Exploitation of a Bacterium-Encoded Lytic Transglycosylase by a Human Oral Lytic Phage To Facilitate Infection.

Bacteriophages (phages) are an integral part of the human oral microbiome. Their roles in modulating bacterial physiology and shaping microbial communities have been discussed but remain understudied due to limited isolation and characterization of oral phage. Here, we report the isolation of LC001, a lytic phage targeting human oral Schaalia odontolytica (formerly known as Actinomyces odontolyticus) strain XH001. We showed that LC001 attached to and infected surface-grown, but not planktonic, XH001 cells, and it displayed remarkable host specificity at the strain level. Whole-genome sequencing of spontaneous LC001-resistant, surface-grown XH001 mutants revealed that the majority of the mutants carry nonsense or frameshift mutations in XH001 gene APY09_05145 (renamed ltg-1), which encodes a putative lytic transglycosylase (LT). The mutants are defective in LC001 binding, as revealed by direct visualization of the significantly reduced attachment of phage particles to the XH001 spontaneous mutants compared that to the wild type. Meanwhile, targeted deletion of ltg-1 produced a mutant that is defective in LC001 binding and resistant to LC001 infection even as surface-grown cells, while complementation of ltg-1 in the mutant background restored the LC001-sensitive phenotype. Intriguingly, similar expression levels of ltg-1 were observed in surface-grown and planktonic XH001, which displayed LC001-binding and nonbinding phenotypes, respectively. Furthermore, the overexpression of ltg-1 failed to confer an LC001-binding and -sensitive phenotype to planktonic XH001. Thus, our data suggested that rather than directly serving as a phage receptor, ltg-1-encoded LT may increase the accessibility of phage receptor, possibly via its enzymatic activity, by cleaving the peptidoglycan structure for better receptor exposure during peptidoglycan remodeling, a function that can be exploited by LC001 to facilitate infection. IMPORTANCE The evidence for the presence of a diverse and abundant phage population in the host-associated oral microbiome came largely from metagenomic analysis or the observation of virus-like particles within saliva/plaque samples, while the isolation of oral phage and investigation of their interaction with bacterial hosts are limited. Here, we report the isolation of LC001, the first lytic phage targeting oral Schaalia odontolytica. Our study suggested that LC001 may exploit the host bacterium-encoded lytic transglycosylase function to gain access to the receptor, thus facilitating its infection.

Fusobacterium nucleatum secretes amyloid-like FadA to enhance pathogenicity.

Fusobacterium nucleatum (Fn) is a Gram-negative oral commensal, prevalent in various human diseases. It is unknown how this common commensal converts to a rampant pathogen. We report that Fn secretes an adhesin (FadA) with amyloid properties via a Fap2-like autotransporter to enhance its virulence. The extracellular FadA binds Congo Red, Thioflavin-T, and antibodies raised against human amyloid ß42. Fn produces amyloid-like FadA under stress and disease conditions, but not in healthy sites or tissues. It functions as a scaffold for biofilm formation, confers acid tolerance, and mediates Fn binding to host cells. Furthermore, amyloid-like FadA induces periodontal bone loss and promotes CRC progression in mice, with virulence attenuated by amyloid-binding compounds. The uncleaved signal peptide of FadA is required for the formation and stability of mature amyloid FadA fibrils. We propose a model in which hydrophobic signal peptides serve as "hooks" to crosslink neighboring FadA filaments to form a stable amyloid-like structure. Our study provides a potential mechanistic link between periodontal disease and CRC and suggests anti-amyloid therapies as possible interventions for Fn-mediated disease processes.

Fusobacteriumnucleatum Adheres to Clostridioides difficile via the RadD Adhesin to Enhance Biofilm Formation in Intestinal Mucus.

