Streptococcus mutans is considered a major causative of tooth decay due to it’s ability to rapidly metabolize carbohydrates such as sucrose. One prominent excreted end product of sucrose metabolism is lactic acid. Lactic acid causes a decrease in the pH of the oral environment with subsequent demineralization of the tooth enamel. Biologically relevant bacteria-induced enamel demineralization was studied.
Optical profiling was used to measure tooth enamel decay with vertical resolution under one nanometer and lateral features with optical resolution as a result of S. mutans biofilm exposure. Comparison measurements were made using AFM.
After 72 hr of biofilm exposure the enamel displayed an 8-fold increase in the observed roughness average, (Ra), as calculated over the entire measured array. Similarly, the average root mean square (RMS) roughness, RRMS, of the enamel before and after biofilm exposure for 3 days displayed a 7-fold increase. Further, the direct effect of chemically induced enamel demineralization using biologically relevant organic acids was shown. Optical profiles of the enamel surface after addition of a 30% lactic acid solution showed a significant alteration in the surface topography with a corresponding increase in respective surface roughness statistics. Similar measurements with 10% citric acid over seconds and minutes give insight into the demineralization process by providing quantitative measures for erosion rates: comparing surface height and roughness as metrics.
The strengths of optical profilometry as an analytical tool for understanding and analyzing biologically relevant processes such as biofilm induced tooth enamel demineralization were demonstrated.
enamel erosion; optical profilometry; biofilm; Streptococcus mutans; enamel demineralization; citric acid; lactic acid; AFM
Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/ remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals creation of an acidic environment favors growth of acid enduring and acid generating species, which causes further reduction in the plaque pH. In this study we developed a prototype antimicrobial peptide capable of achieving high activity exclusively at low environmental pH to target bacterial species like Streptococcus mutans that produce acid and thrive under the low pH conditions detrimental for tooth integrity. The features of clavanin A, a naturally occurring peptide rich in histidine and phenylalanine residues with pH-dependent antimicrobial activity, served as a design basis for these prototype “acid-activated peptides” (AAPs). Employing the major cariogenic species S. mutans as a model system, the two AAPs characterized in this study exhibited a striking pH-dependent antimicrobial activity which correlated well with the calculated charge distribution. This type of peptide represents a potential new way to combat dental caries.
Targeted antimicrobial therapy; pH dependent antimicrobial activity; biofilm; Streptococcus mutans
Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell-surface associated exopolysaccharide (EPS) is essential for S motility and the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base-pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA+ background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.
Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell surface-associated exopolysaccharide (EPS) is essential for S motility and that the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA+ background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.
Since the initial observations of oral bacteria within dental plaque by van Leeuwenhoek using his primitive microscopes in 1680, an event that is generally recognized as the advent of oral microbiological investigation, oral microbiology has gone through phases of “reductionism” and “holism”. From the small beginnings of the Miller and Black period, in which microbiologists followed Koch’s postulates, took the reductionist approach to try to study the complex oral microbial community by analyzing individual species; to the modern era when oral researchers embrace “holism” or “system thinking”, adopt new concepts such as interspecies interaction, microbial community, biofilms, poly-microbial diseases, oral microbiological knowledge has burgeoned and our ability to identify the resident organisms in dental plaque and decipher the interactions between key components has rapidly increased, such knowledge has greatly changed our view of the oral microbial flora, provided invaluable insight into the etiology of dental and periodontal diseases, opened the door to new approaches and techniques for developing new therapeutic and preventive tools for combating oral poly-microbial diseases.
Streptococcus mutans is considered a primary pathogen for human dental caries. Its ability to produce a variety of peptide antibiotics called mutacins may play an important role in its invasion and establishment in the dental biofilm. S. mutans strain UA140 produces two types of mutacins, the lantibiotic mutacin I and the non-lantibitoc mutacin IV. In a previous study, we constructed a random insertional-mutation library to screen for genes involved in regulating mutacin I production, and found 25 genes/operons that have a positive effect on mutacin I production. In this study, we continued our previous work to identify genes that are negatively involved in mutacin I production. By using a high phosphate BHI plate that inhibited mutacin I production of the wild-type, we isolated 77 clones that consistently produced mutacin I under repressive conditions. From the 34 clones that we were able to obtain a sequence, 17 unique genes were identified. These genes encompass a variety of functional groups including the central metabolism, surface binding, sugar transport, and unknown functions. Some of the 17 mutations were further characterized and shown to increase mutacin gene expression during growth when it is usually not expressed in the wild-type. These results further demonstrate an intimate and intricate connection between mutacin production and the overall cellular homeostasis.
