Dysregulation of Cell Envelope Homeostasis in Staphylococcus aureus Exposed to Solvated Lignin.

Lignin is an aromatic plant cell wall polymer that facilitates water transport through the vasculature of plants and is generated in large quantities as an inexpensive by-product of pulp and paper manufacturing and biorefineries. Although lignin's ability to reduce bacterial growth has been reported previously, its hydrophobicity complicates the ability to examine its biological effects on living cells in aqueous growth media. We recently described the ability to solvate lignin in Good's buffers with neutral pH, a breakthrough that allowed examination of lignin's antimicrobial effects against the human pathogen Staphylococcus aureus. These analyses showed that lignin damages the S. aureus cell membrane, causes increased cell clustering, and inhibits growth synergistically with tunicamycin, a teichoic acid synthesis inhibitor. In the present study, we examined the physiological and transcriptomic responses of S. aureus to lignin. Intriguingly, lignin restored the susceptibility of genetically resistant S. aureus isolates to penicillin and oxacillin, decreased intracellular pH, impaired normal cell division, and rendered cells more resistant to detergent-induced lysis. Additionally, transcriptome sequencing (RNA-Seq) differential expression (DE) analysis of lignin-treated cultures revealed significant gene expression changes (P < 0.05 with 5% false discovery rate [FDR]) related to the cell envelope, cell wall physiology, fatty acid metabolism, and stress resistance. Moreover, a pattern of concurrent up- and downregulation of genes within biochemical pathways involved in transmembrane transport and cell wall physiology was observed, which likely reflects an attempt to tolerate or compensate for lignin-induced damage. Together, these results represent the first comprehensive analysis of lignin's antibacterial activity against S. aureus. IMPORTANCE S. aureus is a leading cause of skin and soft tissue infections. The ability of S. aureus to acquire genetic resistance to antibiotics further compounds its ability to cause life-threatening infections. While the historical response to antibiotic resistance has been to develop new antibiotics, bacterial pathogens are notorious for rapidly acquiring genetic resistance mechanisms. As such, the development of adjuvants represents a viable way of extending the life span of current antibiotics to which pathogens may already be resistant. Here, we describe the phenotypic and transcriptomic response of S. aureus to treatment with lignin. Our results demonstrate that lignin extracted from sugarcane and sorghum bagasse restores S. aureus susceptibility to ß-lactams, providing a premise for repurposing these antibiotics in treatment of resistant S. aureus strains, possibly in the form of topical lignin/ß-lactam formulations.

Characterization of the Streptococcus mutans SMU.1703c-SMU.1702c Operon Reveals Its Role in Riboflavin Import and Response to Acid Stress.

Streptococcus mutans utilizes numerous metabolite transporters to obtain essential nutrients in the "feast or famine" environment of the human mouth. S. mutans and most other streptococci are considered auxotrophic for several essential vitamins including riboflavin (vitamin B2), which is used to generate key cofactors and to perform numerous cellular redox reactions. Despite the well-known contributions of this vitamin to central metabolism, little is known about how S. mutans obtains and metabolizes B2 The uncharacterized protein SMU.1703c displays high sequence homology to the riboflavin transporter RibU. Deletion of SMU.1703c hindered S. mutans growth in complex and defined medium in the absence of saturating levels of exogenous riboflavin, whereas deletion of cotranscribed SMU.1702c alone had no apparent effect on growth. Expression of SMU.1703c in a Bacillus subtilis riboflavin auxotroph functionally complemented growth in nonsaturating riboflavin conditions. S. mutans was also able to grow on flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) in an SMU.1703c-dependent manner. Deletion of SMU.1703c and/or SMU.1702c impacted S. mutans acid stress tolerance, as all mutants showed improved growth at pH 5.5 compared to that of the wild type when medium was supplemented with saturating riboflavin. Cooccurrence of SMU.1703c and SMU.1702c, a hypothetical PAP2 family acid phosphatase gene, appears unique to the streptococci and may suggest a connection of SMU.1702c to the acquisition or metabolism of flavins within this genus. Identification of SMU.1703c as a RibU-like riboflavin transporter furthers our understanding of how S. mutans acquires essential micronutrients within the oral cavity and how this pathogen successfully competes within nutrient-starved oral biofilms.IMPORTANCE Dental caries form when acid produced by oral bacteria erodes tooth enamel. This process is driven by the fermentative metabolism of cariogenic bacteria, most notably Streptococcus mutans Nutrient acquisition is key in the competitive oral cavity, and many organisms have evolved various strategies to procure carbon sources or necessary biomolecules. B vitamins, such as riboflavin, which many oral streptococci must scavenge from the oral environment, are necessary for survival within the competitive oral cavity. However, the primary mechanism and proteins involved in this process remain uncharacterized. This study is important because it identifies a key step in S. mutans riboflavin acquisition and cofactor generation, which may enable the development of novel anticaries treatment strategies via selective targeting of metabolite transporters.

