Glycosyltransferase-Mediated Biofilm Matrix Dynamics and Virulence of Streptococcus mutans.

Streptococcus mutans is a key cariogenic bacterium responsible for the initiation of tooth decay. Biofilm formation is a crucial virulence property. We discovered a putative glycosyltransferase, SMU_833, in S. mutans capable of modulating dynamic interactions between two key biofilm matrix components, glucan and extracellular DNA (eDNA). The deletion of smu_833 decreases glucan and increases eDNA but maintains the overall biofilm biomass. The decrease in glucan is caused by a reduction in GtfB and GtfC, two key enzymes responsible for the synthesis of glucan. The increase in eDNA was accompanied by an elevated production of membrane vesicles, suggesting that SMU_833 modulates the release of eDNA via the membrane vesicles, thereby altering biofilm matrix constituents. Furthermore, glucan and eDNA were colocalized. The complete deletion of gtfBC from the smu_833 mutant significantly reduced the biofilm biomass despite the elevated eDNA, suggesting the requirement of minimal glucans as a binding substrate for eDNA within the biofilm. Despite no changes in overall biofilm biomass, the mutant biofilm was altered in biofilm architecture and was less acidic in vitro Concurrently, the mutant was less virulent in an in vivo rat model of dental caries, demonstrating that SMU_833 is a new virulence factor. Taken together, we conclude that SMU_833 is required for optimal biofilm development and virulence of S. mutans by modulating extracellular matrix components. Our study of SMU_833-modulated biofilm matrix dynamics uncovered a new target that can be used to develop potential therapeutics that prevent and treat dental caries.IMPORTANCE Tooth decay, a costly and painful disease affecting the vast majority of people worldwide, is caused by the bacterium Streptococcus mutans The bacteria utilize dietary sugars to build and strengthen biofilms, trapping acids onto the tooth's surface and causing demineralization and decay of teeth. As knowledge of our body's microbiomes increases, the need for developing therapeutics targeted to disease-causing bacteria has arisen. The significance of our research is in studying and identifying a novel therapeutic target, a dynamic biofilm matrix that is mediated by a new virulence factor and membrane vesicles. The study increases our understanding of S. mutans virulence and also offers a new opportunity to develop effective therapeutics targeting S. mutans In addition, the mechanisms of membrane vesicle-mediated biofilm matrix dynamics are also applicable to other biofilm-driven infectious diseases.

AgTC and AgETL: open-source tools to enhance data collection and management for plant science research.

Advancements in phenotyping technology have enabled plant science researchers to gather large volumes of information from their experiments, especially those that evaluate multiple genotypes. To fully leverage these complex and often heterogeneous data sets (i.e. those that differ in format and structure), scientists must invest considerable time in data processing, and data management has emerged as a considerable barrier for downstream application. Here, we propose a pipeline to enhance data collection, processing, and management from plant science studies comprising of two newly developed open-source programs. The first, called AgTC, is a series of programming functions that generates comma-separated values file templates to collect data in a standard format using either a lab-based computer or a mobile device. The second series of functions, AgETL, executes steps for an Extract-Transform-Load (ETL) data integration process where data are extracted from heterogeneously formatted files, transformed to meet standard criteria, and loaded into a database. There, data are stored and can be accessed for data analysis-related processes, including dynamic data visualization through web-based tools. Both AgTC and AgETL are flexible for application across plant science experiments without programming knowledge on the part of the domain scientist, and their functions are executed on Jupyter Notebook, a browser-based interactive development environment. Additionally, all parameters are easily customized from central configuration files written in the human-readable YAML format. Using three experiments from research laboratories in university and non-government organization (NGO) settings as test cases, we demonstrate the utility of AgTC and AgETL to streamline critical steps from data collection to analysis in the plant sciences.

Intraspecies interactions of Streptococcus mutans impact biofilm architecture and virulence determinants in childhood dental caries.

