Competence-stimulating Peptide Research Articles

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.

Developing multispecies quorum-sensing modulators based on the Streptococcus mitis competence-stimulating peptide

Bacteria utilize quorum sensing (QS) to coordinate many group behaviors. As such, QS has attracted significant attention as a potential mean to attenuate bacterial infectivity without introducing selective pressure for resistance development. Streptococcus mitis, a human commensal, acts as a genetic diversity reservoir for Streptococcus pneumoniae, a prevalent human pathogen. S.mitis possesses a typical comABCDE competence regulon QS circuitry; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon QS circuitry in S.mitis are yet to be explored. We set out to delineate the competence regulon QS circuitry in S.mitis, including confirming the identity of the native CSP signal, evaluating the molecular mechanism that governs CSP interactions with histidine kinase receptor ComD leading to ComD activation, and defining the regulatory roles of the competence regulon QS circuitry in initiating various S.mitis phenotypes. Our analysis revealed important structure-activity relationship insights of the CSP signal and facilitated the development of novel CSP-based QS modulators. Our analysis also revealed the involvement of the competence regulon in modulating competence development and biofilm formation. Furthermore, our analysis revealed that the native S.mitis CSP signal can modulate QS response in S.pneumoniae. Capitalizing on this crosstalk, we developed a multispecies QS modulator that activates both the pneumococcus ComD receptors and the S.mitis ComD-2 receptor with high potencies. The novel scaffolds identified herein can be utilized to evaluate the effects temporal QS modulation has on S.mitis as it inhabits its natural niche.

Structural basis of peptide secretion for Quorum sensing by ComA

Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as Streptococcus pneumoniae, ComA is a conserved efflux pump responsible for the maturation and secretion of peptide signals, including the competence-stimulating peptide (CSP), yet its structure and function remain unclear. Here, we functionally characterize ComA as an ABC transporter with high ATP affinity and determined its cryo-EM structures in the presence or absence of CSP or nucleotides. Our findings reveal a network of strong electrostatic interactions unique to ComA at the intracellular gate, a putative binding pocket for two CSP molecules, and negatively charged residues facilitating CSP translocation. Mutations of these residues affect ComA’s peptidase activity in-vitro and prevent CSP export in-vivo. We demonstrate that ATP-Mg2+ triggers the outward-facing conformation of ComA for CSP release, rather than ATP alone. Our study provides molecular insights into the QS signal peptide secretion, highlighting potential targets for QS-targeting drugs.

Noncanonical Streptococcus sanguinis ComCDE circuitry integrates environmental cues in transformation outcome decision

Natural competence is the principal driver of streptococcal evolution. While acquisition of new traits could facilitate rapid fitness improvement for bacteria, entry into the competent state is a highly orchestrated event, involving an interplay between various pathways. We present a new type of competence-predation coordination mechanism in Streptococcus sanguinis. Unlike other streptococci that mediate competence through the ComABCDE regulon, several key components are missing in the S.sanguinis ComCDE circuitry. We assembled two synthetic biology devices linking competence-stimulating peptide (CSP) cleavage and export with a quantifiable readout to unravel the unique features of the S.sanguinis circuitry. Our results revealed the ComC precursor cleavage pattern and the two host ABC transporters implicated in the export of the S.sanguinis CSP. Moreover, we discovered a ComCDE-dependent bacteriocin locus. Overall, this study presents a mechanism for commensal streptococci to maximize transformation outcome in a fluid environment through extensive circuitry rewiring.

The ComRS-SigX Pathway Regulates Natural Transformation in Streptococcus ferus.

