Ribosomal-processing cysteine protease homolog modulates Streptococcus mutans glucan production and interkingdom interactions.

Glucan-dependent biofilm formation is a crucial process in the establishment of Streptococcus mutans as a cariogenic oral microbe. The process of glucan formation has been investigated in great detail, with glycosyltransferases GtfB, GtfC, and GtfD shown to be indispensable for the synthesis of glucans from sucrose. Glucan production can be visualized during biofilm formation through fluorescent labeling, and its abundance, as well as the effect of glucans on general biofilm architecture, is a common phenotype to study S. mutans virulence regulation. Here, we describe an entirely new phenotype associated with glucan production, caused by a mutation in the open reading frame SMU_848, which is located in an operon encoding ribosome-associated proteins. This mutation led to the excess production and accumulation of glucan-containing droplets on the surface of biofilms formed on agar plates after prolonged incubation. While not characterized in S. mutans, SMU_848 shows homology to the phage-related ribosomal protease Prp, essential in cleaving off the N-terminal extension of ribosomal protein L27 for functional ribosome assembly in Staphylococcus aureus. We present a further characterization of SMU_848/Prp, demonstrating that the deletion of this gene leads to significant changes in S. mutans gtfBC expression. Surprisingly, it also profoundly impacts the interkingdom interaction between S. mutans and Candida albicans, a relevant dual-species interaction implicated in severe early childhood caries. The presented data support a potential broader role for SMU_848/Prp, possibly extending its functionality beyond the ribosomal network to influence important ecological processes. IMPORTANCE: Streptococcus mutans is an important member of the oral biofilm and is implicated in the initiation of caries. One of the main virulence mechanisms is the glucan-dependent formation of biofilms. We identified a new player in the regulation of glucan production, SMU_848, which is part of an operon that also encodes for ribosomal proteins L27 and L21. A mutation in SMU_848, which encodes a phage-related ribosomal protease Prp, leads to a significant accumulation of glucan-containing droplets on S. mutans biofilms, a previously unknown phenotype. Further investigations expanded our knowledge about the role of SMU_848 beyond its role in glucan production, including significant involvement in interkingdom interactions, thus potentially playing a global role in the virulence regulation of S. mutans.

Plasmalogen, a glycerophospholipid crucial for Streptococcus mutans acid tolerance and colonization.

Plasmalogen is a specific glycerophospholipid present in both animal and bacterial organisms. It plays a crucial function in eukaryotic cellular processes and is closely related to several human diseases, including neurological disorders and cancers. Nonetheless, the precise biological role of plasmalogen in bacteria is not well understood. In this study, we identified SMU_438c as the enzyme responsible for plasmalogen production in Streptococcus mutans under anaerobic conditions. The heterologous expression of SMU_438c in a plasmalogen-negative strain, Streptococcus sanguinis, resulted in the production of plasmalogen, indicating that this enzyme is sufficient for plasmalogen production. Additionally, the plasmalogen-deficient S. mutans exhibited significantly lower acid tolerance and diminished its colonization in Drosophila flies compared to the wild-type strain and complemented strain. In summary, our data suggest that plasmalogen plays a vital role in bacterial stress tolerance and in vivo colonization. IMPORTANCE: This study sheds light on the biological role of plasmalogen, a specific glycerophospholipid, in bacteria, particularly in Streptococcus mutans. Plasmalogens are known for their significant roles in eukaryotic cells and have been linked to human diseases like neurological disorders and cancers. The enzyme SMU_438c, identified as essential for plasmalogen production under anaerobic conditions, was crucial for acid tolerance and in vivo colonization in Drosophila by S. mutans, underscoring its importance in bacterial stress response and colonization. These findings bridge the knowledge gap in bacterial physiology, highlighting plasmalogen's role in microbial survival and offering potential insights into microbial pathogenesis and host-microbe interactions.

The transcription regulator BrsR serves as a network hub of natural competence protein-protein interactions in Streptococcus mutans.

