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Gnotobiotic animal models reconventionalized under controlled laboratory conditions with multi-species bacterial communities are commonly used to study host-microbiota interactions under presumably more reproducible conditions than conventional animals. The usefulness of these models is however limited by inter-animal variability in bacterial colonization and our general lack of understanding of the inter-individual fluctuation and spatio-temporal dynamics of microbiota assemblies at the micron to millimeter scale. Here, we show underreported variability in gnotobiotic models by analyzing differences in gut colonization efficiency, bacterial composition, and host intestinal mucus production between conventional and gnotobiotic zebrafish larvae re-conventionalized with a mix of 9 bacteria isolated from conventional microbiota. Despite similar bacterial community composition, we observed high variability in the spatial distribution of bacteria along the intestinal tract in the reconventionalized model. We also observed that, whereas bacteria abundance and intestinal mucus per fish were not correlated, reconventionalized fish had lower intestinal mucus compared to conventional animals, indicating that the stimulation of mucus production depends on the microbiota composition. Our findings, therefore, suggest that variable colonization phenotypes affect host physiology and impact the reproducibility of experimental outcomes in studies that use gnotobiotic animals. This work provides insights into the heterogeneity of gnotobiotic models and the need to accurately assess re-conventionalization for reproducibility in host-microbiota studies.

SummaryGerm cells differentiate into oocytes that become totipotent upon fertilization. How the highly specialized oocyte acquires this distinct cell fate is poorly understood. During Drosophila oogenesis, H3K9me3 histone methyltransferase SETDB1 translocates from the cytoplasm to the nucleus of germ cells concurrent with oocyte specification. Here, we discovered that nuclear SETDB1 is required to silence a cohort of differentiation-promoting genes by mediating their heterochromatinization. Intriguingly, SETDB1 is also required for the upregulation of 18 of the ~30 nucleoporins (Nups) that comprise the nucleopore complex (NPC). NPCs in turn anchor SETDB1-dependent heterochromatin at the nuclear periphery to maintain H3K9me3 and gene silencing in the egg chambers. Aberrant gene expression due to loss of SETDB1 or Nups results in loss of oocyte identity, cell death and sterility. Thus, a feedback loop between heterochromatin and NPCs promotes transcriptional reprogramming at the onset of oocyte specification that is critical to establish oocyte identity.

The human oral cavity harbors diverse communities of microbes that live as biofilms: highly ordered, surface-associated assemblages of microbes embedded in an extracellular matrix. Oral microbial communities contribute to human health by fine-tuning immune responses and reducing dietary nitrate. Dental caries and periodontal disease are together the most prevalent microbially-mediated human diseases, worldwide. Both of these oral diseases are known to be caused not by the introduction of exogenous pathogens to the oral environment, but rather by a homeostasis breakdown that leads to changes in the structure of the microbial communities present in states of health. Both dental caries and periodontal disease are mediated by synergistic interactions within communities and both diseases are further driven by specific host inputs: diet and behavior in the case of dental caries and immune system interactions in the case of periodontal disease. Changes in community structure (taxonomic identity and abundance) are well documented during the transition from health to disease. In this review, changes in biofilm physical structure during the transition from oral health to disease and the concomitant relationship between structure and community function will be emphasized.

Ecologists have long recognized the importance of spatial scale in understanding structure-function relationships among communities of organisms within their environment. Here, we review historical and contemporary studies of dental plaque community structure in the context of three distinct scales: the micro (1-10 µm), meso (10-100 µm) and macroscale (100 µm to ≥1 cm). Within this framework, we analyze the compositional nature of dental plaque at the macroscale, the molecular interactions of microbes at the microscale, and the emergent properties of dental plaque biofilms at the mesoscale. Throughout our analysis of dental plaque across spatial scales, we draw attention to disease and health-associated structure-function relationships and include a discussion of host immune involvement in the mesoscale structure of periodontal disease-associated biofilms. We end with a discussion of two filamentous organisms, Fusobacterium nucleatum and Corynebacterium matruchotii, and their relevant contributions in structuring dental plaque biofilms.