The ability to take up and incorporate foreign DNA via natural transformation is a well-known characteristic of some species of Streptococcus, and is a mechanism that rapidly allows for the acquisition of antibacterial resistance. Here, we describe that the understudied species Streptococcus ferus is also capable of natural transformation and uses a system analogous to that identified in Streptococcus mutans. S. mutans natural transformation is under the control of the alternative sigma factor sigX (also known as comX), whose expression is induced by two types of peptide signals: CSP (competence stimulating peptide, encoded by comC) and XIP (sigX-inducing peptide, encoded by comS). These systems induce competence via either the two-component signal-transduction system ComDE or the RRNPP transcriptional regulator ComR, respectively. Protein and nucleotide homology searches identified putative orthologs of comRS and sigX in S. ferus, but not homologs of S. mutans blpRH (also known as comDE). We demonstrate that natural transformation in S. ferus is induced by a small, double-tryptophan containing sigX-inducing peptide (XIP), akin to that of S. mutans, and requires the presence of the comR and sigX orthologs for efficient transformation. Additionally, we find that natural transformation is induced in S. ferus by both the native XIP and the XIP variant of S. mutans, implying that cross talk between the two species is possible. This process has been harnessed to construct gene deletions in S. ferus and provides a method to genetically manipulate this understudied species. IMPORTANCE Natural transformation is the process by which bacteria take up DNA and allows for acquisition of new genetic traits, including those involved in antibiotic resistance. This study demonstrates that the understudied species Streptococcus ferus is capable of natural transformation using a peptide-pheromone system like that previously identified in Streptococcus mutans and provides a framework for future studies concerning this organism.

Interkingdom Detection of Bacterial Quorum-Sensing Molecules by Mammalian Taste Receptors

Bitter and sweet taste G protein-coupled receptors (known as T2Rs and T1Rs, respectively) were originally identified in type II taste cells on the tongue, where they signal perception of bitter and sweet tastes, respectively. Over the past ~15 years, taste receptors have been identified in cells all over the body, demonstrating a more general chemosensory role beyond taste. Bitter and sweet taste receptors regulate gut epithelial function, pancreatic β cell secretion, thyroid hormone secretion, adipocyte function, and many other processes. Emerging data from a variety of tissues suggest that taste receptors are also used by mammalian cells to “eavesdrop” on bacterial communications. These receptors are activated by several quorum-sensing molecules, including acyl-homoserine lactones and quinolones from Gram-negative bacteria such as Pseudomonas aeruginosa, competence stimulating peptides from Streptococcus mutans, and D-amino acids from Staphylococcus aureus. Taste receptors are an arm of immune surveillance similar to Toll-like receptors and other pattern recognition receptors. Because they are activated by quorum-sensing molecules, taste receptors report information about microbial population density based on the chemical composition of the extracellular environment. This review summarizes current knowledge of bacterial activation of taste receptors and identifies important questions remaining in this field.

The Antibacterial Effect of Cannabigerol toward Streptococcus mutans Is Influenced by the Autoinducers 21-CSP and AI-2

Bacteria can communicate through an intercellular signaling system referred to as quorum sensing (QS). The QS system involves the production of autoinducers that interact with their respective receptors, leading to the induction of specific signal transduction pathways. The QS systems of the oral cariogenic Streptococcus mutans regulate the maturation of biofilms and affect its virulent properties. We have previously shown that the non-psychoactive compound cannabigerol (CBG) of the Cannabis sativa L. plant has anti-bacterial and anti-biofilm activities towards S. mutans. Here we were interested in investigating the effect of the two QS systems ComCDE and LuxS on the susceptibility of S. mutans to CBG and the anti-QS activities of CBG. This was assessed by using various comCDE and luxS mutant strains and complementation with the respective autoinducers, competence stimulating peptide (CSP) and (S)-4,5-dihydroxy-2,3-pentandione (DPD, pre-AI-2). We found that S. mutans comCDE knockout strains were more sensitive to the anti-bacterial actions of CBG compared to the WT strain. Exogenously added 21-CSP prevented the anti-bacterial actions caused by CBG on the ΔcomC, ΔcomE and ΔluxS mutants, while having no effect on the susceptibility of the WT and ΔcomCDE strains to CBG. Exogenously added DPD increased the susceptibility of WT and ΔluxS to CBG. Vice versa, CBG significantly reduced the 21-CSP-induced expression of comCDE genes and ComE-regulated genes and suppressed the expression of luxS with concomitant reduction in AI-2 production. DPD induced the expression of comCDE genes and ComE-regulated genes, and this induction was repressed by CBG. 21-CSP alone had no significant effect on luxS gene expression, while ΔcomCDE strains showed reduced AI-2 production. In conclusion, our study shows that the susceptibility of S. mutans to CBG is affected by the ComCDE and LuxS QS pathways, and CBG is a potential anti-QS compound for S. mutans. Additionally, we provide evidence for crosstalk between the ComCDE and LuxS QS systems.

