Surface colonization by Flavobacterium johnsoniae promotes its survival in a model microbial community.

Flavobacterium johnsoniae is a ubiquitous soil and rhizosphere bacterium, but despite its abundance, the factors contributing to its success in communities are poorly understood. Using a model microbial community, The Hitchhikers of the Rhizosphere (THOR), we determined the effects of colonization on the fitness of F. johnsoniae in the community. Insertion sequencing, a massively parallel transposon mutant screen, on sterile sand identified 25 genes likely to be important for surface colonization. We constructed in-frame deletions of candidate genes predicted to be involved in cell membrane biogenesis, motility, signal transduction, and transport of amino acids and lipids. All mutants poorly colonized sand, glass, and polystyrene and produced less biofilm than the wild type, indicating the importance of the targeted genes in surface colonization. Eight of the nine colonization-defective mutants were also unable to form motile biofilms or zorbs, thereby suggesting that the affected genes play a role in group movement and linking stationary and motile biofilm formation genetically. Furthermore, we showed that the deletion of colonization genes in F. johnsoniae affected its behavior and survival in THOR on surfaces, suggesting that the same traits are required for success in a multispecies microbial community. Our results provide insight into the mechanisms of surface colonization by F. johnsoniae and form the basis for further understanding its ecology in the rhizosphere. IMPORTANCE: Microbial communities direct key environmental processes through multispecies interactions. Understanding these interactions is vital for manipulating microbiomes to promote health in human, environmental, and agricultural systems. However, microbiome complexity can hinder our understanding of the underlying mechanisms in microbial community interactions. As a first step toward unraveling these interactions, we explored the role of surface colonization in microbial community interactions using The Hitchhikers Of the Rhizosphere (THOR), a genetically tractable model community of three bacterial species, Flavobacterium johnsoniae, Pseudomonas koreensis, and Bacillus cereus. We identified F. johnsoniae genes important for surface colonization in solitary conditions and in the THOR community. Understanding the mechanisms that promote the success of bacteria in microbial communities brings us closer to targeted manipulations to achieve outcomes that benefit agriculture, the environment, and human health.

Microbiome composition modulates secondary metabolism in a multispecies bacterial community.

Bacterial secondary metabolites are a major source of antibiotics and other bioactive compounds. In microbial communities, these molecules can mediate interspecies interactions and responses to environmental change. Despite the importance of secondary metabolites in human health and microbial ecology, little is known about their roles and regulation in the context of multispecies communities. In a simplified model of the rhizosphere composed of Bacillus cereus, Flavobacterium johnsoniae, and Pseudomonas koreensis, we show that the dynamics of secondary metabolism depend on community species composition and interspecies interactions. Comparative metatranscriptomics and metametabolomics reveal that the abundance of transcripts of biosynthetic gene clusters (BGCs) and metabolomic molecular features differ between monocultures or dual cultures and a tripartite community. In both two- and three-member cocultures, P. koreensis modified expression of BGCs for zwittermicin, petrobactin, and other secondary metabolites in B. cereus and F. johnsoniae, whereas the BGC transcriptional response to the community in P. koreensis itself was minimal. Pairwise and tripartite cocultures with P. koreensis displayed unique molecular features that appear to be derivatives of lokisin, suggesting metabolic handoffs between species. Deleting the BGC for koreenceine, another P. koreensis metabolite, altered transcript and metabolite profiles across the community, including substantial up-regulation of the petrobactin and bacillibactin BGCs in B. cereus, suggesting that koreenceine represses siderophore production. Results from this model community show that bacterial BGC expression and chemical output depend on the identity and biosynthetic capacity of coculture partners, suggesting community composition and microbiome interactions may shape the regulation of secondary metabolism in nature.

THOR's Hammer: the Antibiotic Koreenceine Drives Gene Expression in a Model Microbial Community.