The development of drug-free therapy for prevention of dental caries.

PURPOSE: The purpose of this study was to develop a novel, drug-free therapy that can reduce the over-accumulation of cariogenic bacteria on dental surfaces. METHODS: We designed and synthesized a polyethylene glycol (PEG)-based hydrophilic copolymer functionalized with a pyrophosphate (PPi) tooth-binding anchor using "click" chemistry. The polymer was then evaluated for hydroxyapatite (HA) binding kinetics and capability of reducing bacteria adhesion to artificial tooth surface. RESULTS: The PPi-PEG copolymer can effectively inhibit salivary protein adsorption after rapid binding to an artificial tooth surface. As a result, the in vitro S. mutans adhesion study showed that the PPi-PEG copolymer can inhibit saliva protein-promoted S. mutans adhesion through the creation of a neutral, hydrophilic layer on the artificial tooth surface. CONCLUSIONS: The results suggested the potential application of a PPi-PEG copolymer as a drug-free alternative to current antimicrobial therapy for caries prevention.

The development of dentotropic micelles with biodegradable tooth-binding moieties.

PURPOSE: Development of dentotropic (tooth-binding) micelle formulations to improved efficacy and safety of antimicrobial therapy for dental plaque prevention and treatment. METHODS: Because of their excellent biocompatibility and biodegradability, diphosphoserine peptide and pyrophosphate were selected as the tooth-binding moieties to replace alendronate, which was used previously. Diphosphoserine peptide was conjugated to Pluronic P123 using "click" chemistry, whereas pyrophosphate was attached to P123 through an ester bond. The tooth-binding micelles (TBMs) were prepared by self-assembly of the modified P123 with the antimicrobial agent triclosan. The influence of human saliva and/or its components on TBMs' drug-releasing profile, tooth-binding potential and binding stability was evaluated in vitro. S. mutans UA159 biofilm formed on hydroxyapatite (HA) discs was used to evaluate the TBMs' therapeutic potential. RESULTS: Saliva does not affect triclosan release from TBMs. More than 60% of TBMs' HA binding capacity was maintained in the presence of saliva. Less than 5% of TBMs bound to HA was released over 24 h in human saliva, protease or phosphatase, suggesting the retention properties of the TBMs will not be compromised due to the biodegradable nature of the binding moieties. In both in vitro biofilm prevention and treatment studies, the TBM treated group showed significantly lower CFU per HA disc compared to the controls (2-log reduction, p < 0.05). CONCLUSION: The data from these studies suggest that the novel dentotropic micelle formulations bearing biodegradable tooth-binding moieties can be used as an effective and safe delivery tool for antimicrobials to improve dental plaque prevention and treatment.

Triclosan-loaded tooth-binding micelles for prevention and treatment of dental biofilm.

