The present study was focused on evaluating the potential of Emblica officinalis against cariogenic properties of Streptococcus mutans, a causative microorganism for caries. The effect of crude extract and ethanolic fraction from Emblica officinalis fruit was analysed against S. mutans. The sub-MIC concentrations of crude and ethanolic fraction of E. officinalis were evaluated for its cariogenic properties such as acid production, biofilm formation, cell-surface hydrophobicity, glucan production, sucrose-dependent and independent adherence. Its effect on biofilm architecture was also investigated with the help of confocal and scanning electron microscopy (SEM). Moreover, expression of genes involved in biofilm formation was also studied by quantitative RT- PCR. This study showed 50% reduction in adherence at concentrations 156 µg/ and 312.5 µg/ml of crude extract and ethanolic fraction respectively. However, the biofilm was reduced to 50% in the presence of crude extract (39.04 µg/ml) and ethanolic fraction (78.08 µg/ml). Furthermore, effective reduction was observed in the glucan synthesis and cell surface hydrophobicity. The qRT-PCR revealed significant suppression of the genes involved in its virulence. Confocal and scanning electron microscopy clearly depicted the obliteration of biofilm structure with reference to control. Hence, this study reveals the potential of E. officinalis fruit extracts as an alternative and complementary medicine for dental caries by inhibiting the virulence factors of Streptococcus mutans.
Streptococcus mutans is a major cariogenic bacterium. It has adapted to the biofilm lifestyle, which is essential for pathogenesis of dental caries. We aimed to identify small molecules that can inhibit cariogenic S. mutans and to discover lead structures that could give rise to therapeutics for dental caries. In this study, we screened a focused small-molecule library of 506 compounds. Eight small molecules which inhibited S. mutans at a concentration of 4 μM or less but did not affect cell growth or biofilm formation of commensal bacteria, represented by Streptococcus sanguinis and Streptococcus gordonii, in monospecies biofilms were identified. The active compounds share similar structural properties, which are characterized by a 2-aminoimidazole (2-AI) or 2-aminobenzimidazole (2-ABI) subunit. In multispecies biofilm models, the most active compound also inhibited cell survival and biofilm formation of S. mutans but did not affect commensal streptococci. This inhibitor downregulated the expression of six biofilm-associated genes, ftf, pac, relA, comDE, gbpB, and gtfB, in planktonic S. mutans cells, while it downregulated the expression of only ftf, pac, and relA in the biofilm cells of S. mutans. The most potent compound also inhibited production of two key adhesins of S. mutans, antigen I/II and glucosyltransferase (GTF). However, the compound did not alter the expression of the corresponding genes in both S. sanguinis and S. gordonii, indicating that it possesses a selective inhibitory activity against S. mutans.
Microbial cell-cell interactions in the oral flora are believed to play an integral role in the development of dental plaque and ultimately, its pathogenicity. The effects of other species of oral bacteria on biofilm formation and virulence gene expression by Streptococcus mutans, the primary etiologic agent of dental caries, were evaluated using a dual-species biofilm model and RealTime-PCR analysis.
As compared to mono-species biofilms, biofilm formation by S. mutans was significantly decreased when grown with Streptococcus sanguinis, but was modestly increased when co-cultivated with Lactobacillus casei. Co-cultivation with S. mutans significantly enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the respective mono-species biofilms. RealTime-PCR analysis showed that expression of spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that synthesizes α1,6-linked glucan polymers from sucrose and starch carbohydrates) and gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was decreased significantly when S. mutans were co-cultivated with L. casei. Similar results were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased. No significant differences were observed in expression of the selected genes when S. mutans was co-cultivated with S. sanguinis.
These results suggest that the presence of specific oral bacteria differentially affects biofilm formation and virulence gene expression by S. mutans.
The association of specific bioactive flavonoids and terpenoids with fluoride can modulate the development of cariogenic biofilms by simultaneously affecting the synthesis of exopolysaccharides (EPS) and acid production by Streptococcus mutans, which enhanced the cariostatic effectiveness of fluoride in vivo. In the present study, we further investigated whether the biological actions of combinations of myricetin (flavonoid), tt-farnesol (terpenoid) and fluoride can influence the expression of specific genes of S. mutans within biofilms and their structural organization using real-time PCR and confocal fluorescence microscopy.
