Risk Assessment of Industrial Microbes Using a Terrestrial Mesocosm Platform.

Industrial microbes and bio-derived products have emerged as an integral component of the bioeconomy, with an array of agricultural, bioenergy, and biomedical applications. However, the rapid development of microbial biotechnology raises concerns related to environmental escape of laboratory microbes, detection and tracking thereof, and resultant impact upon native ecosystems. Indeed, though wild-type and genetically modified microbes are actively deployed in industrial bioprocesses, an understanding of microbial interactivity and impact upon the environment is severely lacking. In particular, the persistence and sustained ecosystem impact of industrial microbes following laboratory release or unintentional laboratory escape remains largely unexplored. Herein, we investigate the applicability of soil-sorghum mesocosms for the ecological risk assessment of the industrial microbe, Saccharomyces cerevisiae. We developed and applied a suite of diagnostic and bioinformatic analyses, including digital droplet PCR, microscopy, and phylogenomic analyses to assess the impacts of a terrestrial ecosystem perturbation event over a 30-day time course. The platform enables reproducible, high-sensitivity tracking of S. cerevisiae in a complex soil microbiome and analysis of the impact upon abiotic soil characteristics and soil microbiome population dynamics and diversity. The resultant data indicate that even though S. cerevisiae is relatively short-lived in the soil, a single perturbation event can have sustained impact upon mesocosm soil composition and underlying microbial populations in our system, underscoring the necessity for more comprehensive risk assessment and development of mitigation and biocontainment strategies in industrial bioprocesses.

Improved Combinatorial Assembly and Barcode Sequencing for Gene-Sized DNA Constructs.

Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion challenges the analysis of multigene interactions. The incorporation of DNA barcodes to mark genes coupled with next-generation sequencing offers a solution to this challenge. We describe improvements for a key method in this space, CombiGEM, to broaden its application to assembling typical gene-sized DNA fragments and to reduce the cost of sequencing for prevalent small-scale projects. The expanded reach of the method beyond currently targeted small RNA genes promotes the discovery and incorporation of gene synergy in natural and engineered processes such as biocontainment, the production of desired compounds, and previously uncharacterized fundamental biological mechanisms.

Disruption of Genes Encoding Putative Zwitterionic Capsular Polysaccharides of Diverse Intestinal Bacteroides Reduces the Induction of Host Anti-Inflammatory Factors.

Bacterial zwitterionic capsular polysaccharides (ZPS), such as polysaccharide A (PSA) of the intestinal commensal Bacteroides fragilis, have been shown to modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). We previously used a genomic screen to identify diverse host-associated bacteria with the predicted genetic capacity to produce ZPSs related to PSA of B. fragilis and hypothesized that genetic disruption (KO) of a key functional gene within these operons would reduce the anti-inflammatory activity of these bacteria. We found that ZPS-KO bacteria in two common gut commensals, Bacteroides uniformis and Bacteroides cellulosilyticus, had a reduced ability to induce Tregs and IL-10 in stimulations of human peripheral blood mononuclear cells (PBMCs). Additionally, we found that macrophage stimulated with either wildtype B. fragilis or B. uniformis produced significantly more IL-10 than KOs, indicating a potentially novel function of ZPS of shifting the cytokine response in macrophages to a more anti-inflammatory state. These findings support the hypothesis that these related ZPS may represent a shared strategy to modulate host immune responses.

Dietary fat promotes antibiotic-induced Clostridioides difficile mortality in mice.

Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea, and emerging evidence has linked dietary components with CDI pathogenesis, suggesting that dietary modulation may be an effective strategy for prevention. Here, we show that mice fed a high-fat/low-fiber "Western-type" diet (WD) had dramatically increased mortality in a murine model of antibiotic-induced CDI compared to a low-fat/low-fiber (LF/LF) diet and standard mouse chow controls. We found that the WD had a pro- C. difficile bile acid composition that was driven in part by higher levels of primary bile acids that are produced to digest fat, and a lower level of secondary bile acids that are produced by the gut microbiome. This lack of secondary bile acids was associated with a greater disturbance to the gut microbiome with antibiotics in both the WD and LF/LF diet compared to mouse chow. Mice fed the WD also had the highest level of toxin TcdA just prior to the onset of mortality, but not of TcdB or increased inflammation. These findings indicate that dietary intervention to decrease fat may complement previously proposed dietary intervention strategies to prevent CDI in high-risk individuals.

Biotechnology for secure biocontainment designs in an emerging bioeconomy.