Microbial interactions dictate the structure and function of microbiomes, but the complexity of natural communities can obscure the individual interactions. Model microbial communities constructed with genetically tractable strains known to interact in natural settings can untangle these networks and reveal underpinning mechanisms. Our model system, The Hitchhikers of the Rhizosphere (THOR), is composed of three species-Bacillus cereus, Flavobacterium johnsoniae, and Pseudomonas koreensis-that co-isolate from field-grown soybean roots. Comparative metatranscriptomics on THOR revealed global patterns of interspecies transcriptional regulation. When grown in pairs, each member of THOR exhibits unique signaling behavior. In the community setting, gene expression is dominated by pairwise interactions with Pseudomonas koreensis mediated either directly or indirectly by its production of the antibiotic koreenceine-the apparent "hammer" of THOR. In pairwise interactions, the koreenceine biosynthetic cluster is responsible for 85 and 22% of differentially regulated genes in F. johnsoniae and B. cereus, respectively. Although both deletion of the koreenceine locus and reduction of P. koreensis inoculum size increase F. johnsoniae populations, the transcriptional response of P. koreensis is only activated when it is a relative minority member at the beginning of coculture. The largest group of upregulated P. koreensis genes in response to F. johnsoniae are those without functional annotation, indicating that focusing on genes important for community interactions may offer a path toward functional assignments for unannotated genes. This study illustrates the power of comparative metatranscriptomics of microorganisms encountering increasing microbial complexity for understanding community signal integration, antibiotic responses, and interspecies communication. IMPORTANCE The diversity, ubiquity, and significance of microbial communities is clear. However, the predictable and reliable manipulation of microbiomes to impact human, environmental, and agricultural health remains a challenge. Effective remodeling of microbiomes will be enabled by understanding the interspecies interactions that govern community processes. The extreme complexity of most microbiomes has impeded characterization of the relevant interactions. Investigating the genetics and biochemistry of simplified, model microbiomes could unearth specific interactions and generate predictions about community-governing principles. Here, we use one such model community to quantify changes in gene expression of individual species as they encounter stimuli from one or more species, directly mapping combinatorial interspecies interactions. A surprising amount of gene expression is regulated by a single molecule, the antibiotic koreenceine, which appears to impact gene regulation across community networks.

Social motility of biofilm-like microcolonies in a gliding bacterium.

Bacterial biofilms are aggregates of surface-associated cells embedded in an extracellular polysaccharide (EPS) matrix, and are typically stationary. Studies of bacterial collective movement have largely focused on swarming motility mediated by flagella or pili, in the absence of a biofilm. Here, we describe a unique mode of collective movement by a self-propelled, surface-associated biofilm-like multicellular structure. Flavobacterium johnsoniae cells, which move by gliding motility, self-assemble into spherical microcolonies with EPS cores when observed by an under-oil open microfluidic system. Small microcolonies merge, creating larger ones. Microscopic analysis and computer simulation indicate that microcolonies move by cells at the base of the structure, attached to the surface by one pole of the cell. Biochemical and mutant analyses show that an active process drives microcolony self-assembly and motility, which depend on the bacterial gliding apparatus. We hypothesize that this mode of collective bacterial movement on solid surfaces may play potential roles in biofilm dynamics, bacterial cargo transport, or microbial adaptation. However, whether this collective motility occurs on plant roots or soil particles, the native environment for F. johnsoniae, is unknown.

Tiny Earth: A Big Idea for STEM Education and Antibiotic Discovery.

The world faces two seemingly unrelated challenges-a shortfall in the STEM workforce and increasing antibiotic resistance among bacterial pathogens. We address these two challenges with Tiny Earth, an undergraduate research course that excites students about science and creates a pipeline for antibiotic discovery.

Obstructive sleep apnea and CPAP therapy alter distinct transcriptional programs in subcutaneous fat tissue.

Obstructive sleep apnea (OSA) has been linked to dysregulated metabolic states, and treatment of sleep apnea may improve these conditions. Subcutaneous adipose tissue is a readily samplable fat depot that plays an important role in regulating metabolism. However, neither the pathophysiologic consequences of OSA nor the effects of continuous positive airway pressure (CPAP) in altering this compartment's molecular pathways are understood. This study aimed to systematically identify subcutaneous adipose tissue transcriptional programs modulated in OSA and in response to its effective treatment with CPAP. Two subject groups were investigated: Study Group 1 was comprised of 10 OSA and 8 controls; Study Group 2 included 24 individuals with OSA studied at baseline and following CPAP. For each subject, genome-wide gene expression measurement of subcutaneous fat was performed. Differentially activated pathways elicited by OSA (Group 1) and in response to its treatment (Group 2) were determined using network and Gene Set Enrichment Analysis (GSEA). In Group 2, treatment of OSA with CPAP improved apnea-hypopnea index, daytime sleepiness, and blood pressure, but not anthropometric measures. In Group 1, GSEA revealed many up-regulated gene sets in OSA subjects, most of which were involved in immuno-inflammatory (e.g. interferon-γ signaling), transcription, and metabolic processes such as adipogenesis. Unexpectedly, CPAP therapy in Group 2 subjects was also associated with up-regulation of several immune pathways as well as cholesterol biosynthesis. Collectively, our findings demonstrate that OSA alters distinct inflammatory and metabolic programs in subcutaneous fat, but these transcriptional signatures are not reversed with short-term effective therapy.

