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1.  Targeted Killing of Streptococcus mutans by a Pheromone-Guided “Smart” Antimicrobial Peptide 
Antimicrobial Agents and Chemotherapy  2006;50(11):3651-3657.
Within the repertoire of antibiotics available to a prescribing clinician, the majority affect a broad range of microorganisms, including the normal flora. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. To address this problem, our laboratory has recently developed a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs), based on the fusion of a species-specific targeting peptide domain with a wide-spectrum antimicrobial peptide domain. In the current study, we focused on achieving targeted killing of Streptococcus mutans, a cavity-causing bacterium that resides in a multispecies microbial community (dental plaque). In particular, we explored the possibility of utilizing a pheromone produced by S. mutans, namely, the competence stimulating peptide (CSP), as a STAMP targeting domain to mediate S. mutans-specific delivery of an antimicrobial peptide domain. We discovered that STAMPs constructed with peptides derived from CSP were potent against S. mutans grown in liquid or biofilm states but did not affect other oral streptococci tested. Further studies showed that an 8-amino-acid region within the CSP sequence is sufficient for targeted delivery of the antimicrobial peptide domain to S. mutans. The STAMPs presented here are capable of eliminating S. mutans from multispecies biofilms without affecting closely related noncariogenic oral streptococci, indicating the potential of these molecules to be developed into “probiotic” antibiotics which could selectively eliminate pathogens while preserving the protective benefits of a healthy normal flora.
doi:10.1128/AAC.00622-06
PMCID: PMC1635210  PMID: 17060534
2.  Systematic Approach to Optimizing Specifically Targeted Antimicrobial Peptides against Streptococcus mutans▿  
Previously we reported a novel strategy of “targeted killing” through the design of narrow-spectrum molecules known as specifically targeted antimicrobial peptides (STAMPs) (R. Eckert et al., Antimicrob. Agents Chemother. 50:3651-3657, 2006; R. Eckert et al., Antimicrob. Agents Chemother. 50:1480-1488, 2006). Construction of these molecules requires the identification and the subsequent utilization of two conjoined yet functionally independent peptide components: the targeting and killing regions. In this study, we sought to design and synthesize a large number of STAMPs targeting Streptococcus mutans, the primary etiologic agent of human dental caries, in order to identify candidate peptides with increased killing speed and selectivity compared with their unmodified precursor antimicrobial peptides (AMPs). We hypothesized that a combinatorial approach, utilizing a set number of AMP, targeting, and linker regions, would be an effective method for the identification of STAMPs with the desired level of activity. STAMPs composed of the Sm6 S. mutans binding peptide and the PL-135 AMP displayed selectivity at MICs after incubation for 18 to 24 h. A STAMP where PL-135 was replaced by the B-33 killing domain exhibited both selectivity and rapid killing within 1 min of exposure and displayed activity against multispecies biofilms grown in the presence of saliva. These results suggest that potent and selective STAMP molecules can be designed and improved via a tunable “building-block” approach.
doi:10.1128/AAC.01391-09
PMCID: PMC2863653  PMID: 20211885
3.  A Novel Target-Specific, Salt-Resistant Antimicrobial Peptide against the Cariogenic Pathogen Streptococcus mutans▿† 
Antimicrobial Agents and Chemotherapy  2011;55(11):5205-5213.
In this study, we constructed and evaluated a target-specific, salt-resistant antimicrobial peptide (AMP) that selectively targeted Streptococcus mutans, a leading cariogenic pathogen. The rationale for creating such a peptide was based on the addition of a targeting domain of S. mutans ComC signaling peptide pheromone (CSP) to a killing domain consisting of a portion of the marine-derived, broad-spectrum AMP pleurocidin to generate a target-specific AMP. Here, we report the results of our assessment of such fusion peptides against S. mutans and two closely related species. The results showed that nearly 95% of S. mutans cells lost viability following exposure to fusion peptide IMB-2 (5.65 μM) for 15 min. In contrast, only 20% of S. sanguinis or S. gordonii cells were killed following the same exposure. Similar results were also observed in dual-species mixed cultures of S. mutans with S. sanguinis or S. gordonii. The peptide-guided killing was further confirmed in S. mutans biofilms and was shown to be dose dependent. An S. mutans mutant defective in the CSP receptor retained 60% survival following exposure to IMB-2, suggesting that the targeted peptide predominantly bound to the CSP receptor to mediate killing in the wild-type strain. Our work confirmed that IMB-2 retained its activity in the presence of physiological or higher salt concentrations. In particular, the fusion peptide showed a synergistic killing effect on S. mutans with a preventive dose of NaF. In addition, IMB-2 was relatively stable in the presence of saliva containing 1 mM EDTA and did not cause any hemolysis. We also found that replacement of serine-14 by histidine improved its activity at lower pH. Because of its effectiveness, salt resistance, and minimal toxicity to host cells, this novel target-specific peptide shows promise for future development as an anticaries agent.
doi:10.1128/AAC.05175-11
PMCID: PMC3194993  PMID: 21844316
4.  Targeted Antimicrobial Therapy Against Streptococcus mutans Establishes Protective Non-cariogenic Oral Biofilms and Reduces Subsequent Infection 
Aim
Dental biofilms are complex communities composed largely of harmless bacteria. Certain pathogenic species including Streptococcus mutans (S. mutans) can become predominant when host factors such as dietary sucrose intake imbalance the biofilm ecology. Current approaches to control S. mutans infection are not pathogen-specific and eliminate the entire oral community along with any protective benefits provided. Here, we tested the hypothesis that removal of S. mutans from the oral community through targeted antimicrobial therapy achieves protection against subsequent S. mutans colonization.
