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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.
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Proteínas de Bactérias , Biofilmes , Glucanos , Streptococcus mutans , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Streptococcus mutans/enzimologia , Biofilmes/crescimento & desenvolvimento , Glucanos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ribossomos/metabolismo , Mutação , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genéticaRESUMO
Parvimonas micra is a pathobiont of humans that is often found in abundance at sites of mucosal inflammation as well as within malignant tumors. Here, we report the complete genome sequence of P. micra strain JM503A, which is a genetically tractable clinical isolate derived from a human odontogenic abscess specimen.
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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.
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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.
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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.
Assuntos
Placa Dentária , Fusobacterium nucleatum , Fusobacterium , Humanos , Fusobacterium nucleatum/genética , Abscesso , BocaRESUMO
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.
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Proteínas de Bactérias , Streptococcus mutans , Proteínas de Bactérias/genética , Streptococcus mutans/genética , Proteólise , Luciferases/metabolismo , Espectrometria de MassasRESUMO
Microenvironmental changes in the chemical surrounding of bacterial cells might have a profound impact on the ecology of biofilms. However, quantifying total amount of picomoles of analyte from a miniscule number of bacteria is an analytical challenge. Here we provide a novel microliter volume hydrogel based electrochemical cell platform suitable of coulometrically measuring hydrogen peroxide (H2O2) produced by less than 100 cells of Streptococcus sanguinis, a relevant member of the healthy oral microbiome. A morpholine moiety was incorporated into the polymer structure of the hydrogel to create a controlled microenvironment at biological pH. We calculated the buffering capacity of this hydrogel as 0.257 ± 0.135 molHNO3molMEA×ΔpH over the pH range of 7.2-6.2 by using a novel method designed for buffering hydrogels. The H2O2 sensors coated in microliter volume of buffering hydrogel showed no change in sensitivity within the pH range of 7.0-3.0, allowing for H2O2 measurements of S. sanguinis independent of any acid they produce. The novel platform was able to measure down to 22.7 ± 3.5 pmol H2O2 produced by less than 100 bacterial cells, which would otherwise not be attainable in large solution-based assays. These findings indicate that this is a suitable platform for quantifying metabolites from sub-milligram biological samples and may even be suitable for direct analysis of raw biofilms samples with little to no sample pretreatment.
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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.
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Firmicutes , Microbiota , Humanos , Firmicutes/genética , Mutagênese , Clonagem MolecularRESUMO
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.
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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.
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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.
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Microbiota , Simbiose , Humanos , Bactérias/genética , Boca/microbiologia , FungosRESUMO
It is widely acknowledged that the human-associated microbial community influences host physiology, systemic health, disease progression, and even behavior. There is currently an increased interest in the oral microbiome, which occupies the entryway to much of what the human initially encounters from the environment. In addition to the dental pathology that results from a dysbiotic microbiome, microbial activity within the oral cavity exerts significant systemic effects. The composition and activity of the oral microbiome is influenced by (1) host-microbial interactions, (2) the emergence of niche-specific microbial "ecotypes," and (3) numerous microbe-microbe interactions, shaping the underlying microbial metabolic landscape. The oral streptococci are central players in the microbial activity ongoing in the oral cavity, due to their abundance and prevalence in the oral environment and the many interspecies interactions in which they participate. Streptococci are major determinants of a healthy homeostatic oral environment. The metabolic activities of oral Streptococci, particularly the metabolism involved in energy generation and regeneration of oxidative resources vary among the species and are important factors in niche-specific adaptations and intra-microbiome interactions. Here we summarize key differences among streptococcal central metabolic networks and species-specific differences in how the key glycolytic intermediates are utilized.
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Bactérias , Microbiota , Humanos , Bactérias/metabolismo , Streptococcus , Redes e Vias Metabólicas , Interações entre Hospedeiro e MicrorganismosRESUMO
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.