Genetically modified organisms (GMOs) have emerged as an integral component of a sustainable bioeconomy, with an array of applications in agriculture, bioenergy, and biomedicine. However, the rapid development of GMOs and associated synthetic biology approaches raises a number of biosecurity concerns related to environmental escape of GMOs, detection thereof, and impact upon native ecosystems. A myriad of genetic safeguards have been deployed in diverse microbial hosts, ranging from classical auxotrophies to global genome recoding. However, to realize the full potential of microbes as biocatalytic platforms in the bioeconomy, a deeper understanding of the fundamental principles governing microbial responsiveness to biocontainment constraints, and interactivity of GMOs with the environment, is required. Herein, we review recent analytical biotechnological advances and strategies to assess biocontainment and microbial bioproductivity, as well as opportunities for predictive systems biodesigns towards securing a viable bioeconomy.

Blood type and the microbiome- untangling a complex relationship with lessons from pathogens.

The complex communities of microbes that constitute the human microbiome are influenced by host and environmental factors. Here, we address how a fundamental aspect of human biology, blood type, contributes to shaping this microscopic ecosystem. Although this question remains largely unexplored, we glean insights from decades of work describing relationships between pathogens and blood type. The bacterial strategies, molecular mechanisms, and host responses that shaped those relationships may parallel those that characterize how blood type and commensals interact. Understanding these nuanced interactions will expand our capacity to analyze and manipulate the human microbiome.

Human Cytomegalovirus UL111A and US27 Gene Products Enhance the CXCL12/CXCR4 Signaling Axis via Distinct Mechanisms.

Human cytomegalovirus (HCMV) is a prevalent pathogen that establishes lifelong infection in the host. Virus persistence is aided by extensive manipulation of the host immune system, particularly cytokine and chemokine signaling pathways. The HCMV UL111A gene encodes cmvIL-10, an ortholog of human interleukin-10 that has many immunomodulatory effects. We found that cmvIL-10 increased signaling outcomes from human CXCR4, a chemokine receptor with essential roles in hematopoiesis and immune cell trafficking, in response to its natural ligand CXCL12. Calcium flux and chemotaxis to CXCL12 were significantly greater in the presence of cmvIL-10 in monocytes, epithelial cells, and fibroblasts that express CXCR4. cmvIL-10 effects on CXCL12/CXCR4 signaling required the IL-10 receptor and Stat3 activation. Heightened signaling occurred both in HCMV-infected cells and in uninfected bystander cells, suggesting that cmvIL-10 may broadly influence chemokine networks by paracrine signaling during infection. Moreover, CXCL12/CXCR4 signaling was amplified in HCMV-infected cells compared to mock-infected cells even in the absence of cmvIL-10. Enhanced CXCL12/CXCR4 outcomes were associated with expression of the virally encoded chemokine receptor US27, and CXCL12/CXCR4 activation was reduced in cells infected with a deletion mutant lacking US27 (TB40/E-mCherry-US27Δ). US27 effects were Stat3 independent but required close proximity to CXCR4 in cell membranes of either HCMV-infected or US27-transfected cells. Thus, HCMV encodes two proteins, cmvIL-10 and US27, that exhibit distinct mechanisms for enhancing CXCR4 signaling. Either individually or in combination, cmvIL-10 and US27 may enable HCMV to exquisitely manipulate CXCR4 signaling to alter host immune responses and modify cell trafficking patterns during infection.IMPORTANCE The human chemokine system plays a central role in host defense, as evidenced by the many strategies devised by viruses for manipulating it. Human cytomegalovirus (HCMV) is widespread in the human population, but infection rarely causes disease except in immunocompromised hosts. We found that two different HCMV proteins, cmvIL-10 and US27, act through distinct mechanisms to upregulate the signaling activity of a cellular chemokine receptor, CXCR4. cmvIL-10 is a secreted viral cytokine that affects CXCR4 signaling in both infected and uninfected cells, while US27 is a component of the virus particle and impacts CXCR4 activity only in infected cells. Both cmvIL-10 and US27 promote increased intracellular calcium signaling and cell migration in response to chemokine CXCL12 binding to CXCR4. Our results demonstrate that HCMV exerts fine control over the CXCL12/CXCR4 pathway, which could lead to enhanced virus dissemination, altered immune cell trafficking, and serious health implications for HCMV patients.

Striking a Balance with Help from our Little Friends - How the Gut Microbiota Contributes to Immune Homeostasis.

The trillions of microbes that inhabit the human gut (the microbiota) together with the host comprise a complex ecosystem, and like any ecosystem, health relies on stability and balance. Some of the most important members of the human microbiota are those that help maintain this balance via modulation of the host immune system. Gut microbes, through both molecular factors (such as capsular components) and by-products of their metabolism (such as Short Chain Fatty Acids (SCFAs)), can influence both innate and adaptive components of the immune system, in ways that can drive both effector, and regulatory responses. Here we review how commensal microbes can specifically promote a dynamic balance of these immune responses in the mammalian gut.

Diverse Intestinal Bacteria Contain Putative Zwitterionic Capsular Polysaccharides with Anti-inflammatory Properties.