BACKGROUND & AIMS: Although Clostridioides difficile infection (CDI) is known to involve the disruption of the gut microbiota, little is understood regarding how mucus-associated microbes interact with C difficile. We hypothesized that select mucus-associated bacteria would promote C difficile colonization and biofilm formation. METHODS: To create a model of the human intestinal mucus layer and gut microbiota, we used bioreactors inoculated with healthy human feces, treated with clindamycin and infected with C difficile with the addition of human MUC2-coated coverslips. RESULTS: C difficile was found to colonize and form biofilms on MUC2-coated coverslips, and 16S rRNA sequencing showed a unique biofilm profile with substantial cocolonization with Fusobacterium species. Consistent with our bioreactor data, publicly available data sets and patient stool samples showed that a subset of patients with C difficile infection harbored high levels of Fusobacterium species. We observed colocalization of C difficile and F nucleatum in an aggregation assay using adult patients and stool of pediatric patients with inflammatory bowel disease and in tissue sections of patients with CDI. C difficile strains were found to coaggregate with F nucleatum subspecies in vitro; an effect that was inhibited by blocking or mutating the adhesin RadD on Fusobacterium and removal of flagella on C difficile. Aggregation was shown to be unique between F nucleatum and C difficile, because other gut commensals did not aggregate with C difficile. Addition of F nucleatum also enhanced C difficile biofilm formation and extracellular polysaccharide production. CONCLUSIONS: Collectively, these data show a unique interaction of between pathogenic C difficile and F nucleatum in the intestinal mucus layer.

Klebsiella and Providencia emerge as lone survivors following long-term starvation of oral microbiota.

It is well-understood that many bacteria have evolved to survive catastrophic events using a variety of mechanisms, which include expression of stress-response genes, quiescence, necrotrophy, and metabolic advantages obtained through mutation. However, the dynamics of individuals leveraging these abilities to gain a competitive advantage in an ecologically complex setting remain unstudied. In this study, we observed the saliva microbiome throughout the ecological perturbation of long-term starvation, allowing only the species best equipped to access and use the limited resources to survive. During the first several days, the community underwent a death phase that resulted in a ∼50-100-fold reduction in the number of viable cells. Interestingly, after this death phase, only three species, Klebsiella pneumoniae, Klebsiella oxytoca, and Providencia alcalifaciens, all members of the family Enterobacteriaceae, appeared to be transcriptionally active and recoverable. Klebsiella are significant human pathogens, frequently resistant to multiple antibiotics, and recently, ectopic colonization of the gut by oral Klebsiella was documented to induce dysbiosis and inflammation. MetaOmics analyses provided several leads for further investigation regarding the ecological success of the Enterobacteriaceae. The isolates accumulated single nucleotide polymorphisms in known growth advantage in stationary phase alleles and produced natural products closely resembling antimicrobial cyclic depsipeptides. The results presented in this study suggest that pathogenic Enterobacteriaceae persist much longer than their more benign neighbors in the salivary microbiome when faced with starvation. This is particularly significant, given that hospital surfaces contaminated with oral fluids, especially sinks and drains, are well-established sources of outbreaks of drug-resistant Enterobacteriaceae.

The impact of fixed orthodontic appliances and clear aligners on the oral microbiome and the association with clinical parameters: A longitudinal comparative study.

INTRODUCTION: Orthodontic treatment interferes with oral hygiene and promotes plaque retention, which leads to gingival inflammation and enamel demineralization. Although removable clear aligners (CAs) are designed to improve oral hygiene compared with fixed appliances (FAs), comprehensive studies comparing their respective effects on the oral microbiome are limited. This longitudinal study investigated the microbial changes during orthodontic treatment with FA and CA in correlation with clinical parameters. METHODS: Clinical parameters and supragingival plaque were collected from 12 study participants for the FA or CA treatment groups at baseline and at least twice at the 1, 3, 6, and 12-month follow-up appointments. The plaque was also harvested from the aligner tray for the CA group. Microbiome composition was determined via 16S rRNA gene sequencing, compared between groups, and correlated with clinical parameters. RESULTS: Plaque (PI) and gingival indexes (GI) increased significantly in the FA but not the CA group. Beta but not alpha diversities of the microbial communities were distinct between the 2 treatment groups, even though genus-level differences were not significant except for Leptotrichia. The CA tray harbors a unique plaque community. Elevated PI and GI in the FA group correlated with a higher abundance of disease-related genera. CONCLUSIONS: Orthodontic treatments trigger appliance-related plaque community shifts from baseline, and the CA tray environment attracts distinct microbial communities. In comparison with FA, the use of CA resulted in better oral health index outcomes, which is reflected by the corresponding PI and GI-associated oral microbial communities.