Identification of a Novel Gene Involved in Cell-to-cell Communication-induced Cell Death and eDNA Production in Streptococcus mutans.

Streptococcus mutans is a major caries-causing bacterium that forms firmly attached biofilms on tooth surfaces. Biofilm formation by S. mutans consists of polysaccharide-dependent and polysaccharide-independent processes. Among polysaccharide-independent processes, extracellular DNA (eDNA) mediates the initial attachment of cells to surfaces. We previously reported that the secreted peptide signal, competence-stimulating peptide (CSP) induced cell death in a subpopulation of cells, leading to autolysis-mediated eDNA release. The autolysin gene lytF, the expression of which is stimulated by CSP, has been shown to mediate CSP-dependent cell death, while cell death was not entirely abolished in the lytF deletion mutant, indicating the involvement of other factors. To identify novel genes involved in CSP-dependent cell death, we herein compared transcriptomes between live and dead cells derived from an isogenic population. The results obtained revealed the accumulation of several mRNAs in dead cells. The deletion of SMU_1553c, a putative bacteriocin gene, resulted in significant reductions in CSP-induced cell death and eDNA production levels from those in the parental strain. Moreover, in the double mutant strain of lytF and SMU_1553c, cell death and eDNA production in response to synthetic CSP were completely abolished under both planktonic and biofilm conditions. These results indicate that SMU_1553c is a novel cell death-related factor that contributes to CSP-dependent cell death and eDNA production.

Post-vaccine epidemiology of serotype 3 pneumococci identifies transformation inhibition through prophage-driven alteration of a non-coding RNA

BackgroundThe respiratory pathogen Streptococcus pneumoniae (the pneumococcus) is a genetically diverse bacterium associated with over 101 immunologically distinct polysaccharide capsules (serotypes). Polysaccharide conjugate vaccines (PCVs) have successfully eliminated multiple targeted serotypes, yet the mucoid serotype 3 has persisted despite its inclusion in PCV13. This capsule type is predominantly associated with a single globally disseminated strain, GPSC12 (clonal complex 180).MethodsA genomic epidemiology study combined previous surveillance datasets of serotype 3 pneumococci to analyse the population structure, dynamics, and differences in rates of diversification within GPSC12 during the period of PCV introductions. Transcriptomic analyses, whole genome sequencing, mutagenesis, and electron microscopy were used to characterise the phenotypic impact of loci hypothesised to affect this strain’s evolution.ResultsGPSC12 was split into clades by a genomic analysis. Clade I, the most common, rarely underwent transformation, but was typically infected with the prophage ϕOXC141. Prior to the introduction of PCV13, this clade’s composition shifted towards a ϕOXC141-negative subpopulation in a systematically sampled UK collection. In the post-PCV13 era, more rapidly recombining non-Clade I isolates, also ϕOXC141-negative, have risen in prevalence. The low in vitro transformation efficiency of a Clade I isolate could not be fully explained by the ~100-fold reduction attributable to the serotype 3 capsule. Accordingly, prophage ϕOXC141 was found to modify csRNA3, a non-coding RNA that inhibits the induction of transformation. This alteration was identified in ~30% of all pneumococci and was particularly common in the unusually clonal serotype 1 GPSC2 strain. RNA-seq and quantitative reverse transcriptase PCR experiments using a genetically tractable pneumococcus demonstrated the altered csRNA3 was more effective at inhibiting production of the competence-stimulating peptide pheromone. This resulted in a reduction in the induction of competence for transformation.ConclusionThis interference with the quorum sensing needed to induce competence reduces the risk of the prophage being deleted by homologous recombination. Hence the selfish prophage-driven alteration of a regulatory RNA limits cell-cell communication and horizontal gene transfer, complicating the interpretation of post-vaccine population dynamics.