Candida albicans as an opportunistic pathogen exploits the host immune system and causes a variety of life-threatening infections. The polymorphic nature of this fungus gives it tremendous advantage to breach mucosal barriers and cause oral and disseminated infections. Similar to C. albicans, Enterococcus faecalis is a major opportunistic pathogen, which is of critical concern in immunocompromised patients. There is increasing evidence that E. faecalis co-exists with C. albicans in the human body in disease samples. While the interactive profiles between these two organisms have been studied on abiotic substrates and mouse models, studies on their interactions on human oral mucosal surfaces are non-existent. Here, for the first time, we comprehensively characterized the interactive profiles between laboratory and clinical isolates of C. albicans (SC5314 and BF1) and E. faecalis (OG1RF and P52S) on an organotypic oral mucosal model. Our results demonstrated that the dual species biofilms resulted in profound surface erosion and significantly increased microbial invasion into mucosal compartments, compared to either species alone. Notably, several genes of C. albicans involved in tissue adhesion, hyphal formation, fungal invasion, and biofilm formation were significantly upregulated in the presence of E. faecalis. By contrast, E. faecalis genes involved in quorum sensing, biofilm formation, virulence, and mammalian cell invasion were downregulated. This study highlights the synergistic cross-kingdom interactions between E. faecalis and C. albicans in mucosal tissue invasion.

Candida albicans as an opportunistic pathogen exploits the host immune system and causes a variety of life-threatening infections. The polymorphic nature of this fungus gives it tremendous advantage to breach mucosal barriers and cause a variety of oral and disseminated infections. Enterococcus faecalis, another opportunistic pathogen co-exists with C. albicans in several niches in the human body, including the oral cavity and gastrointestinal tract. However, interactions between E. faecalis and C. albicans on oral mucosal surfaces remain unknown. Here, for the first time, we comprehensively characterized the interactive profiles between laboratory and clinical isolates of C. albicans (SC5314 and BF1) and E. faecalis (OG1RF and 846) on an organotypic oral mucosal model. Our results demonstrated that the two species formed robust biofilms on the mucosal tissue surface with profound surface erosion and fungal invasion. Specifically, this effect was more pronounced in the laboratory isolates than in the clinical isolates. Notably, several genes of C. albicans involved in tissue adhesion, hyphal formation, fungal invasion, and biofilm formation were significantly upregulated in the presence of E. faecalis. This study highlights the strain-dependent cross-kingdom interactions between E. faecalis and C. albicans on oral mucosa, demonstrating the requisite to study more substrate-dependent polymicrobial interactions.

Eukaryotic cells are organized into membrane-bound organelles. These organelles communicate with one another through vesicular trafficking pathways and membrane contact sites (MCSs). MCSs are sites of close apposition between two or more organelles that play diverse roles in the exchange of metabolites, lipids and proteins. Organelle interactions at MCSs also are important for organelle division and biogenesis. For example, the division of several organelles, including mitochondria and endosomes, seem to be regulated by contacts with the endoplasmic reticulum (ER). Moreover, the biogenesis of autophagosomes and peroxisomes involves contributions from the ER and multiple other cellular compartments. Thus, organelle-organelle interactions allow cells to alter the shape and activities of their membrane-bound compartments, allowing them to cope with different developmental and environmental conditions.

Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort.Overall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma specifically, a separate group of taxa are identified, in which Acidovorax is enriched in smokers. Acidovorax temporans is identified within tumor sections by fluorescent in situ hybridization and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibit higher abundance among the subset of squamous cell carcinoma cases with TP53 mutations, an association not seen in adenocarcinomas.The results of this comprehensive study show both microbiome-gene and microbiome-exposure interactions in squamous cell carcinoma lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium that is higher in relative abundance in smoking-associated tumors of this type. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.

Fluorescent proteins and vital dyes are invaluable tools for studying dynamic processes within living cells. However, the ability to distinguish more than a few different fluorescent reporters in a single sample is limited by the spectral overlap of available fluorophores. Here, we present a protocol for imaging live cells labeled with six fluorophores simultaneously. A confocal microscope with a spectral detector is used to acquire images, and linear unmixing algorithms are applied to identify the fluorophores present in each pixel of the image. We describe the application of this method to visualize the dynamics of six different organelles, and to quantify the contacts between organelles. However, this method can be used to image any molecule amenable to tagging with a fluorescent probe. Thus, multispectral live-cell imaging is a powerful tool for systems-level analysis of cellular organization and dynamics. © 2018 by John Wiley & Sons, Inc.