Genome evolution is an essential and stringently regulated aspect of biological fitness. For bacteria, natural competence is one of the principal mechanisms of genome evolution and is frequently subject to multiple layers of regulation derived from a plethora of environmental and physiological stimuli. Here, we present a regulatory mechanism that illustrates how such disparate stimuli can be integrated into the Streptococcus mutans natural competence phenotype. S. mutans possesses an intriguing, but poorly understood ability to coordinately control its independently regulated natural competence and bacteriocin genetic pathways as a means to acquire DNA released from closely related, bacteriocin-susceptible streptococci. Our results reveal how the bacteriocin-specific transcription activator BrsR directly mediates this coordination by serving as an anti-adaptor protein responsible for antagonizing the proteolysis of the inherently unstable, natural competence-specific alternative sigma factor ComX. This BrsR ability functions entirely independent of its transcription regulator function and directly modulates the timing and severity of the natural competence phenotype. Additionally, many of the DNA uptake proteins produced by the competence system were surprisingly found to possess adaptor abilities, which are employed to terminate the BrsR regulatory circuit via negative feedback. BrsR-competence protein heteromeric complexes directly inhibit nascent brsR transcription as well as stimulate the Clp-dependent proteolysis of extant BrsR proteins. This study illustrates how critical genetic regulatory abilities can evolve in a potentially limitless variety of proteins without disrupting their conserved ancestral functions. These unrecognized regulatory abilities are likely fundamental for transducing information through complex genetic networks.

The Streptococcus mutans irvA gene encodes a trans-acting riboregulatory mRNA.

In both prokaryotes and eukaryotes, insight into gene function is typically obtained by in silico homology searches and/or phenotypic analyses of strains bearing mutations within open reading frames. However, the studies herein illustrate how mRNA function is not limited to the expression of a cognate protein. We demonstrate that a stress-induced protein-encoding mRNA (irvA) from the dental caries pathogen Streptococcus mutans directly modulates target mRNA (gbpC) stability through seed pairing interactions. The 5' untranslated region of irvA mRNA is a trans riboregulator of gbpC and a critical activator of the DDAG stress response, whereas IrvA functions independently in the regulation of natural competence. The irvA riboregulatory domain controls GbpC production by forming irvA-gbpC hybrid mRNA duplexes that prevent gbpC degradation by an RNase J2-mediated pathway. These studies implicate a potentially ubiquitous role for typical protein-encoding mRNAs as riboregulators, which could alter current concepts in gene regulation.

Genome-Wide Identification of Novel sRNAs in Streptococcus mutans.

Streptococcus mutans is a major pathobiont involved in the development of dental caries. Its ability to utilize numerous sugars and to effectively respond to environmental stress promotes S. mutans proliferation in oral biofilms. Because of their quick action and low energetic cost, noncoding small RNAs (sRNAs) represent an ideal mode of gene regulation in stress response networks, yet their roles in oral pathogens have remained largely unexplored. We identified 15 novel sRNAs in S. mutans and show that they respond to four stress-inducing conditions commonly encountered by the pathogen in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. To better understand the role of sRNAs in S. mutans, we further explored the function of the novel sRNA SmsR4. Our data demonstrate that SmsR4 regulates the enzyme IIA (EIIA) component of the sorbitol phosphotransferase system, which transports and phosphorylates the sugar alcohol sorbitol. The fine-tuning of EIIA availability by SmsR4 likely promotes S. mutans growth while using sorbitol as the main carbon source. Our work lays a foundation for understanding the role of sRNAs in regulating gene expression in stress response networks in S. mutans and highlights the importance of the underexplored phenomenon of posttranscriptional gene regulation in oral bacteria. IMPORTANCE Small RNAs (sRNAs) are important gene regulators in bacteria, but the identities and functions of sRNAs in Streptococcus mutans, the principal bacterium involved in the formation of dental caries, are unknown. In this study, we identified 15 putative sRNAs in S. mutans and show that they respond to four common stress-inducing conditions present in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. We further show that the novel sRNA SmsR4 likely modulates sorbitol transport into the cell by regulating SMU_313 mRNA, which encodes the EIIA subunit of the sorbitol phosphotransferase system. Gaining a better understanding of sRNA-based gene regulation may provide new opportunities to develop specific inhibitors of S. mutans growth, thereby improving oral health.

