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BackgroundEndodontic 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.ObjectiveThis study aims to investigate the potential interaction between F. nucleatum and E. faecalis via phenotypical and genetic approaches.MethodsPhysical 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.ResultsE. 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.ConclusionsThis 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.

Here, we profiled putative phages of Saccharibacteria, which are of particular importance as Saccharibacteria influence some human oral diseases. We additionally profiled putative phages of Gracilibacteria and Absconditabacteria, two Candidate Phyla Radiation (CPR) lineages of interest given their use of an alternative genetic code. Among the phages identified in this study, some are targeted by spacers from both CPR and non-CPR bacteria and others by both bacteria that use the standard genetic code as well as bacteria that use an alternative genetic code. These findings represent new insights into possible phage replication strategies and have relevance for phage therapies that seek to manipulate microbiomes containing CPR bacteria.

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.

The ultra-small, obligate parasitic epibiont, TM7x, the first and only current member of the long-elusive Saccharibacteria (formerly the TM7 phylum) phylum to be cultivated, was isolated in co-culture with its bacterial host, Actinomyces odontolyticus subspecies actinosynbacter, XH001. Initial phenotypic characterization of the TM7x-associated XH001 co-culture revealed enhanced biofilm formation in the presence of TM7x compared to XH001 as monoculture. Genomic analysis and previously published transcriptomic profiling of XH001 also revealed the presence of a putative AI-2 quorum sensing (QS) operon, which was highly upregulated upon association of TM7x with XH001. This analysis revealed that the most highly induced gene in XH001 was an lsrB ortholog, which encodes a putative periplasmic binding protein for the auto inducer (AI)-2 QS signaling molecule. Further genomic analyses suggested the lsrB operon in XH001 is a putative hybrid AI-2/ribose transport operon as well as the existence of a luxS ortholog, which encodes the AI-2 synthase. In this study, the potential role of AI-2 QS in the epibiotic-parasitic relationship between XH001 and TM7x in the context of biofilm formation was investigated. A genetic system for XH001 was developed to generate lsrB and luxS gene deletion mutants in XH001. Phenotypic characterization demonstrated that deletion mutations in either lsrB or luxS did not affect XH001's growth dynamic, mono-species biofilm formation capability, nor its ability to associate with TM7x. TM7x association with XH001 induced lsrB gene expression in a luxS-dependent manner. Intriguingly, unlike wild type XH001, which displayed significantly increased biofilm formation upon establishing the epibiotic-parasitic relationship with TM7x, XH001ΔlsrB, and XH001ΔluxS mutants failed to achieve enhanced biofilm formation when associated with TM7x. In conclusion, we demonstrated a significant role for AI-2 QS in modulating dual-species biofilm formation when XH001 and TM7x establish their epibiotic-parasitic relationship.

Although great strides have been made in understanding the complex bacterial community inhabiting the human oral cavity, for a variety of (mainly technical) reasons the ecological contributions of oral fungi, viruses, phages, and the candidate phyla radiation (CPR) group of ultrasmall bacteria have remained understudied. Several recent reports have illustrated the diversity and importance of these organisms in the oral cavity, while TM7x and Candida albicans have served as crucial paradigms for CPR species and oral fungi, respectively. A comprehensive understanding of the oral microbiota and its influence on host health and disease will require a holistic view that emphasizes interactions among different residents within the oral community, as well as their interaction with the host.

Bacteriophages (phages) play critical roles in modulating microbial ecology. Within the human microbiome, the factors influencing the long-term coexistence of phages and bacteria remain poorly investigated. Saccharibacteria (formerly TM7) are ubiquitous members of the human oral microbiome. These ultrasmall bacteria form episymbiotic relationships with their host bacteria and impact their physiology. Here, we showed that during surface-associated growth, a human oral Saccharibacteria isolate (named TM7x) protects its host bacterium, a Schaalia odontolytica strain (named XH001) against lytic phage LC001 predation. RNA-Sequencing analysis identified in XH001 a gene cluster with predicted functions involved in the biogenesis of cell wall polysaccharides (CWP), whose expression is significantly down-regulated when forming a symbiosis with TM7x. Through genetic work, we experimentally demonstrated the impact of the expression of this CWP gene cluster on bacterial-phage interaction by affecting phage binding. In vitro coevolution experiments further showed that the heterogeneous populations of TM7x-associated and TM7x-free XH001, which display differential susceptibility to LC001 predation, promote bacteria and phage coexistence. Our study highlights the tripartite interaction between the bacterium, episymbiont, and phage. More importantly, we present a mechanism, i.e., episymbiont-mediated modulation of gene expression in host bacteria, which impacts their susceptibility to phage predation and contributes to the formation of "source-sink" dynamics between phage and bacteria in biofilm, promoting their long-term coexistence within the human microbiome. [ABSTRACT FROM AUTHOR]

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.