BackgroundLung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesized that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from controls (n=33) and cancer cases (n=143), we conducted 16S rRNA bacterial gene sequencing, with RNA-seq data from lung cancer cases in The Cancer Genome Atlas (n=1112) serving as the validation cohort.ResultsOverall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma (SCC) specifically, a separate group of taxa were identified, in which Acidovorax was enriched in smokers (P =0.0013). Acidovorax temporans was identified by fluorescent in situ hybridization within tumor sections, and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibited higher abundance among the subset of SCC cases with TP53 mutations, an association not seen in adenocarcinomas (AD).ConclusionsThe results of this comprehensive study show both a microbiome-gene and microbiome-exposure interactions in SCC lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can damage epithelial function, have a unique bacterial consortia which is higher in relative abundance in smoking-associated SCC. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.

Specific interbacterial adhesion, termed coaggregation, is well established for three early colonizers of the plaque biofilm: streptococci, actinomyces, and veillonellae. However, little is known about interactions of other early colonizers and about the extent of interactions within the bacterial community from a single host. To address these gaps, subject-specific culture collections from two individuals were established using an intraoral biofilm retrieval device. Molecular taxonomy (Human Oral Microbe Identification Microarray [HOMIM]) analysis of biofilm samples confirmed the integrity and completeness of the collections. HOMIM analysis verified the isolation of Streptococcus gordonii and S. anginosus from only one subject, as well as isolation of a previously uncultivated streptococcal phylotype from the other subject. Strains representative of clonal diversity within each collection were further characterized. Greater than 70% of these streptococcal strains from each subject coaggregated with at least one other coisolate. One-third of the strains carry a known coaggregation mediator: receptor polysaccharide (RPS). Almost all nonstreptococcal isolates coaggregated with other coisolates. Importantly, certain Rothia strains demonstrated more coaggregations with their coisolated bacteria than did any Streptococcus or Actinomyces strain, and certain Haemophilus isolates participated in twice as many. Confocal microscopy of undisturbed biofilms showed that Rothia and Haemophilus each occur in small multispecies microcolonies. However, in confluent high-biomass regions, Rothia occurred in islands whereas Haemophilus was distributed throughout. Together, the data demonstrate that coaggregation networks within an individual's oral microflora are extensive and that Rothia and Haemophilus can be important initiators of cell-cell interactions in the early biofilm. IMPORTANCE Extensive involvement of specific interbacterial adhesion in dental plaque biofilm formation has been postulated based on in vitro coaggregation between oral bacteria from culture collections that are not subject specific. In the present study, subject-specific culture collections were obtained from early plaque biofilm of two volunteers, and coaggregations within each culture collection were assayed. Coaggregations, several of which involved a coaggregation-mediating cell surface molecule known from well-studied streptococci, were widespread. Unexpectedly, the little-studied organisms Haemophilus and Rothia participated in the greatest numbers of interactions with community members; these two organisms showed different distributions within the undisturbed biofilm. The data show that coaggregation networks encompass most organisms within the biofilm community of each individual, and they indicate prominent participation of organisms such as Haemophilus and Rothia in early plaque biofilm formation.

Although the number of phylotypes present in a microbial community may number in the hundreds or more, until recently, fluorescence in situ hybridization has been used to label, at most, only a handful of different phylotypes in a single sample. We recently developed a technique, CLASI-FISH for combinatorial labeling and spectral imaging – fluorescence in situ hybridization, to greatly expand the number of distinguishable taxa in a single FISH experiment. The CLASI technique involves labeling microbes of interest with combinations of probes coupled with spectral imaging to allow the use of fluorophores with highly overlapping excitation and emission spectra. Here, we present the basic principles and theory of CLASI-FISH along with some guidelines for performing CLASI-FISH experiments. We further include a protocol for creating fluorescence spectral reference standards, a vital component of successful CLASI-FISH.

