Episymbiotic Saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence.

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.

Acquisition of the arginine deiminase system benefits epiparasitic Saccharibacteria and their host bacteria in a mammalian niche environment.

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.

The short-term impact of comprehensive caries treatment on the supragingival microbiome of severe early childhood caries.

BACKGROUND: Children affected by severe early childhood caries (S-ECC) usually need comprehensive caries treatment due to the extensive of caries. How the oral microbiome changes after caries therapy within the short-term warrant further study. AIM: This study aimed to investigate the short-term impact of comprehensive caries treatment on the supragingival plaque microbiome of S-ECC children. DESIGN: Thirty-three children aged 2-4 years with severe caries (dt > 7) were recruited. Comprehensive caries treatment was performed under general anesthesia in one session and included restoration, pulp treatment, extraction, and fluoride application. Supragingival plaque was sampled pre- and 1-month posttreatment. The genomic DNA of the supragingival plaque was extracted, and bacterial 16S ribosomal RNA gene sequencing was performed. RESULTS: Our data showed that the microbial community evenness significantly decreased posttreatment. Furthermore, comprehensive caries treatment led to more diverse microbial structures among the subjects. The interbacterial interactions reflected by the microbial community's co-occurrence network tended to be less complex posttreatment. Caries treatment increased the relative abundance of Corynebacterium matruchotii, Corynebacterium durum, Actinomyces naeslundii, and Saccharibacteria HMT-347, as well as Aggregatibacter HMT-458 and Haemophilus influenzae. Meanwhile, the relative abundance of Streptococcus mutans, three species from Leptotrichia, Neisseria bacilliformis, and Provotella pallens significantly decreased posttreatment. CONCLUSION: Our results suggested that comprehensive caries treatment may contribute to the reconstruction of a healthier supragingival microbiome.

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.

Coordinated modulation of long non-coding RNA ASBEL and curcumin co-delivery through multicomponent nanocomplexes for synchronous triple-negative breast cancer theranostics.

BACKGROUND: Abnormally regulated long non-coding RNAs (lncRNAs) functions in cancer emphasize their potential to serve as potential targets for cancer therapeutic intervention. LncRNA ASBEL has been identified as oncogene and an anti-sense transcript of tumor-suppressor gene of BTG3 in triple-negative breast cancer (TNBC). RESULTS: Herein, multicomponent self-assembled polyelectrolyte nanocomplexes (CANPs) based on the polyelectrolytes of bioactive hyaluronic acid (HA) and chitosan hydrochloride (CS) were designed and prepared for the collaborative modulation of oncogenic lncRNA ASBEL with antago3, an oligonucleotide antagonist targeting lncRNA ASBEL and hydrophobic curcumin (Cur) co-delivery for synergetic TNBC therapy. Antago3 and Cur co-incorporated CANPs were achieved via a one-step assembling strategy with the cooperation of noncovalent electrostatic interactions, hydrogen-bonding, and hydrophobic interactions. Moreover, the multicomponent assembled CANPs were ulteriorly decorated with a near-infrared fluorescence (NIRF) Cy-5.5 dye (FCANPs) for synchronous NIRF imaging and therapy monitoring performance. Resultantly, MDA-MB-231 cells proliferation, migration, and invasion were efficiently inhibited, and the highest apoptosis ratio was induced by FCANPs with coordination patterns. At the molecular level, effective regulation of lncRNA ASBEL/BTG3 and synchronous regulation of Bcl-2 and c-Met pathways could be observed. CONCLUSION: As expected, systemic administration of FCANPs resulted in targeted and preferential accumulation of near-infrared fluorescence signal and Cur in the tumor tissue. More attractively, systemic FCANPs-mediated collaborative modulating lncRNA ASBEL/BTG3 and Cur co-delivery significantly suppressed the MDA-MB-231 xenograft tumor growth, inhibited metastasis and extended survival rate with negligible systemic toxicity. Our present study represented an effective approach to developing a promising theranostic platform for combating TNBC in a combined therapy pattern.

CD16 CAR-T cells enhance antitumor activity of CpG ODN-loaded nanoparticle-adjuvanted tumor antigen-derived vaccinevia ADCC approach.

