Subgingival host-microbiome metatranscriptomic changes following scaling and root planing in grade II/III periodontitis.

AIM: To assess the effects of scaling and root planing (SRP) on the dynamics of gene expression by the host and the microbiome in subgingival plaque samples. MATERIALS AND METHODS: Fourteen periodontitis patients were closely monitored in the absence of periodontal treatment for 12 months. During this period, comprehensive periodontal examination and subgingival biofilm sample collection were performed bi-monthly. After 12 months, clinical attachment level (CAL) data were compiled and analysed using linear mixed models (LMM) fitted to longitudinal CAL measurements for each tooth site. LMM classified the sites as stable (S), progressing (P), or fluctuating (F). After the 12-month visit, subjects received SRP, and at 15 months they received comprehensive examination and supportive periodontal therapy. Those procedures were repeated at the 18-month visit, when patients were also sampled. Each patient contributed with one S, one P, and one F site collected at the 12- and 18-month visits. Samples were analysed using Dual RNA-Sequencing to capture host and bacterial transcriptomes simultaneously. RESULTS: Microbiome and host response behaviour were specific to the site's progression classification (i.e., S, P, or F). Microbial profiles of pre- and post-treatment samples exhibited specific microbiome changes, with progressing sites showing the most significant changes. Among them, Porphyromonas gingivalis was reduced after treatment, while Fusobacterium nucleatum showed an increase in proportion. Transcriptome analysis of the host response showed that interleukin (IL)-17, TNF signalling pathways, and neutrophil extracellular trap formation were the primary immune response activities impacted by periodontal treatment. CONCLUSIONS: SRP resulted in a significant "rewiring" of host and microbial activities in the progressing sites, while restructuring of the microbiome was minor in stable and fluctuating sites.

Long-term dynamics of the human oral microbiome during clinical disease progression.

BACKGROUND: Oral microbiome dysbiosis is linked to overt inflammation of tooth-supporting tissues, leading to periodontitis, an oral condition that can cause tooth and bone loss. Microbiome dysbiosis has been described as a disruption in the symbiotic microbiota composition's stability that could adversely affect the host's health status. However, the precise microbiome dynamics that lead to dysbiosis and the progression of the disease are largely unknown. The objective of our study was to investigate the long-term dynamics of periodontitis progression and its connection to dysbiosis. RESULTS: We studied three different teeth groups: sites that showed disease progression, sites that remained stable during the study, and sites that exhibited a cyclic deepening followed by spontaneous recovery. Time-series analysis revealed that communities followed a characteristic succession of bacteria clusters. Stable and fluctuating sites showed high asynchrony in the communities (i.e., different species responding dissimilarly through time) and a reordering of the communities where directional changes dominated (i.e., sample distance increases over time) in the stable sites but not in the fluctuating sites. Progressing sites exhibited low asynchrony and convergence (i.e., samples distance decreases over time). Moreover, new species were more likely to be recruited in stable samples if a close relative was not recruited previously. In contrast, progressing and fluctuating sites followed a neutral recruitment model, indicating that competition between closely related species is a significant component of species-species interactions in stable samples. Finally, periodontal treatment did not select similar communities but stabilized α-diversity, centered the abundance of different clusters to the mean, and increased community rearrangement. CONCLUSIONS: Here, we show that ecological principles can define dysbiosis and explain the evolution and outcomes of specific microbial communities of the oral microbiome in periodontitis progression. All sites showed an ecological succession in community composition. Stable sites were characterized by high asynchrony, a reordering of the communities where directional changes dominated, and new species were more likely to be recruited if a close relative was not recruited previously. Progressing sites were characterized by low asynchrony, community convergence, and a neutral model of recruitment. Finally, fluctuating sites were characterized by high asynchrony, community convergence, and a neutral recruitment model.

Genetic and Biochemical Characterization of the Na+/H+ Antiporters of Pseudomonas aeruginosa.