Early childhood dental caries (ECC) is the most common chronic disease among children, especially among low socioeconomic populations. Streptococcus mutans is most frequently associated with initiation of ECC. Although many studies report children with multiple S. mutans strains (i.e., genotypes) have greater odds of developing ECC, studies investigating intraspecies interactions in dental caries are lacking. This study investigates the impact of intraspecies interactions on cariogenic and fitness traits of clinical S. mutans isolates using in vitro and in vivo approaches. Association analysis evaluated if presence of multiple S. mutans genotypes within the first year of colonization was associated with caries. Initially, clinical S. mutans isolates from 10 children were evaluated. S. mutans strains (G09 and G18, most prevalent) isolated from one child were used for subsequent analysis. Biofilm analysis was performed for single and mixed cultures to assess cariogenic traits, including biofilm biomass, intra-polysaccharide, pH, and glucan. Confocal laser scanning microscopy (CLSM) and time-lapse imaging were used to evaluate spatial and temporal biofilm dynamics, respectively. A Drosophila model was used to assess colonization in vivo. Results showed the mean biofilm pH was significantly lower in co-cultured biofilms versus monoculture. Doubling of S. mutans biofilms was observed by CLSM and in vivo colonization in Drosophila for co-cultured S. mutans. Individual strains occupied specific domains in co-culture and G09 contributed most to increased co-culture biofilm thickness and colonization in Drosophila. Biofilm formation and acid production displayed distinct signatures in time-lapsed experiments. This study illuminates that intraspecies interactions of S. mutans significantly impacts biofilm acidity, architecture, and colonization.IMPORTANCEThis study sheds light on the complex dynamics of a key contributor to early childhood dental caries (ECC) by exploring intraspecies interactions of different S. mutans strains and their impact on cariogenic traits. Utilizing clinical isolates from children with ECC, the research highlights significant differences in biofilm architecture and acid production in mixed versus single genotype cultures. The findings reveal that co-cultured S. mutans strains exhibit increased cell density and acidity, with individual strains occupying distinct domains. These insights, enhanced by use of time-lapsed confocal laser scanning microscopy and a Drosophila model, offer a deeper understanding of ECC pathogenesis and potential avenues for targeted interventions.

Intraspecies interactions of Streptococcus mutans impact biofilm architecture and virulence determinants in childhood dental caries.

Early childhood dental caries (ECC) is the most common chronic disease among children with a heavy disease burden among low socioeconomic populations. Streptococcus mutans is most frequently associated with initiation of ECC. Many studies report children with multiple S. mutans strains (i.e., genotypes) having greater odds of developing ECC, studies investigating intraspecies interactions in dental caries are lacking. In this study, the impact of intraspecies interactions on cariogenic and fitness traits of clinical S. mutans isolates are investigated using in-vitro and in-vivo approaches. Initially clinical S. mutans isolates of 10 children from a longitudinal epidemiological study were evaluated. S. mutans strains (G09 and G18, most prevalent) isolated from one child were used for subsequent analysis. Association analysis was used to determine if presence of multiple S. mutans genotypes within the first-year of colonization was associated with caries. Biofilm analysis was performed for single and mixed cultures to assess cariogenic traits, including biofilm biomass, intra-polysaccharide, pH, and glucan. Confocal Laser Scanning Microscopy (CLSM) and time-lapse imaging were used to evaluate spatial and temporal biofilm dynamics, respectively. A Drosophila model was used to assess colonization in-vivo. Mean biofilm pH was significantly lower in co-cultured biofilms as compared with monoculture biofilms. Doubling of S. mutans in-vitro biofilms was observed by CLSM and in-vivo colonization in Drosophila for co-cultured S. mutans. Individual strains occupied specific domains in co-culture and G09 contributed most to increased co-culture biofilm thickness and colonization in Drosophila. Biofilm formation and acid production displayed distinct signatures in time-lapsed experiments.

Quantitative Proteomics Uncovers the Interaction between a Virulence Factor and Mutanobactin Synthetases in Streptococcus mutans.

