Lantibiotics Produced by Oral Inhabitants as a Trigger for Dysbiosis of Human Intestinal Microbiota.

Lantibiotics are a type of bacteriocin produced by Gram-positive bacteria and have a wide spectrum of Gram-positive antimicrobial activity. In this study, we determined that Mutacin I/III and Smb (a dipeptide lantibiotic), which are mainly produced by the widespread cariogenic bacterium Streptococcus mutans, have strong antimicrobial activities against many of the Gram-positive bacteria which constitute the intestinal microbiota. These lantibiotics also demonstrate resistance to acid and temperature. Based on these features, we predicted that lantibiotics may be able to persist into the intestinal tract maintaining a strong antimicrobial activity, affecting the intestinal microbiota. Saliva and fecal samples from 69 subjects were collected to test this hypothesis and the presence of lantibiotics and the composition of the intestinal microbiota were examined. We demonstrate that subjects possessing lantibiotic-producing bacteria in their oral cavity exhibited a tendency of decreased species richness and have significantly reduced abundance of the phylum Firmicutes in their intestinal microbiota. Similar results were obtained in the fecal microbiota of mice fed with S. mutans culture supernatant containing the lantibiotic bacteriocin Mutacin I. These results showed that lantibiotic bacteriocins produced in the oral cavity perturb the intestinal microbiota and suggest that oral bacteria may be one of the causative factors of intestinal microbiota dysbiosis.

Pharmacological, Toxicological, and Dose Range Assessment of OG716, a Novel Lantibiotic for the Treatment of Clostridium difficile-Associated Infection.

Lantibiotics present an attractive scaffold for the development of novel antibiotics. We report here a novel lantibiotic for the treatment of Clostridium difficile infection. The lead compounds were selected from a library of over 700 single- and multiple-substitution variants of the lantibiotic mutacin 1140 (MU1140). The best performers in vitro and in vivo were further used to challenge Golden Syrian hamsters orally in a Golden Syrian hamster model of Clostridium difficile-associated disease (CDAD) in a dose-response format, resulting in the selection of OG716 as the lead compound. This lantibiotic was characterized by a 50% effective dose of 23.85 mg/kg of body weight/day (10.97 µmol/kg/day) in this model. Upon oral administration of the maximum feasible dose (≥1,918 mg/kg/day), no observable toxicities or side effects were noted, and no effect on intestinal motility was observed. Compartmentalization to the gastrointestinal tract was confirmed. MU1140-derived variants offer a large pipeline for the development of novel antibiotics for the treatment of several indications and are particularly attractive considering their novel mechanism of action. Based on the currently available data, OG716 has an acceptable profile for further development for the treatment of CDAD.

Preclinical evaluation of the maximum tolerated dose and toxicokinetics of enteric-coated lantibiotic OG253 capsules.

Clostridium difficile associated disease (CDAD) is the leading infectious cause of antibiotic-associated diarrhea and colitis in the United States. Both the incidence and severity of CDAD have been increased over the past two decades. We evaluated the maximum tolerated dose (MTD) and toxicokinetics of OG253, a novel lantibiotic in development for the treatment of CDAD. OG253 was orally administered to Wistar Han rats as enteric-coated capsules in a one-day dose escalation study, followed by a seven-day repeated dose toxicokinetics study. All three doses of OG253 (6.75, 27 and 108 mg/day) were generally well-tolerated with no treatment-related clinical signs, alterations in body weight or food consumption in both one-day acute tolerability and seven-days repeated dose tolerability and toxicokinetics study. OG253 capsule administration neither significantly alter the weight of organs nor affect the hematology, coagulation, clinical biochemistry parameters and urine pH compared to placebo capsule administered rats. LC-MS/MS analysis did not detect OG253 in the plasma, indicating that OG253 is not absorbed into the blood from the rat gastrointestinal tract. Glandular atrophy of the rectal mucosa was noticed in two out of six rats administered with a high dose of OG253. Surprisingly, we found that OG253 treatment significantly lowered both serum cholesterol and triglyceride levels in both sexes of rats. Overall, there was a 29.8 and 61.38% decrease in the serum cholesterol and triglyceride levels, respectively as compared to placebo-treated rats. The well-tolerated high dose of OG253 (425.7 mg/kg/day) is recommended as the MTD for safety and efficacy studies. Further preclinical study is needed to evaluate the safety profile of OG253 under longer exposure.