Using confocal and lattice light sheet microscopy, the authors perform systems-level analysis of the organelle interactome in live cells, allowing them to visualize the frequency and locality of up to five-way interactions between different organelles. Various cell components, or organelles, make contacts that are not mediated by trafficking vesicles, and which result in changes to their physical behaviour, biochemical composition and functionality. Imaging is a powerful tool for studying inter-organelle contact sites, but work by Jennifer Lippincott-Schwartz and colleagues take such analysis to a new level. Using confocal and lattice light sheet microscopy, as well as a multispectral image acquisition and analysis method, they perform systems-level analysis of the organelle interactome in live cells. The approach allows them to visualize the frequency and locality of up to five-way interactions among six different organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet), providing unexpected insights into the dynamics of these interactions. The method could prove a useful tool for further analysis of non-vesicular communication within the cell. The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling1,2,3,4,5. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions5,6,7,8. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image9. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet10 instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics.

Microbes in nature frequently function as members of complex multitaxon communities, but the structural organization of these communities at the micrometer level is poorly understood because of limitations in labeling and imaging technology. We report here a combinatorial labeling strategy coupled with spectral image acquisition and analysis that greatly expands the number of fluorescent signatures distinguishable in a single image. As an imaging proof of principle, we first demonstrated visualization of Escherichia coli labeled by fluorescence in situ hybridization (FISH) with 28 different binary combinations of eight fluorophores. As a biological proof of principle, we then applied this Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH) strategy using genus- and family-specific probes to visualize simultaneously and differentiate 15 different phylotypes in an artificial mixture of laboratory-grown microbes. We then illustrated the utility of our method for the structural analysis of a natural microbial community, namely, human dental plaque, a microbial biofilm. We demonstrate that 15 taxa in the plaque community can be imaged simultaneously and analyzed and that this community was dominated by early colonizers, including species of Streptococcus , Prevotella , Actinomyces , and Veillonella . Proximity analysis was used to determine the frequency of inter- and intrataxon cell-to-cell associations which revealed statistically significant intertaxon pairings. Cells of the genera Prevotella and Actinomyces showed the most interspecies associations, suggesting a central role for these genera in establishing and maintaining biofilm complexity. The results provide an initial systems-level structural analysis of biofilm organization.

Fluorescence in situ hybridization (FISH) combined with spectral analysis was performed to image specific bacteria from seawater using probes targeting the V6 hypervariable region of small subunit ribosomal RNA (SSU rRNA), corresponding to positions 984 to 1047 of E. coli 16S rRNA gene. For each target, we designed two probes, each with a distinct fluorescent reporter and a unique hybridization site. Spectral imaging analysis of bacteria incubated with a pair of specific probes and a general bacterial probe enabled the detection of three probe-conferred spectra in each target cell, increasing the reliability of target identification in a diverse bacterial community.

The number of fluorescent labels that can unambiguously be distinguished in a single image when acquired through band pass filters is severely limited by the spectral overlap of available fluorophores. The recent development of spectral microscopy and the application of linear unmixing algorithms to spectrally recorded image data have allowed simultaneous imaging of fluorophores with highly overlapping spectra. However, the number of distinguishable fluorophores is still limited by the unavoidable decrease in signal to noise ratio when fluorescence signals are fractionated over multiple wavelength bins. Here we present a spectral image analysis algorithm to greatly expand the number of distinguishable objects labeled with binary combinations of fluorophores. Our algorithm utilizes a priori knowledge about labeled specimens and imposes a binary label constraint on the unmixing solution. We have applied our labeling and analysis strategy to identify microbes labeled by fluorescence in situ hybridization and here demonstrate the ability to distinguish 120 differently labeled microbes in a single image.

Discoveries spanning several decades have pointed to vital membrane lipid trafficking pathways involving both vesicular and non-vesicular carriers. But the relative contributions for distinct membrane delivery pathways in cell growth and organelle biogenesis continue to be a puzzle. This is because lipids flow from many sources and across many paths via transport vesicles, non-vesicular transfer proteins, and dynamic interactions between organelles at membrane contact sites. This forum presents our latest understanding, appreciation, and queries regarding the lipid transport mechanisms necessary to drive membrane expansion during organelle biogenesis and cell growth. ispartof: BMC BIOLOGY vol:15 issue:1 ispartof: location:England status: published

Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier function and increases susceptibility to infections. Herein, we hypothesized that somatic mutations together with cigarette smoke create a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from controls (n=33) or cancer cases (n=143), we conducted 16S rRNA gene sequencing (MiSeq), with RNA-seq data from lung cancer cases in The Cancer Genome Atlas (n=1112) serving as the validation cohort. We demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue, indicating a shift in the overall microbial community in lung cancer patients as compared to those without cancer. Lung cancer cases were classified by the relative abundance of two taxa, Variovorax and Streptococcus, with an increase in Variovorax abundance in tumors as compared to non-tumor adjacent paired lung tissue (FDR corrected P=0.013). The species of Variovorax was identified histologically, and also by two additional 16S rRNA strategies (Resphera Insight analysis and PacBio sequencing). A group of taxa were associated with squamous cell carcinoma (SCC), of which Acidovorax were enriched in smokers (P =0.0013). Further, these taxa, including Acidovorax, exhibited higher abundance among the subset of SCC cases with TP53 mutations, an association not seen in adenocarcinomas (AD). Therefore, we observed a microbiome-gene and a microbiome-exposure interaction in SCC lung cancer tissue. Together, these results open the door for future biomarker research that could be used to improve screening and direct mechanistic studies of lung cancer therapy. Citation Format: Leigh Greathouse, James White, Valery Bliskovsky, Ashley Vargas, Eric Polley, Elise Bowman, Mohammed Khan, Ana Robles, Brid Ryan, Amiran Dzutsev, Giorgio Trinchieri, Marbin Pineda, Sven Bilke, Paul Meltzer, Marina Walther-Antonio, Garth Ehrlich, Joshua Mell, Joshua Earl, Sergey Balashov, Archana Bhat, Alex Valm, Clayton Deming, Sean Conlan, Julia Oh, Julie Segre, Curtis Harris. Microbiome-TP53 gene interaction in human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4925. doi:10.1158/1538-7445.AM2017-4925

We have investigated systematically the effects of short-term exposure to main stream cigarette smoke condensates (CSC-MS) on basal and inducible functional capacities of murine peritoneal exudate macrophages. Macrophages treated with CSC-MS form granules that fluoresce orange under blue excitation, consistent with the speculation that they are polycyclic aromatic hydrocarbons (PAH). CSC-MS selectively suppressed interferon gamma (IFN gamma) induction of four macrophage functional capacities: enhanced phagocytosis of immunoglobulin-opsonized sheep red blood cells, TPA-induced H2O2 production, class II major histocompatibility complex expression, and nitric oxide synthesis. In contrast, two macrophage functions that are not induced by IFN gamma, basal electron transport and LPS-induced TNF alpha production, were enhanced by treatment with CSC-MS. These results suggest that the suppressive effects of CSC-MS on macrophage responsiveness were selective and were not due to nonspecific inhibition of general functions such as RNA or protein synthesis. Since macrophage responsiveness to IFN gamma can result in induction of functional capacities that are fundamental to immunity, the data suggest that CSC-MS maybe deleterious to the general health of the smoker.

The number of fluorescent proteins, organic fluorophores, and inorganic fluorescent biomarkers is ever increasing. However, the ability to unambiguously distinguish more than a few different labels in a single fluorescence image is severely hampered by the excitation cross-talk and signal bleed-through of fluorophores with highly overlapping excitation and emission spectra. Here, we report the development of a fluorescence labeling, imaging, and analysis method to greatly expand the number of identifiable labels in a single image. The CLASI method involves labeling targets with specific combinations of fluorophore reporters. Commercially available microscopes with spectral detection capabilities are used to image the combinatorially-labeled specimens. Novel computational algorithms are used to analyze spectrally-recorded image data. We have developed a linear unmixing algorithm constrained to identify specific combinations of fluorophores. Our novel algorithms allow the concatenation of spectral data acquired with several different excitation wavelengths, either in parallel or sequentially. A goodness-of-fit is reported for each spectral combination, either in every pixel or for every particle identified in the image. We have applied the CLASI method to the study of the composition and spatial arrangement of complex microbial communities. Using fluorescence in situ hybridization with oligonucleotide probes specific for 16S rRNA sequences, we demonstrate that we can distinguish 120 differently labeled microbes in a mixture using binary combinations of 16 fluorophores.