Pseudomonas aeruginosa has four Na+/H+ antiporters that interconvert and balance Na+ and H+ gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we constructed four strains, each of which has only one antiporter, i.e., NhaB, NhaP, NhaP2, and Mrp. We also constructed a quadruple deletion mutant that has no Na+/H+ antiporters. Although the antiporters of P. aeruginosa have been studied previously, the strains constructed here present the opportunity to characterize their kinetic properties in their native membranes and their roles in the physiology of P. aeruginosa. The strains expressing only NhaB or Mrp, the two electrogenic antiporters, were able to grow essentially like the wild-type strain across a range of Na+ concentrations and pH values. Strains with only NhaP or NhaP2, which are electroneutral, grew more poorly at increasing Na+ concentrations, especially at high pH values, with the strain expressing NhaP being more sensitive. The strain with no Na+/H+ antiporters was extremely sensitive to the Na+ concentration and showed essentially no Na+(Li+)/H+ antiporter activity, but it retained most K+/H+ antiporter activity of the wild-type strain at pH 7.5 and approximately one-half at pH 8.5. We also used the four strains that each express one of the four antiporters to characterize the kinetic properties of each transporter. Transcriptome sequencing analysis of the quadruple deletion strain showed widespread changes, including changes in pyocyanin synthesis, biofilm formation, and nitrate and glycerol metabolism. Thus, the strains constructed for this study will open a new door to understanding the physiological roles of these proteins and their activities in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa has four Na+/H+ antiporters that connect and interconvert its Na+ and H+ gradients. We have constructed four deletion mutants, each of which has only one of the four Na+/H+ antiporters. These strains made it possible to study the properties and physiological roles of each antiporter independently in its native membrane. Mrp and NhaB are each able to sustain growth over a wide range of pH values and Na+ concentrations, whereas the two electroneutral antiporters, NhaP and NhaP2, are most effective at low pH values. We also constructed a quadruple mutant lacking all four antiporters, in which the H+ and Na+ gradients are disconnected. This will make it possible to study the role of the two gradients independently.

Potassium is a key signal in host-microbiome dysbiosis in periodontitis.

Dysbiosis, or the imbalance in the structural and/or functional properties of the microbiome, is at the origin of important infectious inflammatory diseases such as inflammatory bowel disease (IBD) and periodontal disease. Periodontitis is a polymicrobial inflammatory disease that affects a large proportion of the world's population and has been associated with a wide variety of systemic health conditions, such as diabetes, cardiovascular and respiratory diseases. Dysbiosis has been identified as a key element in the development of the disease. However, the precise mechanisms and environmental signals that lead to the initiation of dysbiosis in the human microbiome are largely unknown. In a series of previous in vivo studies using metatranscriptomic analysis of periodontitis and its progression we identified several functional signatures that were highly associated with the disease. Among them, potassium ion transport appeared to be key in the process of pathogenesis. To confirm its importance we performed a series of in vitro experiments, in which we demonstrated that potassium levels a increased the virulence of the oral community as a whole and at the same time altering the immune response of gingival epithelium, increasing the production of TNF-α and reducing the expression of IL-6 and the antimicrobial peptide human ß-defensin 3 (hBD-3). These results indicate that levels of potassium in the periodontal pocket could be an important element in of dysbiosis in the oral microbiome. They are a starting point for the identification of key environmental signals that modify the behavior of the oral microbiome from a symbiotic community to a dysbiotic one.

Small RNA Transcriptome of the Oral Microbiome during Periodontitis Progression.

The oral microbiome is one of the most complex microbial communities in the human body, and due to circumstances not completely understood, the healthy microbial community becomes dysbiotic, giving rise to periodontitis, a polymicrobial inflammatory disease. We previously reported the results of community-wide gene expression changes in the oral microbiome during periodontitis progression and identified signatures associated with increasing severity of the disease. Small noncoding RNAs (sRNAs) are key players in posttranscriptional regulation, especially in fast-changing environments such as the oral cavity. Here, we expanded our analysis to the study of the sRNA metatranscriptome during periodontitis progression on the same samples for which mRNA expression changes were analyzed. We observed differential expression of 12,097 sRNAs, identifying a total of 20 Rfam sRNA families as being overrepresented in progression and 23 at baseline. Gene ontology activities regulated by the differentially expressed (DE) sRNAs included amino acid metabolism, ethanolamine catabolism, signal recognition particle-dependent cotranslational protein targeting to membrane, intron splicing, carbohydrate metabolism, control of plasmid copy number, and response to stress. In integrating patterns of expression of protein coding transcripts and sRNAs, we found that functional activities of genes that correlated positively with profiles of expression of DE sRNAs were involved in pathogenesis, proteolysis, ferrous iron transport, and oligopeptide transport. These findings represent the first integrated sequencing analysis of the community-wide sRNA transcriptome of the oral microbiome during periodontitis progression and show that sRNAs are key regulatory elements of the dysbiotic process leading to disease.

