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.

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.

Stratification of Fusobacterium nucleatum by local health status in the oral cavity defines its subspecies disease association.

The ubiquitous inflammophilic oral pathobiont Fusobacterium nucleatum (Fn) is widely recognized for its strong association with inflammatory dysbiotic diseases and cancer. Fn is subdivided into four subspecies, which are historically considered functionally interchangeable in the oral cavity. To test this assumption, we analyzed patient-matched dental plaque and odontogenic abscess clinical specimens and examined whether an inflammatory environment selects for/against particular Fn subspecies. Dental plaque harbored a greater diversity of fusobacteria, with Fn. polymorphum dominating, whereas odontogenic abscesses were exceptionally biased for the largely uncharacterized organism Fn. animalis. Comparative genomic analyses revealed significant genotypic distinctions among Fn subspecies that correlate with their preferred ecological niches and support a taxonomic reassignment of each as a distinct Fusobacterium species. Despite originating as a low-abundance organism in dental plaque, Fn. animalis typically outcompetes other oral fusobacteria within the inflammatory abscess environment, which may explain its prevalence in other oral and extraoral diseases.

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.

Mass spectrometry and split luciferase complementation assays reveal the MecA protein interactome of Streptococcus mutans.

MecA is a highly conserved adaptor protein encoded by prokaryotes from the Bacillota phylum. MecA mutants exhibit similar pleiotropic defects in a variety of organisms, although most of these phenotypes currently lack a mechanistic basis. MecA mediates ClpCP-dependent proteolysis of its substrates, but only several such substrates have been reported in the literature and there are suggestions that proteolysis-independent regulatory mechanisms may also exist. Here, we provide the first comprehensive characterization of the MecA interactome and further assess its regulatory role in Clp-dependent proteolysis. Untargeted coimmunoprecipitation assays coupled with mass spectrometry revealed that the MecA ortholog from the oral pathobiont Streptococcus mutans likely serves as a major protein interaction network hub by potentially complexing with >100 distinct protein substrates, most of which function in highly conserved metabolic pathways. The interactome results were independently verified using a newly developed prokaryotic split luciferase complementation assay (SLCA) to detect MecA protein-protein interactions in vivo. In addition, we further develop a new application of SLCA to support in vivo measurements of MecA relative protein binding affinities. SLCA results were independently verified using targeted coimmunoprecipitation assays, suggesting the general utility of this approach for prokaryotic protein-protein interaction studies. Our results indicate that MecA indeed regulates its interactome through both Clp-dependent proteolysis as well as through an as-yet undefined proteolysis-independent mechanism that may affect more than half of its protein interactome. This suggests a significant aspect of the MecA regulatory function still has yet to be discovered.IMPORTANCEDespite multiple decades of study, the regulatory mechanism and function of MecA have remained largely a mystery. The current study provides the first detailed roadmap to investigate these functions in other medically significant bacteria. Furthermore, this study developed new genetic approaches to assay prokaryotic protein-protein interactions via the split luciferase complementation assay (SLCA). SLCA technology is commonly employed in eukaryotic genetic research but has not yet been established for studies of bacterial protein-protein interactions. The SLCA protein binding affinity assay described here is a new technological advance exclusive to the current study and has not been reported elsewhere.

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.

The prevalence of Fusobacterium nucleatum subspecies in the oral cavity stratifies by local health status.

The ubiquitous inflammophilic pathobiont Fusobacterium nucleatum is widely recognized for its strong association with a variety of human dysbiotic diseases such as periodontitis and oral/extraoral abscesses, as well as multiple types of cancer. F. nucleatum is currently subdivided into four subspecies: F. nucleatum subspecies nucleatum (Fn. nucleatum), animalis (Fn. animalis), polymorphum (Fn. polymorphum), and vincentii/fusiforme (Fn. vincentii). Although these subspecies have been historically considered as functionally interchangeable in the oral cavity, direct clinical evidence is largely lacking for this assertion. Consequently, we assembled a collection of oral clinical specimens to determine whether F. nucleatum subspecies prevalence in the oral cavity stratifies by local oral health status. Patient-matched clinical specimens of both disease-free dental plaque and odontogenic abscess were analyzed with newly developed culture-dependent and culture-independent approaches using 44 and 60 oral biofilm/tooth abscess paired specimens, respectively. Most oral cavities were found to simultaneously harbor multiple F. nucleatum subspecies, with a greater diversity present within dental plaque compared to abscesses. In dental plaque, Fn. polymorphum is clearly the dominant organism, but this changes dramatically within odontogenic abscesses where Fn. animalis is heavily favored over all other fusobacteria. Surprisingly, the most commonly studied F. nucleatum subspecies, Fn. nucleatum, is only a minor constituent in the oral cavity. To gain further insights into the genetic basis for these phenotypes, we subsequently performed pangenome, phylogenetic, and functional enrichment analyses of oral fusobacterial genomes using the Anvi'o platform, which revealed significant genotypic distinctions among F. nucleatum subspecies. Accordingly, our results strongly support a taxonomic reassignment of each F. nucleatum subspecies into distinct Fusobacterium species. Of these, Fn. animalis should be considered as the most clinically relevant at sites of active inflammation, despite being among the least characterized oral fusobacteria.

Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform.

The human microbiome is predominantly composed of facultative and obligate anaerobic bacteria that live in hypoxic/anoxic polymicrobial biofilm communities. Given the oxidative sensitivity of large fractions of the human microbiota, green fluorescent protein (GFP) and related genetically-encoded fluorophores only offer limited utility for live cell imaging due the oxygen requirement for chromophore maturation. Consequently, new fluorescent imaging modalities are needed to study polymicrobial interactions and microbiome-host interactions within anaerobic environments. The fluorescence-activating and absorption shifting tag (FAST) is a rapidly developing genetically-encoded fluorescent imaging technology that exhibits tremendous potential to address this need. In the FAST system, fluorescence only occurs when the FAST protein is complexed with one of a suite of cognate small molecule fluorogens. To expand the utility of FAST imaging, we sought to develop a modular platform (Click-FAST) to democratize fluorogen engineering for personalized use cases. Using Click-FAST, investigators can quickly and affordably sample a vast chemical space of compounds, potentially imparting a broad range of desired functionalities to the parental fluorogen. In this work, we demonstrate the utility of the Click-FAST platform using a novel fluorogen, PLBlaze-alkyne, which incorporates the widely available small molecule ethylvanillin as the hydroxybenzylidine head group. Different azido reagents were clicked onto PLBlaze-alkyne and shown to impart useful characteristics to the fluorogen, such as selective bacterial labeling in mixed populations as well as fluorescent signal enhancement. Conjugation of an 80 Å PEG molecule to PLBlaze-alkyne illustrates the broad size range of functional fluorogen chimeras that can be employed. This PEGylated fluorogen also functions as an exquisitely selective membrane permeability marker capable of outperforming propidium iodide as a fluorescent marker of cell viability.

Molecular commensalism-how to investigate underappreciated health-associated polymicrobial communities.

The study of human commensal bacteria began with the first observation of prokaryotes >340 years ago. Since then, the study of human-associated microbes has been justifiably biased toward the study of infectious pathogens. However, the role of commensal microbes has in recent years begun to be understood with some appreciation of them as potential protectors of host health rather than bystanders. As our understanding of these valuable microbes grows, it highlights how much more remains to be learned about them and their roles in maintaining health. We note here that a thorough framework for the study of commensals, both in vivo and in vitro is overall lacking compared to well-developed methodologies for pathogens. The modification and application of methods for the study of pathogens can work well for the study of commensals but is not alone sufficient to properly characterize their relationships. This is because commensals live in homeostasis with the host and within complex communities. One difficulty is determining which commensals have a quantifiable impact on community structure and stability as well as host health, vs benign microbes that may indeed serve only as bystanders. Human microbiomes are composed of bacteria, archaea, fungi, and viruses. This review focuses particularly on oral bacteria, yet many of the principles of commensal impacts on host health observed in the mouth can translate well to other host sites. Here, we discuss the value of commensals, the shortcomings involved in model systems for their study, and some of the more notable impacts they have upon not only each other but host health.

Environmental influences on Streptococcus sanguinis membrane vesicle biogenesis.

Membrane vesicles are produced by Gram-negative and Gram-positive bacteria. While membrane vesicles are potent elicitors of eukaryotic cells and involved in cell-cell communication, information is scarce about their general biology in the context of community members and the environment. Streptococcus sanguinis, a Gram-positive oral commensal, is prevalent in the oral cavity and well-characterized for its ability to antagonize oral pathobionts. We have found that production and dissemination of membrane vesicles by S. sanguinis is dependent on environmental and community factors. Co-culture with interacting commensal Corynebacterium durum, as well as with the periodontal pathobiont Filifactor alocis had no effect on S. sanguinis vesicle number and size, whereas the periodontal pathobiont Porphyromonas gingivalis abolished S. sanguinis vesicle production. Using both correlation and differential expression analyses to examine the transcriptomic changes underlying vesicle production, we found that differential expression of genes encoding proteins related to the cytoplasmic membrane and peptidoglycan correlate with the abundance of membrane vesicles. Proteomic characterizations of the vesicle cargo identified a variety of proteins, including those predicted to influence host interactions or host immune responses. Cell culture studies of gingival epithelial cells demonstrated that both crude and highly purified membrane vesicles could induce the expression of IL-8, TNF-α, IL-1ß, and Gro-α within 6 hours of inoculation at levels comparable to whole cells. Our findings suggest that production of membrane vesicles by S. sanguinis is heavily influenced by community and environmental factors and plays an important role in communication with host cells.