Regulatory control of the Streptococcus mutans HdrRM LytTR Regulatory System functions via a membrane sequestration mechanism.

Bacteria sense and respond to environmental changes via several broad categories of sensory signal transduction systems. Recently, we described the key features of a previously unrecognized, but widely conserved class of prokaryotic sensory system that we refer to as the LytTR Regulatory System (LRS). Our previous studies suggest that most, if not all, prokaryotic LRS membrane proteins serve as inhibitors of their cognate transcription regulators, but the inhibitory mechanisms employed have thus far remained a mystery. Using the Streptococcus mutans HdrRM LRS as a model, we demonstrate how the LRS membrane protein HdrM inhibits its cognate transcription regulator HdrR by tightly sequestering HdrR in a membrane-localized heteromeric HdrR/M complex. Membrane sequestration of HdrR prevents the positive feedback autoregulatory function of HdrR, thereby maintaining a low basal expression of the hdrRM operon. However, this mechanism can be antagonized by ectopically expressing a competitive inhibitor mutant form of HdrR that lacks its DNA binding ability while still retaining its HdrM interaction. Our results indicate that sequestration of HdrR is likely to be the only mechanism required to inhibit its transcription regulator function, suggesting that endogenous activation of the HdrRM LRS is probably achieved through a modulation of the HdrR/M interaction.

LytTR Regulatory Systems: A potential new class of prokaryotic sensory system.

The most commonly studied prokaryotic sensory signal transduction systems include the one-component systems, phosphosignaling systems, extracytoplasmic function (ECF) sigma factor systems, and the various types of second messenger systems. Recently, we described the regulatory role of two separate sensory systems in Streptococcus mutans that jointly control bacteriocin gene expression, natural competence development, as well as a cell death pathway, yet they do not function via any of the currently recognized signal transduction paradigms. These systems, which we refer to as LytTR Regulatory Systems (LRS), minimally consist of two proteins, a transcription regulator from the LytTR Family and a transmembrane protein inhibitor of this transcription regulator. Here, we provide evidence suggesting that LRS are a unique uncharacterized class of prokaryotic sensory system. LRS exist in a basal inactive state. However, when LRS membrane inhibitor proteins are inactivated, an autoregulatory positive feedback loop is triggered due to LRS regulator protein interactions with direct repeat sequences located just upstream of the -35 sequences of LRS operon promoters. Uncharacterized LRS operons are widely encoded by a vast array of Gram positive and Gram negative bacteria as well as some archaea. These operons also contain unique direct repeat sequences immediately upstream of their operon promoters indicating that positive feedback autoregulation is a globally conserved feature of LRS. Despite the surprisingly widespread occurrence of LRS operons, the only characterized examples are those of S. mutans. Therefore, the current study provides a useful roadmap to investigate LRS function in the numerous other LRS-encoding organisms.

Functional definition of NrtR, a remnant regulator of NAD+ homeostasis in the zoonotic pathogen Streptococcus suis.

NAD+ is an enzyme cofactor required for the 3 domains of life. However, little is known about the NAD+ biosynthesis and salvage pathways in the opportunistic pathogen Streptococcus suis. A genome-wide search allows us to identify the NAD+ salvage pathway encoded by an operon of nadR-pnuC-nrtR (from SSU05_1973 to SSU05_1971 on the reverse strand) in the S. suis 05ZYH33 that causes streptococcal toxin shock-like syndrome. The regulator of this pathway is Nudix-related transcriptional regulator (NrtR), a transcription regulator of the Nudix family comprising an N-terminal Nudix-like effector domain, and a C-terminal DNA-binding winged helix-turn-helix-like domain. Intriguingly, the S. suis NrtR naturally contains a single amino acid substitution (K92E) in the catalytic site of its Nudix domain that renders it catalytically inactive but does not influence its ability to bind DNA. Despite its lack of enzymatic activity, DNA-binding activity of NrtR is antagonized by the effector ADP-ribose. Furthermore, nrtR knockout in S. suis serotype 2 reduces its capacity to form biofilms and attenuates its virulence in a mouse infection model. Genome mining indicates that nrtR appears in a strain-specific manner whose occupancy is correlated to bacterial infectivity. Unlike the paradigmatic member of NrtR family having 2 unrelated functions (Nudix hydrolase and DNA binding), S. suis 2 retains a single regulatory role in the modulation of NAD+ salvage. This control of NAD+ homeostasis contributes to S. suis virulence.-Wang, Q., Hassan, B. H., Lou, N., Merritt, J., Feng, Y. Functional definition of NrtR, a remnant regulator of NAD+ homeostasis in the zoonotic pathogen Streptococcus suis.