Methodology
Controlled amounts of S. mutans were mixed with S. mutans-free saliva, grown into biofilms and visualized by antibody staining and cfu quantization. Two specifically-targeted antimicrobial peptides (STAMPs) against S. mutans were tested for their ability to reduce S. mutans biofilm incorporation upon treatment of the inocula. The resulting biofilms were also evaluated for their ability to resist subsequent exogenous S. mutans colonization.
Results
S. mutans colonization was considerably reduced (9 ± 0.4 fold reduction, P=0.01) when the surface was preoccupied with saliva-derived biofilms. Furthermore, treatment with S. mutans-specific STAMPs yielded S. mutans-deficient biofilms with significant protection against further S. mutans colonization (5 minutes treatment: 38 ± 13 fold reduction P=0.01; 16 hours treatment: 96 ± 28 fold reduction P=0.07).
Conclusion
S. mutans infection is reduced by the presence of existing biofilms. Thus maintaining a healthy or “normal” biofilm through targeted antimicrobial therapy (such as the STAMPs) could represent an effective strategy for the treatment and prevention of S. mutans colonization in the oral cavity and caries progression.
doi:10.4248/IJOS10024
PMCID: PMC3733586  PMID: 20737932
targeted antimicrobial therapy; antimicrobial peptide; biofilm; Streptococcus mutans; protective colonization; caries
5.  Spectroscopic and Computational Study of Melittin, Cecropin A, and the Hybrid Peptide CM15 
The Journal of Physical Chemistry. B  2012;116(35):10600-10608.
Antimicrobial peptides (AMPs), such as cecropin A from silk moth, are key components of the innate immune system. They are effective defensive weapons against invading pathogens, yet they do not target host eukaryotic cells. In contrast, peptide toxins, such as honeybee melittin, are nondiscriminating and target both eukaryotic and prokaryotic cells. An AMP-toxin hybrid peptide that is composed of cecropin A and melittin (CM15) improves upon the antimicrobial activity of cecropin A without displaying the nonspecific, hemolytic properties of melittin. Here we report fluorescence and UV resonance Raman spectra of melittin, cecropin A, and CM15 with the goal of elucidating peptide-membrane interactions that help guide specificity. We have probed the potency for membrane disruption, local environment and structure of the single tryptophan residue, backbone conformation near the peptide hinge, and amide backbone structure of the peptides in lipid environments that mimic eukaryotic and prokaryotic membranes. These experimental results suggest that melittin inserts deeply into the bilayer, whereas cecropin A remains localized to the lipid headgroup region. A surprising finding is that CM15 is a potent membrane-disruptor despite its largely unfolded conformation. A molecular dynamics analysis complements these data and demonstrates the ability of CM15 to associate favorably with membranes as an unfolded peptide. This combined experimental–computational study suggests that new models for peptide–membrane interactions should be considered.
doi:10.1021/jp304021t
PMCID: PMC3434763  PMID: 22845179
6.  Antimicrobial Properties and Membrane-Active Mechanism of a Potential α-Helical Antimicrobial Derived from Cathelicidin PMAP-36 
PLoS ONE  2014;9(1):e86364.
Antimicrobial peptides (AMPs), which present in the non-specific immune system of organism, are amongst the most promising candidates for the development of novel antimicrobials. The modification of naturally occurring AMPs based on their residue composition and distribution is a simple and effective strategy for optimization of known AMPs. In this study, a series of truncated and residue-substituted derivatives of antimicrobial peptide PMAP-36 were designed and synthesized. The 24-residue truncated peptide, GI24, displayed antimicrobial activity comparable to the mother peptide PMAP-36 with MICs ranging from 1 to 4 µM, which is lower than the MICs of bee venom melittin. Although GI24 displayed high antimicrobial activity, its hemolytic activity was much lower than melittin, suggesting that GI24 have optimal cell selectivity. In addition, the crucial site of GI24 was identified through single site-mutation. An amino acid with high hydrophobicity at position 23 played an important role in guaranteeing the high antimicrobial activity of GI24. Then, lipid vesicles and whole bacteria were employed to investigate the membrane-active mechanisms. Membrane-simulating experiments showed that GI24 interacted strongly with negatively charged phospholipids and weakly with zwitterionic phospholipids, which corresponded well with the data of its biological activities. Membrane permeabilization and flow cytometry provide the evidence that GI24 killed microbial cells by permeabilizing the cell membrane and damaging membrane integrity. GI24 resulted in greater cell morphological changes and visible pores on cell membrane as determined using scanning electron microscopy (SEM) and transmission electron microscope (TEM). Taken together, the peptide GI24 may provide a promising antimicrobial agent for therapeutic applications against the frequently-encountered bacteria.
doi:10.1371/journal.pone.0086364
PMCID: PMC3897731  PMID: 24466055
7.  Studies on the Mode of Action of the Antifungal Hexapeptide PAF26 
Antimicrobial Agents and Chemotherapy  2006;50(11):3847-3855.