The periodontal pathogen Porphyromonas gingivalis induces expression of transposases and cell death of Streptococcus mitis in a biofilm model.

Oral microbial communities are extremely complex biofilms with high numbers of bacterial species interacting with each other (and the host) to maintain homeostasis of the system. Disturbance in the oral microbiome homeostasis can lead to either caries or periodontitis, two of the most common human diseases. Periodontitis is a polymicrobial disease caused by the coordinated action of a complex microbial community, which results in inflammation of tissues that support the teeth. It is the most common cause of tooth loss among adults in the United States, and recent studies have suggested that it may increase the risk for systemic conditions such as cardiovascular diseases. In a recent series of papers, Hajishengallis and coworkers proposed the idea of the "keystone-pathogen" where low-abundance microbial pathogens (Porphyromonas gingivalis) can orchestrate inflammatory disease by turning a benign microbial community into a dysbiotic one. The exact mechanisms by which these pathogens reorganize the healthy oral microbiome are still unknown. In the present manuscript, we present results demonstrating that P. gingivalis induces S. mitis death and DNA fragmentation in an in vitro biofilm system. Moreover, we report here the induction of expression of multiple transposases in a Streptococcus mitis biofilm when the periodontopathogen P. gingivalis is present. Based on these results, we hypothesize that P. gingivalis induces S. mitis cell death by an unknown mechanism, shaping the oral microbiome to its advantage.

A Porphyromonas gingivalis hypothetical protein controlled by the type I-C CRISPR-Cas system is a novel adhesin important in virulence.

The ability of many human pathogens to infect requires their ability to adhere to the host surfaces as a first step in the process. Porphyromonas gingivalis, a keystone oral pathogen, uses adhesins to adhere to the surface of the gingival epithelium and other members of the oral microbiome. In a previous study, we identified several proteins potentially linked to virulence whose mRNA levels are regulated by CRISPR-Cas type I-C. Among those, PGN_1547 was highly upregulated in the CRISPR-Cas 3 mutant. PGN_1547 is annotated as a hypothetical protein. Employing homology searching, our data support that PGN_1547 resembles an auto-transporter adhesin of P. gingivalis based on containing the DUF2807 domain. To begin to characterize the function of PGN_1547, we found that a deletion mutant displayed a significant decrease in virulence using a Galleria mellonela model. Furthermore, this mutant was significantly impaired in forming biofilms and attaching to the macrophage-like cell THP-1. Luminex revealed that the PGN_1547 mutant elicited a less robust cytokine and chemokine response from THP-1 cells, and TLR2 predominantly sensed that recombinant PGN_1547. Taken together, these findings broaden our understanding of the toolbox of virulence factors possessed by P. gingivalis. Importantly, PGN_1547, a hypothetical protein, has homologs in another member of the order Bacteroidales whose function is unknown, and our results could shed light on the role of this family of proteins as auto-transport adhesins in this phylogenetic group.IMPORTANCEPeriodontal diseases are among humans' most common infections, and besides their effect on the oral cavity, they have been associated with systemic inflammatory conditions. Among members of the oral microbiome implicated in the development of periodontitis, Porphyromonas gingivalis is considered a keystone pathogen. We have identified a new adhesin that acts as a virulence factor, PGN_1547, which contains the DUF2807 domain, which belongs to the putative auto-transporter adhesin, head GIN domain family. Deletion of this gene lowers the virulence of P. gingivalis and impacts the ability of P. gingivalis to form biofilm and attach to host cells. Furthermore, the broad distribution of these receptors in the order Bacteroidales suggests their importance in colonization by this important group of organisms.

Lessons learned and unlearned in periodontal microbiology.