Effect of biofilm exposure on marginal integrity of composite restorations.

PURPOSE: To evaluate the effect of bacterial exposure on the marginal integrity of dentin-resin interfaces for composites with and without bioactive glass (BAG). METHODS: Cavity preparations of 5 mm width and 1.5 mm depth were machined into dentin disks by means of a computer controlled milling system. After applying the bonding agent, cavity preparations (n=3-5) were restored by incremental technique with experimental resin composites (50:50 BisGMA/TEGDMA: 72wt% filler) with different filler compositions: control - 67 wt% silanated strontium glass and 5wt% aerosol-silica filler and BAG - 57 wt% silanated strontium glass and 15 wt% BAG-65 wt% silica. Samples were then stored in sterile Todd-Hewitt media or co-incubated with Streptococcus mutans (UA 159), at 37°C, 5% CO2 for 1-2 weeks. For samples co-incubated with a living biofilm, a luciferase assay was performed in order to assess its viability. Surfaces were impressed before and after each storage condition and replicas examined in a scanning electron microscope. Using image analysis software (Image J), the discontinuous margins percentage (%DM) was quantitatively assessed. Data were analyzed using two-way ANOVA followed by Tukey's test (α= 0.05). RESULTS: Gap size ranged between 7-23 µm. The bacterial exposure significantly increased the %DM in both groups predominantly due to the formation of new gap regions. There was no difference between control and BAG composites regarding %DM and the biofilm viability. Bacterial exposure promoted degradation of composite restoration marginal integrity, with no difference between composites with and without BAG. CLINICAL SIGNIFICANCE: The samples incubated with living biofilm had a higher gap percentage in the margins, confirming the negative effect of cariogenic bacteria on margin degradation. The parameters defined for such synergy can help to understand the multi-factorial aspect of marginal discontinuity and therefore, predict the behavior of composite restorations subjected to the challenging oral environment.

Distinct Regulatory Role of Carbon Catabolite Protein A (CcpA) in Oral Streptococcal spxB Expression.

Pyruvate oxidase (SpxB)-dependent H2O2 production is under the control of carbon catabolite protein A (CcpA) in the oral species Streptococcus sanguinis and Streptococcus gordonii Interestingly, both species react differently to the presence of the preferred carbohydrate source glucose. S. gordonii CcpA-dependent regulation of spxB follows classical carbon catabolite repression. Conversely, spxB expression in S. sanguinis is not influenced by glucose but is repressed by CcpA. Here, we constructed strains expressing the heterologous versions of CcpA or the spxB promoter region to learn if the distinct regulation of spxB expression is transferable from S. gordonii to S. sanguinis and vice versa. While cross-species binding of CcpA to the spxB promoter is conserved in vitro, we were unable to swap the species-specific regulation. This suggests that a regulatory mechanism upstream of CcpA most likely is responsible for the observed difference in spxB expression. Moreover, the overall ecological significance of differential spxB regulation in the presence of various glucose concentrations was tested with additional oral streptococcus isolates and demonstrated that carbohydrate-dependent and carbohydrate-independent mechanisms exist to control expression of spxB in the oral biofilm. Overall, our data demonstrate the unexpected finding that metabolic pathways between two closely related oral streptococcal species can be regulated differently despite an exceptionally high DNA sequence identity.IMPORTANCE Polymicrobial diseases are the result of interactions among the residential microbes, which can lead to a dysbiotic community. Streptococcus sanguinis and Streptococcus gordonii are considered commensal species that are present in the healthy dental biofilm. Both species are able to produce significant amounts of H2O2 via the enzymatic action of the pyruvate oxidase SpxB. H2O2 is able to inhibit species associated with oral diseases. SpxB and its gene-regulatory elements present in both species are highly conserved. Nonetheless, a differential response to the presence of glucose was observed. Here, we investigate the mechanisms that lead to this differential response. Detailed knowledge of the regulatory mechanisms will aid in a better understanding of oral disease development and how to prevent dysbiosis.

