Xuesong He

Interspecies Interactions within Oral Microbial Communities

SUMMARY While reductionism has greatly advanced microbiology in the past 400 years, assembly of smaller pieces just could not explain the whole! Modern microbiologists are learning “system thinking” and “holism.” Such an approach is changing our understanding of microbial physiology and our ability to diagnose/treat microbial infections. This review uses oral microbial communities as a focal point to describe this new trend. With the common name “dental plaque,” oral microbial communities are some of the most complex microbial floras in the human body, consisting of more than 700 different bacterial species. For a very long time, oral microbiologists endeavored to use reductionism to identify the key genes or key pathogens responsible for oral microbial pathogenesis. The limitations of reductionism forced scientists to begin adopting new strategies using emerging concepts such as interspecies interaction, microbial community, biofilms, polymicrobial disease, etc. These new research directions indicate that the whole is much more than the simple sum of its parts, since the interactions between different parts resulted in many new physiological functions which cannot be observed with individual components. This review describes some of these interesting interspecies-interaction scenarios.

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

Significance TM7 is one of the most enigmatic bacterial phyla among the uncultivated candidate phyla referred to as “microbial dark matter,” and it has potential pathogenic associations. We revealed molecular insights into its uncultivability and pathogenicity, as well its unique epibiotic and parasitic lifestyle phases. These novel discoveries shed significant light on the biological, ecological, and medical importance of TM7, as well as providing useful information for culturing other TM7 and currently uncultivable bacteria that may evade standard cultivation approaches. 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.

Using DGGE profiling to develop a novel culture medium suitable for oral microbial communities

More than 700 bacterial species have been detected in the human oral cavity. They form highly organized microbial communities and are responsible for many oral infectious diseases, such as dental caries and periodontal disease. The prevention and treatment of these diseases require a comprehensive knowledge of oral microbial communities, which largely relies on culture-dependent methods to provide detailed phenotypic and physiological analysis of these communities. However, most of the currently available laboratory media can only selectively support the growth of a limited number of bacterial species within these communities, and fail to sustain the original oral microbial diversity. In this study, using denaturing gradient gel electrophoresis (DGGE) as an index to systematically survey and analyse the selectivity of commonly used laboratory media, we developed a new medium (SHI medium) by combining the ingredients of several selected media that can support different subpopulations within the original oral microbial community derived from pooled saliva. DGGE and 454 pyrosequencing analysis showed that SHI medium was capable of supporting a more diversified community with a microbial profile closer to that of the original oral microbiota. Furthermore, 454 pyrosequencing revealed that SHI medium supported the growth of many oral species that have not before been cultured. Crystal violet assay and the confocal laser scanning microscope analysis indicated that, compared with other media, SHI medium is able to support a more complex saliva-derived biofilm with higher biomass yield and more diverse species. This DGGE-guided method could also be used to develop novel media for other complex microbial communities.

An in vitro biofilm model system maintaining a highly reproducible species and metabolic diversity approaching that of the human oral microbiome.

BackgroundOur knowledge of microbial diversity in the human oral cavity has vastly expanded during the last two decades of research. However, much of what is known about the behavior of oral species to date derives from pure culture approaches and the studies combining several cultivated species, which likely does not fully reflect their function in complex microbial communities. It has been shown in studies with a limited number of cultivated species that early oral biofilm development occurs in a successional manner and that continuous low pH can lead to an enrichment of aciduric species. Observations that in vitro grown plaque biofilm microcosms can maintain similar pH profiles in response to carbohydrate addition as plaque in vivo suggests a complex microbial community can be established in the laboratory. In light of this, our primary goal was to develop a robust in vitro biofilm-model system from a pooled saliva inoculum in order to study the stability, reproducibility, and development of the oral microbiome, and its dynamic response to environmental changes from the community to the molecular level.ResultsComparative metagenomic analyses confirmed a high similarity of metabolic potential in biofilms to recently available oral metagenomes from healthy subjects as part of the Human Microbiome Project. A time-series metagenomic analysis of the taxonomic community composition in biofilms revealed that the proportions of major species at 3 hours of growth are maintained during 48 hours of biofilm development. By employing deep pyrosequencing of the 16S rRNA gene to investigate this biofilm model with regards to bacterial taxonomic diversity, we show a high reproducibility of the taxonomic carriage and proportions between: 1) individual biofilm samples; 2) biofilm batches grown at different dates; 3) DNA extraction techniques and 4) research laboratories.ConclusionsOur study demonstrates that we now have the capability to grow stable oral microbial in vitro biofilms containing more than one hundred operational taxonomic units (OTU) which represent 60-80% of the original inoculum OTU richness. Previously uncultivated Human Oral Taxa (HOT) were identified in the biofilms and contributed to approximately one-third of the totally captured 16S rRNA gene diversity. To our knowledge, this represents the highest oral bacterial diversity reported for an in vitro model system so far. This robust model will help investigate currently uncultivated species and the known virulence properties for many oral pathogens not solely restricted to pure culture systems, but within multi-species biofilms.