Exploiting Conserved Quorum Sensing Signals in Streptococcus mutans and Streptococcus pneumoniae

Bacterial species of the Streptococcus genera are considered either commensal bacteria or potential pathogens, according to their metabolic evolution and production of quorum sensing (QS)-controlled virulence factors. S. mutans, in particular, has become one of the best-studied examples of bacteria that are able to get along or cheat commensal species, even of the same genera. S. mutans and S. pneumoniae share homolog QS pathways and a competence stimulating peptide (CSP) for regulating bacteriocin production. Intriguingly, the abundance of S. pneumoniae and S. mutans alternates in complex microbial communities, thus opening the role for the fratricide communication of homolog QS systems. Since the inhibition of the QS has been proposed in treating bacterial infections, in this study, we designed and synthesized analogs of S. pneumoniae CSP with precise residual modifications. We reported that S. pneumoniae CSP analogs reduced the expression of genes involved in the QS of S. mutans and biofilm formation without affecting bacterial growth. The CSP analogs inhibited bacteriocin production in S. mutans, as reported by co-cultures with commensal bacteria of the oral cavity. The peptide CSP1AA, bearing substitutions in the residues involved in QS receptor recognition and activation, reported the most significant quorum-quenching activities. Our findings provide new insights into specific chemical drivers in the CSP sequences controlling the interconnection between S. mutans and S. pneumoniae. We think that the results reported in this study open the way for new therapeutic interventions in controlling the virulence factors in complex microbial communities such as the oral microbiota.

Markerless Genome Editing in Competent Streptococci.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.

Investigating the Streptococcus sinensis competence regulon through a combination of transcriptome analysis and phenotypic evaluation.

Streptococcus sinensis is a recently identified member of the Mitis group of streptococci. This species has been associated with infective endocarditis; however its mechanisms of pathogenesis and virulence are not fully understood. This study aimed to investigate the influence of the competence-stimulating peptide (CSP) and the competence regulon quorum-sensing circuitry (ComABCDE) on subsequent gene transcription and expression, as well as resultant phenotypes. In this study we confirmed the native CSP identity, ascertained when endogenous CSP was produced and completed a transcriptome-wide analysis of all genes following CSP exposure. RNA sequencing analysis revealed the upregulation of genes known to be associated with competence, biofilm formation and virulence. As such, a variety of phenotypic assays were utilized to assess the correlation between increased mRNA expression and potential phenotype response, ultimately gaining insight into the effects of CSP on both gene expression and developed phenotypes. The results indicated that the addition of exogenous CSP aided in competence development and successful transformation, yielding an average transformation efficiency comparable to that of other Mitis group streptococci. Additional studies are needed to further delineate the effects of CSP exposure on biofilm formation and virulence. Overall, this study provides novel information regarding S. sinensis and provides a substantial foundation on which this species and its role in disease pathogenesis can be further investigated.