Insight into the Effect of Small RNA srn225147 on Mutacin IV in Streptococcus mutans.

Streptococcus mutans (S. mutans) is a serious microbe causing dental caries. Mutacin IV is an effective bacteriocin produced by S. mutans to antagonize numerous non-mutans streptococcal species. However, the posttranscriptional regulation of mutacin IV remains unclear. This study aimed to analyze the effect of small RNA srn225147 on mutacin IV. The functional prediction suggested that srn225147 is involved in the production of mutacin IV, an important secondary metabolite. According to RNAhybrid and RNAPredator prediction, the mutacin IV formation-associated gene comD is a target of srn225147. We further analyzed the roles of srn225147 and comD in 20 S. mutans clinical strains with high production of mutacin IV (High-IV group) and lacking mutacin IV (None-IV group). Levels of comD expression were significantly higher in the High-IV group, whereas the Non-IV group showed relatively higher expression of srn225147, with a negative correlation observed between srn225147 and comD. Moreover, compared to the mimic negative control (NC) group, comD expression was decreased at 400-fold srn225147 overexpression but increased at approximately 1400-fold overexpression. Although the production of mutacin IV in the 1400-fold change srn225147 mimic group was larger than that in the 400-fold change mimic group, there was no significant difference in the production of mutacin IV between the srn225147 mimic group and mimic NC group. These results indicate that srn225147 has a two-way regulation effect on the expression of comD but that its regulation in the production of mutacin IV is weak.

Characterization of the Trehalose Utilization Operon in Streptococcus mutans Reveals that the TreR Transcriptional Regulator Is Involved in Stress Response Pathways and Toxin Production.

Streptococcus mutans, the organism most frequently associated with the development of dental caries, is able to utilize a diverse array of carbohydrates for energy metabolism. One such molecule is trehalose, a disaccharide common in human foods, which has been recently implicated in enhancing the virulence of epidemic strains of the pathogen Clostridium difficile In this study, mutants with deletions of all three genes in the putative S. mutans trehalose utilization operon were characterized, and the genes were shown to be required for wild-type levels of growth when trehalose was the only carbohydrate source provided. Interestingly, the TreR transcriptional regulator appeared to be critical for responding to oxidative stress and for mounting a protective stress tolerance response following growth at moderately acidic pH. mRNA sequencing (RNA-seq) of a treR deletion mutant suggested that in S. mutans, TreR acts as a trehalose-sensing activator of transcription of the tre operon, rather than as a repressor, as described in other species. In addition, deletion of treR caused the downregulation of a number of genes involved in genetic competence and bacteriocin production, supporting the results of a recent study linking trehalose and the S. mutans competence pathways. Finally, deletion of treR compromised the ability of S. mutans to inhibit the growth of the competing species Streptococcus gordonii and Lactococcus lactis Taking the results together, this study solidifies the role of the S. mutans tre operon in trehalose utilization and suggests novel functions for the TreR regulator, including roles in the stress response and competitive fitness.IMPORTANCES. mutans is the primary etiologic agent of dental caries, which globally is the most common chronic disease. S. mutans must be able to outcompete commensal organisms in its dental plaque niche in order to establish persistence and pathogenesis. To that end, S. mutans metabolizes a diverse array of carbohydrates to generate acid and impede its acid-sensitive neighbors. Additionally, S. mutans utilizes quorum signaling through genetic competence-associated pathways to induce production of toxins to kill its rivals. This study definitively shows that the S. mutans trehalose utilization operon is required for growth in trehalose. Furthermore, this study suggests that the S. mutans TreR transcriptional regulator has a novel role in virulence through regulation of genes involved in genetic competence and toxin production.

Efficacious Analogs of the Lantibiotic Mutacin 1140 against a Systemic Methicillin-Resistant Staphylococcus aureus Infection.