Glycerol metabolism supports oral commensal interactions.

During oral biofilm development, interspecies interactions drive species distribution and biofilm architecture. To understand what molecular mechanisms determine these interactions, we used information gained from recent biogeographical investigations demonstrating an association of corynebacteria with streptococci. We previously reported that Streptococcus sanguinis and Corynebacterium durum have a close relationship through the production of membrane vesicle and fatty acids leading to S. sanguinis chain elongation and overall increased fitness supporting their commensal state. Here we present the molecular mechanisms of this interspecies interaction. Coculture experiments for transcriptomic analysis identified several differentially expressed genes in S. sanguinis. Due to its connection to fatty acid synthesis, we focused on the glycerol-operon. We further explored the differentially expressed type IV pili genes due to their connection to motility and biofilm adhesion. Gene inactivation of the glycerol kinase glpK had a profound impact on the ability of S. sanguinis to metabolize C. durum secreted glycerol and impaired chain elongation important for their interaction. Investigations on the effect of type IV pili revealed a reduction of S. sanguinis twitching motility in the presence of C. durum, which was caused by a decrease in type IV pili abundance on the surface of S. sanguinis as determined by SEM. In conclusion, we identified that the ability to metabolize C. durum produced glycerol is crucial for the interaction of C. durum and S. sanguinis. Reduced twitching motility could lead to a closer interaction of both species, supporting niche development in the oral cavity and potentially shaping symbiotic health-associated biofilm communities.

Adaptation of Prokaryotic Toxins for Negative Selection and Cloning-Independent Markerless Mutagenesis in Streptococcus Species.

The Streptococcus mutans genetic system offers a variety of strategies to rapidly engineer targeted chromosomal mutations. Previously, we reported the first S. mutans negative selection system that functions in a wild-type background. This system utilizes induced sensitivity to the toxic amino acid analog p-chlorophenylalanine (4-CP) as a negative selection mechanism and was developed for counterselection-based cloning-independent markerless mutagenesis (CIMM). While we have employed this system extensively for our ongoing genetic studies, we have encountered a couple limitations with the system, mainly its narrow host range and the requirement for selection on a toxic substrate. Here, we report the development of a new negative selection system that addresses both limitations, while still retaining the utility of the previous 4-CP-based markerless mutagenesis system. We placed a variety of toxin-encoding genes under the control of the xylose-inducible gene expression cassette (Xyl-S) and found the Fst-sm and ParE toxins to be suitable candidates for inducible negative selection. We combined the inducible toxins with an antibiotic resistance gene to create several different counterselection cassettes. The most broadly useful of these contained a wild-type fst-sm open reading frame transcriptionally fused to a point mutant form of the Xyl-S expression system, which we subsequently named IFDC4. IFDC4 was shown to exhibit exceptionally low background resistance, with 3- to 4-log reductions in cell number observed when plating on xylose-supplemented medium. IFDC4 also functioned similarly in multiple strains of S. mutans as well as with Streptococcus gordonii and Streptococcus sanguinis. We performed CIMM with IFDC4 and successfully engineered a variety of different types of markerless mutations in all three species. The counterselection strategy described here provides a template approach that should be adaptable for the creation of similar counterselection systems in many other bacteria. IMPORTANCE Multiple medically significant Streptococcus species, such as S. mutans, have highly sophisticated genetic systems available, largely as a consequence of their amenability to genetic manipulation via natural competence. Despite this, few options are available for the creation of markerless mutations in streptococci, especially within wild-type strains. Markerless mutagenesis is a critical tool for genetic studies, as it allows the user to explore many fundamental questions that are not easily addressable using marked mutagenesis. Here, we describe a new approach for streptococcal markerless mutagenesis that offers a variety of advantages over the current approach, which employs induced sensitivity to the toxic substrate 4-CP. The approach employed here should be readily adaptable for the creation of similar markerless mutagenesis systems in other organisms.

Who is in the driver's seat? Parvimonas micra: An understudied pathobiont at the crossroads of dysbiotic disease and cancer.