During Drosophila development, the formin actin nucleator Cappuccino (Capu) helps build a cytoplasmic actin mesh throughout the oocyte. Loss of Capu leads to female sterility, presumably because polarity determinants fail to localize properly in the absence of the mesh. To gain deeper insight into how Capu builds this actin mesh, we systematically characterized seven capu alleles, which have missense mutations in Capu's formin homology 2 (FH2) domain. We report that all seven alleles have deleterious effects on fly fertility and the actin mesh in vivo but have strikingly different effects on Capu's biochemical activity in vitro. Using a combination of bulk and single- filament actin-assembly assays, we find that the alleles differentially affect Capu's ability to nucleate and processively elongate actin filaments. We also identify a unique "loop" in the lasso region of Capu's FH2 domain. Removing this loop enhances Capu's nucleation, elongation, and F-actin-bundling activities in vitro. Together our results on the loop and the seven missense mutations provides mechanistic insight into formin function in general and Capu's role in the Drosophila oocyte in particular.

The Drosophila formin Cappuccino (Capu) creates an actin mesh-like structure that traverses the oocyte during midoogenesis. This mesh is thought to prevent premature onset of fast cytoplasmic streaming which normally happens during late-oogenesis. Proper cytoskeletal organization and cytoplasmic streaming are crucial for localization of polarity determinants such as osk, grk, bcd, and nanos mRNAs. Capu mutants disrupt these events, leading to female sterility. Capu is regulated by another nucleator, Spire, as well as by autoinhibition in vitro. Studies in vivo confirm that Spire modulates Capu's function in oocytes; however, how autoinhibition contributes is still unclear. To study the role of autoinhibition in flies, we expressed a Capu construct that is missing the Capu Inhibitory Domain, CapuΔN. Consistent with a gain of activity due to loss of autoinhibition, the actin mesh was denser in CapuΔN oocytes. Further, cytoplasmic streaming was delayed and fertility levels decreased. Localization of osk mRNA in early stages, and bcd and nanos in late stages, were disrupted in CapuΔN-expressing oocytes. Finally, evidence that these phenotypes were due to a loss of autoinhibition comes from coexpression of the N-terminal half of Capu with CapuΔN, which suppressed the defects in actin, cytoplasmic streaming and fertility. From these results, we conclude that Capu can be autoinhibited during Drosophila oocyte development.

Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.

Formins are a conserved family of proteins known to enhance actin polymerization. Most formins are regulated by an intramolecular interaction. The Drosophila formin, Cappuccino (Capu), was believed to be an exception. Capu does not contain conserved autoinhibitory domains and can be regulated by a second protein, Spire. We report here that Capu is, in fact, autoinhibited. The N-terminal half of Capu (Capu-NT) potently inhibits nucleation and binding to the barbed end of elongating filaments by the C-terminal half of Capu (Capu-CT). Hydrodynamic analysis indicates that Capu-NT is a dimer, similar to the N-termini of other formins. These data, combined with those from circular dichroism, suggest, however, that it is structurally distinct from previously described formin inhibitory domains. Finally, we find that Capu-NT binds to a site within Capu-CT that overlaps with the Spire-binding site, the Capu-tail. We propose models for the interaction between Spire and Capu in light of the fact that Capu can be regulated by autoinhibition.

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. [ABSTRACT FROM AUTHOR]

Saccharibacteria are a group of widespread and genetically diverse ultrasmall bacteria with highly reduced genomes that belong to the Candidate Phyla Radiation. Comparative genomic analyses suggest convergent evolution of key functions enabling the adaptation of environmental Saccharibacteria to mammalian microbiomes. Currently, our understanding of this environment-to-mammal niche transition within Saccharibacteria and their obligate episymbiotic association with host bacteria is limited. Here, we identified a complete arginine deiminase system (ADS), found in further genome streamlined mammal-associated Saccharibacteria but missing in their environmental counterparts, suggesting acquisition during environment-to-mammal niche transition. Using TM7x, the first cultured Saccharibacteria strain from the human oral microbiome and its host bacterium Actinomyces odontolyticus, we experimentally tested the function and impact of the ADS. We demonstrated that by catabolizing arginine and generating adenosine triphosphate, the ADS allows metabolically restrained TM7x to maintain higher viability and infectivity when disassociated from the host bacterium. Furthermore, the ADS protects TM7x and its host bacterium from acid stress, a condition frequently encountered within the human oral cavity due to bacterial metabolism of dietary carbohydrates. Intriguingly, with a restricted host range, TM7x forms obligate associations with Actinomyces spp. lacking the ADS but not those carrying the ADS, suggesting the acquired ADS may also contribute to partner selection for cooperative episymbiosis within a mammalian microbiome. These data present experimental characterization of a mutualistic interaction between TM7x and their host bacteria, and illustrate the benefits of acquiring a novel pathway in the transition of Saccharibacteria to mammalian microbiomes. [ABSTRACT FROM AUTHOR]