Quorum quenching of Streptococcus mutans via the nano-quercetin-based antimicrobial photodynamic therapy as a potential target for cariogenic biofilm

BackgroundQuorum sensing (QS) system can regulate the expression of virulence factors and biofilm formation in Streptococcus mutans. Antimicrobial photodynamic therapy (aPDT) inhibits quorum quenching (QQ), and can be used to prevent microbial biofilm. We thereby aimed to evaluate the anti-biofilm potency and anti-metabolic activity of nano-quercetin (N-QCT)-mediated aPDT against S. mutans. Also, in silico evaluation of the inhibitory effect of N-QCT on the competence-stimulating peptide (CSP) of S. mutans was performed to elucidate the impact of aPDT on various QS-regulated genes.MethodsCytotoxicity and intracellular reactive oxygen species (ROS) generation were assessed following synthesis and confirmation of N-QCT. Subsequently, the minimum biofilm inhibitory concentration (MBIC) of N-QCT against S. mutans and anti-biofilm effects of aPDT were assessed using colorimetric assay and plate counting. Molecular modeling and docking analysis were performed to confirm the connection of QCT to CSP. The metabolic activity of S. mutans and the expression level of various genes involved in QS were evaluated by flow cytometry and reverse transcription quantitative real-time PCR, respectively.ResultsSuccessful synthesis of non-toxic N-QCT was confirmed through several characterization tests. The MBIC value of N-QCT against S. mutans was 128 μg/mL. Similar to the crystal violet staining, the results log10 CFU/mL showed a significant degradation of preformed biofilms in the group treated with aPDT compared to the control group (P < 0.05). Following aPDT, metabolic activity of S. mutans also decreased by 85.7% (1/2 × MBIC of N-QCT) and 77.3% (1/4 × MBIC of N-QCT), as compared to the control values (P < 0.05). In silico analysis showed that the QCT molecule was located in the site formed by polypeptide helices of CSP. The relative expression levels of the virulence genes were significantly decreased in the presence of N-QCT-mediated aPDT (P < 0.05).ConclusionsThe combination of N-QCT with blue laser as a QQ-strategy leads to maximum ROS generation, disrupts the microbial biofilm of S. mutans, reduces metabolic activity, and downregulates the expression of genes involved in the QS pathway by targeting genes of the QS signaling system of S. mutans.

Attenuating the Streptococcus pneumoniae Competence Regulon Using Urea-Bridged Cyclic Dominant-Negative Competence-Stimulating Peptide Analogs.

Streptococcus pneumoniae (pneumococcus) is a prevalent human pathogen that utilizes the competence regulon quorum sensing circuitry to acquire antibiotic resistance and initiate its attack on the human host. Therefore, targeting the competence regulon can be applied as an anti-infective approach with minimal pressure for resistance development. Herein, we report the construction of a library of urea-bridged cyclic dominant-negative competence-stimulating peptide (dnCSP) derivatives and their evaluation as competitive inhibitors of the competence regulon. Our results reveal the first pneumococcus dual-action CSPs that inhibit the group 1 pneumococcus competence regulon while activating the group 2 pneumococcus competence regulon. Structural analysis indicates that the urea-bridge cyclization stabilizes the bioactive α-helix conformation, while in vivo studies using a mouse model of infection exhibit that the lead dual-action dnCSP, CSP1-E1A-cyc(Dab6Dab10), attenuates group 1-mediated mortality without significantly reducing the bacterial burden. Overall, our results pave the way for developing novel therapeutics against this notorious pathogen.

Pharmacological Evaluation of Synthetic Dominant-Negative Peptides Derived from the Competence-Stimulating Peptide of Streptococcus pneumoniae.

The competence regulon of Streptococcus pneumoniae (pneumococcus) is a quorum-sensing circuitry that regulates the ability of this pathogen to acquire antibiotic resistance or perform serotype switching, leading to vaccine-escape serotypes, via horizontal gene transfer, as well as initiate virulence. Induction of the competence regulon is centered on binding of the competence-stimulating peptide (CSP) to its cognate receptor, ComD. We have recently synthesized multiple dominant-negative peptide analogs capable of inhibiting competence induction and virulence in S. pneumoniae. However, the pharmacodynamics and safety profiles of these peptide drug leads have not been characterized. Therefore, in this study, we compared the biostability of cyanine-7.5-labeled wild-type CSPs versus dominant-negative peptide analogs (dnCSPs) spatiotemporally by using an IVIS Spectrum in vivo imaging system. Moreover, in vitro cytotoxicity and in vivo toxicity were evaluated. We conclude that our best peptide analog, CSP1-E1A-cyc(Dap6E10), is an attractive therapeutic agent against pneumococcal infection with superior safety and pharmacokinetics profiles.