PURPOSE: To develop tooth-binding micelle formulations of triclosan for the prevention and treatment of dental caries. METHODS: Alendronate (ALN) was conjugated to the chain termini of different Pluronic copolymers to confer tooth-binding ability to the micelles. Using 3 different formulation methods, Pluronics and ALN-modified Pluronics were used to prepare triclosan-loaded tooth-binding micelles. The formulation parameters were optimized for triclosan solubility, particle size, hydroxyapatite (HA) binding capability and in vitro drug release profile. The optimized formulation was tested on an in vitro biofilm model. RESULTS: Direct dissolution was selected as the best formulation method. Triclosan-loaded tooth-binding micelles were able to inhibit initial biofilm growth of Streptococcus mutans UA159 by 6-log CFU/HA disc compared to the untreated control. These tooth-binding micelles were also able to reduce the viability of preformed biofilm by 4-log CFU/HA disc compared to the untreated control. CONCLUSIONS: Triclosan-loaded tooth-binding micelle formulations have been successfully developed and optimized in this study. These micelle formulations demonstrated promising anti-cariogenic bacteria capabilities and may find applications in the prevention and treatment of dental caries.

The Pta-AckA Pathway Regulates LrgAB-Mediated Pyruvate Uptake in Streptococcus mutans.

Pyruvate forms the central node of carbon metabolism and promotes growth as an alternative carbon source during starvation. We recently revealed that LrgAB functions as a stationary phase pyruvate uptake system in Streptococcus mutans, the primary causative agent of human dental caries, but its underlying regulatory mechanisms are still not clearly understood. This study was aimed at further characterizing the regulation of LrgAB from a metabolomic perspective. We utilized a series of GFP quantification, growth kinetics, and biochemical assays. We disclosed that LrgAB is critical for pyruvate uptake especially during growth under low-glucose stress. Inactivation of the Pta-Ack pathway, responsible for the conversion of acetyl-CoA to acetate, completely inhibits stationary phase lrgAB induction and pyruvate uptake, and renders cells insensitive to external pyruvate as a signal. Inactivation of Pfl, responsible for the conversion of pyruvate to acetyl-CoA under anaerobic conditions, also affected stationary phase pyruvate uptake. This study explores the metabolic components of pyruvate uptake regulation through LrgAB, and highlights its potential as a metabolic stimulator, contributing to the resuscitation and survival of S. mutans cells during nutritional stress.

Regulation of cid and lrg expression by CodY in Streptococcus mutans.

The ability of Streptococcus mutans to persist in a variety of adverse environments and to emerge as a numerically dominant member of stable oral biofilm communities are essential elements for its cariogenicity. The S. mutans Cid/Lrg system has been studied as a key player in the integration of complex environmental signals into regulatory networks that modulate virulence and cell homeostasis. Cid/Lrg has also been shown to be closely associated with metabolic pathways of this organism, due to distinct patterns of cid and lrg expression in response to growth phase and glucose/oxygen levels. In this study, a comparison of cid and lrg promoter regions with conserved CodY (a regulator which responds to starvation stress)-binding motifs revealed the presence of a potential CodY-binding site, which is arranged similarly in both cid and lrg promoters. Electrophoretic mobility shift assays (EMSAs) and promoter reporter assays demonstrated that expression of the cid and lrg operons is directly mediated by the global transcriptional regulator CodY. DNase I footprinting analyses confirmed the predicted binding sequences for CodY in both the cid and the lrg promoter regions. Overexpression of CodY had no obvious effect on lrgAB expression, but deficiency of CodY still affected lrgAB expression in a lytST-overexpressing strain, suggesting that CodY is required for the full regulation of lrgAB by LytST. We also demonstrated that both CodY and CcpA are involved in regulating pyruvate flux and utilization. Collectively, these data show that CodY directly regulates cid and lrg expression, and together with CcpA (previously shown to directly regulate cid and lrg promoters) contributes to coordinating pyruvate uptake and utilization in response to both the external environment and the cellular metabolic status.