Introducing THOR, a Model Microbiome for Genetic Dissection of Community Behavior.

The quest to manipulate microbiomes has intensified, but many microbial communities have proven to be recalcitrant to sustained change. Developing model communities amenable to genetic dissection will underpin successful strategies for shaping microbiomes by advancing an understanding of community interactions. We developed a model community with representatives from three dominant rhizosphere taxa, the Firmicutes, Proteobacteria, and Bacteroidetes We chose Bacillus cereus as a model rhizosphere firmicute and characterized 20 other candidates, including "hitchhikers" that coisolated with B. cereus from the rhizosphere. Pairwise analysis produced a hierarchical interstrain-competition network. We chose two hitchhikers, Pseudomonas koreensis from the top tier of the competition network and Flavobacterium johnsoniae from the bottom of the network, to represent the Proteobacteria and Bacteroidetes, respectively. The model community has several emergent properties, induction of dendritic expansion of B. cereus colonies by either of the other members, and production of more robust biofilms by the three members together than individually. Moreover, P. koreensis produces a novel family of alkaloid antibiotics that inhibit growth of F. johnsoniae, and production is inhibited by B. cereus We designate this community THOR, because the members are the hitchhikers of the rhizosphere. The genetic, genomic, and biochemical tools available for dissection of THOR provide the means to achieve a new level of understanding of microbial community behavior.IMPORTANCE The manipulation and engineering of microbiomes could lead to improved human health, environmental sustainability, and agricultural productivity. However, microbiomes have proven difficult to alter in predictable ways, and their emergent properties are poorly understood. The history of biology has demonstrated the power of model systems to understand complex problems such as gene expression or development. Therefore, a defined and genetically tractable model community would be useful to dissect microbiome assembly, maintenance, and processes. We have developed a tractable model rhizosphere microbiome, designated THOR, containing Pseudomonas koreensis, Flavobacterium johnsoniae, and Bacillus cereus, which represent three dominant phyla in the rhizosphere, as well as in soil and the mammalian gut. The model community demonstrates emergent properties, and the members are amenable to genetic dissection. We propose that THOR will be a useful model for investigations of community-level interactions.

Gut microbiota density influences host physiology and is shaped by host and microbial factors.

To identify factors that regulate gut microbiota density and the impact of varied microbiota density on health, we assayed this fundamental ecosystem property in fecal samples across mammals, human disease, and therapeutic interventions. Physiologic features of the host (carrying capacity) and the fitness of the gut microbiota shape microbiota density. Therapeutic manipulation of microbiota density in mice altered host metabolic and immune homeostasis. In humans, gut microbiota density was reduced in Crohn's disease, ulcerative colitis, and ileal pouch-anal anastomosis. The gut microbiota in recurrent Clostridium difficile infection had lower density and reduced fitness that were restored by fecal microbiota transplantation. Understanding the interplay between microbiota and disease in terms of microbiota density, host carrying capacity, and microbiota fitness provide new insights into microbiome structure and microbiome targeted therapeutics. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

CPAP Adherence Predictors in a Randomized Trial of Moderate-to-Severe OSA Enriched With Women and Minorities.

BACKGROUND: Suboptimal CPAP adherence in OSA clinical trials involving predominantly white men limits interpretability and generalizability. We examined predictors of CPAP adherence in a clinical trial enriched with minorities. METHODS: The Sleep Apnea Stress Study-a randomized, double-blind, sham-controlled trial of patients with moderate-to-severe OSA-included participants with complete 8-week adherence data (n = 138). Overnight 14-channel polysomnography, anthropometry, socioeconomic status, mood questionnaires, and week 1 CPAP adherence were analyzed via adjusted linear models relative to CPAP adherence (average minutes per night usage). RESULTS: Overall, age was 51 ± 12 years, 55% of the patients were male, 55% were white, BMI was 36.7 ± 7.7 kg/m2, and median apnea-hypopnea index was 20 (interquartile range, 13-37). In univariate analyses adherence increased with randomization to active CPAP (81 min; 95% CI, 30-132), increasing age (35 min/decade; 95% CI, 13-57), white race (78 min, 95% CI, 26-129), and per hour of week 1 adherence (41 min, 95% CI, 32-51). Active CPAP (48 min, 95% CI, 6-91), increasing age (27 min/decade, 95% CI, 10-44), and higher 1-week adherence (36 min/h, 95% CI, 27-46) were significantly associated with improved adherence in multivariable analyses. Subgroup analyses showed stronger associations of adherence with treatment arm in whites and increasing age in minorities. Increasing age and white race were more strongly associated with adherence in women. CONCLUSIONS: In this trial with near-even sex distribution and high ethnic minority representation, we identified CPAP assignment, increasing age, and early adherence to be associated with improved adherence in addition to sex-specific and race-specific adherence differences. These results can inform targeted clinical trial adherence optimization strategies. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT00607893; URL: www.clinicaltrials.gov.