The small antimicrobial peptide PAF26 (Ac-RKKWFW-NH2) has been identified by a combinatorial approach and shows preferential activity toward filamentous fungi. In this work, we investigated the mode of action and inhibitory effects of PAF26 on the fungus Penicillium digitatum. The dye Sytox Green was used to demonstrate that PAF26 induced cell permeation. However, microscopic observations showed that sub-MIC concentrations of PAF26 produced both alterations of hyphal morphology (such as altered polar growth and branching) and chitin deposition in areas of no detectable permeation. Analysis of dose-response curves of inhibition and permeation suggested that growth inhibition is not solely a consequence of permeation. In order to shed light on the mode of PAF26 action, its antifungal properties were compared with those of melittin, a well-known pore-forming peptide that kills through cytolysis. While the 50% inhibitory concentrations and MICs of the two peptides against P. digitatum mycelium were comparable, they differed markedly in their fungicidal activities toward conidia and their hemolytic activities toward human red blood cells. Kinetic studies showed that melittin quickly induced Penicillium cell permeation, while PAF26-induced Sytox Green uptake was significantly slower and less efficient. Therefore, the ultimate growth inhibition and morphological alterations induced by PAF26 for P. digitatum are not likely a result of conventional pore formation. Fluorescently labeled PAF26 was used to demonstrate its specific in vivo interaction and translocation inside germ tubes and hyphal cells, at concentrations as low as 0.3 μM (20 times below the MIC), at which no inhibitory, morphological, or permeation effects were observed. Interestingly, internalized PAF26 could bind to cellular RNAs, since in vitro nonspecific RNA binding activity of PAF26 was demonstrated by electrophoretic mobility shift assays. We propose that PAF26 is a short, de novo-designed penetratin-type peptide that has multiple detrimental effects on target fungi, which ultimately result in permeation and killing.
doi:10.1128/AAC.00650-06
PMCID: PMC1635192  PMID: 17065623
8.  The Tea Catechin Epigallocatechin Gallate Suppresses Cariogenic Virulence Factors of Streptococcus mutans▿  
Streptococcus mutans, the primary etiologic agent of dental caries, possesses a series of virulence factors associated with its cariogenicity. Alternatives to traditional antimicrobial treatment, agents selectively inhibiting the virulence factors without necessarily suppressing the resident oral species, are promising. The anticariogenic properties of tea have been suggested in experimental animals and humans. Tea polyphenols, especially epigallocatechin gallate (EGCg), have been shown to inhibit the growth and glucosyltransferases activity of S. mutans. However, their effects on biofilm and cariogenic virulence factors of oral streptococci other than glucosyltransferases have not been well documented. In this study, we investigated the biological effect of EGCg on the virulence factors of S. mutans associated with its acidogenicity and acidurity. The antimicrobial effects of EGCg on S. mutans biofilm grown in chemically defined medium were also examined. EGCg inhibited growth of S. mutans planktonic cells at an MIC of 31.25 μg/ml and a minimal bactericidal concentration (MBC) of 62.5 μg/ml. EGCg also inhibited S. mutans biofilm formation at 15.6 μg/ml (minimum concentration that showed at least 90% inhibition of biofilm formation) and reduced viability of the preformed biofilm at 625 μg/ml (sessile MIC80). EGCg at sub-MIC levels inhibited acidogenicity and acidurity of S. mutans cells. Analysis of the data obtained from real-time PCR showed that EGCg significantly suppressed the ldh, eno, atpD, and aguD genes of S. mutans UA159. Inhibition of the enzymatic activity of F1Fo-ATPase and lactate dehydrogenase was also noted (50% inhibitory concentration between 15.6 and 31.25 μg/ml). These findings suggest that EGCg is a natural anticariogenic agent in that it exhibits antimicrobial activity against S. mutans and suppresses the specific virulence factors associated with its cariogenicity.
doi:10.1128/AAC.01016-10
PMCID: PMC3067078  PMID: 21149622
9.  A genomic approach highlights common and diverse effects and determinants of susceptibility on the yeast Saccharomyces cerevisiae exposed to distinct antimicrobial peptides 
BMC Microbiology  2010;10:289.
Background
The mechanism of action of antimicrobial peptides (AMP) was initially correlated with peptide membrane permeation properties. However, recent evidences indicate that action of a number of AMP is more complex and involves specific interactions at cell envelopes or with intracellular targets. In this study, a genomic approach was undertaken on the model yeast Saccharomyces cerevisiae to characterize the antifungal effect of two unrelated AMP.
Results
Two differentiated peptides were used: the synthetic cell-penetrating PAF26 and the natural cytolytic melittin. Transcriptomic analyses demonstrated distinctive gene expression changes for each peptide. Quantitative RT-PCR confirmed differential expression of selected genes. Gene Ontology (GO) annotation of differential gene lists showed that the unique significant terms shared by treatment with both peptides were related to the cell wall (CW). Assays with mutants lacking CW-related genes including those of MAPK signaling pathways revealed genes having influence on sensitivity to peptides. Fluorescence microscopy and flow cytometry demonstrated PAF26 interaction with cells and internalization that correlated with cell killing in sensitive CW-defective mutants such as Δecm33 or Δssd1. GO annotation also showed differential responses between peptides, which included ribosomal biogenesis, ARG genes from the metabolism of amino groups (specifically induced by PAF26), or the reaction to unfolded protein stress. Susceptibility of deletion mutants confirmed the involvement of these processes. Specifically, mutants lacking ARG genes from the metabolism of arginine pathway were markedly more resistant to PAF26 and had a functional CW. In the deletant in the arginosuccinate synthetase (ARG1) gene, PAF26 interaction occurred normally, thus uncoupling peptide interaction from cell killing. The previously described involvement of the glycosphingolipid gene IPT1 was extended to the peptides studied here.