Periodontal diseases are initiated by bacterial species living in polymicrobial biofilms at or below the gingival margin and progress largely as a result of the inflammation elicited by specific subgingival species. In the past few decades, efforts to understand the periodontal microbiota have led to an exponential increase in information about biofilms associated with periodontal health and disease. In fact, the oral microbiota is one of the best-characterized microbiomes that colonize the human body. Despite this increased knowledge, one has to ask if our fundamental concepts of the etiology and pathogenesis of periodontal diseases have really changed. In this article we will review how our comprehension of the structure and function of the subgingival microbiota has evolved over the years in search of lessons learned and unlearned in periodontal microbiology. More specifically, this review focuses on: (i) how the data obtained through molecular techniques have impacted our knowledge of the etiology of periodontal infections; (ii) the potential role of viruses in the etiopathogenesis of periodontal diseases; (iii) how concepts of microbial ecology have expanded our understanding of host-microbe interactions that might lead to periodontal diseases; (iv) the role of inflammation in the pathogenesis of periodontal diseases; and (v) the impact of these evolving concepts on therapeutic and preventive strategies to periodontal infections. We will conclude by reviewing how novel systems-biology approaches promise to unravel new details of the pathogenesis of periodontal diseases and hopefully lead to a better understanding of their mechanisms.

Effect of periodontal pathogens on the metatranscriptome of a healthy multispecies biofilm model.

Oral bacterial biofilms are highly complex microbial communities with up to 700 different bacterial taxa. We report here the use of metatranscriptomic analysis to study patterns of community gene expression in a multispecies biofilm model composed of species found in healthy oral biofilms (Actinomyces naeslundii, Lactobacillus casei, Streptococcus mitis, Veillonella parvula, and Fusobacterium nucleatum) and the same biofilm plus the periodontopathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The presence of the periodontopathogens altered patterns in gene expression, and data indicate that transcription of protein-encoding genes and small noncoding RNAs is stimulated. In the healthy biofilm hypothetical proteins, transporters and transcriptional regulators were upregulated while chaperones and cell division proteins were downregulated. However, when the pathogens were present, chaperones were highly upregulated, probably due to increased levels of stress. We also observed a significant upregulation of ABC transport systems and putative transposases. Changes in Clusters of Orthologous Groups functional categories as well as gene set enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that in the absence of pathogens, only sets of proteins related to transport and secondary metabolism were upregulated, while in the presence of pathogens, proteins related to growth and division as well as a large portion of transcription factors were upregulated. Finally, we identified several small noncoding RNAs whose predicted targets were genes differentially expressed in the open reading frame libraries. These results show the importance of pathogens controlling gene expression of a healthy oral community and the usefulness of metatranscriptomic techniques to study gene expression profiles in complex microbial community models.

Microbial community gene expression in ocean surface waters.

Metagenomics is expanding our knowledge of the gene content, functional significance, and genetic variability in natural microbial communities. Still, there exists limited information concerning the regulation and dynamics of genes in the environment. We report here global analysis of expressed genes in a naturally occurring microbial community. We first adapted RNA amplification technologies to produce large amounts of cDNA from small quantities of total microbial community RNA. The fidelity of the RNA amplification procedure was validated with Prochlorococcus cultures and then applied to a microbial assemblage collected in the oligotrophic Pacific Ocean. Microbial community cDNAs were analyzed by pyrosequencing and compared with microbial community genomic DNA sequences determined from the same sample. Pyrosequencing-based estimates of microbial community gene expression compared favorably to independent assessments of individual gene expression using quantitative PCR. Genes associated with key metabolic pathways in open ocean microbial species-including genes involved in photosynthesis, carbon fixation, and nitrogen acquisition-and a number of genes encoding hypothetical proteins were highly represented in the cDNA pool. Genes present in the variable regions of Prochlorococcus genomes were among the most highly expressed, suggesting these encode proteins central to cellular processes in specific genotypes. Although many transcripts detected were highly similar to genes previously detected in ocean metagenomic surveys, a significant fraction ( approximately 50%) were unique. Thus, microbial community transcriptomic analyses revealed not only indigenous gene- and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.

UV hyper-resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase.