Asymmetric regulation of quorum-sensing receptors drives autoinducer-specific gene expression programs in Vibrio cholerae.

Quorum sensing (QS) is a mechanism of chemical communication that bacteria use to monitor cell-population density and coordinate group behaviors. QS relies on the production, detection, and group-wide response to extracellular signal molecules called autoinducers. Vibrio cholerae employs parallel QS circuits that converge into a shared signaling pathway. At high cell density, the CqsS and LuxPQ QS receptors detect the intra-genus and inter-species autoinducers CAI-1 and AI-2, respectively, to repress virulence factor production and biofilm formation. We show that positive feedback, mediated by the QS pathway, increases CqsS but not LuxQ levels during the transition into QS-mode, which amplifies the CAI-1 input into the pathway relative to the AI-2 input. Asymmetric feedback on CqsS enables responses exclusively to the CAI-1 autoinducer. Because CqsS exhibits the dominant QS signaling role in V. cholerae, agonism of CqsS with synthetic compounds could be used to control pathogenicity and host dispersal. We identify nine compounds that share no structural similarity to CAI-1, yet potently agonize CqsS via inhibition of CqsS autokinase activity.

Investigation of piperazine benzamides as human ß3 adrenergic receptor agonists for the treatment of overactive bladder.

The synthesis of a novel class of piperazine benzamide (reverse amides) targeting the human ß3-adrenergic receptor for the treatment of overactive bladder (OAB) is described. The SAR studies directed towards maintaining well established ß3 potency and selectivities while improving the overall pharmacokinetic profile in the reverse amide class will be evaluated. The results and consequences associated with functional activity at the norepinephrine transporter (NET) will also be discussed.

Nanocarriers from GRAS Zein Proteins to Encapsulate Hydrophobic Actives.

One factor limiting the expansion of nanomedicines has been the high cost of the materials and processes required for their production. We present a continuous, scalable, low cost nanoencapsulation process, Flash Nanoprecipitation (FNP) that enables the production of nanocarriers (NCs) with a narrow size distribution using zein corn proteins. Zein is a low cost, GRAS protein (having the FDA status of "Generally Regarded as Safe") currently used in food applications, which acts as an effective encapsulant for hydrophobic compounds using FNP. The four-stream FNP configuration allows the encapsulation of very hydrophobic compounds in a way that is not possible with previous precipitation processes. We present the encapsulation of several model active compounds with as high as 45 wt % drug loading with respect to zein concentration into ∼100 nm nanocarriers. Three examples are presented: (1) the pro-drug antioxidant, vitamin E-acetate, (2) an anticholera quorum-sensing modulator CAI-1 ((S)-3-hydroxytridecan-4-one; CAI-1 that reduces Vibrio cholerae virulence by modulating cellular communication), and (3) hydrophobic fluorescent dyes with a range of hydrophobicities. The specific interaction between zein and the milk protein, sodium caseinate, provides stabilization of the NCs in PBS, LB medium, and in pH 2 solutions. The stability and size changes in the three media provide information on the mechanism of assembly of the zein/active/casein NC.

Structure, Regulation, and Inhibition of the Quorum-Sensing Signal Integrator LuxO.

In a process called quorum sensing, bacteria communicate with chemical signal molecules called autoinducers to control collective behaviors. In pathogenic vibrios, including Vibrio cholerae, the accumulation of autoinducers triggers repression of genes responsible for virulence factor production and biofilm formation. The vibrio autoinducer molecules bind to transmembrane receptors of the two-component histidine sensor kinase family. Autoinducer binding inactivates the receptors' kinase activities, leading to dephosphorylation and inhibition of the downstream response regulator LuxO. Here, we report the X-ray structure of LuxO in its unphosphorylated, autoinhibited state. Our structure reveals that LuxO, a bacterial enhancer-binding protein of the AAA+ ATPase superfamily, is inhibited by an unprecedented mechanism in which a linker that connects the catalytic and regulatory receiver domains occupies the ATPase active site. The conformational change that accompanies receiver domain phosphorylation likely disrupts this interaction, providing a mechanistic rationale for LuxO activation. We also determined the crystal structure of the LuxO catalytic domain bound to a broad-spectrum inhibitor. The inhibitor binds in the ATPase active site and recapitulates elements of the natural regulatory mechanism. Remarkably, a single inhibitor molecule may be capable of inhibiting an entire LuxO oligomer.