Conclusions
Reinforcement of CW is a general response common after exposure to distinct AMP, and likely contributes to shield cells from peptide interaction. However, a weakened CW is not necessarily indicative of a higher sensitivity to AMP. Additional processes modulate susceptibility to specific peptides, exemplified in the involvement of the metabolism of amino groups in the case of PAF26. The relevance of the response to unfolded protein stress or the sphingolipid biosynthesis, previously reported for other unrelated AMP, was also independently confirmed.
doi:10.1186/1471-2180-10-289
PMCID: PMC2996382  PMID: 21078184
10.  Lactobacillus paracasei DSMZ16671 Reduces Mutans Streptococci: A Short-Term Pilot Study 
Reducing the burden of pathogenic mutans streptococci is a goal of oral health. Lactobacillus paracasei DSMZ16671, even after heat-killing, specifically co-aggregates mutans streptococci in vitro and retains this activity in human saliva. In rats, it reduces mutans streptococcal colonization of teeth and caries scores. This pilot study sought to assess the potential of heat-killed L. paracasei DSMZ16671 (pro-t-action®) to reduce levels of salivary mutans streptococci in humans, using sugar-free candies as a delivery vehicle. A randomized, placebo-controlled, double-blind in vivo study of three groups examined the short-term effect of sugar-free candies containing 0 (placebo), 1, or 2 mg/candy piece of heat-killed L. paracasei DSMZ16671 on the levels of salivary mutans streptococci determined before and after consumption of the candies. The candies were consumed 4 times during 1.5 consecutive days. Compared to the placebo group, the test groups’ saliva had significantly reduced mutans streptococci as an immediate effect. These results suggest the use of heat-killed L. paracasei DSMZ16671 in suckable candies as a method to reduce mutans streptococci in the mouth and, thereby, caries risk. We think this a new concept and strategy for caries prevention and management.
doi:10.1007/s12602-013-9148-9
PMCID: PMC3825618  PMID: 24273614
Dental caries; Humans; Sugar-free candy; Lactobacillus pro-t-action; Co-aggregation; Saliva
11.  Enhancement of Antimicrobial Activity against Pseudomonas aeruginosa by Coadministration of G10KHc and Tobramycin▿  
Antimicrobial Agents and Chemotherapy  2006;50(11):3833-3838.
Pseudomonas aeruginosa is a common opportunistic human pathogen that is associated with life-threatening acute infections and chronic airway colonization during cystic fibrosis. Previously, we converted the wide-spectrum antimicrobial peptide novispirin G10 into a selectively-targeted antimicrobial peptide (STAMP), G10KHc. Compared to novispirin G10, the STAMP had an enhanced ability to kill Pseudomonas mendocina. In this study, we explored the activity of G10KHc against P. aeruginosa. G10KHc was found to be highly active (as active as tobramycin) against P. aeruginosa clinical isolates. Most interestingly, we observed a synergistic-like enhancement in killing activity when biofilms and planktonic cultures of P. aeruginosa were cotreated with G10KHc and tobramycin. The data indicate that the mechanism of enhanced activity may involve increased tobramycin uptake due to G10KHc-mediated cell membrane disruption. These results suggest that G10KHc may be useful against P. aeruginosa during acute and chronic infection states, especially when it is coadministered with tobramycin.
doi:10.1128/AAC.00509-06
PMCID: PMC1635211  PMID: 16940063
12.  Inhibited biofilm formation and improved antibacterial activity of a novel nanoemulsion against cariogenic Streptococcus mutans in vitro and in vivo 
The aim of this study was to prepare a novel nanoemulsion loaded with poorly water-soluble chlorhexidine acetate (CNE) to improve its solubility, and specifically enhance the antimicrobial activity against Streptococcus mutans in vitro and in vivo. In this study, a novel CNE nanoemulsion with an average size of 63.13 nm and zeta potential of −67.13 mV comprising 0.5% CNE, 19.2% Tween 80, 4.8% propylene glycol, and 6% isopropyl myristate was prepared by the phase inversion method. Important characteristics such as the content, size, zeta potential, and pH value of CNE did not change markedly, stored at room temperature for 1 year. Also, compared with chlorhexidine acetate water solution (CHX), the release profile results show that the CNE has visibly delayed releasing effect in both phosphate-buffered saline and artificial saliva solutions (P<0.005). The minimum inhibitory concentration and minimum bactericidal concentration of CHX for S. mutans (both 0.8 μg/mL) are both two times those of CNE (0.4 μg/mL). Besides, CNE of 0.8 μg/mL exhibited fast-acting bactericidal efficacy against S. mutans, causing 95.07% death within 5 minutes, compared to CHX (73.33%) (P<0.01). We observed that 5 mg/mL and 2 mg/mL CNE were both superior to CHX, significantly reducing oral S. mutans numbers and reducing the severity of carious lesions in Sprague Dawley rats (P<0.05), in an in vivo test. CNE treatment at a concentration of 0.2 μg/mL inhibited biofilm formation more effectively than CHX, as indicated by the crystal violet staining method, scanning electron microscopy, and atomic force microscopy. The cell membrane of S. mutans was also severely disrupted by 0.2 μg/mL CNE, as indicated by transmission electron microscopy. These results demonstrated that CNE greatly improved the solubility and antimicrobial activity of this agent against S. mutans both in vitro and in vivo. This novel nanoemulsion is a promising medicine for preventing and curing dental caries.
