimetric method based on the reading of the optical density (OD) of cell suspension and coloring crystal violet (CV) which permits indirect quantification of the biofilm formation by staining with crystal violet. Then were examined characteristics of biofilms formed by 24 hours, through the analysis simple optical microscope. The results for the biofilm were subjected to ANOVA with Bonferroni post-test, with a statistical difference of p<0.01.Our results suggest that soluble substances produced by S. oralis, S. sanguinis and S. parasanguinis induce the formation of hyphae of C. tropicalis without interfering with planktonic growth.In addition to dramatically decrease biofilm development of oral yeast when in contact with SSP and SSS. This reinforces the idea that there is great heterogeneity within polymicrobial biofilms, especially between yeasts and bacteria. And the result of this interaction depends on the conditions which these micro-organisms will be subjected.

Streptococcus sanguinis is a leading cause of infective endocarditis. Bacterial adhesion to platelets is likely important in the pathogenesis of infective endocarditis. Bacterial cell wall-anchored (Cwa) proteins may mediate this adhesion. To begin to test this hypothesis, S. sanguinis adhesion to platelets was examined in vitro. The requirement of 12 Cwa proteins for S. sanguinis-platelet adhesion was individually assessed, measuring adhesion of purified platelets to polystyrene wells coated with S. sanguinis strain SK36 or 12 isogenic Cwa protein mutants. Significantly fewer platelets adhered to wells coated with one mutant strain, VT1614. However, results of a whole-cell enzyme-linked immunosorbent assay (ELISA) showed that 8 mutants, including VT1614, adhered in significantly lower numbers to wells than did SK36. After accounting for unequal bacterial numbers, we determined there was no significant difference in platelet adhesion among the strains. This suggests that none of the Cwa proteins examined were required for S. sanguinis-platelet adhesion.