Plasticity of the Pyruvate Node Modulates Hydrogen Peroxide Production and Acid Tolerance in Multiple Oral Streptococci.

Commensal Streptococcus sanguinis and Streptococcus gordonii are pioneer oral biofilm colonizers. Characteristic for both is the SpxB-dependent production of H2O2, which is crucial for inhibiting competing biofilm members, especially the cariogenic species Streptococcus mutans H2O2 production is strongly affected by environmental conditions, but few mechanisms are known. Dental plaque pH is one of the key parameters dictating dental plaque ecology and ultimately oral health status. Therefore, the objective of the current study was to characterize the effects of environmental pH on H2O2 production by S. sanguinis and S. gordoniiS. sanguinis H2O2 production was not found to be affected by moderate changes in environmental pH, whereas S. gordonii H2O2 production declined markedly in response to lower pH. Further investigation into the pyruvate node, the central metabolic switch modulating H2O2 or lactic acid production, revealed increased lactic acid levels for S. gordonii at pH 6. The bias for lactic acid production at pH 6 resulted in concomitant improvement in the survival of S. gordonii at low pH and seems to constitute part of the acid tolerance response of S. gordonii Differential responses to pH similarly affect other oral streptococcal species, suggesting that the observed results are part of a larger phenomenon linking environmental pH, central metabolism, and the capacity to produce antagonistic amounts of H2O2IMPORTANCE Oral biofilms are subject to frequent and dramatic changes in pH. S. sanguinis and S. gordonii can compete with caries- and periodontitis-associated pathogens by generating H2O2 Therefore, it is crucial to understand how S. sanguinis and S. gordonii adapt to low pH and maintain their competitiveness under acid stress. The present study provides evidence that certain oral bacteria respond to environmental pH changes by tuning their metabolic output in favor of lactic acid production, to increase their acid survival, while others maintain their H2O2 production at a constant level. The differential control of H2O2 production provides important insights into the role of environmental conditions for growth competition of the oral flora.

Negative Regulation of Ectoine Uptake and Catabolism in Sinorhizobium meliloti: Characterization of the EhuR Gene.

Ectoine has osmoprotective effects on Sinorhizobium meliloti that differ from its effects in other bacteria. Ectoine does not accumulate in S. meliloti cells; instead, it is degraded. The products of the ehuABCD-eutABCDE operon were previously discovered to be responsible for the uptake and catabolism of ectoine in S. meliloti However, the mechanism by which ectoine is involved in the regulation of the ehuABCD-eutABCDE operon remains unclear. The ehuR gene, which is upstream of and oriented in the same direction as the ehuABCD-eutABCDE operon, encodes a member of the MocR/GntR family of transcriptional regulators. Quantitative reverse transcription-PCR and promoter-lacZ reporter fusion experiments revealed that EhuR represses transcription of the ehuABCD-eutABCDE operon, but this repression is inhibited in the presence of ectoine. Electrophoretic mobility shift assays and DNase I footprinting assays revealed that EhuR bound specifically to the DNA regions overlapping the -35 region of the ehuA promoter and the +1 region of the ehuR promoter. Surface plasmon resonance assays further demonstrated direct interactions between EhuR and the two promoters, although EhuR was found to have higher affinity for the ehuA promoter than for the ehuR promoter. In vitro, DNA binding by EhuR could be directly inhibited by a degradation product of ectoine. Our work demonstrates that EhuR is an important negative transcriptional regulator involved in the regulation of ectoine uptake and catabolism and is likely regulated by one or more end products of ectoine catabolism. IMPORTANCE: Sinorhizobium meliloti is an important soil bacterium that displays symbiotic interactions with legume hosts. Ectoine serves as a key osmoprotectant for S. meliloti However, ectoine does not accumulate in the cells; rather, it is degraded. In this study, we characterized the transcriptional regulation of the operon responsible for ectoine uptake and catabolism in S. meliloti We identified and characterized the transcription repressor EhuR, which is the first MocR/GntR family member found to be involved in the regulation of compatible solute uptake and catabolism. More importantly, we demonstrated for the first time that an ectoine catabolic end product could modulate EhuR DNA-binding activity. Therefore, this work provides new insights into the unique mechanism of ectoine-induced osmoprotection in S. meliloti.