Video abstract
doi:10.2147/IJN.S72920
PMCID: PMC4296965  PMID: 25624759
nanoemulsion; chlorhexidine acetate; Streptococcus mutans; antibacterial
13.  Bactericidal Effects of a Fusion Protein of Llama Heavy-Chain Antibodies Coupled to Glucose Oxidase on Oral Bacteria 
Enzymes such as lactoperoxidase and glucose oxidase (GOx) are used as antimicrobial agents in oral care products. Their low specificities and substantiveness can be reduced by covalent coupling of antimicrobial molecules to antibodies. Variable domains (VHH) derived from llama heavy-chain antibodies are particularly suited for such an approach. The antibodies are composed solely of heavy-chain dimers; therefore, production of active fusion proteins by using molecular biology-based techniques is less complicated than production by use of conventional antibodies. In this study, a fusion protein consisting of VHH and GOx was constructed and expressed by Saccharomyces cerevisiae. A llama was immunized with Streptococcus mutans strain HG982. Subsequently, B lymphocytes were isolated and cDNA fragments encoding the VHH fragments were obtained by reverse transcription-PCR. After construction of a VHH library in Escherichia coli and screening of the library against mutans group streptococci and Streptococcus sanguinis strains, we found two VHH fragments with high specificities for S. mutans strains. A GOx gene was linked to the two VHH genes and cloned into S. cerevisiae yeasts. The yeasts expressed and secreted the recombinant proteins into the growth medium. The test of binding of fusion proteins to oral bacteria through their VHH fragments showed that S. mutans had been specifically targeted by GOx-S120, one of the fusion protein constructs. A low concentration of the fusion protein was also able to selectively kill S. mutans within 20 min in the presence of lactoperoxidase and potassium iodide. These findings demonstrate that the fusion protein GOx-VHH is potentially valuable in the selective killing of target bacteria such as S. mutans.
doi:10.1128/AAC.48.9.3390-3395.2004
PMCID: PMC514777  PMID: 15328101
14.  The Antimicrobial Peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm 
PLoS Pathogens  2008;4(10):e1000190.
Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides.
Author Summary
In most healthy individuals, the yeast Candida albicans is found within the oral cavity as part of the normal microflora. Though under immunocompromising conditions, this benign microbe can become an opportunistic pathogen causing oral candidiasis (i.e. thrush). Although antifungal drugs are typically efficacious, the paucity of drugs and their increasing usage has led to rising drug resistance. Thus, researchers have begun to look at alternative therapeutics, such as the candidacidal salivary peptide histatin-5. To date, little is known about the initial binding and subsequent internalization that facilitates Hst-5's killing activity. Thus, our study attempted to determine how Hst-5 is internalized into C. albicans. It was thought that Hst-5 is transported into the cytoplasm without disruption of the plasma membrane. However, our study finds that Hst-5, under physiological concentrations, disrupts the plasma membrane and is rapidly translocated into the cytoplasm, leading to cell death. Interestingly, the internalization of Hst-5 is initiated from a single spatially restricted site on the plasma membrane rather than multiple breaches on the cell surface. This novel mechanism of membrane disruption provides new insights into how Hst-5 and other antimicrobial peptides may be acting on pathogenic microorganisms.
doi:10.1371/journal.ppat.1000190
PMCID: PMC2568956  PMID: 18974864
15.  Clinical Efficacy of a Specifically Targeted Antimicrobial Peptide Mouth Rinse: Targeted Elimination of Streptococcus mutans and Prevention of Demineralization 
Caries Research  2011;45(5):415-428.
Background/Aims
Streptococcus mutans, the major etiological agent of dental caries, has a measurable impact on domestic and global health care costs. Though persistent in the oral cavity despite conventional oral hygiene, S. mutans can be excluded from intact oral biofilms through competitive exclusion by other microorganisms. This suggests that therapies capable of selectively eliminating S. mutans while limiting the damage to the normal oral flora might be effective long-term interventions to fight cariogenesis. To meet this challenge, we designed C16G2, a novel synthetic specifically targeted antimicrobial peptide with specificity for S. mutans. C16G2 consists of a S. mutans-selective ‘targeting region’ comprised of a fragment from S. mutans competence stimulation peptide (CSP) conjoined to a ‘killing region’ consisting of a broad-spectrum antimicrobial peptide (G2). In vitro studies have indicated that C16G2 has robust efficacy and selectivity for S. mutans, and not other oral bacteria, and affects targeted bacteria within seconds of contact.
Methods
In the present study, we evaluated C16G2 for clinical utility in vitro, followed by a pilot efficacy study to examine the impact of a 0.04% (w/v) C16G2 rinse in an intra-oral remineralization/demineralization model.
Results and Conclusions
C16G2 rinse usage was associated with reductions in plaque and salivary S. mutans, lactic acid production, and enamel demineralization. The impact on total plaque bacteria was minimal. These results suggest that C16G2 is effective against S. mutans in vivo and should be evaluated further in the clinic.
doi:10.1159/000330510
PMCID: PMC3169368  PMID: 21860239
Antimicrobial; Antimicrobial peptide; Caries; Demineralization; Dental plaque; Lactic acid; Mouth rinse; Oral therapeutic; Selective antibiotic; Selective therapeutic; Specifically targeted antimicrobial peptide; Streptococcus mutans; Targeted antimicrobial
16.  Proteases of an Early Colonizer Can Hinder Streptococcus mutans Colonization in vitro 
Journal of Dental Research  2011;90(4):501-505.