BACKGROUND: Combinatorial immunotherapy strategies for enhancing the responsiveness of immune system have shown great promise for cancer therapy. Engineered nanoformulation incorporated toll-like receptor (TLR) 9 agonist CpG ODN has shown more positive results in suppressing tumor growth and can significantly enhance other immunotherapy activity with combinatorial effects due to the innate and adaptive immunostimulatory effects of CpG. RESULTS: In the present work, protamine sulfate (PS) and carboxymethyl ß-glucan (CMG) were used as nanomaterials to form nanoparticles through a self-assembly approach for CpG ODN encapsulation to generate CpG ODN-loaded nano-adjuvant (CNPs), which was subsequently mixed with the mixture of mouse melanoma-derived antigens of tumor cell lysates (TCL) and neoantigens to develop vaccine for anti-tumor immunotherapy. The obtained results showed that CNPs was able to effectively deliver CpG ODN into murine bone marrow-derived dendritic cells (DC) in vitro, and remarkably stimulate the maturation of DC cells with proinflammatory cytokine secretion. In addition, in vivo analysis showed that CNPs enhanced anti-tumor activity of PD1 antibody and CNPs-adjuvanted vaccine based on the mixture antigens of melanoma TCL and melanoma-specific neoantigen could not only induce anti-melanoma cellular immune responses, but also elicit melanoma specific humoral immune responses, which significantly inhibited xenograft tumor growth. Furthermore, CD16 CAR-T cells were generated by expressing CD16-CAR in CD3+CD8+ murine T cells. CONCLUSION: Our results eventually showed that anti-melanoma antibodies induced by CNPs-adjuvanted TCL vaccines were able to collaborate with CD16-CAR-T cells to generate an enhanced targeted anti-tumor effects through ADCC (antibody dependent cell cytotoxicity) approach. CD16 CAR-T cells has thus a great potential to be an universal promising strategy targeting on solid tumor synergistic immunotherapy via co-operation with TCL-based vaccine.

Suicide in China: Community Attitudes and Stigma.

China accounts for an estimated third of the world's suicides, yet individuals experiencing suicidality typically do not seek out or receive treatment. This study examines community perceptions and public stigma toward suicide. In Shanghai, China 186 adults were recruited to participate in a survey with an experimental vignette describing a suicidal individual, manipulated on gender and age, followed by questions eliciting attitudes toward suicide. Most participants agreed that the suicidal subject had a serious problem, with seriousness of the problem decreasing with participant's age. Participants reported moderate levels of public stigma. More stigma was found toward adolescent subjects rather than adult. Male subjects were perceived as being more likely to change than females. The public's accurate view of suicide without biases could help prevent suicide from getting worse. Public perceptions regarding a suicidal individual's likelihood to change could lead to stigma reduction, which can subsequently help with effective crisis intervention.

Transcriptome of Epibiont Saccharibacteria Nanosynbacter lyticus Strain TM7x During the Establishment of Symbiosis.

Saccharibacteria Nanosynbacter lyticus strain TM7x is a member of the broadly distributed candidate phylum radiation. These bacteria have ultrasmall cell sizes, have reduced genomes, and live as epibionts on the surfaces of other bacteria. The mechanisms by which they establish and maintain this relationship are not yet fully understood. The transcriptomes of the epibiont TM7x and its host bacteria Schaalia odontolytica strain XH001 were captured across the establishment of symbiosis during both the initial interaction and stable symbiosis. The results showed a dynamic interaction with large shifts in gene expression for both species between the initial encounter and stable symbiosis, notably in transporter genes. During stable symbiosis, the host XH001 showed higher gene expression for peptidoglycan biosynthesis, mannosylation, cell cycle and stress-related genes, whereas it showed lower expression of chromosomal partitioning genes. This was consistent with the elongated cell shape seen in XH001 infected with TM7x and our discovery that infection resulted in thickened cell walls. Within TM7x, increased pili, type IV effector genes, and arginine catabolism/biosynthesis gene expression during stable symbiosis implied a key role for these functions in the interaction. Consistent with its survival and persistence in the human microbiome as an obligate epibiont with reduced de novo biosynthetic capacities, TM7x also showed higher levels of energy production and peptidoglycan biosynthesis, but lower expression of stress-related genes, during stable symbiosis. These results imply that TM7x and its host bacteria keep a delicate balance in order to sustain an episymbiotic lifestyle. IMPORTANCE Nanosynbacter lyticus type strain TM7x is the first cultivated member of the Saccharibacteria and the candidate phyla radiation (CPR). It was discovered to be ultrasmall in cell size with a highly reduced genome that establishes an obligate epibiotic relationship with its host bacterium. The CPR is a large, monophyletic radiation of bacteria with reduced genomes that includes Saccharibacteria. The vast majority of the CPR have yet to be cultivated, and our insights into these unique organisms to date have been derived from only a few Saccharibacteria species. Being obligate parasites, it is unknown how these ultrasmall Saccharibacteria, which are missing many de novo biosynthetic pathways, are maintained at a high prevalence within the human microbiome as well as in the environment.