Twice-daily treatment (one-minute exposure) during biofilm formation affected the gene expression by S. mutans both at early (49-h) and later (97-h) stages of biofilm development. Biofilms treated with combination of agents displayed lower mRNA levels for gtfB and gtfD (associated with exopolysaccharides synthesis) and aguD (associated with S. mutans acid tolerance) than those treated with vehicle-control (p < 0.05). Furthermore, treatment with combination of agents markedly affected the structure-architecture of S. mutans biofilms by reducing the biovolume (biomass) and proportions of both EPS and bacterial cells across the biofilm depth, especially in the middle and outer layers (vs. vehicle-control, p < 0.05). The biofilms treated with combination of agents were also less acidogenic, and had reduced amounts of extracellular insoluble glucans and intracellular polysaccharides than vehicle-treated biofilms (p < 0.05).
The data show that the combination of naturally-occurring agents with fluoride effectively disrupted the expression of specific virulence genes, structural organization and accumulation of S. mutans biofilms, which may explain the enhanced cariostatic effect of our chemotherapeutic approach.
Streptococcus mutans is implicated as a major etiological agent in human dental caries, and one of the important virulence properties of this organism is its ability to form biofilms (dental plaque) on tooth surfaces. We examined the role of autoinducer-2 (AI-2) on S. mutans biofilm formation by constructing a GS-5 luxS-null mutant. Biofilm formation by the luxS mutant in 0.5% sucrose defined medium was found to be markedly attenuated compared to the wild type. Scanning electron microscopy also revealed that biofilms of the luxS mutant formed larger clumps in sucrose medium compared to the parental strain. Therefore, the expression of glucosyltransferase genes was examined and the gtfB and gtfC genes, but not the gtfD gene, in the luxS mutant were upregulated in the mid-log growth phase. Furthermore, we developed a novel two-compartment system to monitor AI-2 production by oral streptococci and periodontopathic bacteria. The biofilm defect of the luxS mutant was complemented by strains of S. gordonii, S. sobrinus, and S. anginosus; however, it was not complemented by S. oralis, S. salivarius, or S. sanguinis. Biofilm formation by the luxS mutant was also complemented by Porphyromonas gingivalis 381 and Actinobacillus actinomycetemcomitans Y4 but not by a P. gingivalis luxS mutant. These results suggest that the regulation of the glucosyltransferase genes required for sucrose-dependent biofilm formation is regulated by AI-2. Furthermore, these results provide further confirmation of previous proposals that quorum sensing via AI-2 may play a significant role in oral biofilm formation.
Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxSSm) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.
Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxS in biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutans is a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxS for S. mutans was identified using the data available in the S. mutans genome project (http://www.genome.ou.edu/smutans.html). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxS negative phenotype of Escherichia coli DH5α. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxS gene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxS mutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxS mutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S. mutans.
Streptococcus mutans (S. mutans), harboring biofilm formation, considered as a main aetiological factor of dental caries. Gtf genes play an important role in S. mutans biofilm formation. The purpose of this study was to investigate the effect of Lactobacillus acidophilus–derived biosurfactant on S. mutans biofilm formation and gtfB/C expression level (S. mutans standard strain ATCC35668 and isolated S. mutans strain (22) from dental plaque).
The Lactobacillus acidophilus (L. acidophilus) DSM 20079 was selected as a probiotic strain to produce biosurfactant. The FTIR analysis of its biosurfactant showed that it appears to have a protein-like component. Due to the release of such biosurfactants, L. acidophilus was able to interfere in the adhesion and biofilm formation of the S. mutans to glass slide. It also could make streptococcal chains shorter. Using realtime RT-PCR quantitation method made it clear that gtfB and gtfC gene expression were decreased in the presence of L. acidophilus–derived biosurfactant fraction.
Several properties of S. mutans cells (the surface properties, biofilm formation, adhesion ability and gene expression) were changed after L. acidophilus- derived biosurfactant treatment. It is also concluded that biosurfacant treatment can provide an optional way to control biofilm development. On the basis of our findings, we can suggest that the prepared biosurfactant may interfere with adhesion processes of S. mutans to teeth surfaces, provided additional evaluation produce satisfactory results.
Biofilm formation; biosurfactant; gtfB/C; Lactobacillus acidophilus; real-time RT PCR; Streptococcus mutans
A biofilm is a complex community of microorganisms that develop on surfaces in diverse environments. The thickness of the biofilm plays a crucial role in the physiology of the immobilized bacteria. The most cariogenic bacteria, mutans streptococci, are common inhabitants of a dental biofilm community. In this study, DNA-microarray analysis was used to identify differentially expressed genes associated with the thickness of S. mutans biofilms.