Recent advances in our understanding of microbiome composition at sites of inflammatory dysbiosis have triggered a substantial interest in a variety of historically understudied bacteria, especially among fastidious obligate anaerobes. A plethora of new evidence suggests that these microbes play outsized roles in establishing synergistic polymicrobial infections at many different sites in the human body. Parvimonas micra is a prime example of such an organism. Despite being almost completely uncharacterized at the genetic level, it is one of the few species commonly detected in abundance at multiple mucosal sites experiencing either chronic or acute inflammatory diseases, and more recently, it has been proposed as a discriminating biomarker for multiple types of malignancies. In the absence of disease, P. micra is commonly found in low abundance, typically residing within the oral cavity and gastrointestinal tract. P. micra exhibits the typical features of an inflammophilic organism, meaning its growth actually benefits from active inflammation and inflammatory tissue destruction. In this mini-review, we will describe our current understanding of this underappreciated but ubiquitous pathobiont, specifically focusing upon the role of P. micra in polymicrobial inflammatory dysbiosis and cancer as well as the key emerging questions regarding its pathobiology. Through this timely work, we highlight Parvimonas micra as a significant driver of disease and discuss its unique position at the crossroads of dysbiosis and cancer.

Illuminating the oral microbiome and its host interactions: tools and approaches for molecular microbiology studies.

Advancements in DNA sequencing technologies within the last decade have stimulated an unprecedented interest in the human microbiome, largely due the broad diversity of human diseases found to correlate with microbiome dysbiosis. As a direct consequence of these studies, a vast number of understudied and uncharacterized microbes have been identified as potential drivers of mucosal health and disease. The looming challenge in the field is to transition these observations into defined molecular mechanistic studies of symbiosis and dysbiosis. In order to meet this challenge, many of these newly identified microbes will need to be adapted for use in experimental models. Consequently, this review presents a comprehensive overview of the molecular microbiology tools and techniques that have played crucial roles in genetic studies of the bacteria found within the human oral microbiota. Here, we will use specific examples from the oral microbiome literature to illustrate the biology supporting these techniques, why they are needed in the field, and how such technologies have been implemented. It is hoped that this information can serve as a useful reference guide to help catalyze molecular microbiology studies of the many new understudied and uncharacterized species identified at different mucosal sites in the body.

Illuminating the oral microbiome and its host interactions: tools and approaches for molecular ecological studies.

A more comprehensive understanding of oral diseases like caries and periodontitis is dependent on an intimate understanding of the microbial ecological processes that are responsible for disease development. With this review, we provide a comprehensive overview of relevant molecular ecology techniques that have played critical roles in the current understanding of human oral biofilm development, interspecies interactions, and microbiome biogeography. The primary focus is on relevant technologies and examples available in the oral microbiology literature. However, most, if not all, of the described technologies should be readily adaptable for studies of microbiomes from other mucosal sites in the body. Therefore, this review is intended to serve as a reference guide used by microbiome researchers as they inevitably transition into molecular mechanistic studies of the many significant phenotypes observed clinically.

Effect of S. mutans biofilm on the hybrid ceramic-resin cement bond strength assessed by different methods

Aim: The purpose of this study was to investigate the biofilm effect on the hybrid ceramic-resin cement bond strength (BS) by comparing two methods. Methods: Teeth were distributed into groups (n=5), according to the resin cement (Maxcem Elite-(MC) or NX3 Nexus-(NX)) and degradation method (24h or 7 days in distilled water; 7 or 30 days incubated with biofilm and 30 days in sterile media). Treated surfaces of Vita Enamic blocks (5x6x7mm) were luted to treated or no treated dentin surfaces and light-cured. After 24h, beams were obtained (1x1x10mm) and stored accordingly. The flexural bond strength (FBS) was assessed by four-point bending test. Additional beams were obtained from new teeth (n=5), stored for 24h or 7 days in distilled water, and submitted to a microtensile bond strength (µTBS) assay. Failure modes were determined by scanning electron microscopy (100X). The flexure strength of the cements (n=10) was assessed by a four-point bending test. Data were analyzed by 1 and 2-ways ANOVA, and Tukey's test (α=0.05). Results: There was no significant difference between the degradation methods for the FBS groups. For the µTBS, the significant difference was as follows: NX 7days > NX 24h > MC 7days = MC 24h. Failure mode was mainly adhesive and mixed, but with an increase of cohesive within cement and pre-failures for the MC groups assessed by µTBS. NX had better performance than MC, regardless of the method. Conclusions: The biofilm had no effect on the materials BS and FBS test was a useful method to evaluate BS of materials with poor performance

Oral microbiome diversity: The curious case of Corynebacterium sp. isolation.