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.

Mental health help-seeking in China.

BACKGROUND: In China, mental health disorders are considered the leading causes of disability, yet treatment-seeking behaviors among individuals with mental health problems are deficient. AIMS: This study sought to examine attitudes and participant characteristics associated with help-seeking among adults residing in China's Shanghai metropolitan area. METHODS: This study employed a convenience cross-sectional sampling strategy and recruited 500 participants in public places in Shanghai. The survey administered in Mandarin was comprised of two sections: a series of demographic questions and standardized instruments measuring stigma and help-seeking attitudes. RESULTS: Findings indicate that beliefs about seeking professional help for mental health are influenced by knowing someone with a mental health problem. In addition, men who were older, had a child, and were married endorsed more openness to help-seeking for mental health needs, underscoring the importance of life experience as an essential variable when considering attitudes toward help-seeking. CONCLUSIONS: Findings support future research identifying the mechanisms by which these life experiences impact individuals' help-seeking attitudes.

Rapid evolution of decreased host susceptibility drives a stable relationship between ultrasmall parasite TM7x and its bacterial host.

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 long-term 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 long-term 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.

Human mesenchymal stem cells-derived conditioned medium inhibits hypoxia-induced death of neonatal porcine islets by inducing autophagy.

BACKGROUND: The dysfunction of islet grafts is generally attributed to hypoxia-induced damage. Mesenchymal stem cells (MSCs) are currently thought to effectively protect cells from various risk factors via regulating autophagy. In our study, we investigated if human umbilical cord-derived MSCs could ameliorate hypoxia-induced apoptosis in porcine islets by modulating autophagy, and we explored the underlying mechanisms. METHODS: Neonatal porcine islet cell clusters (NICCs) were cultured with human umbilical cord-derived MSC conditioned medium (huc-MSC-CM) and RPMI-1640 medium (control) under hypoxic conditions (1% O2 ) in vitro. NICCs were treated with 3-methyladenine (3-MA) and chloroquine (CQ) to examine the role of huc-MSC-CM in regulating autophagy. Finally, the levels of several cytokines secreted by huc-MSCs were detected by ELISAs, and the corresponding inhibitors were applied to investigate which cytokine mediates the protective effects of huc-MSC-CM. The effects of huc-MSC-CM on NICCs viability and autophagy were examined using AO/PI staining, flow cytometry analysis, transmission electron microscopy (TEM) and confocal fluorescence microscopy analysis. The insulin secretion of NICCs was tested with an insulin immunoradiometric assay kit. RESULTS: Compared to the control, the huc-MSC-CM treatment improved the viability of NICCs, inhibited apoptosis, increased autophagic activity and the levels of PI3K class III and phosphorylated Akt, while the ratio of phosphorylated mTOR/mTOR was reduced. These changes were reversed by CQ and 3-MA treatments. High concentrations of IL-6 were detected in hu-MSC-CM. Furthermore, recombinant IL-6 pre-treatment exerted similar effects as huc-MSC-CM, and these effects were reversed by a specific inhibitor of IL-6 (Sarilumab). CONCLUSIONS: Our results demonstrated that huc-MSC-CM improved islet viability and function by increasing autophagy through the PI3K/Akt/mTOR pathway under hypoxic conditions. Additionally, IL-6 plays an important role in the function of huc-MSC-CM.

Pseudomonas aeruginosa MutL promotes large chromosomal deletions through non-homologous end joining to prevent bacteriophage predation.

Pseudomonas aeruginosa is an opportunistic pathogen with a relatively large genome, and has been shown to routinely lose genomic fragments during environmental selection. However, the underlying molecular mechanisms that promote chromosomal deletion are still poorly understood. In a recent study, we showed that by deleting a large chromosomal fragment containing two closely situated genes, hmgA and galU, P. aeruginosa was able to form 'brown mutants', bacteriophage (phage) resistant mutants with a brown color phenotype. In this study, we show that the brown mutants occur at a frequency of 227 ± 87 × 10-8 and contain a deletion ranging from ∼200 to ∼620 kb. By screening P. aeruginosa transposon mutants, we identified mutL gene whose mutation constrained the emergence of phage-resistant brown mutants. Moreover, the P. aeruginosa MutL (PaMutL) nicking activity can result in DNA double strand break (DSB), which is then repaired by non-homologous end joining (NHEJ), leading to chromosomal deletions. Thus, we reported a noncanonical function of PaMutL that promotes chromosomal deletions through NHEJ to prevent phage predation.