Omics and interspecies interaction.

Interspecies interactions are key determinants in biofilm behavior, ecology, and architecture. The cellular responses of microorganisms to each other at transcriptional, proteomic, and metabolomic levels ultimately determine the characteristics of biofilm and the corresponding implications for health and disease. Advances in omics technologies have revolutionized our understanding of microbial community composition and their activities as a whole. Large-scale analyses of the complex interaction between the many microbial species residing within a biofilm, however, are currently still hampered by technical and bioinformatics challenges. Thus, studies of interspecies interactions have largely focused on the transcriptional and proteomic changes that occur during the contact of a few prominent species, such as Porphyromonas gingivalis, Streptococcus mutans, Candida albicans, and a few others, with selected partner species. Expansion of available tools is necessary to grow the revealing, albeit limited, insight these studies have provided into a profound understanding of the nature of individual microbial responses to the presence of others. This will allow us to answer important questions including: Which intermicrobial interactions orchestrate the myriad of cooperative, synergistic, antagonistic, manipulative, and other types of relationships and activities in the complex biofilm environment, and what are the implications for oral health and disease?

Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

Histone Lys-specific demethylases (KDMs) play a key role in many biological processes through epigenetic mechanisms. However, the role of KDMs in inflammatory responses to oral bacterial infection is poorly understood. Here, we show a novel regulatory role of KDM3C in inflammatory responses to oral bacterial infection. KDM3C expression is transiently suppressed in human and mouse macrophages exposed to LPS from Porphyromonas gingivalis (Pg LPS). Loss of KDM3C in both human and mouse macrophages led to notable induction of proinflammatory cytokines in response to Pg LPS stimulation. Also, KDM3C depletion led to strong induction of p65 phosphorylation and accelerated nuclear translocation in cells exposed to Pg LPS. Kdm3C knockout (KO) in mice led to increased alveolar bone destruction upon induction of experimental periodontitis or pulp exposure compared with those of the wild-type (WT) littermates. The Kdm3C KO mice also revealed an increased number of osteoclasts juxtaposed to the bony lesions. We also confirmed enhanced osteoclastogenesis by bone marrow-derived macrophages isolated from the Kdm3C KO compared with the WT controls. These findings suggest an anti-inflammatory function of KDM3C in regulating the inflammatory responses against oral bacterial infection through suppression of NF-κB signaling and osteoclastogenesis.-Lee, J. Y., Mehrazarin, S., Alshaikh, A., Kim, S., Chen, W., Lux, R., Gwack, Y., Kim, R. H., Kang, M. K. Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

The Oral Bacterium Fusobacterium nucleatum Binds Staphylococcus aureus and Alters Expression of the Staphylococcal Accessory Regulator sarA.

Staphylococcus aureus, an opportunistic pathogen member of the nasal and skin microbiota, can also be found in human oral samples and has been linked to infectious diseases of the oral cavity. As the nasal and oral cavities are anatomically connected, it is currently unclear whether S. aureus can colonize the oral cavity and become part of the oral microbiota, or if its presence in the oral cavity is simply transient. To start addressing this question, we assessed S. aureus ability to directly bind selected members of the oral microbiota as well as its ability to integrate into a human-derived complex oral microbial community in vitro. Our data show that S. aureus forms aggregates with Fusobacterium nucleatum and Porphyromonas gingivalis and that it can incorporate into the human-derived in vitro oral community. Further analysis of the F. nucleatum-S. aureus interaction revealed that the outer-membrane adhesin RadD is partially involved in aggregate formation and that the RadD-mediated interaction leads to an increase in expression of the staphylococcal global regulator gene sarA. Our findings lend support to the notion that S. aureus can become part of the complex microbiota of the human mouth, which could serve as a reservoir for S. aureus. Furthermore, direct interaction with key members of the oral microbiota could affect S. aureus pathogenicity contributing to the development of several S. aureus associated oral infections.