Understanding LrgAB Regulation of Streptococcus mutans Metabolism.

Lack of LrgAB renders cariogenic Streptococcus mutans more sensitive to oxidative stress, as well as limits the capacity of this organism to re-uptake pyruvate upon starvation. This study was aimed at investigating the ecological and metabolic contribution of LrgAB to competitive fitness, using S. mutans strains, that either lack or overexpress lrgAB. These experiments revealed that impaired aerobic growth of the ΔlrgAB mutant can be effectively restored by supplementation of pyruvate, and that perturbated expression of lrgAB significantly affects pyruvate flux and the conversion of pyruvate to acetyl-CoA by the Pdh pathway, verifying that LrgAB is closely linked to pyruvate catabolism. In vitro competition assays revealed that LrgAB plays an important role in S. mutans competition with H2O2-producing S. gordonii, an interaction which can also be modulated by external pyruvate. However, no obvious competitive disadvantage was observed against S. gordonii by either the S. mutans lrgAB mutant or lrgAB overexpression strain in vivo using a mouse caries model. Organic acid analysis of mouse dental biofilms revealed that metabolites produced by the host and/or dental plaque microbiota could complement the deficiency of a lrgAB mutant, and favored S. mutans establishment compared to S. gordonii. Collectively, these results reinforce the importance of the oral microbiota and the metabolic environment in the oral cavity battleground, and highlight that pyruvate uptake through LrgAB may be crucial for interspecies competition that drives niche occupancy.

Tooth-binding micelles for dental caries prevention.

Maintenance of the effective local concentration of antimicrobials on the tooth surface is critical for the management of cariogenic bacteria in the oral cavity. We report on the design of a simple tooth-binding micellar drug delivery platform that would effectively bind to tooth surfaces. To achieve tooth-binding ability, the chain termini of biocompatible Pluronic copolymers were modified with a biomineral-binding moiety (i.e., alendronate). The micelles formulated with this polymer were shown to be able to swiftly (<1 min) bind to hydroxyapatite (HA; a model tooth surface) and gradually release the encapsulated model antimicrobial (farnesol). These tooth-binding micelles were negatively charged and had an average effective hydrodynamic diameter of less than 100 nm. In vitro biofilm inhibition studies demonstrated that the farnesol-containing tooth-binding micelles were able to provide significantly stronger inhibition of Streptococcus mutans UA159 biofilm formation on HA discs than the untreated blank control micelles (P < 0.0001). Upon further optimization, this delivery platform could provide an effective tool for caries prevention and treatment.

Genomic instability of TnSMU2 contributes to Streptococcus mutans biofilm development and competence in a cidB mutant.

Streptococcus mutans is a key pathogenic bacterium in the oral cavity and a primary contributor to dental caries. The S. mutans Cid/Lrg system likely contributes to tolerating stresses encountered in this environment as cid and/or lrg mutants exhibit altered oxidative stress sensitivity, genetic competence, and biofilm phenotypes. It was recently noted that the cidB mutant had two stable colony morphologies: a "rough" phenotype (similar to wild type) and a "smooth" phenotype. In our previously published work, the cidB rough mutant exhibited increased sensitivity to oxidative stress, and RNAseq identified widespread transcriptomic changes in central carbon metabolism and oxidative stress response genes. In this current report, we conducted Illumina-based genome resequencing of wild type, cidB rough, and cidB smooth mutants and compared their resistance to oxidative and acid stress, biofilm formation, and competence phenotypes. Both cidB mutants exhibited comparable aerobic growth inhibition on agar plates, during planktonic growth, and in the presence of 1 mM hydrogen peroxide. The cidB smooth mutant displayed a significant competence defect in BHI, which was rescuable by synthetic CSP. Both cidB mutants also displayed reduced XIP-mediated competence, although this reduction was more pronounced in the cidB smooth mutant. Anaerobic biofilms of the cidB smooth mutant displayed increased propidium iodide staining, but corresponding biofilm CFU data suggest this phenotype is due to cell damage and not increased cell death. The cidB rough anaerobic biofilms showed altered structure relative to wild type (reduced biomass and average thickness) which correlated with decreased CFU counts. Sequencing data revealed that the cidB smooth mutant has a unique "loss of read coverage" of ~78 kb of DNA, corresponding to the genomic island TnSMU2 and genes flanking its 3' end. It is therefore likely that the unique biofilm and competence phenotypes of the cidB smooth mutant are related to its genomic changes in this region.