Elucidating the Role and Structure-Activity Relationships of the Streptococcus oligofermentans Competence-Stimulating Peptide.

Streptococcus oligofermentans is an early colonizer of the oral microbiome with documented bactericidal activity against the oral pathogen Streptococcus mutans. S. oligofermentans has been observed to possess the typical comABCDE competence regulon found within most oral streptococci; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon is yet to be explored. Herein, we have both confirmed the identity of the S. oligofermentans CSP and utilized a wide range of phenotypic assays to characterize its regulatory role in competence, biofilm formation, and hydrogen peroxide formation. To determine the importance of each amino acid residue in CSP/ComD binding, we performed systematic replacement of amino acid residues within the S. oligofermentans CSP and developed a luciferase-based reporter system to assess the ability of these mutated analogues to modulate the competence regulon. Additionally, we performed CD analysis on mutated CSP analogues to determine the correlation between the peptide secondary structure and QS activation. To further explore S. oligofermentans' potential as a biotherapeutic against S. mutans infection, lead QS activators and inhibitors were used in interspecies competition assays to assess the effect of QS modulation on interactions between these two species. Lastly, we have documented a lack of S. oligofermentans-induced cytotoxicity, highlighting the potential of this native flora as a biotherapeutic with minimal health risks.

Short-chain fatty acids inhibit the biofilm formation of Streptococcus gordonii through negative regulation of competence-stimulating peptide signaling pathway.

Streptococcus gordonii, a Gram-positive commensal bacterium, is an opportunistic pathogen closely related to initiation and progression of various oral diseases, such as periodontitis and dental caries. Its biofilm formation is linked with the development of such diseases by enhanced resistance against antimicrobial treatment or host immunity. In the present study, we investigated the effect of short-chain fatty acids (SCFAs) on the biofilm formation of S. gordonii. SCFAs, including sodium acetate (NaA), sodium propionate (NaP), and sodium butyrate (NaB), showed an effective inhibitory activity on the biofilm formation of S. gordonii without reduction in bacterial growth. SCFAs suppressed S. gordonii biofilm formation at early time points whereas SCFAs did not affect its preformed biofilm. A quorum-sensing system mediated by competence-stimulating peptide (CSP) is known to regulate biofilm formation of streptococci. Interestingly, SCFAs substantially decreased mRNA expression of comD and comE, which are CSP-sensor and its response regulator responsible for CSP pathway, respectively. Although S. gordonii biofilm formation was enhanced by exogenous synthetic CSP treatment, such effect was not observed in the presence of SCFAs. Collectively, these results suggest that SCFAs have an anti-biofilm activity on S. gordonii through inhibiting comD and comE expression which results in negative regulation of CSP quorum-sensing system. SCFAs could be an effective anti-biofilm agent against S. gordonii for the prevention of oral diseases.