Murein Hydrolase LytF of Streptococcus sanguinis and the Ecological Consequences of Competence Development.

The overall health of the oral cavity is dependent on proper homeostasis between health-associated bacterial colonizers and bacteria known to promote dental caries. Streptococcus sanguinis is a health-associated commensal organism, a known early colonizer of the acquired tooth pellicle, and is naturally competent. We have shown that LytF, a competence-controlled murein hydrolase, is capable of inducing the release of extracellular DNA (eDNA) from oral bacteria. Precipitated LytF and purified LytF were used as treatments against planktonic cultures and biofilms. Larger amounts of eDNA were released from cultures treated with protein samples containing LytF. Additionally, LytF could affect biofilm formation and cellular morphology. Biofilm formation was significantly decreased in the lytF-complemented strain, in which increased amounts of LytF are present. The same strain also exhibited cell morphology defects in both planktonic cultures and biofilms. Furthermore, the LytF cell morphology phenotype was reproducible in wild-type cells using purified LytF protein. In sum, our findings demonstrate that LytF can induce the release of eDNA from oral bacteria, and they suggest that, without proper regulation of LytF, cells display morphological abnormalities that contribute to biofilm malformation. In the context of the oral biofilm, LytF may play important roles as part of the competence and biofilm development programs, as well as increasing the availability of eDNA.IMPORTANCEStreptococcus sanguinis, a commensal organism in the oral cavity and one of the pioneer colonizers of the tooth surface, is associated with the overall health of the oral environment. Our laboratory showed previously that, under aerobic conditions, S. sanguinis can produce H2O2 to inhibit the growth of bacterial species that promote dental caries. This production of H2O2 by S. sanguinis also induces the release of eDNA, which is essential for proper biofilm formation. Under anaerobic conditions, S. sanguinis does not produce H2O2 but DNA is still released. Determining how S. sanguinis releases DNA is thus essential to understand biofilm formation in the oral cavity.

Let there be bioluminescence: development of a biophotonic imaging platform for in situ analyses of oral biofilms in animal models.

In the current study, we describe a novel biophotonic imaging-based reporter system that is particularly useful for the study of virulence in polymicrobial infections and interspecies interactions within animal models. A suite of luciferase enzymes was compared using three early colonizing species of the human oral flora (Streptococcus mutans, Streptococcus gordonii and Streptococcus sanguinis) to determine the utility of the different reporters for multiplexed imaging studies in vivo. Using the multiplex approach, we were able to track individual species within a dual-species oral infection model in mice with both temporal and spatial resolution. We also demonstrate how biophotonic imaging of multiplexed luciferase reporters could be adapted for real-time quantification of bacterial gene expression in situ. By creating an inducible dual-luciferase expressing reporter strain of S. mutans, we were able to exogenously control and measure expression of nlmAB (encoding the bacteriocin mutacin IV) within mice to assess its importance for the persistence ability of S. mutans in the oral cavity. The imaging system described in the current study circumvents many of the inherent limitations of current animal model systems, which should now make it feasible to test hypotheses that were previously impractical to model.

RNases J1 and J2 are critical pleiotropic regulators in Streptococcus mutans.