Comparative transcriptome analyses indicated that expression of 29 genes was differentially altered in 400- vs. 100-microns depth and 39 genes in 200- vs. 100-microns biofilms. Only 10 S. mutans genes showed differential expression in both 400- vs. 100-microns and 200- vs. 100-microns biofilms. All of these genes were upregulated.
As sucrose is a predominant factor in oral biofilm development, its influence was evaluated on selected genes expression in the various depths of biofilms. The presence of sucrose did not noticeably change the regulation of these genes in 400- vs. 100-microns and/or 200- vs. 100-microns biofilms tested by real-time RT-PCR.
Furthermore, we analyzed the expression profile of selected biofilm thickness associated genes in the luxS- mutant strain. The expression of those genes was not radically changed in the mutant strain compared to wild-type bacteria in planktonic condition. Only slight downregulation was recorded in SMU.2146c, SMU.574, SMU.609, and SMU.987 genes expression in luxS- bacteria in biofilm vs. planktonic environments.
These findings reveal genes associated with the thickness of biofilms of S. mutans. Expression of these genes is apparently not regulated directly by luxS and is not necessarily influenced by the presence of sucrose in the growth media.
Streptococcus mutans, the primary etiological agent of human dental caries, has developed multiple mechanisms to colonize and form biofilms on the tooth surface. The brpA gene codes for a predicted surface-associated protein with apparent roles in biofilm formation, autolysis, and cell division. In this study, we used two models to further characterize the biofilm-forming characteristics of a BrpA-deficient mutant, strain TW14. Compared to those of the parent strain, UA159, TW14 formed long chains and sparse microcolonies on hydroxylapatite disks but failed to accumulate and form three-dimensional biofilms when grown on glucose as the carbohydrate source. The biofilm formation defect was also readily apparent by confocal laser scanning microscopy when flow cells were used to grow biofilms. When subjected to acid killing at pH 2.8 for 45 min, the survival rate of strain TW14 was more than 1 log lower than that of the wild-type strain. TW14 was at least 3 logs more susceptible to killing by 0.2% hydrogen peroxide than was UA159. The expression of more than 200 genes was found by microarray analysis to be altered in cells lacking BrpA (P < 0.01). These results suggest that the loss of BrpA can dramatically influence the transcriptome and significantly affects the regulation of acid and oxidative stress tolerance and biofilm formation in S. mutans, which are key virulence attributes of the organism.
Biofilm formation by Streptococcus mutans is considered as its principal virulence factor, causing dental caries. Mutants of S. mutans defective in biofilm
formation were generated and analyzed to study the collective role of proteins in its formation. Mutants were characterized on the basis of adherence to
saliva-coated surface, and biofilm formation. The confocal laser microscopy and scanning electron microscopy images showed that the control biofilms
had cluster of cells covered by layer of exo-polysaccharide while the biofilms of mutants were thin and spaced. Two-dimensional protein electrophoresis
data analysis identified 57 proteins that are either up (44 proteins) or down (13 proteins) regulated. These data points to the importance of up and down
regulated proteins in the formation of biofilm in Streptococcus mutans.
The importance of Streptococcus mutans in the etiology of dental caries has been well documented. However, there is growing recognition that the cariogenic potential of dental plaque may be determined by the composite interactions of the total plaque bacteria rather than solely the virulence properties of a single organism. This study will examine how the interactions of S. mutans with other biofilm constituents may influence the cariogenicity of plaque samples.
In order to begin to investigate the effects of nonmutans streptococci on the cariogenic potential of S. mutans, we have examined the effects of Streptococcus gordonii on the virulence properties of the former organisms. These studies have indicated that S.gordonii can attenuate several potential virulence properties of S. mutans including bacteriocin production, genetic transformation, and biofilm formation. Therefore, modulation of the interactions between plaque bacteria might be a novel approach for attenuating dental caries initiation.
The complete genome sequence of Streptococcus mutans, a bacterial pathogen commonly associated with human dental caries, was published in 2002. The streamlined genome (2.03Mb) revealed an organism that was well adapted to its obligately host-associated existence in multispecies biofilms on tooth surfaces; a dynamic environment that undergoes rapid and substantial environmental fluctuations. However, S. mutans lacks many of the sensing systems and alternative sigma factors that bacteria often use to coordinate gene expression in response to stress and changes in their environment. Over the past seven years, functional genomics and proteomics have enhanced our understanding of how S. mutans has integrated the stress regulon and global transcriptional regulators to integrate responses to environmental fluctuations with modulation of virulence in a way that ensures persistence in the oral cavity and capitalizes on conditions that are favorable for the development of dental caries. Here, we highlight advances on dissection of the stress regulon of S. mutans and its intimate interrelationship with pathogenesis.
Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (P<0.05). Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other organisms. Our data provide insights about how S. mutans optimizes its metabolism and adapts/survives within the mixed-species community in response to a dynamically changing environment. This reflects the intricate physiological processes linked to expression of virulence by this bacterium within complex biofilms.
Bacteria can detect, transmit, and react to signals from the outside world by using two-component systems (TCS) and serine-threonine kinases and phosphatases. Streptococcus mutans contains one serine-threonine kinase, encoded by pknB. A gene encoding a serine-threonine phosphatase, pppL, is located upstream of pknB. In this study, the phenotypes of pknB and pppL single mutants and a pknB pppL double mutant were characterized. All mutants exhibited a reduction in genetic transformability and biofilm formation, showed abnormal cell shapes, grew slower than the wild-type strain in several complex media, and exhibited reduced acid tolerance. The mutants had reduced cariogenic capacity but no significant defects in colonization in a rat caries model. Whole-genome transcriptome analysis revealed that a pknB mutant showed reduced expression of genes involved in bacteriocin production and genetic competence. Among the genes that were differentially regulated in the pknB mutant, several were likely to be involved in cell wall metabolism. One such gene, SMU.2146c, and two genes encoding bacteriocins were shown to also be downregulated in a vicK mutant, which encodes a sensor kinase involved in the response to oxidative stress. Collectively, the results lead us to speculate that PknB may modulate the activity of the two-component signal transduction systems VicKR and ComDE. Real-time reverse transcriptase PCR (RT-PCR) showed that genes downregulated in the pknB mutant were upregulated in the pppL mutant, indicating that PppL serves to counteract PknB.
Bacteria form complex and highly elaborate surface adherent communities known as biofilms which are held together by a self-produced extracellular matrix. We have previously shown that by adopting a biofilm mode of existence in vivo, the Gram negative bacterial pathogens Bordetella bronchiseptica and Bordetella pertussis are able to efficiently colonize and persist in the mammalian respiratory tract. In general, the bacterial biofilm matrix includes polysaccharides, proteins and extracellular DNA (eDNA). In this report, we investigated the function of DNA in Bordetella biofilm development. We show that DNA is a significant component of Bordetella biofilm matrix. Addition of DNase I at the initiation of biofilm growth inhibited biofilm formation. Treatment of pre-established mature biofilms formed under both static and flow conditions with DNase I led to a disruption of the biofilm biomass. We next investigated whether eDNA played a role in biofilms formed in the mouse respiratory tract. DNase I treatment of nasal biofilms caused considerable dissolution of the biofilm biomass. In conclusion, these results suggest that eDNA is a crucial structural matrix component of both in vitro and in vivo formed Bordetella biofilms. This is the first evidence for the ability of DNase I to disrupt bacterial biofilms formed on host organs.
The Streptococcus mutans atlA gene encodes an autolysin required for biofilm maturation and biogenesis of a normal cell surface. We found that the capacity to form biofilms by S. mutans, one of the principal causative agents of dental caries, was dramatically impaired by growth of the organism in an aerated environment and that cells exposed to oxygen displayed marked changes in surface protein profiles. Inactivation of the atlA gene alleviated repression of biofilm formation in the presence of oxygen. Also, the formation of long chains, a characteristic of AtlA-deficient strains, was less evident in cells grown with aeration. The SMu0629 gene is immediately upstream of atlA and encodes a product that contains a C-X-X-C motif, a characteristic of thiol-disulfide oxidoreductases. Inactivation of SMu0629 significantly reduced the levels of AtlA protein and led to resistance to autolysis. The SMu0629 mutant also displayed an enhanced capacity to form biofilms in the presence of oxygen compared to that of the parental strain. The expression of SMu0629 was shown to be under the control of the VicRK two-component system, which influences oxidative stress tolerance in S. mutans. Disruption of vicK also led to inhibition of processing of AtlA, and the mutant was hyperresistant to autolysis. When grown under aerobic conditions, the vicK mutant also showed significantly increased biofilm formation compared to strain UA159. This study illustrates the central role of AtlA and VicK in orchestrating growth on surfaces and envelope biogenesis in response to redox conditions.