Mutacin 1140, a member of the epidermin family of type AI lantibiotics, has a broad spectrum of activity against Gram-positive bacteria. It blocks cell wall synthesis by binding to lipid II. Although it has rapid bactericidal effects and potent activity against Gram-positive pathogens, its rapid clearance and short half-life in vivo limit its development in the clinic. In this study, we evaluated the effect of charged and dehydrated residues on the pharmacokinetics of mutacin 1140. The dehydrated residues were determined to contribute to the stability of mutacin 1140, while alanine substitutions for the lysine or arginine residues improved the pharmacological properties of the antibiotic. Analogs K2A and R13A had significantly lower clearances, leading to higher plasma concentrations over time. They also had improved bioactivities against several pathogenic bacteria. In a murine systemic methicillin-resistant Staphylococcus aureus (MRSA) infection model, a 10-mg/kg single intravenous bolus injection of the K2A and R13A analogs (1:1 ratio) protected 100% of the infected mice, while a 2.5-mg/kg dose resulted in 50% survival. The 10-mg/kg treatment group had a significant reduction in bacteria load in the livers and kidneys compared to that in the vehicle control group. The study provides lead compounds for the future development of antibiotics used to treat systemic Gram-positive infections.

Modifying the Lantibiotic Mutacin 1140 for Increased Yield, Activity, and Stability.

Mutacin 1140 belongs to the epidermin family of type AI lantibiotics. This family has a broad spectrum of activity against Gram-positive bacteria. The binding of mutacin 1140 to lipid II leads to the inhibition of cell wall synthesis. Pharmacokinetic experiments with type AI lantibiotics are generally discouraging for clinical applications due to the short half-life of these compounds. The unprotected dehydrated and protease-susceptible residues outside the lanthionine rings may play a role in the short half-life in physiological settings. Previous mutagenesis work on mutacin 1140 has been limited to the lanthionine-forming residues, the C-terminally decarboxylated residue, and single amino acid substitutions at residues Phe1, Trp4, Dha5, and Arg13. To study the importance of the dehydrated (Dha5 and Dhb14) and protease-susceptible (Lys2 and Arg13) residues within mutacin 1140 for stability and bioactivity, each of these residues was evaluated for its impact on production and inhibitory activity. More than 15 analogs were purified, enabling direct comparison of the activities against a select panel of Gram-positive bacteria. The efficiency of the posttranslational modification (PTM) machinery of mutacin 1140 is highly restricted on its substrate. Analogs in the various intermediate stages of PTMs were observed as minor products following single point mutations at the 2nd, 5th, 13th, and 14th positions. The combination of alanine substitutions at the Dha5 and Dhb14 positions abolished mutacin 1140 production, while the production was restored by substitution of a Gly residue at one of these positions. Analogs with improved activity, productivity, and proteolytic stability were identified.IMPORTANCE Our findings show that the efficiency of mutacin 1140 PTMs is highly dependent on the core peptide sequence. Analogs in various intermediate stages of PTMs can be transported by the bacterium, which indicates that PTMs and transport are finely tuned for the native mutacin 1140 core peptide. Only certain combinations of amino acid substitutions at the Dha5 and Dhb14 dehydrated residue positions were tolerated. Observation of glutamylated core peptide analogs shows that dehydrations occur in a glutamate-dependent manner. Interestingly, mutations at positions outside rings A and B, the lipid II binding domain, would interfere with lipid II binding. Purified mutacin 1140 analogs have various activities and selectivities against different genera of bacteria, supporting the effort to generate analogs with higher specificity against pathogenic bacteria. The discovery of analogs with improved inhibitory activity against pathogenic bacteria, increased stability in the presence of protease, and higher product yields may promote the clinical development of this unique antimicrobial compound.

Screening for inhibitors of mutacin synthesis in Streptococcus mutans using fluorescent reporter strains.

BACKGROUND: Within the polymicrobial dental plaque biofilm, bacteria kill competitors by excreting mixtures of bacteriocins, resulting in improved fitness and survival. Inhibiting their bacteriocin synthesis might therefore be a useful strategy to eliminate specific pathogens. We used Streptococcus mutans, a highly acidogenic inhabitant of dental plaque, as a model and searched for natural products that reduced mutacin synthesis. To this end we fused the promoter of mutacin VI to the GFP+ gene and integrated the construct into the genome of S. mutans UA159 by single homologous recombination. RESULTS: The resulting reporter strain 423p - gfp + was used to screen 297 secondary metabolites from different sources, mainly myxobacteria and fungi, for their ability to reduce the fluorescence of the fully induced reporter strain by > 50% while growth was almost unaffected (> 90% of control). Seven compounds with different chemical structures and different modes of action were identified. Erinacine C was subsequently validated and shown to inhibit transcription of all three mutacins of S. mutans. The areas of the inhibition zones of the sensor strains S. sanguinis and Lactococcus lactis were reduced by 35% to 61% in comparison to controls in the presence of erinacine C, demonstrating that the amount of active mutacins in the culture supernatants of S. mutans was reduced. Erinacines are cyathane diterpenes that were extracted from cultures of the edible mushroom Hericium erinaceus. They have anti-inflammatory, antimicrobial and neuroprotective effects. For erinacine C, a new biological activity was found here. CONCLUSIONS: We demonstrate the successful development of a whole-cell fluorescent reporter for the screening of natural compounds and report that erinacine C suppresses mutacin synthesis in S. mutans without affecting cell viability.