Precision-guided antimicrobial peptide as a targeted modulator of human microbial ecology.

One major challenge to studying human microbiome and its associated diseases is the lack of effective tools to achieve targeted modulation of individual species and study its ecological function within multispecies communities. Here, we show that C16G2, a specifically targeted antimicrobial peptide, was able to selectively kill cariogenic pathogen Streptococcus mutans with high efficacy within a human saliva-derived in vitro oral multispecies community. Importantly, a significant shift in the overall microbial structure of the C16G2-treated community was revealed after a 24-h recovery period: several bacterial species with metabolic dependency or physical interactions with S. mutans suffered drastic reduction in their abundance, whereas S. mutans' natural competitors, including health-associated Streptococci, became dominant. This study demonstrates the use of targeted antimicrobials to modulate the microbiome structure allowing insights into the key community role of specific bacterial species and also indicates the therapeutic potential of C16G2 to achieve a healthy oral microbiome.

Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle.

The candidate phylum TM7 is globally distributed and often associated with human inflammatory mucosal diseases. Despite its prevalence, the TM7 phylum remains recalcitrant to cultivation, making it one of the most enigmatic phyla known. In this study, we cultivated a TM7 phylotype (TM7x) from the human oral cavity. This extremely small coccus (200-300 nm) has a distinctive lifestyle not previously observed in human-associated microbes. It is an obligate epibiont of an Actinomyces odontolyticus strain (XH001) yet also has a parasitic phase, thereby killing its host. This first completed genome (705 kb) for a human-associated TM7 phylotype revealed a complete lack of amino acid biosynthetic capacity. Comparative genomics analyses with uncultivated environmental TM7 assemblies show remarkable conserved gene synteny and only minimal gene loss/gain that may have occurred as TM7x adapted to conditions within the human host. Transcriptomic and metabolomic profiles provided the first indications, to our knowledge, that there is signaling interaction between TM7x and XH001. Furthermore, the induction of TNF-α production in macrophages by XH001 was repressed in the presence of TM7x, suggesting its potential immune suppression ability. Overall, our data provide intriguing insights into the uncultivability, pathogenicity, and unique lifestyle of this previously uncharacterized oral TM7 phylotype.

Effect of titanium and zirconia dental implant abutments on a cultivable polymicrobial saliva community.

STATEMENT OF PROBLEM: Peri-implantitis is considered the most important biological complication responsible for late implant failure. The physical chemical properties intrinsic to each material can affect the first step to biofilm development and is an important precursor to the adaptive behavior of pathogenic bacteria species. PURPOSE: The purpose of this in vitro study was to evaluate the effect of 2 commercially available implant abutment materials on the adhesion phase and biofilm formation. MATERIAL AND METHODS: Disks (8 mm in diameter, 2 mm thick) of machined pure titanium (Ti) and yttrium-stabilized zirconia (ZrO2) materials were used to mimic implant abutments. The physical chemical surface properties were investigated using different approaches. Initial adherent bacteria and biofilm formation were evaluated after 16 and 48 hours by incubating the disks in a rich medium containing representative saliva-derived oral microbial community. Unpaired t test, 2 tailed, was used to compare the groups. RESULTS: Ti presented lower hydrophobicity and surface free energy values than the ZrO2, and 6.1-fold fewer bacteria adhered to the Ti. After 48 hours, detailed quantitative analysis showed that biofilm biomass and biofilm density were lower on the Ti disks than on ZrO2. The quantity of phylotypes on the Ti and ZrO2 surfaces was relatively similar during the attachment and early biofilm formation periods. CONCLUSIONS: Although no difference in the bacteria profile was observed between both materials independent of the time point, the highest level of colonization was on ZrO2.

FAD-I, a Fusobacterium nucleatum Cell Wall-Associated Diacylated Lipoprotein That Mediates Human Beta Defensin 2 Induction through Toll-Like Receptor-1/2 (TLR-1/2) and TLR-2/6.