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

Significance The lack of effective tools to modulate individual species within a complex microbial community poses a major barrier to studying human microbiome and its associated diseases. We showed targeted removal of human cariogenic Streptococcus mutans within an in vitro oral multispecies community using a high-efficacy antimicrobial peptide—C16G2—as well as drastic reconstruction of the microbial structure following treatment. The findings suggest the potential of using targeted antimicrobials to modulate microbiome and study the ecological function of specific bacterial species. 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.

Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of Pseudomonas aeruginosa bacteriophages PaP1 and JG004.

The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.

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.

Achieving Probiotic Effects via Modulating Oral Microbial Ecology

Unlike many pathogens are foreign invaders, oral “pathogens” such as Streptococcus mutans are part of the “normal” oral microbial flora. While they express certain pathogenic properties, the balance of synergistic and antagonistic interactions determines whether these commensal pathogens cause damage or not. Recognition of these microbial ecology based pathogeneses argues for new strategies for disease treatment and prevention. Probiotics, potentially beneficial live bacteria or yeasts, have been used to combat dental caries. This includes the application of S. mutans types that cannot produce acids or other bacteria that interfere with the pathogenic effects of S. mutans. While these approaches show therapeutic effects against S. mutans experimentally, the conversion into commercial products remains a challenge, due to safety and shelf life issues. New high-tech approaches, such as quorum sensing interference of pathogenic bacteria or targeted antimicrobial therapies, offer novel ways to achieve probiotic effects against dental caries.

Chromosomal DNA deletion confers phage resistance to Pseudomonas aeruginosa

Bacteria develop a broad range of phage resistance mechanisms, such as prevention of phage adsorption and CRISPR/Cas system, to survive phage predation. In this study, Pseudomonas aeruginosa PA1 strain was infected with lytic phage PaP1, and phage-resistant mutants were selected. A high percentage (~30%) of these mutants displayed red pigmentation phenotype (Red mutant). Through comparative genomic analysis, one Red mutant PA1r was found to have a 219.6 kb genomic fragment deletion, which contains two key genes hmgA and galU related to the observed phenotypes. Deletion of hmgA resulted in the accumulation of a red compound homogentisic acid; while A galU mutant is devoid of O-antigen, which is required for phage adsorption. Intriguingly, while the loss of galU conferred phage resistance, it significantly attenuated PA1r in a mouse infection experiment. Our study revealed a novel phage resistance mechanism via chromosomal DNA deletion in P. aeruginosa.

Intercellular communications in multispecies oral microbial communities

The oral cavity contains more than 700 microbial species that are engaged in extensive cell–cell interactions. These interactions contribute to the formation of highly structured multispecies communities, allow them to perform physiological functions, and induce synergistic pathogenesis. Co-adhesion between oral microbial species influences their colonization of oral cavity and effectuates, to a large extent, the temporal and spatial formation of highly organized polymicrobial community architecture. Individual species also compete and collaborate with other neighboring species through metabolic interactions, which not only modify the local microenvironment such as pH and the amount of oxygen, making it more suitable for the growth of other species, but also provide a metabolic framework for the participating microorganisms by maximizing their potential to extract energy from limited substrates. Direct physical contact of bacterial species with its neighboring co-habitants within microbial community could initiate signaling cascade and achieve modulation of gene expression in accordance with different species it is in contact with. In addition to communication through cell–cell contact, quorum sensing (QS) mediated by small signaling molecules such as competence-stimulating peptides (CSPs) and autoinducer-2 (AI-2), plays essential roles in bacterial physiology and ecology. This review will summarize the evidence that oral microbes participate in intercellular communications with co-inhabitants through cell contact-dependent physical interactions, metabolic interdependencies, as well as coordinative signaling systems to establish and maintain balanced microbial communities.