Contribution of Streptococcus Mutans Virulence Factors and Saliva Agglutinating Capacity to Caries Susceptibility in Children: A Preliminary Study.

BACKGROUND: Many factors contribute to caries development in humans, such as diet, host factors - including different saliva components - and the presence of acidogenic bacteria in the dental biofilm, particularly Streptococcus mutans (S. mutans). Despite the influence of S. mutans in caries, this bacterium is also prevalent among healthy individuals, suggesting the contribution of genetic variation on the cariogenic potential. Based on this hypothesis, the present work investigated the influence of S. mutans virulence factors and saliva agglutinating capacity on caries susceptibility in children. STUDY DESIGN: Saliva samples of 24 children from low income families (13 caries-free and 11 caries-active individuals) were collected and tested for their ability to agglutinate S. mutans. The bacteria were isolated from these samples and analyzed for the presence of the gene coding for mutacin IV (mut IV). Biofilm formation and acid tolerance were also investigated in both groups (caries-free and caries-active). RESULTS: Saliva samples from caries-free children showed an increased capacity to agglutinate S. mutans (p=0.006). Also, bacteria isolated from the caries-free group formed less biofilm when compared to the caries-active group (p=0.04). The presence of mut IV gene did not differ between bacteria isolated from caries-free and caries-active individuals, nor did the ability to tolerate an acidic environment, which was the same for the two groups. CONCLUSIONS: Altogether, the results suggest that the adhesive properties of S. mutans and the agglutinating capacity of the saliva samples correlated with the presence of caries lesions in children.

Carboxyl Analogue of Mutacin 1140, a Scaffold for Lead Antibacterial Discovery.

Mutacin 1140 belongs to the epidermin group of lantibiotics. Epidermin class lantibiotics are ribosomally synthesized and posttranslationally modified antibiotics with potent activity against Gram-positive bacteria. In particular, this class is effective at targeting drug-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium tuberculosis, and Clostridium difficile A C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) residue is derived from a decarboxylation of a terminal cysteine that is involved in lanthionine ring formation. Studies on mutacin 1140 have revealed new insight into the structural importance of the C-terminal AviCys residue. A C-terminal carboxyl analogue of mutacin 1140 was engineered. Capping the C-terminal carboxyl group with a primary amine restores bioactivity and affords a novel opportunity to synthesize new analogues. A C-terminal fluorescein-labeled mutacin 1140 analogue traps lipid II into a large lipid II lantibiotic complex, similar to that observed in vivo for the lantibiotic nisin. A C-terminal carboxyl analogue of mutacin 1140 competitively inhibits the activity of native mutacin 1140 and nisin. The presence of a C-terminal carboxyl group prevents the formation of the large lipid II lantibiotic complexes but does not prevent the binding of the lantibiotic to lipid II.IMPORTANCE This study addressed the importance of the C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) residue for antibacterial activity. We have learned that the posttranslational modification for making the AviCys residue is presumably important for the lateral assembly mechanism of activity that traps lipid II into a large complex. The C-terminal carboxyl analogue of this class of lantibiotics is agreeable to the addition of a wide variety of substrates. The addition of fluorescein enabled in vivo visualization of the epidermin class of lantibiotics in action. These results are significant because, as we demonstrate, the presence of the AviCys residue is not essential for bioactivity, but, more importantly, the removal of the carboxyl group is essential. The ability to make a C-terminal carboxyl analogue that is modifiable will facilitate the synthesis of novel analogues of the epidermin class of lantibiotics that can be developed for new applications.

Frequency of Mutacin Gene Types in Streptococcus mutans Isolated From Oral Potentially Malignant Disorder (OPMD) Patients.