Developing a laboratory model of oral polymicrobial communities is essential for in vitro studies of the transition from healthy to diseased oral plaque. SHI medium is an enriched growth medium capable of supporting in vitro biofilms with similar diversity to healthy supragingival inocula; however, this medium does not maintain the diversity of gram‐negative bacteria more associated with subgingival plaque. Here, we systematically modified SHI medium components to investigate the impacts of varying nutrients and develop a medium capable of supporting a specific disease‐state subgingival community. A diseased subgingival plaque sample was inoculated in SHI medium with increasing concentrations of sucrose (0%, 0.1%, 0.5%), fetal bovine serum (FBS) (0%, 10%, 20%, 30%, 50%), and mucin (0.1, 2.5, 8.0 g/L) and grown for 48 hrs, then the 16S rRNA profiles of the resulting biofilms were examined. In total, these conditions were able to capture 89 of the 119 species and 43 of the 51 genera found in the subgingival inoculum. Interestingly, biofilms grown in high sucrose media, although dominated by acidogenic Firmicutes with a low final pH, contained several uncultured taxa from the genus Treponema, information that may aid culturing these periodontitis‐associated fastidious organisms. Biofilms grown in a modified medium (here named subSHI‐v1 medium) with 0.1% sucrose and 10% FBS had a high diversity closest to the inoculum and maintained greater proportions of many gram‐negative species of interest from the subgingival periodontal pocket (including members of the genera Prevotella and Treponema, and the Candidate Phyla Radiation phylum Saccharibacteria), and therefore best represented the disease community. [ABSTRACT FROM AUTHOR]

Around one-quarter of bacterial diversity comprises a single radiation with reduced genomes, known collectively as the Candidate Phyla Radiation. Recently, we coisolated TM7x, an ultrasmall strain of the Candidate Phyla Radiation phylum Saccharibacteria, with its bacterial host Actinomyces odontolyticus strain XH001 from human oral cavity and stably maintained as a coculture. Our current work demonstrates that within the coculture, TM7x cells establish a longterm parasitic association with host cells by infecting only a subset of the population, which stay viable yet exhibit severely inhibited cell division. In contrast, exposure of a naïve A. odontolyticus isolate, XH001n, to TM7x cells leads to high numbers of TM7x cells binding to each host cell, massive host cell death, and a host population crash. However, further passaging reveals that XH001n becomes less susceptible to TM7x over time and enters a long-term stable relationship similar to that of XH001. We show that this reduced susceptibility is driven by rapid host evolution that, in contrast to many forms of phage resistance, offers only partial protection. The result is a stalemate where infected hosts cannot shed their parasites; nevertheless, parasite load is sufficiently low that the host population persists. Finally, we show that TM7x can infect and form stable longterm relationships with other species in a single clade of Actinomyces, displaying a narrow host range. This system serves as a model to understand how parasitic bacteria with reduced genomes such as those of the Candidate Phyla Radiation have persisted with their hosts and ultimately expanded in their diversity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Oral Saccharibacteria Nanosynbacter lyticus strain TM7× lives as an ultrasmall epibiont on the surface of its host, Schaalia odontolytica strain XH001. Establishing this interaction is a poorly understood multi-step process. The recovery phase marks a shift in the TM7×/host interaction, switching from the early killing phase, with extensive host cell death, to a stable symbiosis phase where the host and epibiont can grow together., Results: Transcriptomes of TM7× and host, XH001, were captured during the recovery phase and compared to uninfected host and the early host/epibiont interaction (initial encounter). XH001 showed increased expression for rhamnose cell wall components and for the precursor to peptidoglycan while TM7× showed increases in the peptidoglycan pathway. Transporter expression was generally increased for both organisms during recovery compared to the initial encounter, though, XH001 showed lower amino acid transporter expression. Consistent with host parasitism, XH001 showed increased expression of various stress-related genes during recovery while TM7× showed reduced stress. TM7× displayed higher expression of type IV pili, consistent with increased attachment to new hosts., Conclusion: As TM7× is a member of the broadly distributed Candidate Phyla Radiation with small genomes lacking numerous biosynthetic pathways, this study provides further insights into how these epibionts interact and modulate their host bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)