Numerous reports have indicated the important role of human normal flora in the prevention of microbial pathogenesis and disease. Evidence suggests that infections at mucosal surfaces result from the outgrowth of subpopulations or clusters within a microbial community and are not linked to one pathogenic organism alone. To preserve the protective normal flora while treating the majority of infective bacteria in the community, a tuneable therapeutic is necessary that can discriminate between benign bystanders and multiple pathogenic organisms. Here we describe the proof-of-principle for such a multitargeted antimicrobial: a multiple-headed specifically-targeted antimicrobial peptide (MH-STAMP). The completed MH-STAMP, M8(KH)-20, displays specific activity against targeted organisms in vitro (Pseudomonas aeruginosa and Streptococcus mutans) and can remove both species from a mixed planktonic culture with little impact against untargeted bacteria. These results demonstrate that a functional, dual-targeted molecule can be constructed from a wide-spectrum antimicrobial peptide precursor.
Antimicrobial peptide; Targeted therapeutic; Streptococcus mutans; Pseudomonas aeruginosa; Peptide synthesis; Novel antibiotic; STAMP; Specifically-targeted antimicrobial peptide; MH-STAMP
Glycosylation is important for a number of biological processes and is perhaps the most abundant and complicated of the known post-translational modifications found on proteins. This work combines two-dimensional polyacrylamide gel electrophoresis (2-DE) and lectin blotting to map the salivary glycome, and mass spectrometry to identity the proteins that are associated with the glycome map. A panel of 15 lectins that recognize six sugar-specific categories was used to visualize the type and extent of glycosylation in saliva from two healthy male individuals. Lectin blots were compared to 2-D gels stained either with Sypro Ruby (protein stain) or Pro-Q Emerald 488 (glycoprotein stain). Each lectin shows a distinct pattern, even those belonging to the same sugar-specific category. In addition, the glycosylation profiles generated from the lectin blots show that most of the salivary proteins are glycosylated and that the pattern is more widespread than is demonstrated by the glycoprotein stained gel. Finally, the co-reactivity between two lectins was measured to determine the glycan structures that are most and least often associated with one another along with the population variation of the lectin reactivity for 66 individuals.
Glycosylation; human whole saliva; lectin blotting; two-dimensional gel electrophoresis
Several small (<25aa) peptides have been designed based on the sequence of the dentin phosphoprotein, one of the major noncollagenous proteins thought to be involved in the mineralization of the dentin extracellular matrix during tooth development. These peptides, consisting of multiple repeats of the tripeptide aspartate-serine-serine (DSS), bind with high affinity to calcium phosphate compounds and, when immobilized, can recruit calcium phosphate to peptide-derivatized polystyrene beads or to demineralized human dentin surfaces. The affinity of binding to hydroxyapatite surfaces increases with the number of (DSS)n repeats, and though similar repeated sequences—(NTT)n, (DTT)n, (ETT)n, (NSS)n, (ESS)n, (DAA)n, (ASS)n, and (NAA)n—also showed HA binding activity, it was generally not at the same level as the natural sequence. Binding of the (DSS)n peptides to sectioned human teeth was shown to be tissue-specific, with high levels of binding to the mantle dentin, lower levels of binding to the circumpulpal dentin, and little or no binding to healthy enamel. Phosphorylation of the serines of these peptides was found to affect the avidity, but not the affinity, of binding. The potential utility of these peptides in the detection of carious lesions, the delivery of therapeutic compounds to mineralized tissues, and the modulation of remineralization is discussed.
Dentin phosphoprotein; Peptide; Mineralization
Unlike many pathogens are foreign invaders, oral “pathogens” such as Streptococcus mutans are part of the “normal” oral microbial flora. While they express certain pathogenic properties, the balance of synergistic and antagonistic interactions determines whether these çommensal pathogens cause damage or not. Recognition of these microbial ecology based pathogeneses argues for new strategies for disease treatment and prevention.
Probiotics, potentially beneficial live bacteria or yeasts, have been used to combat dental caries. This includes the application of S. mutans types that cannot produce acids or other bacteria that interfere with the pathogenic effects of S. mutans. While these approaches show therapeutic effects against S. mutans experimentally, the conversion into commercial products remains a challenge, due to safety and shelf life issues. New high-tech approaches, such as quorum sensing interference of pathogenic bacteria or targeted antimicrobial therapies, offer novel ways to achieve probiotic effects against dental caries.
Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.