Objectives Mutacins are potent virulent factors attributing to the virulence in Streptococcus mutans leading to oro-dental diseases, and oral potentially malignant disorders (OPMDs) are considered a premalignant condition of the oro-mucosal layers in the oral cavity. The purpose of this study was to phenotypically characterize S. mutans from the clinical samples of patients with OPMD and to assess the frequency of mutacin genes in comparison with healthy individuals. Methods Saliva samples (n=60) were collected from three different groups and the samples were incubated at 37°C for 48 hours in Mutans-Sanguis agar. After incubation, the isolates were identified phenotypically for S. mutans and the frequency of mutacin genes and its types were assessed by polymerase chain reaction (PCR). Results S. mutans was found to be more prevalent in the OPMD cases (45%) followed by healthy individuals with caries (15%). Mutacin genes were expressed in all the groups except Group 3 (healthy individuals) without caries. Mutacin I was expressed the highest in Group 1 and Group 2 with 88% and 62.5, respectively, and mutacin III was expressed the least in all groups with 0% expression. Conclusion The findings of the study show the presence of mutacin gene types in the clinical strains of S. mutans in association with OPMD and caries. Further experimental evidence may be required to assess the frequency and to design a novel drug targeting the same.

Nattokinase, a Subtilisin-like Alkaline-Serine Protease, Reduces Mutacin Activity by Inactivating the Competence-Stimulating Peptide in Streptococcus mutans.

Streptococcus mutans is a major cariogenic organism because of its ability to form biofilms on tooth surfaces. Bacteriocins produced by S. mutans (known as mutacins) are indirect pathogenic factors that play a role in the persistence of this microbe in the oral environment. Nattokinase, a subtilisin-like alkaline serine protease, potently inhibits biofilm formation without affecting S. mutans growth. However, effective strategies utilizing nattokinase to control mutacin production by S. mutans are lacking. In this study, we evaluated the effect of nattokinase on mutacin activity in 46 strains of S. mutans with different mutacin genotypes isolated from the dental plaques of pediatric patients with caries. Nattokinase reduced the activity of mutacin against oral streptococci at a concentration of 1 mg/mL in all clinical isolates. Furthermore, nattokinase reduced the expression of non-lantibiotic mutacin structural genes (nlmABCD) and inactivated the extracellular competence-stimulating peptide involved in comDE activation, which regulates non-lantibiotic mutacin gene expression. These results suggest that nattokinase may reduce the virulence of S. mutans and could potentially be used as a new caries-preventive agent as an alternative to conventional drug treatments.

Synthesis of a Novel Lantibiotic Using Mutacin II Biosynthesis Apparatus.

Owing to extensive metagenomic studies, we now have access to numerous sequences of novel bacteriocin-like antimicrobial peptides encoded by various cultivable and noncultivable bacteria. However, relatively rarely, we even have access to these cultivable strains to examine the potency and the targets of the predicted bacteriocins. In this study, we evaluated a heterologous biosynthetic system to produce biologically active nonnative novel lantibiotics, which are modified bacteriocins. We chose Streptococcus mutans, a dental pathogen, as the host organism because it is genetically easy to manipulate and is inherently a prolific producer of various bacteriocins. We chose the S. mutans T8 strain as the host, which produces the lantibiotic mutacin II, to express 10 selected homologs of mutacin II identified from GenBank. These lantibiotic peptides either are novel or have been studied very minimally. The core regions of the selected lantibiotic peptides were fused to the leader sequence of the mutacin II peptide and integrated into the chromosome such that the core region of the native mutacin II was replaced with the new core sequences. By this approach, using the mutacin II biosynthesis machinery, we obtained one bioactive novel lantibiotic peptide with 52% different residues compared to the mutacin II core region. This unknown lantibiotic is encoded by Streptococcus agalactiae and Streptococcus ovuberis strains. Since this peptide displays some homology with nukacin ISK-1, we named it nukacin Spp. 2. This study demonstrated that the mutacin II biosynthesis machinery can be successfully used as an efficient system for the production of biologically active novel lantibiotics. IMPORTANCE In this study, we report for the first time that Streptococcus mutans can be used as a host to produce various nonnative lantibiotics. We showed that in the T8 strain, we could produce bioactive lacticin 481 and nukacin ISK-1, both of which are homologs of mutacin II, using T8's modification and secretion apparatus. Similarly, we also synthesized a novel bioactive lantibiotic, which we named nukacin Spp. 2.

Gene Rearrangement and Modification of Immunity Factors Are Correlated with the Insertion of Bacteriocin Cassettes in Streptococcus mutans.