Streptococcus mutans is the primary cariogen that produces several virulence factors that are modulated by a competence-stimulating peptide (CSP) signaling system. In this study, we sought to determine if proteases produced by early dental plaque colonizers such as Streptococcus gordonii interfere with the subsequent colonization of S. mutans BM71 on the existing streptococcal biofilms. We demonstrated that S. mutans BM71 colonized much less efficiently in vitro on streptococcal biofilms than on Actinomyces naeslundii biofilms. Several oral streptococci, relative to A. naeslundii, produced proteases that inactivated the S. mutans CSP. We further demonstrated that cell protein extracts from S. gordonii, but not from A. naeslundii, interfered with S. mutans BM71 colonization. In addition, S. mutans BM71 colonized more efficiently on the sgc protease knockout mutant of S. gordonii than on the parent biofilms. In conclusion, proteases of early colonizers can interfere with subsequent colonization by S. mutans in vitro.
doi:10.1177/0022034510388808
PMCID: PMC3144128  PMID: 21088146
Streptococcus mutans; Streptococcus gordonii; Actinomyces naeslundii; biofilm; protease; quorum sensing
17.  Design and activity of a ‘dual-targeted’ antimicrobial peptide 
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.
doi:10.1016/j.ijantimicag.2008.11.013
PMCID: PMC2696886  PMID: 19188046
Antimicrobial peptide; Targeted therapeutic; Streptococcus mutans; Pseudomonas aeruginosa; Peptide synthesis; Novel antibiotic; STAMP; Specifically-targeted antimicrobial peptide; MH-STAMP
18.  A Conserved Streptococcal Membrane Protein, LsrS, Exhibits a Receptor-Like Function for Lantibiotics 
Journal of Bacteriology  2014;196(8):1578-1587.
Streptococcus mutans strain GS-5 produces a two-peptide lantibiotic, Smb, which displays inhibitory activity against a broad spectrum of bacteria, including other streptococci. For inhibition, lantibiotics must recognize specific receptor molecules present on the sensitive bacterial cells. However, so far no such receptor proteins have been identified for any lantibiotics. In this study, using a powerful transposon mutagenesis approach, we have identified in Streptococcus pyogenes a gene that exhibits a receptor-like function for Smb. The protein encoded by that gene, which we named LsrS, is a membrane protein belonging to the CAAX protease family. We also found that nisin, a monopeptide lantibiotic, requires LsrS for its optimum inhibitory activity. However, we found that LsrS is not required for inhibition by haloduracin and galolacticin, both of which are two-peptide lantibiotics closely related to Smb. LsrS appears to be a well-conserved protein that is present in many streptococci, including S. mutans. Inactivation of SMU.662, an LsrS homolog, in S. mutans strains UA159 and V403 rendered the cells refractory to Smb-mediated killing. Furthermore, overexpression of LsrS in S. mutans created cells more susceptible to Smb. Although LsrS and its homolog contain the CAAX protease domain, we demonstrate that inactivation of the putative active sites on the LsrS protein has no effect on its receptor-like function. This is the first report describing a highly conserved membrane protein that displays a receptor-like function for lantibiotics.
doi:10.1128/JB.00028-14
PMCID: PMC3993366  PMID: 24509319
19.  Interaction of melittin peptides with perfluorocarbon nanoemulsion particles 
The journal of physical chemistry. B  2011;115(51):15271-15279.
Melittin, an anti-microbial peptide, forms pores in biological membranes and triggers cell death. Therefore it has potential as an anti-cancer therapy. However, until recently, the therapeutic application of melittin has been impractical because a suitable platform for delivery was not available. Recently, we showed that phospholipid stabilized perfluorooctylbromide- based nanoemulsion particles (PFOB-NEPs) were resistant to destruction by melittin and enabled specific delivery of melittin to tumor cells, killing them and reducing tumor growth. Earlier prior work also showed that melittin adsorbed onto the stabilizing phospholipid monolayer of PFOB-NEP but did not disrupt the phospholipid monolayer or produce “cracking” of the PFOB-NEPs. The present work identifies the important structural motifs for melittin binding to PFOB-NEPs through a series of atomistic molecular dynamics simulations. The conformational ensemble of melittin bound to PFOB-NEP lipid monolayer was compared to structure from a control simulation of melittin bound to a lipid bilayer to identify several differences in melittin-lipid interactions between the two systems. First, melittin was deeply buried in the hydrophobic tail region of bilayer, while its depth was attenuated in the PFOB-NEP monolayer. Second, a helical conformation was the major secondary structure in the bilayer, but the fraction of helix was reduced in the PFOB-NEP. Finally, the overall pattern for the direct interaction of melittin with surrounding lipids was similar between liposome and PFOB-NEP, but the level of interaction was slightly decreased in the PFOB-NEP. These results suggest that melittin interacts with the monolayer of PFOB-NEP in a way that is similar way to its interaction with bilayers but that deeper penetration into the hydrophobic interior is inhibited.
doi:10.1021/jp209543c
PMCID: PMC3245340  PMID: 22050303
Perfluorocarbon; nanoemulsions; melittin; molecular dynamics
20.  A Lactotransferrin Single Nucleotide Polymorphism Demonstrates Biological Activity That Can Reduce Susceptibility to Caries 
Infection and Immunity  2013;81(5):1596-1605.