Exposure to solar radiation can cause mortality in natural communities of pico-phytoplankton, both at the surface and to a depth of at least 30 m. DNA damage is a significant cause of death, mainly due to cyclobutane pyrimidine dimer formation, which can be lethal if not repaired. While developing a UV mutagenesis protocol for the marine cyanobacterium Prochlorococcus, we isolated a UV-hyper-resistant variant of high light-adapted strain MED4. The hyper-resistant strain was constitutively upregulated for expression of the mutT-phrB operon, encoding nudix hydrolase and photolyase, both of which are involved in repair of DNA damage that can be caused by UV light. Photolyase (PhrB) breaks pyrimidine dimers typically caused by UV exposure, using energy from visible light in the process known as photoreactivation. Nudix hydrolase (MutT) hydrolyses 8-oxo-dGTP, an aberrant form of GTP that results from oxidizing conditions, including UV radiation, thus impeding mispairing and mutagenesis by preventing incorporation of the aberrant form into DNA. These processes are error-free, in contrast to error-prone SOS dark repair systems that are widespread in bacteria. The UV-hyper-resistant strain contained only a single mutation: a 1 bp deletion in the intergenic region directly upstream of the mutT-phrB operon. Two subsequent enrichments for MED4 UV-hyper-resistant strains from MED4 wild-type cultures gave rise to strains containing this same 1 bp deletion, affirming its connection to the hyper-resistant phenotype. These results have implications for Prochlorococcus DNA repair mechanisms, genome stability and possibly lysogeny.

A priori estimation of sequencing effort in complex microbial metatranscriptomes.

Metatranscriptome analysis or the analysis of the expression profiles of whole microbial communities has the additional challenge of dealing with a complex system with dozens of different organisms expressing genes simultaneously. An underlying issue for virtually all metatranscriptomic sequencing experiments is how to allocate the limited sequencing budget while guaranteeing that the libraries have sufficient depth to cover the breadth of expression of the community. Estimating the required sequencing depth to effectively sample the target metatranscriptome using RNA-seq is an essential first step to obtain robust results in subsequent analysis and to avoid overexpansion, once the information contained in the library reaches saturation. Here, we present a method to calculate the sequencing effort using a simulated series of metatranscriptomic/metagenomic matrices. This method is based on an extrapolation rarefaction curve using a Weibull growth model to estimate the maximum number of observed genes as a function of sequencing depth. This approach allowed us to compute the effort at different confidence intervals and to obtain an approximate a priori effort based on an initial fraction of sequences. The analytical pipeline presented here may be successfully used for the in-depth and time-effective characterization of complex microbial communities, representing a useful tool for the microbiome research community.

The Oral Microbiome and Cancer.

There is mounting evidence that members of the human microbiome are highly associated with a wide variety of cancer types. Among oral cancers, oral squamous cell carcinoma (OSCC) is the most prevalent and most commonly studied, and it is the most common malignancy of the head and neck worldwide. However, there is a void regarding the role that the oral microbiome may play in OSCC. Previous studies have not consistently found a characteristic oral microbiome composition associated with OSCC. Although a direct causality has not been proven, individual members of the oral microbiome are capable of promoting various tumorigenic functions related to cancer development. Two prominent oral pathogens, Porphyromonas gingivalis, and Fusobacterium nucleatum can promote tumor progression in mice. P. gingivalis infection has been associated with oro-digestive cancer, increased oral cancer invasion, and proliferation of oral cancer stem cells. The microbiome can influence the evolution of the disease by directly interacting with the human body and significantly altering the response and toxicity to various forms of cancer therapy. Recent studies have shown an association of certain phylogenetic groups with the immunotherapy treatment outcomes of certain tumors. On the other side of the coin, recently it has been a resurgence in interest on the potential use of bacteria to cure cancer. These kinds of treatments were used in the late nineteenth and early twentieth centuries as the first line of defense against cancer in some hospitals but later displaced by other types of treatments such as radiotherapy. Currently, organisms such as Salmonella typhimurium and Clostridium spp. have been used for targeted strategies as potential vectors to treat cancer. In this review, we briefly summarize our current knowledge of the role of the oral microbiome, focusing on its bacterial fraction, in cancer in general and in OSCC more precisely, and a brief description of the potential use of bacteria to target tumors.

Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is a unique genomic entity that provides prokaryotic cells with adaptive and heritable immunity. Initial studies identified CRISPRs as central elements used by bacteria to protect against foreign nucleic acids; however, emerging evidence points to CRISPR involvement in bacterial virulence. The present study aimed to identify the participation of one CRISPR-Cas protein, Cas3, in the virulence of the oral pathogen Porphyromonas gingivalis, an organism highly associated with periodontitis. Our results show that compared to the wild type, a mutant with a deletion of the Cas3 gene, an essential nuclease part of the class 1 type I CRISPR-Cas system, increased the virulence of P. gingivalis In vitro infection modeling revealed only mildly enhanced production of proinflammatory cytokines by THP-1 cells when infected with the mutant strain. Dual transcriptome sequencing (RNA-seq) analysis of infected THP-1 cells showed an increase in expression of genes associated with pathogenesis in response to Δcas3 mutant infection, with the target of Cas3 activities in neutrophil chemotaxis and gene silencing. The importance of cas3 in controlling virulence was corroborated in a Galleria mellonella infection model, where the presence of the Δcas3 mutant resulted in a statistically significant increase in mortality of G. mellonella A time-series analysis of transcription patterning during infection showed that G. mellonella elicited very different immune responses to the wild-type and the Δcas3 mutant strains and revealed a rearrangement of association in coexpression networks. Together, these observations show for the first time that Cas3 plays a significant role in regulating the virulence of P. gingivalis IMPORTANCE Porphyromonas gingivalis is a key pathogen of periodontitis, a polymicrobial disease characterized by a chronic inflammation that destroys the tissues supporting the teeth. Thus, understanding the virulence potential of P. gingivalis is essential to maintaining a healthy oral microbiome. In nonoral organisms, CRISPR-Cas systems have been shown to modulate a variety of microbial processes, including protection from exogenous nucleic acids, and, more recently, have been implicated in bacterial virulence. Previously, our clinical findings identified activation of the CRISPR-Cas system in patient samples at the transition to disease; however, the mechanism of contribution to disease remained unknown. The importance of the present study resides in that it is becoming increasingly clear that CRISPR-associated proteins have broader functions than initially thought and that those functions now include their role in the virulence of periodontal pathogens. Studying a P. gingivalis cas3 mutant, we demonstrate that at least one of the CRISPR-Cas systems is involved in the regulation of virulence during infection.

Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters.

Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food-web.

The Oral Mouse Microbiome Promotes Tumorigenesis in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck worldwide. Dysbiosis of the microbiome has increasingly been linked to the development of different kinds of cancer. Applying 16S rRNA gene sequence analysis and metatranscriptomic analyses, we characterized the longitudinal changes in the profiles and the function of the oral microbiome in a 4-nitroquinoline-1-oxide (4-NQO)-induced model of OSCC in gnotobiotic mice. We characterized the dynamics of the oral microbiome in this model using two different microbiome inocula: one from healthy mice and the other from mice bearing a 4-NQO-induced tumor. Mice colonized with different oral microbiomes and exposed to 4-NQO had increased tumor numbers and sizes compared to controls exposed to 4-NQO but lacking a microbiome. We observed an overall increase in diversity in the tumorigenic samples compared to that in the nontumor group not exposed to 4-NQO. Despite the variability in community dynamics, specific patterns emerged during the progression of the disease. In the two groups that were inoculated with the OSCC-associated microbiome, we observed opposite profiles of abundance in Parabacteroides and Corynebacterium While the percentage of Parabacteroides bacteria decreased in the control group, it increased in the OSCC group, and the opposite was observed for Corynebacterium The metatranscriptomic analysis revealed overexpression of the same metabolic signatures associated with OSCC regardless of the community profile. These included nitrogen transport, response to stress, interspecies interactions, Wnt pathway modulation, and amino acid and lipid biosynthesis. Thus, these results seem to suggest that certain collective physiological activities are critical for microbiome-mediated OSCC progression.IMPORTANCE There is growing evidence that changes in the microbiome are associated with carcinogenesis. To date, no consistent oral microbiome composition associated with OSCC has been identified. Longitudinal and functional studies like the study presented here should yield a better understanding of the role that the oral microbiome plays in OSCC. Our findings, obtained using a germ-free mouse model, indicate that the presence of different oral microbiomes enhances tumorigenesis and increases the final number of tumors in mice. By studying community-wide expression profiles, we found that regardless of the phylogenetic composition of the microbiome, the same metabolic activities were consistently associated with OSCC. Therefore, due to the functional redundancy of the microbiome, the critical element in explaining the contribution of the microbiota in OSCC is the collective physiological activity of the community, thus accounting for the previous inability to identify a consensus community profile or etiologic agents for OSCC.