Oral microbiome sequencing efforts revealed the presence of hundreds of different microbes. Interindividual differences at strain and species resolution suggest that microbiome diversity could lead to mechanistically distinct gene regulation as well as species-related differences in phenotypes. Commonly, gene regulation and related phenotypes are studied in a few selected strains of a particular species with conclusions that are mostly generalized. The aim of this study was to isolate several species of Corynebacterium using an established protocol that led to the previous isolation of C. durum. Characterization of C. durum interspecies interactions revealed a specific mechanism for chain elongation in Streptococcus sanguinis that was the result of corynebacterial fatty acid production and secretion. While the protocol was successfully applied to isolate what we presumed to be additional Corynebacterium based on several phenotypic traits that seem to be identical to C. durum, genome sequencing of the newly isolated strains placed them closer to Actinomyces. Both Corynebacterium and Actinomyces are suborders of the Actinobacteridae and related species. Our study suggests to take several comprehensive strategies into consideration when taxonomically identifying closely related microorganisms. Furthermore, it seems to be important to test common core phenotypes in bacterial ecology to understand the behavior of specific groups of microbes, rather than simply relying upon genome sequence homology to establish relationships in the microbiome.

Development of the First Tractable Genetic System for Parvimonas micra, a Ubiquitous Pathobiont in Human Dysbiotic Disease.

Parvimonas micra is a Gram-positive obligate anaerobe and a typical member of the human microbiome. P. micra is among the most highly enriched species at numerous sites of mucosal dysbiotic disease and is closely associated with the development of multiple types of malignant tumors. Despite its strong association with disease, surprisingly little is known about P. micra pathobiology, which is directly attributable to its longstanding genetic intractability. To address this problem, we directly isolated a collection of P. micra strains from odontogenic abscess clinical specimens and then screened these isolates for natural competence. Amazingly, all of the P. micra clinical isolates exhibited various levels of natural competence, including the reference strain ATCC 33270. By exploiting this ability, we were able to employ cloning-independent methodologies to engineer and complement a variety of targeted chromosomal genetic mutations directly within low-passage-number clinical isolates. To develop a tractable genetic system for P. micra, we first adapted renilla-based bioluminescence for highly sensitive reporter studies. This reporter system was then applied for the development of the novel Theo+ theophylline-inducible riboswitch for tunable gene expression studies over a broad dynamic range. Finally, we demonstrate the feasibility of generating mariner-based transposon sequencing (Tn-seq) libraries for forward genetic screening in P. micra. With the availability of a highly efficient transformation protocol and the current suite of genetic tools, P. micra should now be considered a fully genetically tractable organism suitable for molecular genetic research. The methods presented here provide a clear path to investigate the understudied role of P. micra in polymicrobial infections and tumorigenesis. IMPORTANCE Parvimonas micra is among the most highly enriched species at numerous sites of mucosal dysbiotic disease and is closely associated with numerous cancers. Despite this, little is known about P. micra pathobiology, which is directly attributable to its longstanding genetic intractability. In this study, we provide the first report of P. micra natural competence and describe the only tractable genetic system for this species. The methods presented here will allow for the detailed study of P. micra and its role in infection and tumorigenesis.

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.

Anti-cancer activity of sustained release capsaicin formulations.

Capsaicin (trans-8-methyl-N-vanillyl-6-noneamide) is a hydrophobic, lipophilic vanilloid phytochemical abundantly found in chili peppers and pepper extracts. Several convergent studies show that capsaicin displays robust cancer activity, suppressing the growth, angiogenesis and metastasis of several human cancers. Despite its potent cancer-suppressing activity, the clinical applications of capsaicin as a viable anti-cancer drug have remained problematic due to its poor bioavailability and aqueous solubility properties. In addition, the administration of capsaicin is associated with adverse side effects like gastrointestinal cramps, stomach pain, nausea and diarrhea and vomiting. All these hurdles may be circumvented by encapsulation of capsaicin in sustained release drug delivery systems. Most of the capsaicin-based the sustained release drugs have been tested for their pain-relieving activity. Only a few of these formulations have been investigated as anti-cancer agents. The present review describes the physicochemical properties, bioavailability, and anti-cancer activity of capsaicin-sustained release agents. The asset of such continuous release capsaicin formulations is that they display better solubility, stability, bioavailability, and growth-suppressive activity than the free drug. The encapsulation of capsaicin in sustained release carriers minimizes the adverse side effects of capsaicin. In summary, these capsaicin-based sustained release drug delivery systems have the potential to function as novel chemotherapies, unique diagnostic imaging probes and innovative chemosensitization agents in human cancers.