Investigation of simulated microgravity effects on Streptococcus mutans physiology and global gene expression.

Astronauts have been previously shown to exhibit decreased salivary lysozyme and increased dental calculus and gingival inflammation in response to space flight, host factors that could contribute to oral diseases such as caries and periodontitis. However, the specific physiological response of caries-causing bacteria such as Streptococcus mutans to space flight and/or ground-based simulated microgravity has not been extensively investigated. In this study, high aspect ratio vessel S. mutans simulated microgravity and normal gravity cultures were assessed for changes in metabolite and transcriptome profiles, H2O2 resistance, and competence in sucrose-containing biofilm media. Stationary phase S. mutans simulated microgravity cultures displayed increased killing by H2O2 compared to normal gravity control cultures, but competence was not affected. RNA-seq analysis revealed that expression of 153 genes was up-regulated ≥2-fold and 94 genes down-regulated ≥2-fold during simulated microgravity high aspect ratio vessel growth. These included a number of genes located on extrachromosomal elements, as well as genes involved in carbohydrate metabolism, translation, and stress responses. Collectively, these results suggest that growth under microgravity analog conditions promotes changes in S. mutans gene expression and physiology that may translate to an altered cariogenic potential of this organism during space flight missions.

Pluronics-Formulated Farnesol Promotes Efficient Killing and Demonstrates Novel Interactions with Streptococcus mutans Biofilms.

Streptococcus mutans is the primary causative agent of dental caries, one of the most prevalent diseases in the United States. Previously published studies have shown that Pluronic-based tooth-binding micelles carrying hydrophobic antimicrobials are extremely effective at inhibiting S. mutans biofilm growth on hydroxyapatite (HA). Interestingly, these studies also demonstrated that non-binding micelles (NBM) carrying antimicrobial also had an inhibitory effect, leading to the hypothesis that the Pluronic micelles themselves may interact with the biofilm. To explore this potential interaction, three different S. mutans strains were each grown as biofilm in tissue culture plates, either untreated or supplemented with NBM alone (P85), NBM containing farnesol (P85F), or farnesol alone (F). In each tested S. mutans strain, biomass was significantly decreased (SNK test, p < 0.05) in the P85F and F biofilms relative to untreated biofilms. Furthermore, the P85F biofilms formed large towers containing dead cells that were not observed in the other treatment conditions. Tower formation appeared to be specific to formulated farnesol, as this phenomenon was not observed in S. mutans biofilms grown with NBM containing triclosan. Parallel CFU/ml determinations revealed that biofilm growth in the presence of P85F resulted in a 3-log reduction in viability, whereas F decreased viability by less than 1-log. Wild-type biofilms grown in the absence of sucrose or gtfBC mutant biofilms grown in the presence of sucrose did not form towers. However, increased cell killing with P85F was still observed, suggesting that cell killing is independent of tower formation. Finally, repeated treatment of pre-formed biofilms with P85F was able to elicit a 2-log reduction in viability, whereas parallel treatment with F alone only reduced viability by 0.5-log. Collectively, these results suggest that Pluronics-formulated farnesol induces alterations in biofilm architecture, presumably via interaction with the sucrose-dependent biofilm matrix, and may be a viable treatment option in the prevention and treatment of pathogenic plaque biofilms.