In recent years, it has become increasingly evident that post-transcriptional control mechanisms are the principal source of gene regulation for a large number of prokaryotic genetic pathways, particularly those involved in virulence and environmental adaptation. Post-transcriptional regulation is largely governed by RNA stability, which itself is determined by target accessibility to RNase degradation. In most Firmicutes species, mRNA stability is strongly impacted by the activity of two recently discovered RNases referred to as RNase J1 and RNase J2. Little is known about RNase J1 function in bacteria and even less is known about RNase J2. In the current study, we mutated both RNase J orthologues in Streptococcus mutans to determine their functional roles in the cell. Single and double RNase J mutants were viable, but grew very slowly on agar plates. All of the mutants shared substantial defects in growth, morphology, acid tolerance, natural competence and biofilm formation. However, most of these defects were more severe in the RNase J2 mutant. Phenotypic suppression results also implicate a role for RNase J2 as a regulator of RNase J1 function. Unlike Bacillus subtilis, RNase J2 is a major pleiotropic regulator in S. mutans, which indicates some fundamental differences from B. subtilis in global gene regulation. Key conserved residues among the RNase J2 orthologues of lactic acid bacteria may hint at a greater role for RNase J2 in these species.

Molecular commensalism: how oral corynebacteria and their extracellular membrane vesicles shape microbiome interactions.

Historically, the study of microbe-associated diseases has focused primarily on pathogens, guided by Koch's postulates. This pathogen-centric view has provided a mechanistic understanding of disease etiology and microbial pathogenesis. However, next-generation sequencing approaches have revealed a far more nuanced view of the roles various microbes play in disease, highlighting the importance of microbial diversity beyond individual pathogens. This broader perspective acknowledges the roles of host and microbial communities in disease development and resistance. In particular, the concept of dysbiosis, especially within the oral cavity, has gained attention for explaining the emergence of complex polymicrobial diseases. Such diseases often stem from resident microbes rather than foreign pathogens, complicating their treatment and even clouding our understanding of disease etiology. Oral health is maintained through a delicate balance between commensal microbes and the host, with diseases like caries and periodontal disease arising from pathogenic perturbations of this balance. Commensal microbes, such as certain streptococci and Corynebacterium spp., play crucial roles in maintaining oral health through mechanisms involving hydrogen peroxide production and membrane vesicle secretion, which can inhibit pathogenic species and modulate host immune responses. Recent research focused upon the mechanisms of molecular commensalism has expanded our understanding of these key functions of the commensal microbiome, demonstrating their central role in promoting oral health and preventing disease. These abilities represent a largely untapped reservoir of potential innovative strategies for disease prevention and management, emphasizing the need to bolster a symbiotic microbiome that inherently suppresses pathogenesis.

Optimization of an ultra-bright real-time high-throughput renilla luciferase assay for antibacterial assessment of Streptococcus mutans biofilms.

OBJECTIVE: The present work demonstrates the optimization of a renilla-based real-time, ultra-bright, non-disruptive, high-throughput bioluminescence assay (HTS) to assess the metabolism of intact Streptococcus mutans biofilms and its utility in screening the antibacterial efficacy of experimental nanofilled dental adhesive resins containing varying concentrations of nitrogen-doped titanium dioxide nanoparticles (N_TiO2). METHODS: Optimization of the assay was achieved by screening real-time bioluminescence changes in intact Streptococcus mutans biofilms imposed by the various experimental biofilm growth parameters investigated (bacterial strain, growth media, sucrose concentration, dilution factor, and inoculum volume). The optimized assay was then used to characterize the antibacterial efficacy of experimental nanofilled dental adhesive resins. The assay's ability to discriminate between bacteriostatic and bactericidal approaches was also investigated. RESULTS: Relative Light Units (RLU) values from the HTS optimization were analyzed by multivariate ANOVA (α = 0.05) and coefficients of variation. An optimized HTS bioluminescence assay was developed displaying RLUs values (brightness) that are much more intense when comparing to other previously reported bioluminescence assays, thereby decreasing the error associated with bioluminescence assays and displaying better utility while investigating the functionalities of antimicrobial nanofilled experimental dental adhesive resins with proven long-term properties. SIGNIFICANCE: The present study is anticipated to positively impact subsequent research on dental materials and oral microbiology because it serves as a valuable screening tool in metabolic-based assays with increased sensitivity and robustness. The assay reported is anticipated to be further optimized to be used as a co-reporter for other Luc based assays.

Mutation of the peptide-regulated transcription factor ComR for amidated peptide specificity and heterologous function in Lactiplantibacillus plantarum WCFS1.