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Proteômica , Streptococcus sanguis , Streptococcus sanguis/genética , Streptococcus sanguis/metabolismo , Boca/microbiologia , Gengiva/microbiologia , Bactérias Gram-PositivasRESUMO
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.
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Glicerol , Streptococcus , Glicerol/metabolismo , Streptococcus sanguis/genética , Biofilmes , Simbiose , Streptococcus mutansRESUMO
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.
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Streptococcus , Xilose , Mutagênese , Streptococcus/genética , Mutação , Clonagem MolecularRESUMO
Dental plaque biofilm is a complex ecosystem. The distribution of microbial species in the biofilm is heavily influenced by local chemical interactions that result from diverse metabolic activities and the nature of the released molecules. As a relevant example, H2O2-producing bacteria can antagonize disease-associated bacteria, leading to the maintenance of a healthy oral microbiome. Herein, we report the development of a triple-sensor (redox, pH, and H2O2) scanning electrochemical microscopy (SECM) tip capable of simultaneously mapping the pH and H2O2 concentration produced by a dental plaque-derived multispecies biofilm grown on hydroxyapatite. The pH sensor of the triple SECM tip showed a near Nernstian slope of -71.1 ± 2 mV/pH (N = 3), whereas the H2O2 sensor showed a slope of -0.052 ± 0.002 nA/µM H2O2 at pH 7.2 and a detection limit of 1.0 ± 0.2 µM (N = 7). There is no significant difference in the sensitivities of H2O2 sensors at pH 6.2, 7.2, and 8.2 at 95% CI (N = 7). The pH and H2O2 sensors demonstrated excellent reversibility with response times of 3 and 5 s, respectively, along with reliable stability over 4 h at 37 °C. The sensors did not show any cross talk between pH and H2O2 concentration ([H2O2]) measurements, highlighting the accuracy and versatility of the SECM tip. Simultaneous chemical imaging of pH and [H2O2] across the biofilm revealed a clustered distribution of local H2O2 concentrations, ranging from 0 to 17 µM. Conversely, the local pH remained constant at 7.2. The relation of local chemical profiles and the distribution of bacterial species within the oral microbiome was experimentally investigated in the context of bacterial H2O2 antagonism. The benefit of clustered H2O2 production was that the total area of H2O2 produced by smaller clusters was 67% more than that of a single cluster with the same starting number of bacteria. Thus, this triple SECM tip can potentially be used to study local molecular mechanisms that result in dysbiosis of the oral microbiome.
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Placa Dentária , Peróxido de Hidrogênio , Humanos , Peróxido de Hidrogênio/metabolismo , Microscopia Eletroquímica de Varredura/métodos , Ecossistema , Bactérias/metabolismo , Biofilmes , Concentração de Íons de HidrogênioRESUMO
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.
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Disbiose , Neoplasias , Humanos , Firmicutes/genética , Trato GastrointestinalRESUMO
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.
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Microbiota , Humanos , BiofilmesRESUMO
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.
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Disbiose , Microbiota , Humanos , Microbiota/genética , Bactérias/genética , SimbioseRESUMO
Most bacteria in nature exist in multispecies communities known as biofilms. In the natural habitat where resources (nutrient, space, etc.) are usually limited, individual species must compete or collaborate with other neighboring species in order to perpetuate in the multispecies community. The human oral cavity is colonized by >700 microbial species known as the indigenous microbiota. This indigenous flora normally maintains an ecological balance through antagonistic as well as mutualistic interspecies interactions. However, environmental perturbation may disrupt this balance, leading to overgrowth of pathogenic species which could in turn initiate diseases such as dental caries (tooth decay) and periodontitis (gum disease). Understanding the mechanisms of diversity maintenance may help developing novel approaches to manage these "polymicrobial diseases". In this chapter, we will focus on a well-characterized form of biochemical warfare: bacteriocins produced by Streptococcus mutans, a primary dental caries pathogen, and hydrogen peroxide (H2O2) produced by several oral commensal streptococci. We will describe detailed methodologies on the competition assay, isolation, purification, and characterization of bacteriocins.