Streptococcus mutans, the primary etiological agent of tooth decay, has developed multiple adhesion and virulence factors which enable it to colonize and compete with other bacteria. The putative glycosyltransferase SMU_833 is important for the virulence of S. mutans by altering the biofilm matrix composition and cariogenicity. In this study, we further characterized the smu_833 mutant by evaluating its effects on bacterial fitness. Loss of SMU_833 led to extracellular DNA-dependent bacterial aggregation. In addition, the mutant was more susceptible to oxidative stress and less competitive against H2O2 producing oral streptococci. Quantitative proteomics analysis revealed that SMU_833 deficiency resulted in the significant downregulation of 10 proteins encoded by a biosynthetic gene cluster responsible for the production of mutanobactin, a compound produced by S. mutans which helps it survive oxidative stress. Tandem affinity purification demonstrated that SMU_833 interacts with the synthetic enzymes responsible for the production of mutanobactin. Similar to the smu_833 mutant, the deletion of the mutanobactin gene cluster rendered the mutant less competitive against H2O2-producing streptococci. Our studies revealed a new link between SMU_833 virulence and mutanobactin, suggesting that SMU_833 represents a new virulent target that can be used to develop potential anticaries therapeutics.IMPORTANCEStreptococcus mutans is the major bacterium associated with dental caries. In order to thrive on the highly populated tooth surface and cause disease, S. mutans must be able to protect itself from hydrogen peroxide-producing commensal bacteria and compete effectively against the neighboring microbes. S. mutans produces mutacins, small antimicrobial peptides which help control the population of competing bacterial species. In addition, S. mutans produces a peptide called mutanobactin, which offers S. mutans protection against oxidative stress. Here, we uncover a new link between the putative glycosyltransferase SMU_833 and the mutanobactin-synthesizing protein complex through quantitative proteomic analysis and a tandem-affinity protein purification scheme. Furthermore, we show that SMU_833 mediates bacterial sensitivity to oxidative stress and bacterial ability to compete with commensal streptococci. This study has revealed a previously unknown association between SMU_833 and mutanobactin and demonstrated the importance of SMU_833 in the fitness of S. mutans.

Race and gender differences in how sense of belonging influences decisions to major in STEM.

BACKGROUND: Women and students of color are widely underrepresented in most STEM fields. In order to investigate this underrepresentation, we interviewed 201 college seniors, primarily women and people of color, who either majored in STEM or started but dropped a STEM major. Here we discuss one section of the longer interview that focused on students' sense of belonging, which has been found to be related to retention. In our analysis, we examine the intersections of race and gender with students' sense of belonging, a topic largely absent from the current literature. RESULTS: We found that white men were most likely to report a sense of belonging whereas women of color were the least likely. Further, we found that representation within one's STEM sub-discipline, namely biology versus the physical sciences, impacts sense of belonging for women. Four key factors were found to contribute to sense of belonging for all students interviewed: interpersonal relationships, perceived competence, personal interest, and science identity. CONCLUSIONS: Our findings indicate that students who remain in STEM majors report a greater sense of belonging than those who leave STEM. Additionally, we found that students from underrepresented groups are less likely to feel they belong. These findings highlight structural and cultural features of universities, as well as STEM curricula and pedagogy, that continue to privilege white males.

ZnO nanoparticle preparation route influences surface reactivity, dissolution and cytotoxicity.

ZnO nanoparticles (nZnO) are commonly used in nanotechnology applications despite their demonstrated cytotoxicity against multiple cell types. This underscores the significant need to determine the physicochemical properties that influence nZnO cytotoxicity. In this study, we analyzed six similarly sized nZnO formulations, along with SiO2-coated nZnO, bulk ZnO and ZnSO4 as controls. Four of the nZnO samples were synthesized using various wet chemical methods, while three employed high-temperature flame spray pyrolysis (FSP) techniques. X-ray diffraction and optical analysis demonstrated the lattice parameters and electron band gap of the seven nZnO formulations were similar. However, electrophoretic mobility measures, hydrodynamic size, photocatalytic rate constants, dissolution potential, reactive oxygen species (ROS) production and, more importantly, the cytotoxicity of the variously synthesized nZnO towards Jurkat leukemic and primary CD4+ T cells displayed major differences. Surface structure analysis using FTIR, X-ray photoelectron spectroscopies (XPS) and dynamic light scattering (DLS) revealed significant differences in the surface-bound chemical groups and the agglomeration tendencies of the samples. The wet chemical nZnO, with higher cationic surface charge, faster photocatalytic rates, increased extracellular dissolution and ROS generation demonstrated greater cytotoxicity towards both cell types than those made with FSP techniques. Furthermore, principal component analysis (PCA) suggests that the synthesis procedure employed influences which physicochemical properties contribute more to the cytotoxic response. These results suggest that the synthesis approach results in unique surface chemistries and can be a determinant of cellular cytotoxicity and oxidative stress responses.