To identify the genes regulated by RR11, the regulator of the Streptococcus mutans HK/RR11 two-component system.
Methods and Results
The S. mutans RR11-encoding gene was inactivated, and the effects of gene disruption on the cell's ability to form biofilms under stresses and acquire extracellular DNA were tested. Biofilm was reduced in cells lacking RR11 following exposure to oxidative stress. RR11-defective cells showed approx. 20-fold reduction in transformation efficiency. Microarray used to decipher the RR11-regulated genes in biofilm showed that approx. 5% of the UA159 genome underwent a significant change in expression. RR11 was found to regulate 174 genes, including genes involved in competence, stress-response and cell division.
Target genes controlled by RR11during biofilm growth have been identified by a comparison of transcriptional profiles between an RR11 defective mutant and the parental strain. The results demonstrated that RR11 is involved in the control of diverse cellular processes, including the formation of biofilm under oxidative stress and development of genetic competence.
Significance and Impact of the Study
The regulator of HK/RR11 system controls a large regulon and is an important regulator involved in stress response during S. mutans biofilm growth enabling the survival and persistence of its progeny in the microbial community.
biofilm; competence; DNA microarray; stress response; two-component system
We isolated a single chemical compound from A. continentalis and identified it to be kaurenoic acid (KA) and investigated the influence of anticariogenic properties. Inhibitory effects of KA on cariogenic properties such as growth, acid production, biofilm formation, and the adherence of S. mutans were evaluated. Furthermore, real-time PCR analysis was performed to evaluate the influence of KA on the genetic expression of virulence factors. KA significantly inhibited the growth and acid production of S. mutans at 2–4 μg/mL and 4 μg/mL of KA, respectively. Furthermore, the adherence onto S-HAs was inhibited at 3-4 μg/mL of KA and biofilm formation was significantly inhibited when treated with 3 μg/mL KA and completely inhibited at 4 μg/mL. Also, the inhibitory effect of KA on biofilm formation was confirmed by SEM. In confocal laser scanning microscopy, bacterial viability gradually decreased by KA in a dose dependent manner. Real-time PCR analysis showed that the expressions of gtfB, gtfC, gbpB, spaP, brpA, relA, and vicR were significantly decreased in S. mutans when it was treated with KA. These results suggest that KA from A. continentalis may be a useful agent for inhibiting the cariogenic properties of S. mutans.
Streptococcus mutans has been strongly implicated as the principal etiological agent in dental caries. One of the important virulence properties of these organisms is their ability to form biofilms known as dental plaque on tooth surfaces. Since the roles of sucrose and glucosyltransferases in S. mutans biofilm formation have been well documented, we focused our attention on sucrose-independent factors. We have initially identified several mutants that appear to be defective in biofilm formation on abiotic surfaces by an insertional inactivation mutagenesis strategy applied to S. mutans. A total of 27 biofilm-defective mutants were isolated and analyzed in this study. From these mutants, three genes were identified. One of the mutants was defective in the Bacillus subtilis lytR homologue. Another of the biofilm-defective mutants isolated was a yulF homologue, which encodes a hypothetical protein of B. subtilis whose function in biofilm formation is unknown. The vast majority of the mutants were defective in the comB gene required for competence. We therefore have constructed and examined comACDE null mutants. These mutants were also found to be attenuated in biofilm formation. Biofilm formation by several other regulatory gene mutants were also characterized using an in vitro biofilm-forming assay. These results suggest that competence genes as well as the sgp and dgk genes may play important roles in S. mutans biofilm formation.
The tight control of autolysis by Streptococcus mutans is critical for proper virulence gene expression and biofilm formation. A pair of dicistronic operons, SMU.575/574 (lrgAB) and SMU.1701/1700 (designated cidAB), encode putative membrane proteins that share structural features with the bacteriophage-encoded holin family of proteins, which modulate host cell lysis during lytic infection. Analysis of S. mutans lrg and cid mutants revealed a role for these operons in autolysis, biofilm formation, glucosyltransferase expression and oxidative stress tolerance. Expression of lrgAB was repressed during early exponential phase and was induced over 1000-fold as cells entered late exponential phase, whereas cidAB expression declined from early to late exponential phase. A two-component system encoded immediately upstream of lrgAB (LytST) was required for activation of lrgAB expression, but not for cid expression. In addition to availability of oxygen, glucose levels were revealed to affect lrg and cid transcription differentially and significantly, probably through CcpA (carbon catabolite protein A). Collectively, these findings demonstrate that the Cid/Lrg system can affect several virulence traits of S. mutans, and its expression is controlled by two major environmental signals, oxygen and glucose. Moreover, cid/lrg expression is tightly regulated by LytST and CcpA.