Zwitterionic capsular polysaccharides (ZPSs) are bacterial products that modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). However, only a few diverse bacteria are known to modulate the host immune system via ZPS. We present a genomic screen for bacteria encoding ZPS molecules. We identify diverse host-associated bacteria, including commensals and pathogens with known anti-inflammatory properties, with the capacity to produce ZPSs. Human mononuclear cells stimulated with lysates from putative ZPS-producing bacteria induce significantly greater IL-10 production and higher proportions of Tregs than lysates from non-ZPS-encoding relatives or a commensal strain of Bacteroides cellulosilyticus in which a putative ZPS biosynthetic operon was genetically disrupted. Similarly, wild-type B. cellulosilyticus DSM 14838, but not a close relative lacking a putative ZPS, attenuated experimental colitis in mice. Collectively, this screen identifies bacterial strains that may use ZPSs to interact with the host as well as those with potential probiotic properties.

CXCR4: a virus's best friend?

Viruses are dependent on their hosts for replication and dispersal in the environment; thus, the most successful viruses are those that co-evolve with their hosts. CXCR4 is a cellular chemokine receptor that plays central roles in development, hematopoiesis, and immune surveillance through signaling induced by its ligand, CXCL12. The CXCR4-CXCL12 axis has been besieged by many pathogens that employ a range of strategies to modify or exploit CXCR4 activity. While CXCR4 was identified as a critical co-factor for entry of HIV into CD4+ T cells early on, other viruses may utilize CXCR4 to gain cell entry as well. Moreover, several viruses have been found to modulate CXCR4 expression or alter its functional activity, with direct effects on cell trafficking, immune responses, cell proliferation, and cell survival. Because CXCR4 is targeted by a diverse group of viral pathogens, modification of host CXCR4 signaling activity is emerging as a common theme in virus persistence and is likely to be important for subversion of the host immune system. This review highlights major viral pathogens that use and abuse CXCR4 and explores the possible reasons why this chemokine receptor has become "a virus's best friend".

The US27 gene product of human cytomegalovirus enhances signaling of host chemokine receptor CXCR4.

Human cytomegalovirus (HCMV) is a member of the Herpesviridae family that manipulates host immune responses and establishes life-long latent infection, in part through mimicry of cytokines, chemokines, and chemokine receptors. The HCMV US27 gene product is a putative chemokine receptor with no known ligands. We generated a stable US27 cell line to screen for chemokine ligands but unexpectedly found that US27 potentiated the activity of an endogenous human chemokine receptor, CXCR4. Cells expressing both US27 and CXCR4 exhibited greater calcium mobilization and enhanced chemotaxis in response to CXCL12/SDF-1α than controls. Quantitative RT-PCR revealed a significant increase in CXCR4 expression when US27 was present, and elevated CXCR4 receptor levels were detected via flow cytometry, western blot, and immunofluorescence microscopy. Potentiation of CXCR4 signaling by US27 could represent a novel strategy by which HCMV targets virus-infected cells to the bone marrow in order to expand the reservoir of latently infected cells.

Receptor chimeras demonstrate that the C-terminal domain of the human cytomegalovirus US27 gene product is necessary and sufficient for intracellular receptor localization.

BACKGROUND: Human cytomegalovirus (HCMV) is ubiquitous in the population but generally causes only mild or asymptomatic infection except in immune suppressed individuals. HCMV employs numerous strategies for manipulating infected cells, including mimicry of G-protein coupled receptors (GPCRs). The HCMV US27 gene product is a putative GPCR, yet no ligand or signaling has been identified for this receptor. In the present study, immunofluorescence microscopy was used to examine the cellular distribution of wild type US27, as well as US27 deletion mutants and chimeric receptors. RESULTS: In transiently transfected cells, wild type US27 was found primarily in intracellular compartments, in striking contrast to the cell surface distribution seen for the human cellular chemokine receptor CXCR3. When the N-terminal extracellular domains of the two receptors were swapped, no change in protein localization was observed. However, swapping of the C-terminal intracellular domains resulted in a significant change in receptor distribution. A chimera that contained US27 fused to the C-terminal intracellular tail of CXCR3 exhibited surface distribution similar to that of wild-type CXCR3. When the C-terminal domain of US27 was fused to CXCR3, this chimeric receptor (CXCR3/US27-CT) was found in the same intracellular pattern as wild-type US27. In addition, a US27 mutant lacking the C-terminus (US27ΔCT) failed to accumulate inside the cell and exhibited cell surface distribution. Co-localization with organelle-specific markers revealed that wild-type US27 was found predominantly in the Golgi apparatus and in endosomal compartments, whereas the US27/CXCR3-CT chimera, US27ΔCT and US27Δ348 mutants were not localized to endosomal compartments. CONCLUSIONS: The results indicate that the C-terminal domain of the HCMV US27 protein, which contains a di-leucine endocytic sorting motif, is both necessary and sufficient for intracellular localization, which may also help explain why no cellular ligands have yet been identified for this viral receptor.