Bitter Taste Receptor T2R14 Modulates Gram-Positive Bacterial Internalization and Survival in Gingival Epithelial Cells

Bitter-taste receptors (T2Rs) have emerged as key players in host–pathogen interactions and important modulators of oral innate immunity. Previously, we reported that T2R14 is expressed in gingival epithelial cells (GECs) and interacts with competence stimulating peptides (CSPs) secreted by the cariogenic Streptococcus mutans. The underlying mechanisms of the innate immune responses and physiological effects of T2R14 on Gram-positive bacteria are not well characterized. In this study, we examined the role of T2R14 in internalization and growth inhibitory effects on Gram-positive bacteria, namely Staphylococcus aureus and S. mutans. We utilized CRISPR-Cas9 T2R14 knockdown (KD) GECs as the study model to address these key physiological mechanisms. Our data reveal that the internalization of S. aureus is significantly decreased, while the internalization of S. mutans remains unaffected upon knockdown of T2R14 in GECs. Surprisingly, GECs primed with S. mutans CSP-1 resulted in an inhibition of growth for S. aureus, but not for S. mutans. The GECs infected with S. aureus induced T2R14-dependent human β-defensin-2 (hBD-2) secretion; however, S. mutans–infected GECs did not induce hBD-2 secretion, but induced T2R14 dependent IL-8 secretion. Interestingly, our results show that T2R14 KD affects the cytoskeletal reorganization in GECs, thereby inhibiting S. aureus internalization. Our study highlights the distinct mechanisms and a direct role of T2R14 in influencing physiological responses to Gram-positive bacteria in the oral cavity.

Harnessing Multiple, Nonproteogenic Substitutions to Optimize CSP:ComD Hydrophobic Interactions in Group 1 Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is a human pathobiont that causes drastic antibiotic-resistant infections and is responsible for millions of deaths universally. Pneumococcus pathogenicity relies on the competence stimulating peptide (CSP) - mediated quorum sensing (QS) pathway that controls competence development for genetic transformation and, consequently, the spread of antibiotic resistance and virulence genes. Modulation of QS in S. pneumoniae can therefore be utilized to enervate pneumococcal infectivity as well as minimize the susceptibility for resistance development. In this work, we sought to optimize the interaction of CSP1 with its cognate transmembrane histidine kinase receptor (ComD1) through substitution of proteogenic and non-proteogenic amino acids on the hydrophobic binding face of CSP1. The findings from this study not only provided additional structure-activity data that are significant in optimizing CSP1 potency, but also led to the development of potent QS modulators. These CSP-based QS modulators could be used as privileged scaffolds for the development of antimicrobial agents against pneumococcal infections.

Bitter taste receptor T2R14 detects quorum sensing molecules from cariogenic Streptococcus mutans and mediates innate immune responses in gingival epithelial cells.

Host-pathogen interactions play an important role in defining the outcome of a disease. Recent studies have shown that the bacterial quorum sensing molecules (QSM) can interact with host cell membrane proteins, mainly G protein-coupled receptors (GPCRs), and induce innate immune responses. However, few studies have examined QSM-GPCR interactions and their influence on oral innate immune responses. In this study, we examined the role of bitter taste receptor T2R14 in sensing competence stimulating peptides (CSPs) secreted by cariogenic bacterium Streptococcus mutans and in mediating innate immune responses in gingival epithelial cells (GECs). Transcriptomic and western blot analyses identify T2R14 to be highly expressed in GECs. Our data show that only CSP-1 from S. mutans induces robust intracellular calcium mobilization compared to CSP-2 and CSP-3. By using CRISPR-Cas9, we demonstrate that CSP-1 induced calcium signaling and secretion of cytokines CXCL-8/IL-8, TNF-α, and IL-6 is mediated through T2R14 in GECs. Interestingly, the NF-kB signaling activated by CSP-1 in GECs was independent of T2R14. CSP-1-primed GECs attracted differentiated HL-60 immune cells (dHL-60) and this effect was abolished in T2R14 knock down GECs and also in cells primed with T2R14 antagonist 6-Methoxyflavone (6-MF). Our findings identify S. mutans CSP-1 as a peptide ligand for the T2R family. Our study establishes a novel host-pathogen interaction between cariogenic S. mutans CSP-1 and T2R14 in GECs leading to an innate immune response. Collectively, these findings suggest T2Rs as potential therapeutic targets to modulate innate immune responses upon oral bacterial infections.