An integrative model of internalized stigma and recovery-related outcomes among people diagnosed with schizophrenia in rural China.

PURPOSE: Internalized stigma, an adverse psychological process, severely impedes the lives of people diagnosed with schizophrenia and restricts them from social integration and recovery. The aim of this study was to empirically evaluate an integrative model of relationship between internalized stigma and patients' recovery-related outcomes among people diagnosed with schizophrenia in a rural Chinese community. METHOD: A total of 232 people diagnosed with schizophrenia in Xinjin, Chengdu, participated in this study and completed measures of internalized stigma, social interaction, perceived social support, social functioning, and symptoms. The internalized stigma of mental illness scale (ISMI) was used to measure the internalized stigma. Path analysis was used to test the association between internalized stigma and recovery-related outcomes. RESULTS: There were no significant differences in mean scores of ISMI by gender, age (18-64 years and ≥ 65 years), education, marital status, or economic capacity. Internalized stigma was negatively associated with perceived social support and social interaction. Furthermore, higher level of internalized stigma was associated with impaired social functioning, and a lower level of social functioning was significantly associated with more severe symptoms. CONCLUSION: Internalized stigma is associated with poor social interaction and weakened perceived social support in people diagnosed with schizophrenia, and is linked negatively to outcomes in their recovery. It is essential to tailor interventions related to reducing internalized stigma within a Chinese context and evaluate the effectiveness of anti-stigma intervention on recovery for people diagnosed with schizophrenia.

Influenza Virus Affects Intestinal Microbiota and Secondary Salmonella Infection in the Gut through Type I Interferons.

Human influenza viruses replicate almost exclusively in the respiratory tract, yet infected individuals may also develop gastrointestinal symptoms, such as vomiting and diarrhea. However, the molecular mechanisms remain incompletely defined. Using an influenza mouse model, we found that influenza pulmonary infection can significantly alter the intestinal microbiota profile through a mechanism dependent on type I interferons (IFN-Is). Notably, influenza-induced IFN-Is produced in the lungs promote the depletion of obligate anaerobic bacteria and the enrichment of Proteobacteria in the gut, leading to a "dysbiotic" microenvironment. Additionally, we provide evidence that IFN-Is induced in the lungs during influenza pulmonary infection inhibit the antimicrobial and inflammatory responses in the gut during Salmonella-induced colitis, further enhancing Salmonella intestinal colonization and systemic dissemination. Thus, our studies demonstrate a systemic role for IFN-Is in regulating the host immune response in the gut during Salmonella-induced colitis and in altering the intestinal microbial balance after influenza infection.

A Linear Plasmid-Like Prophage of Actinomyces odontolyticus Promotes Biofilm Assembly.

The human oral cavity is home to a large number of bacteria and bacteriophages (phages). However, the biology of oral phages as members of the human microbiome is not well understood. Recently, we isolated Actinomyces odontolyticus subsp. actinosynbacter strain XH001 from the human oral cavity, and genomic analysis revealed the presence of an intact prophage named xhp1. Here, we demonstrated that xhp1 is a linear plasmid-like prophage, which is a newly identified phage of A. odontolyticus The prophage xhp1 genome is a 35-kb linear double-stranded DNA with 10-bp single-stranded, 3' cohesive ends. xhp1 exists extrachromosomally, with an estimated copy number of 5. Annotation of xhp1 revealed 54 open reading frames, while phylogenetic analysis suggests that it has limited similarity with other phages. xhp1 phage particles can be induced by mitomycin C and belong to the Siphoviridae family, according to transmission electron microscopic examination. The released xhp1 particles can reinfect the xhp1-cured XH001 strain and result in tiny blurry plaques. Moreover, xhp1 promotes XH001 biofilm formation through spontaneous induction and the release of host extracellular DNA (eDNA). In conclusion, we identified a linear plasmid-like prophage of A. odontolyticus, which enhances bacterial host biofilm assembly and could be beneficial to the host for its persistence in the oral cavity.IMPORTANCE The biology of phages as members of the human oral microbiome is understudied. Here, we report the characterization of xhp1, a novel linear plasmid-like prophage identified from a human oral isolate, Actinomyces odontolyticus subsp. actinosynbacter strain XH001. xhp1 can be induced and reinfect xhp1-cured XH001. The spontaneous induction of xhp1 leads to the lysis of a subpopulation of bacterial hosts and the release of eDNA that promotes biofilm assembly, thus potentially contributing to the persistence of A. odontolyticus within the oral cavity.

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.

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.