We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ΔfadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.

Phenotypic and Physiological Characterization of the Epibiotic Interaction Between TM7x and Its Basibiont Actinomyces.

Despite many examples of obligate epibiotic symbiosis (one organism living on the surface of another) in nature, such an interaction has rarely been observed between two bacteria. Here, we further characterize a newly reported interaction between a human oral obligate parasitic bacterium TM7x (cultivated member of Candidatus Saccharimonas formerly Candidate Phylum TM7), and its basibiont Actinomyces odontolyticus species (XH001), providing a model system to study epiparasitic symbiosis in the domain Bacteria. Detailed microscopic studies indicate that both partners display extensive morphological changes during symbiotic growth. XH001 cells manifested as short rods in monoculture, but displayed elongated and hyphal morphology when physically associated with TM7x. Interestingly, these dramatic morphological changes in XH001 were also induced in oxygen-depleted conditions, even in the absence of TM7x. Targeted quantitative real-time PCR (qRT-PCR) analyses revealed that both the physical association with TM7x as well as oxygen depletion triggered up-regulation of key stress response genes in XH001, and in combination, these conditions act in an additive manner. TM7x and XH001 co-exist with relatively uniform cell morphologies under nutrient-replete conditions. However, upon nutrient depletion, TM7x-associated XH001 displayed a variety of cell morphologies, including swollen cell body, clubbed-ends, and even cell lysis, and a large portion of TM7x cells transformed from ultrasmall cocci into elongated cells. Our study demonstrates a highly dynamic interaction between epibiont TM7x and its basibiont XH001 in response to physical association or environmental cues such as oxygen level and nutritional status, as reflected by their morphological and physiological changes during symbiotic growth.

Characterization of aid1, a novel gene involved in Fusobacterium nucleatum interspecies interactions.

The oral opportunistic pathogen Fusobacterium nucleatum is known to interact with a large number of different bacterial species residing in the oral cavity. It adheres to a variety of Gram-positive bacteria, including oral streptococci via the arginine-inhibitable adhesin RadD. In this study, we describe a novel protein encoded by the predicted open reading frame FN1253 that appears to play a role in interspecies interactions of F. nucleatum, particularly with oral streptococci and related Gram-positive species. We designated FN1253 as aid1 (Adherence Inducing Determinant 1). Expression analyses demonstrated that this gene was induced in F. nucleatum single species biofilms, while the presence of representative members of the oral microbiota known to adhere to F. nucleatum triggered its suppression. Inactivation as well as overexpression of aid1 affected the ability of F. nucleatum to coaggregate with oral streptococci and the closely related Enterococcus faecalis, but not other Gram-positive oral species tested. Furthermore, overexpression of aid1 led to a drastic change in the structure of dual species biofilms of F. nucleatum with oral streptococci. Aid1 function was abolished in the presence of arginine and found to be dependent on RadD. Interestingly, differential expression of aid1 did not affect messenger RNA and protein levels of RadD. These findings indicate that RadD-mediated adhesion to oral streptococci involves more complex cellular processes than the simple interaction of adhesins on the surface of partner strains. Aid1 could potentially play an important role in facilitating RadD-mediated interaction with oral streptococci by increasing binding specificity of F. nucleatum to other microbial species.

Fusobacterium nucleatum subsp. nucleatum RadD binds Siglec-7 and inhibits NK cell-mediated cancer cell killing.

Fusobacterium nucleatum is an oral commensal bacterium that can colonize extraoral tumor entities, such as colorectal cancer and breast cancer. Recent studies revealed its ability to modulate the immune response in the tumor microenvironment (TME), promoting cancer progression and metastasis. Importantly, F. nucleatum subsp. animalis was shown to bind to Siglec-7 via lipopolysaccharides, leading to a pro-inflammatory profile in human monocyte-derived dendritic cells. In this study, we show that F. nucleatum subsp. nucleatum RadD binds to Siglec-7 on NK cells, thereby inhibiting NK cell-mediated cancer cell killing. We demonstrate that this binding is dependent on arginine residue R124 in Siglec-7. Finally, we determine that this binding is independent of the known interaction of RadD with IgA. Taken together, our findings elucidate the targeting of Siglec-7 by F. nucleatum subsp. nucleatum RadD as a means to modulate the NK cell response and potentially promoting immune evasion and tumor progression.