Streptococcus mutans is prominently linked to dental caries. Saliva's influence on caries is incompletely understood. Our goal was to identify a salivary protein with anti-S. mutans activity, characterize its genotype, and determine genotypic variants associated with S. mutans activity and reduced caries. An S. mutans affinity column was used to isolate active moieties from saliva obtained from a subject with minimal caries. The bound and eluted protein was identified as lactotransferrin (LTF) by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis and confirmed by Western blotting with LTF antibody. A single nucleotide polymorphism (SNP) that produced a shift from arginine (R) to lysine (K) at amino acid position 47 in the LTF antimicrobial region (rs: 1126478) killed S. mutans in vitro. Saliva from a subject with moderate caries and with the LTF “wild-type” R form at position 47 had no such activity. A pilot genetic study (n = 30) showed that KK subjects were more likely to have anti-S. mutans activity than RR subjects (P = 0.001; relative risk = 3.6; 95% confidence interval [95% CI] = 1.5 to 11.13). Pretreatment of KK saliva with antibody to LTF reduced S. mutans killing in a dose-dependent manner (P = 0.02). KK subjects were less likely to have caries (P = 0.02). A synthetic 11-mer LTF/K peptide killed S. mutans and other caries-related bacteria, while the LTF/R peptide had no effect (P = 0.01). Our results provide functional evidence that the LTF/K variant results in both anti-S. mutans activity and reduced decay. We suggest that the LTF/K variant can influence oral microbial ecology in general and caries-provoking microbes specifically.
doi:10.1128/IAI.01063-12
PMCID: PMC3647982  PMID: 23460521
21.  Protection of gnotobiotic rats against dental caries by passive immunization with bovine milk antibodies to Streptococcus mutans. 
Infection and Immunity  1987;55(10):2341-2347.
A multivalent vaccine consisting of whole cell antigens of seven strains, representing four serotypes (b, c, d and g), of mutans streptococci was used to hyperimmunize a group of cows. Serum samples from these animals contained immunoglobulin G1 (IgG1) antibody activity to seven serotypes (a to g) of mutans streptococci. Whey obtained from the animal with the highest serum antibody activity, which also contained high levels of IgG1 antibody, was used in passive caries immunity studies. Gnotobiotic rats monoinfected with Streptococcus mutans MT8148 serotype c or Streptococcus sobrinus OMZ176 (d) or 6715 (g) and provided a caries-promoting diet containing immune whey had lower plaque scores, numbers of streptococci in plaque, and degree of caries activity than similarly infected animals given a diet containing control whey obtained from nonimmunized cows. To establish the nature of the protective component(s) present in the immune whey, an ultrafiltrate fraction of the whey was prepared. This preparation contained higher levels of IgG1 anti-S. mutans antibody activity than the immune whey. Rats monoinfected with S. mutans MT8148 and provided with a diet supplemented with 0.1% of this fraction exhibited a degree of caries protection similar to that seen in animals provided a diet containing 100% immune whey. In fact, a diet containing as little as 0.01% of the ultrafiltrate fraction gave some degree of protection against oral S. mutans infection. The active component in the immune whey was the IgG1 anti-S. mutans antibody, since rats monoinfected with S. mutans MT8148 and provided a diet supplemented with purified immune whey IgG1 had significantly reduced plaque scores, numbers of S. mutans in plaque, and caries activity compared with control animals. Prior adsorption of the IgG fraction with killed S. mutans MT8148 whole cells removed antibody activity and abrogated caries protection.
PMCID: PMC260710  PMID: 3498689
22.  Influences of trans-trans farnesol, a membrane-targeting sesquiterpenoid, on Streptococcus mutans physiology and survival within mixed-species oral biofilms 
Trans-trans farnesol (tt-farnesol) is a bioactive sesquiterpene alcohol commonly found in propolis (a beehive product) and citrus fruits, which disrupts the ability of Streptococcus mutans (S. mutans) to form virulent biofilms. In this study, we investigated whether tt-farnesol affects cell-membrane function, acid production and/or acid tolerance by planktonic cells and biofilms of S. mutans UA159. Furthermore, the influence of the agent on S. mutans gene expression and ability to form biofilms in the presence of other oral bacteria (Streptococcus oralis (S. oralis) 35037 and Actinomyces naeslundii (A. naeslundii) 12104) was also examined. In general, tt-farnesol (1 mmol-L−1) significantly increased the membrane proton permeability and reduced glycolytic activity of S. mutans in the planktonic state and in biofilms (P<0.05). Moreover, topical applications of 1 mmol-L−1 tt-farnesol twice daily (1 min exposure/treatment) reduced biomass accumulation and prevented ecological shifts towards S. mutans dominance within mixed-species biofilms after introduction of 1% sucrose. S. oralis (a non-cariogenic organism) became the major species after treatments with tt-farnesol, whereas vehicle-treated biofilms contained mostly S. mutans (>90% of total bacterial population). However, the agent did not affect significantly the expression of S. mutans genes involved in acidogenicity, acid tolerance or polysaccharide synthesis in the treated biofilms. Our data indicate that tt-farnesol may affect the competitiveness of S. mutans in a mixed-species environment by primarily disrupting the membrane function and physiology of this bacterium. This naturally occurring terpenoid could be a potentially useful adjunctive agent to the current anti-biofilm/anti-caries chemotherapeutic strategies.
doi:10.4248/IJOS11038
PMCID: PMC3469883  PMID: 21485314
trans-trans farnesol; acid production; acid tolerance; biofilms; proton permeability; Streptococcus mutans
23.  Effective immunity to dental caries: enhancement of salivary anti-Streptococcus mutans antibody responses with oral adjuvants. 
Infection and Immunity  1983;40(2):577-591.