Discovery of benzamides as potent human ß3 adrenergic receptor agonists.

The paper will describe the synthesis and SAR studies that led to the discovery of benzamide (reverse amide) as potent and selective human ß3-adrenergic receptor agonist. Based on conformationally restricted pyrrolidine scaffold we discovered earlier, pyrrolidine benzoic acid intermediate 22 was synthesized. From library synthesis and further optimization efforts, several structurally diverse reverse amides such as 24c and 24i were found to have excellent human ß3-adrenergic potency and good selectivity over the ß1 and ß2 receptors. In addition to human ß1, ß2, ß3 and hERG data, PK of selected compounds will be described.

Discovery of Vibegron: A Potent and Selective ß3 Adrenergic Receptor Agonist for the Treatment of Overactive Bladder.

The discovery of vibegron, a potent and selective human ß3-AR agonist for the treatment of overactive bladder (OAB), is described. An early-generation clinical ß3-AR agonist MK-0634 (3) exhibited efficacy in humans for the treatment of OAB, but development was discontinued due to unacceptable structure-based toxicity in preclinical species. Optimization of a series of second-generation pyrrolidine-derived ß3-AR agonists included reducing the risk for phospholipidosis, the risk of formation of disproportionate human metabolites, and the risk of formation of high levels of circulating metabolites in preclinical species. These efforts resulted in the discovery of vibegron, which possesses improved druglike properties and an overall superior preclinical profile compared to MK-0634. Structure-activity relationships leading to the discovery of vibegron and a summary of its preclinical profile are described.

Modulating Vibrio cholerae quorum-sensing-controlled communication using autoinducer-loaded nanoparticles.

The rise of bacterial antibiotic resistance has created a demand for alternatives to traditional antibiotics. Attractive possibilities include pro- and anti-quorum sensing therapies that function by modulating bacterial chemical communication circuits. We report the use of Flash NanoPrecipitation to deliver the Vibrio cholerae quorum-sensing signal CAI-1 ((S)-3-hydroxytridecan-4-one) in a water dispersible form as nanoparticles. The particles activate V. cholerae quorum-sensing responses 5 orders of magnitude higher than does the identically administered free CAI-1 and are diffusive across in vivo delivery barriers such as intestinal mucus. This work highlights the promise of combining quorum-sensing strategies with drug delivery approaches for the development of next-generation medicines.

Highly Potent, Chemically Stable Quorum Sensing Agonists for Vibrio Cholerae.

In the Vibrio cholerae pathogen, initiation of bacterial quorum sensing pathways serves to suppress virulence. We describe herein a potent and chemically stable small molecule agonist of V. cholerae quorum sensing, which was identified through rational drug design based on the native quorum sensing signal. This novel agonist may serve as a useful lead compound for the control of virulence in V. cholerae.

Design, synthesis, and evaluation of conformationally restricted acetanilides as potent and selective ß3 adrenergic receptor agonists for the treatment of overactive bladder.

A series of conformationally restricted acetanilides were synthesized and evaluated as ß3-adrenergic receptor agonists (ß3-AR) for the treatment of overactive bladder (OAB). Optimization studies identified a five-membered ring as the preferred conformational lock of the acetanilide. Further optimization of both the aromatic and thiazole regions led to compounds such as 19 and 29, which have a good balance of potency and selectivity. These compounds have significantly reduced intrinsic clearance compared to our initial series of pyridylethanolamine ß3-AR agonists and thus have improved unbound drug exposures. Both analogues demonstrated dose dependent ß3-AR mediated responses in a rat bladder hyperactivity model.

Glial cell line-derived neurotrophic factor-secreting human neural progenitors show long-term survival, maturation into astrocytes, and no tumor formation following transplantation into the spinal cord of immunocompromised rats.

Human neural progenitor cells (hNPCs) derived from the fetal cortex can be expanded in vitro and genetically modified through lentiviral transduction to secrete growth factors shown to have a neurotrophic effect in animal models of neurological disease. hNPCs survive and mature following transplantation into the central nervous system of large and small animals including the rat model of amyotrophic lateral sclerosis. Here we report that hNPCs engineered to express glial cell line-derived neurotrophic factor (GDNF) survive long-term (7.5 months) following transplantation into the spinal cord of athymic nude rats and continue to secrete GDNF. Cell proliferation declined while the number of astrocytes increased, suggesting final maturation of the cells over time in vivo. Together these data show that GDNF-producing hNPCs may be useful as a source of cells for long-term delivery of both astrocytes and GDNF to the damaged central nervous system.

EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs.

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.