Bacteriocins have been applied in the food industries and have become promising next-generation antibiotics. Some bacteria produce bacteriocins and possess immunity factors for self-protection. Nisin A, a bacteriocin produced by Lactococcus lactis, shows broad-spectrum activity. However, the evolution and cross-resistance ability of the immunity factors in some species results in reduced susceptibility to bacteriocins. Here, we investigated the elements responsible for nisin A resistance in Streptococcus mutans and their contribution to mutacins (bacteriocins produced by S. mutans) resistance. We classified the nisin A-resistance regions into six types based on the different combinations of 3 immunity factors, mutFEG, nsrX, and mutHIJ, and the presence of mutacin synthesis operon upstream of mutF. Data shows that NsrX effectively acts against nisin A but not mutacins, while the newly identified ABC transporter MutHIJ acts against three mutacins but not nisin A. Three types of MutFEG are identified based on their amino acid sequences: α (in Nsr-types C and D-I), ß (in Nsr-types B and d-III), and γ (in Nsr-type E). MutFEG-α strongly contributes to mutacin I resistance, while MutFEG-ß and MutFEG-γ strongly contribute to mutacin III, IIIb, and nisin A resistance. Additionally, mutFEG-like structures could be found in various streptococcal species isolated from the oral cavity of humans, chimpanzees, monkeys, bears, and hamsters. Our findings suggest that immunity factors rearrange and adapt in the presence of bacteriocins and could be transferred among closely related species, thus altering the bacterial competition within the microflora. IMPORTANCE Streptococcus mutans is an important organism of oral microbiota and associated with dental caries and systemic diseases such as stroke and endocarditis. They produce bacteriocins known as mutacins to compete with other oral bacteria and possess immune factors for self-protection. We found that the nisin A and mutacins resistance patterns correlated with the immunity components and MutFEG variants, and the genetic difference was driven by the insertion of mutacin-synthesis cassettes. Our study provides an understanding of the development of bacteriocin resistance among streptococcal species, which may alter the bacterial interaction and ecology within the oral biofilm.

Evaluation of analogs of mutacin 1140 in systemic and cutaneous methicillin-resistant Staphylococcus aureus infection models in mice.

Mutacin 1140 (Mu1140) is a potent antibiotic against Gram-positive bacteria, such as Staphylococcus aureus. The antibiotic is produced by the oral bacterium Streptococcus mutans and is a member of the epidermin family of type AI lantibiotics. The antibiotic exerts its inhibitory activity by binding to the cell wall precursor lipid II, blocking cell wall synthesis, and by disrupting bacterial membranes. In previous studies, the novel K2A and R13A analogs of Mu1140 have been identified to have superior pharmacokinetic properties compared to native Mu1140. In this study, the use of a combined formulation of the Mu1140 K2A and R13A analogs was shown to be more effective at treating MRSA bacteremia than the native Mu1140 or vancomycin. The analogs were also shown to be effective in treating an MRSA skin infection. The use of K2A and R13A analogs may provide a future alternative for treating serious Gram-positive bacterial infections. In a previous study, the Mu1140 analogs were shown to have significantly longer drug clearance times, leading to higher plasma concentrations over time. These properties warranted further testing to determine whether the analogs are effective for the treatment of systemic MRSA and acute skin infections. In this study, Mu1140 analogs were shown to be more effective than currently available treatments for systemic and skin MRSA infections. Further, the study clearly shows that the new analogs are superior to native Mu1140 for the treatment of a systemic MRSA infection. These findings support continued drug product development efforts using the K2A and R13A Mu1140 analogs, and that these analogs may ameliorate the outcome of serious bacterial infections.

Mutacins of Streptococcus mutans.

The colonization and accumulation of Streptococcus mutans are influenced by various factors in the oral cavity, such as nutrition and hygiene conditions of the host, salivary components, cleaning power and salivary flow and characteristics related with microbial virulence factors. Among these virulence factors, the ability to synthesize glucan of adhesion, glucan-binding proteins, lactic acid and bacteriocins could modify the infection process and pathogenesis of this species in the dental biofilm. This review will describe the role of mutacins in transmission, colonization, and/or establishment of S. mutans, the major etiological agent of human dental caries. In addition, we will describe the method for detecting the production of these inhibitory substances in vitro (mutacin typing), classification and diversity of mutacins and the regulatory mechanisms related to its synthesis.