We have used microarray analysis to study the transcriptome of the bacterial pathogen Bordetella bronchiseptica over the course of five time points representing distinct stages of biofilm development. The results suggest that B. bronchiseptica undergoes a coordinately regulated gene expression program similar to a bacterial developmental process. Expression and subsequent production of the genes encoding flagella, a classical Bvg− phase phenotype, occurs and is under tight regulatory control during B. bronchiseptica biofilm development. Using mutational analysis, we demonstrate that flagella production at the appropriate stage of biofilm development, i.e. production early subsequently followed by repression, is required for robust biofilm formation and maturation. We also demonstrate that flagella are necessary and enhance the initial cell-surface interactions, thereby providing mechanistic information on the initial stages of biofilm development for B. bronchiseptica. Biofilm formation by B. bronchiseptica involves the production of both Bvg-activated and Bvg-repressed factors followed by the repression of factors that inhibit formation of mature biofilms.
Staphylococcus aureus is an important pathogen that forms biofilms. The global regulator sarA is essential for biofilm formation. Since the modulator of sarA (msa) is required for full expression of sarA and regulates several virulence factors, we examined the capacity of the msa mutant to form biofilm.
We found that mutation of msa results in reduced expression of sarA in biofilm and that the msa mutant formed a weak and unstable biofilm. The msa mutant is able to adhere to surfaces and begins to form biofilm but fails to mature indicating that the defect of the msa mutant biofilm is in the accumulation stage but not in primary adhesion.
The msa gene plays an important role in biofilm development which is likely due to its role in modulating the expression of sarA. This finding is significant because it identifies a new gene that plays a role in the development of biofilm.
Biofilms are a protected niche for microorganisms, where they are safe from antibiotic treatment and can create a source of persistent infection. Using two clinically relevant Candida albicans biofilm models formed on bioprosthetic materials, we demonstrated that biofilm formation proceeds through three distinct developmental phases. These growth phases transform adherent blastospores to well-defined cellular communities encased in a polysaccharide matrix. Fluorescence and confocal scanning laser microscopy revealed that C. albicans biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements. In both models, antifungal resistance of biofilm-grown cells increased in conjunction with biofilm formation. The expression of agglutinin-like (ALS) genes, which encode a family of proteins implicated in adhesion to host surfaces, was differentially regulated between planktonic and biofilm-grown cells. The ability of C. albicans to form biofilms contrasts sharply with that of Saccharomyces cerevisiae, which adhered to bioprosthetic surfaces but failed to form a mature biofilm. The studies described here form the basis for investigations into the molecular mechanisms of Candida biofilm biology and antifungal resistance and provide the means to design novel therapies for biofilm-based infections.
Interactions between salivary agglutinin and the adhesin P1 of Streptococcus mutans contribute to bacterial aggregation and mediate sucrose-independent adherence to tooth surfaces. We have examined biofilm formation by S. mutans UA159, and derivative strains carrying mutations affecting the localization or expression of P1, in the presence of fluid-phase or adsorbed saliva or salivary agglutinin preparations. Whole saliva- and salivary agglutinin-induced aggregation of S. mutans was adversely affected by the loss of P1 and sortase (SrtA) but not by the loss of trigger factor (RopA). Fluid-phase salivary agglutinin and, to a lesser extent, immobilized agglutinin inhibited biofilm development by S. mutans in the absence of sucrose, and whole saliva was more effective at decreasing biofilm formation than salivary agglutinin. Inhibition of biofilm development by salivary agglutinin was differently influenced by particular mutations, with the P1-deficient strain displaying a greater inhibition of biofilm development than the SrtA- or RopA-deficient strains. As expected, biofilm-forming capacities of all strains in the presence of salivary preparations were markedly enhanced in the presence of sucrose, although biofilm formation by the mutants was less efficient than that by the parental strain. Aeration strongly inhibited biofilm development, and the presence of salivary components did not restore biofilm formation in aerated conditions. The results disclose a potent ability of salivary constituents to moderate biofilm formation by S. mutans through P1-dependent and P1-independent pathways.