Killing of Escherichia coli by Myxococcus xanthus in aqueous environments requires exopolysaccharide-dependent physical contact.

Nutrient or niche-based competition among bacteria is a widespread phenomenon in the natural environment. Such interspecies interactions are often mediated by secreted soluble factors and/or direct cell-cell contact. As ubiquitous soil bacteria, Myxococcus species are able to produce a variety of bioactive secondary metabolites to inhibit the growth of other competing bacterial species. Meanwhile, Myxococcus spp. also exhibit sophisticated predatory behavior, an extreme form of competition that is often stimulated by close contact with prey cells and largely depends on the availability of solid surfaces. Myxococcus spp. can also be isolated from aquatic environments. However, studies focusing on the interaction between Myxococcus and other bacteria in such environments are still limited. In this study, using the well-studied Myxococcus xanthus DK1622 and Escherichia coli as model interspecies interaction pair, we demonstrated that in an aqueous environment, M. xanthus was able to kill E. coli in a cell contact-dependent manner and that the observed contact-dependent killing required the formation of co-aggregates between M. xanthus and E. coli cells. Further analysis revealed that exopolysaccharide (EPS), type IV pilus, and lipopolysaccharide mutants of M. xanthus displayed various degrees of attenuation in E. coli killing, and it correlated well with the mutants' reduction in EPS production. In addition, M. xanthus showed differential binding ability to different bacteria, and bacterial strains unable to co-aggregate with M. xanthus can escape the killing, suggesting the specific nature of co-aggregation and the targeted killing of interacting bacteria. In conclusion, our results demonstrated EPS-mediated, contact-dependent killing of E. coli by M. xanthus, a strategy that might facilitate the survival of this ubiquitous bacterium in aquatic environments.

Development of a new model system to study microbial colonization on dentures.

PURPOSE: Dentures are often colonized with a variety of microorganisms, including Candida albicans, that contribute to denture stomatitis. Several in vitro models have been previously established to study denture-related microbial colonization and evaluate treatment efficacy of denture cleansers; however, those models typically fail to appreciate the complex topology and heterogeneity of denture surfaces and lack effective ways to accurately measure microbial colonization. The purpose of this study was to study microbial colonization with a new model system based on real dentures, to more realistically mimic in vivo conditions. MATERIALS AND METHODS: Scanning electron microscopy was used to observe topological structures among surfaces from different parts of the denture. Employing C. albicans as a model microorganism, we established microbial colonization on different denture surfaces. Moreover, we applied a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) colorimetric assay to quantify C. albicans colonization on dentures without the necessity of biofilm removal and to evaluate treatment efficacy of denture cleansers. RESULTS: There were significant variations in topological structures among surfaces from different parts of the denture, with the unpolished side having the highest amounts of indentations and pores. The distinct denture surfaces support microbial colonization differently, with the unpolished side containing the highest level of microbial colonization and biofilm formation. Furthermore, the modified MTT colorimetric assay proved to be an accurate assay to measure biofilm formation on dentures and evaluate treatment efficacy of denture cleansers. CONCLUSION: This new denture model system in conjunction with the MTT colorimetric assay is a valuable tool to study denture-related microbiology and treatment approaches.

Antagonistic interaction between two key endodontic pathogens Enterococcus faecalis and Fusobacterium nucleatum.