In the present study, we compared the ability of the soluble adjuvants concanavalin A (ConA), muramyl dipeptide (MDP), and peptidoglycan (PG) to enhance immune responses to orally administered particulate antigens of Streptococcus mutans 6715 in gnotobiotic rats. The isotype and levels of antibody in saliva and in serum from experimental rats were determined by an enzyme-linked immunosorbent assay using S. mutans whole cells (WC) as the coating antigen. The specificities of salivary and serum immunoglobulin A (IgA) antibodies to particulate S. mutans antigens, lipoteichoic acid, S. mutans serotype g carbohydrate, and dextran were also determined. When 50 micrograms of ConA was used as the oral adjuvant with S. mutans 6715 WC immunogen, a slight enhancement of immune responses was obtained. A higher dose of ConA suppressed humoral responses to the immunogen. Enhanced immune responses, especially of the IgA isotype, in both serum and saliva were induced in gnotobiotic rats given MDP and either S. mutans 6715 WC or purified cell walls (CW) by gastric intubation. Elevated IgA antibody levels to CW, lipoteichoic acid, and carbohydrate were observed in rats given S. mutans WC and MDP by gastric intubation, whereas oral immunization with S. mutans CW and MDP resulted in higher antibody levels to CW and carbohydrate and lower levels to lipoteichoic acid when compared with the antibody levels in rats given antigen alone. Rats orally immunized with either S. mutans WC or CW and MDP and challenged with virulent S. mutans 6715 exhibited significantly (P less than or equal to 0.05) lower plaque scores, numbers of viable S. mutans in plaque, and caries scores than did rats immunized with antigen alone or in infected-only controls. In another series of experiments, a PG fraction derived from S. mutans 6715 CW was assessed for adjuvant properties. The oral administration of PG and either S. mutans WC or CW induced good salivary and serum IgA antibody responses. The specificity of the antibodies was similar to that obtained in rats given antigen and MDP. Rats receiving either S. mutans WC or CW and PG and challenged with virulent S. mutans 6715 had lower plaque scores, fewer numbers of viable S. mutans in plaque, and lower caries activity than did infected rats receiving S. mutans WC or CW immunogen alone. These results provide evidence that soluble adjuvants derived from the gram-positive bacterial CW, e.g., MDP and PG, are effective oral adjuvants and augment IgA immune responses to particulate S. mutans antigens which are protective against the mucosally associated disease, dental caries.
PMCID: PMC264894  PMID: 6840853
24.  YlxM Is a Newly Identified Accessory Protein That Influences the Function of Signal Recognition Particle Pathway Components in Streptococcus mutans 
Journal of Bacteriology  2014;196(11):2043-2052.
Streptococcus mutans is a cariogenic oral pathogen whose virulence is determined largely by its membrane composition. The signal recognition particle (SRP) protein-targeting pathway plays a pivotal role in membrane biogenesis. S. mutans SRP pathway mutants demonstrate growth defects, cannot contend with environmental stress, and exhibit multiple changes in membrane composition. This study sought to define a role for ylxM, which in S. mutans and numerous other bacteria resides directly upstream of the ffh gene, encoding a major functional element of the bacterial SRP. YlxM was observed as a produced protein in S. mutans. Its predicted helix-turn-helix motif suggested that it has a role as a transcriptional regulator of components within the SRP pathway; however, no evidence of transcriptional regulation was found. Instead, capture enzyme-linked immunosorbent assay (ELISA), affinity chromatography, and bio-layer interferometry (BLI) demonstrated that S. mutans YlxM interacts with the SRP components Ffh and small cytoplasmic RNA (scRNA) but not with the SRP receptor FtsY. In the absence of FtsY, YlxM increased the GTP hydrolysis activity of Ffh alone and in complex with scRNA. However, in the presence of FtsY, YlxM caused an overall diminution of net GTPase activity. Thus, YlxM appears to modulate GTP hydrolysis, a process necessary for proper recycling of SRP pathway components. The presence of YlxM conferred a significant competitive growth advantage under nonstress and acid stress conditions when wild-type and ylxM mutant strains were cultured together. Our results identify YlxM as a component of the S. mutans SRP and suggest a regulatory function affecting GTPase activity.
doi:10.1128/JB.01465-13
PMCID: PMC4010984  PMID: 24659773
25.  Targeted Antimicrobial Treatment to Re-establish a Healthy Microbial Flora for Long-term Protection 
Advances in Dental Research  2012;24(2):94-97.
Streptococcus mutans has been implicated as the major acid-producing (cariogenic) bacterium. Dietary sugars and other factors may cause an imbalance of oral microflora that enables S. mutans to become dominant in the multi-species biofilms on the tooth surface, which could lead to dental caries. The application of broad-spectrum antimicrobials often results in re-colonization and re-dominance of S. mutans within oral flora, while in contrast, therapies capable of selective elimination of S. mutans from oral microbial communities may help to re-establish the normal flora and provide long-term protection. C16G2, a novel synthetic antimicrobial peptide with specificity for S. mutans, was found to have robust killing efficacy and selectivity for S. mutans in vitro. A subsequent pilot human study found that a single application of C16G2 in the oral cavity (formulated in a mouthrinse vehicle) was associated with a reduction in plaque and salivary S. mutans, lactic acid production, and enamel demineralization during the entire 4-day testing period. C16G2 is now being developed as a new anticaries drug.
doi:10.1177/0022034512453725
PMCID: PMC3420366  PMID: 22899688
microbial ecology; microbiology; microbial genetics; caries; dental biofilm; microbiota

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