The effect of the stress hormone cortisol on the metatranscriptome of the oral microbiome.

Imbalances of the microbiome, also referred to as microbial dysbiosis, could lead to a series of different diseases. One factor that has been shown to lead to dysbiosis of the microbiome is exposure to psychological stressors. Throughout evolution microorganisms of the human microbiome have developed systems for sensing host-associated signals such as hormones associated with those stressors, enabling them to recognize essential changes in their environment, thus changing their expression gene profile to fit the needs of the new environment. The most widely accepted theory explaining the ability of hormones to affect the outcome of an infection involves the suppression of the immune system. Commensal microbiota is involved in stressor-induced immunomodulation, but other biological effects are not yet known. Here we present the impact that cortisol had on the community-wide transcriptome of the oral community. We used a metatranscriptomic approach to obtain first insights into the metabolic changes induced by this stress hormone as well as which members of the oral microbiome respond to the presence of cortisol in the environment. Our findings show that the stress hormone cortisol directly induces shifts in the gene expression profiles of the oral microbiome that reproduce results found in the profiles of expression of periodontal disease and its progression.

Increased virulence of the oral microbiome in oral squamous cell carcinoma revealed by metatranscriptome analyses.

Oral squamous cell carcinoma (OSCC) is the most prevalent and most commonly studied oral cancer. However, there is a void regarding the role that the oral microbiome may play in OSCC. Although the relationship between microbial community composition and OSCC has been thoroughly investigated, microbial profiles of the human microbiome in cancer are understudied. Here we performed a small pilot study of community-wide metatranscriptome analysis to profile mRNA expression in the entire oral microbiome in OSCC to reveal molecular functions associated with this disease. Fusobacteria showed a statistically significantly higher number of transcripts at tumour sites and tumour-adjacent sites of cancer patients compared to the healthy controls analysed. Regardless of the community composition, specific metabolic signatures were consistently found in disease. Activities such as iron ion transport, tryptophanase activity, peptidase activities and superoxide dismutase were over-represented in tumour and tumour-adjacent samples when compared to the healthy controls. The expression of putative virulence factors in the oral communities associated with OSCC showed that activities related to capsule biosynthesis, flagellum synthesis and assembly, chemotaxis, iron transport, haemolysins and adhesins were upregulated at tumour sites. Moreover, activities associated with protection against reactive nitrogen intermediates, chemotaxis, flagellar and capsule biosynthesis were also upregulated in non-tumour sites of cancer patients. Although they are preliminary, our results further suggest that Fusobacteria may be the leading phylogenetic group responsible for the increase in expression of virulence factors in the oral microbiome of OSCC patients.

Functional profiles of coronal and dentin caries in children.

Background: Dental caries results from a dysbiosis of tooth-associated biofilms and frequently extends through enamel into dentin which has a different structure and composition. Objective: To evaluate the metatranscriptome of caries to determine the metabolic potential of caries communities compared with health. Design: Samples from children, caries-free (CF: n = 4) or with coronal (CC: n = 5) or dentin (DC: n = 5) caries were examined for gene expression potential. Functional profiling was performed using HUMAnN2 (HMP Unified Metabolic Analysis Network). Results: There was increased gene expression diversity in DC compared with CC and CF. Genes in CF included alcohol dehydrogenase from Neisseria sicca, methylenetetrahydrofolate reductase from Streptococcus sanguinis and choline kinase from streptococci. Genes in CC mapped mainly to Streptococcus mutans. Arginine deiminase in DC mapped to S. sanguinis and Actinomyces naeslundii. Glycerol kinase genes mapped to S. sanguinis in all groups whereas glycerol kinase in DC were from Rothia, Prevotella and streptococci. Uracil-DNA glycosylase in DC mapped to Prevotella denticola and Actinomyces. Repressor LexA in DC mapped to Scardovia wiggsiae, Dialister invisus and Veillonella parvula. Conclusions: Functional profiling revealed enzyme activities in both caries and caries-free communities and clarified marked differences between coronal and dentin caries in bacterial composition and potential gene expression.

Targeting specific bacteria in the oral microbiome.

A lack of tools that kill selected members of the oral microbiome has hampered the ability to study specific roles of bacteria within bacterial communities. Work by Guo et al. shows the potential of antimicrobial peptides as a tool to assess the role of individual species in the microbial community.