Background: Endodontic infections are known to be caused by pathogenic bacteria. Numerous previous studies found that both Fusobacterium nucleatum and Enterococcus faecalis are associated with endodontic infections, with Fusobacterium nucleatum more abundant in primary infection while Enterococcus faecalis more abundant in secondary infection. Little is known about the potential interactions between different endodontic pathogens. Objective: This study aims to investigate the potential interaction between F. nucleatum and E. faecalis via phenotypical and genetic approaches. Methods: Physical and physiological interactions of F. nucleatum and E. faecalis under both planktonic and biofilm conditions were measured with co-aggregation and competition assays. The mechanisms behind these interactions were revealed with genetic screening and biochemical measurements. Results: E. faecalis was found to physically bind to F. nucleatum under both in vitro planktonic and biofilm conditions, and this interaction requires F. nucleatum fap2, a galactose-inhibitable adhesin-encoding gene. Under our experimental conditions, E. faecalis exhibits a strong killing ability against F. nucleatum by generating an acidic micro-environment and producing hydrogen peroxide (H2O2). Finally, the binding and killing capacities of E. faecalis were found to be necessary to invade and dominate a pre-established in vitro F. nucleatum biofilm. Conclusions: This study reveals multifaceted mechanisms underlying the physical binding and antagonistic interaction between F. nucleatum and E. faecalis, which could play a potential role in the shift of microbial composition in primary and secondary endodontic infections.

The clpB gene is involved in the stress response of Myxococcus xanthus during vegetative growth and development.

The Clp/HSP100 family of molecular chaperones is ubiquitous in both prokaryotes and eukaryotes. These proteins play important roles in refolding, disaggregating and degrading proteins damaged by stress. As a subclass of the Clp/HSP100 family, ClpB has been shown to be involved in various stress responses as well as other functions in bacteria. In the present study, we investigated the role of a predicted ClpB-encoding gene, MXAN5092, in the stress response during vegetative growth and development of Myxococcus xanthus. Transcriptional analysis confirmed induction of this clpB homologue under different stress conditions, and further phenotypic analysis revealed that an in-frame deletion mutant of MXAN5092 was more sensitive to various stress treatments than the wild-type strain during vegetative growth. Moreover, the absence of the MXAN5092 gene resulted in decreased heat tolerance of myxospores, indicating the involvement of this clpB homologue in the stress response during the development of myxospores. The M. xanthus recombinant ClpB (MXAN5092) protein also showed a general chaperone activity in vitro. Overall, our genetic and phenotypic analysis of the predicted ATP-dependent chaperone protein ClpB (MXAN5092) demonstrated that it functions as a chaperone protein and plays an important role in cellular stress tolerance during both vegetative growth and development of M. xanthus.

Adherence to streptococci facilitates Fusobacterium nucleatum integration into an oral microbial community.

The development of multispecies oral microbial communities involves complex intra- and interspecies interactions at various levels. The ability to adhere to the resident bacteria or the biofilm matrix and overcome community resistance are among the key factors that determine whether a bacterium can integrate into a community. Fusobacterium nucleatum is a prevalent Gram-negative oral bacterial species that is able to adhere to a variety of oral microbes and has been implicated in playing an important role in the establishment of multispecies oral microbial community. However, the majority of experiments thus far has focused on the physical adherence between two species as measured by in vitro co-aggregation assays, while the community-based effects on the integration of F. nucleatum into multispecies microbial community remains to be investigated. In this study, we focus on community integration of F. nucleatum. We demonstrated using an established in vitro mice oral microbiota (O-mix) that the viability of F. nucleatum was significantly reduced upon addition to the O-mix due to cell contact-dependent induction of hydrogen peroxide (H(2)O(2)) production by oral community. Interestingly, this inhibitory effect was significantly alleviated when F. nucleatum was allowed to adhere to its known interacting partner species (such as Streptococcus sanguinis) prior to addition. Furthermore, this aggregate formation-dependent protection was absent in the F. nucleatum mutant strain ΔFn1526 that is unable to bind to a number of Gram-positive species. More importantly, this protective effect was also observed during integration of F. nucleatum into a human salivary microbial community (S-mix). These results suggest that by adhering to other oral microbes, F. nucleatum is able to mask the surface components that are recognized by H(2)O(2) producing oral community members. This evasion strategy prevents detection by antagonistic oral bacteria and allows integration into the developing oral microbial community.