There is a growing interest in the use of probiotic bacteria as biosensors for the detection of disease. However, there is a lack of bacterial receptors developed for specific disease biomarkers. Here, we have investigated the use of the peptide-regulated transcription factor ComR from Streptococcus spp. for specific peptide biomarker detection. ComR exhibits a number of attractive features that are potentially exploitable to create a biomolecular switch for engineered biosensor circuitry within the probiotic organism Lactiplantibacillus plantarum WCFS1. Through iterative design-build-test cycles, we developed a genomically integrated, ComR-based biosensor circuit that allowed WCFS1 to detect low nanomolar concentrations of ComR's cognate peptide XIP. By screening a library of ComR proteins with mutant residues substituted at the K100 position, we identified mutations that increased the specificity of ComR toward an amidated version of its cognate peptide, demonstrating the potential for ComR to detect this important class of biomarker.IMPORTANCEUsing bacteria to detect disease is an exciting possibility under active study. Detecting extracellular peptides with specific amino acid sequences would be particularly useful as these are important markers of health and disease (biomarkers). In this work, we show that a probiotic bacteria (Lactiplantibacillus plantarum) can be genetically engineered to detect specific extracellular peptides using the protein ComR from Streptococcus bacteria. In its natural form, ComR allowed the probiotic bacteria to detect a specific peptide, XIP. We then modified XIP to be more like the peptide biomarkers found in humans and engineered ComR so that it activated with this modified XIP and not the original XIP. This newly engineered ComR also worked in the probiotic bacteria, as expected. This suggests that with additional engineering, ComR might be able to activate with human peptide biomarkers and be used by genetically engineered probiotic bacteria to better detect disease.

Employing Cloning-Independent Mutagenesis of Parvimonas micra for the Study of Cell Wall Biogenesis.

The cell wall plays an important structural role for bacteria and is intimately tied to a variety of critical processes ranging from growth and differentiation to pathogenesis. Our understanding of cell wall biogenesis is primarily derived from a relatively small number of heavily studied model organisms. Consequently, these processes can only be inferred for the vast majority of prokaryotes, especially among groups of uncharacterized and/or genetically intractable organisms. Recently, we developed the first tractable genetic system for Parvimonas micra, which is a ubiquitous Gram-positive pathobiont of the human microbiome involved in numerous types of inflammatory infections as well as a variety of malignant tumors. P. micra is also the first, and currently only, member of the entire Tissierellia class of the Bacillota phylum in which targeted genetic manipulation has been demonstrated. Thus, it is now possible to study cell wall biogenesis mechanisms within a member of the Tissierellia, which may also reveal novel aspects of P. micra pathobiology. Herein, we describe a procedure for cloning-independent genetic manipulation of P. micra, including allelic replacement mutagenesis and genetic complementation. The described techniques are also similarly applicable for the study of other aspects of P. micra pathobiology and physiology.

An inflammatory paradox: strategies inflammophilic oral pathobionts employ to exploit innate immunity via neutrophil manipulation.

Inflammatory dysbiotic diseases present an intriguing biological paradox. Like most other infectious disease processes, the alarm bells of the host are potently activated by tissue-destructive pathobionts, triggering a cascade of physiological responses that ultimately mobilize immune cells like neutrophils to sites of active infection. Typically, these inflammatory host responses are critical to inhibit and/or eradicate infecting microbes. However, for many inflammatory dysbiotic diseases, inflammophilic pathobiont-enriched communities not only survive the inflammatory response, but they actually obtain a growth advantage when challenged with an inflammatory environment. This is especially true for those organisms that have evolved various strategies to resist and/or manipulate components of innate immunity. In contrast, members of the commensal microbiome typically experience a competitive growth disadvantage under inflammatory selective pressure, hindering their critical ability to restrict pathobiont proliferation. Here, we examine examples of bacteria-neutrophil interactions from both conventional pathogens and inflammophiles. We discuss some of the strategies utilized by them to illustrate how inflammophilic microbes can play a central role in the positive feedback cycle that exemplifies dysbiotic chronic inflammatory diseases.