[Regulatory role of small RNA srn821978 in mutacin IV expression in Streptococcus mutans].

OBJECTIVE: To analyze the role of small RNA srn821798 in posttranscriptional regulation of mutacin IV expression in Streptococcus mutans. METHODS: The potential target genes of srn821978 were predicted using RNAhybrid, RNAPredator and IntaRNA. We collected 10 Streptococcus mutans (S.muans) strains with high expression of mutacin IV and another 10 S.muans strains that did not express mutacin IV screened by inhibition zone test, and the expression levels of srn821798 and the candidate target genes in these strains were detected by qPCR. Using synthesized mimics and inhibitors of srn821798, we constructed S.muans strains with high or low srn821798 expression via electroporation based on the standard strain of S.muans UA159, and analyzed the expression levels of srn821798 and its candidate target genes in these strains. We also examined the binding ability of srn821798 to its target gene sepM using electrophoresis and a dual- luciferase reporter system. RESULTS: The expression levels of the candidate target genes of srn821798 including sepM, comD, comE, nlmA and nlmB were significantly higher while the expression level of srn821798 was significantly lower in clinical S.muans strains with high expression of mutacin IV than in those without mutacin IV expression (P < 0.05). Although the expression levels of the candidate target genes in strains with up- regulated or down- regulated srn821798 expression did not differ significantly from those in the standard strain, the expression level of sepM showed a trend of differential distribution, and srn821798 was predicted to have a strong binding ability to sepM action site. CONCLUSION: srn821798 may play a regulatory role in the expression of mutacin IV in S.muans, but the underlying mechanism remains to be explored.

[Association of sepM gene mutation with mutacin â £ production by Streptococcus mutans].

OBJECTIVE: To investigate the types of sepM gene mutations and their distribution in clinical isolates of Streptococcus mutans (S. mutans) and explore the association of sepM gene mutation with the capacity of mutacin Ⅳ production by S. mutans. OBJECTIVE: We assessed the capacity of mutacin Ⅳ production in 80 clinical isolates of S. mutans using an inhibition zone assay. The minimum spanning tree and phylogenetic tree of these isolates were constructed using core genome multilocus sequence typing and maximum likelihood method, respectively. GeneMarkS software was used to predict the coding genes of these isolates, and the predicted genes were blasted against the sepM gene sequence of the reference genome UA159 to determine sepM gene mutations and their distribution characteristics in the clinical isolates. The mutation types affecting mutacin Ⅳ production were identified by analyzing the differentially distributed mutations between mutacin Ⅳ-producing isolates and mutacin Ⅳ-free isolates and by comparing the inhibition zones between isolates with sepM gene mutations and those without mutations. OBJECTIVE: Among the 80 clinical isolates of S. mutans, 25 isolates were capable of mutacin Ⅳ production and 55 did not produce mutacin Ⅳ. The minimum spanning tree showed that the allelic differences were less among the mutacin Ⅳproducing isolates than among the mutacin Ⅳ-free isolates, and the origins of the mutacin Ⅳ-producing isolates were similar. We identified a total of 34 single base mutations in the 80 clinical isolates, and among them, C31T (P=0.001), G533A (P < 0.001), C756T (P=0.025), and C1036T (P=0.003) showed significant differential distributions between the mutacin Ⅳ-producing and mutacin Ⅳ-free isolates. These differentially distributed mutations were positively correlated with the capacity of mutacin Ⅳ production of the bacteria. OBJECTIVE: sepM gene mutations that affect the capacity of mutacin Ⅳ production are present in the clinical strains of S. mutans.

Identification of Oral Bacterial Biosynthetic Gene Clusters Associated with Caries.

Small molecules are a primary communication media of the microbial world, and play crucial, yet largely unidentified, roles in microbial ecology and disease pathogenesis. Many small molecules are produced by biosynthetic gene clusters, which can be predicted and analyzed computationally given a genome. A recent study examined the biosynthetic repertoire of the oral microbiome and cross-referenced this information against the disease status of the human host, providing leads for biosynthetic gene clusters, and their natural products, which may be key in the oral microbial ecology affecting dental caries and periodontitis. This chapter provides a step-by-step tutorial to bioinformatically to locate biosynthetic gene clusters within genomes, predict the type of natural products that are produced, and cross-reference the identified biosynthetic gene clusters to microbiomes associated with disease or health.

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.