Dual-Color Bioluminescence Imaging for Simultaneous Monitoring of the Intestinal Persistence of Lactobacillus plantarum and Lactococcus lactis in Living Mice.

Lactic acid bacteria are found in the gastrointestinal tract of mammals and have received tremendous attention due to their health-promoting properties. We report the development of two dual-color luciferase-producing Lactobacillus (Lb.) plantarum and Lactococcus (Lc.) lactis strains for noninvasive simultaneous tracking in the mouse gastrointestinal tract. We previously described the functional expression of the red luciferase mutant (CBRluc) from Pyrophorus plagiophthalamus in Lb. plantarum NCIMB8826 and Lc. lactis MG1363 (C. Daniel, S. Poiret, V. Dennin, D. Boutillier, and B. Pot, Appl Environ Microbiol 79:1086-1094, 2013, http://dx.doi.org/10.1128/AEM.03221-12). In this study, we determined that CBRluc is a better-performing luciferase for in vivo localization of both lactic acid bacteria after oral administration than the green click beetle luciferase mutant construct developed in this study. We further established the possibility to simultaneously detect red- and green-emitting lactic acid bacteria by dual-wavelength bioluminescence imaging in combination with spectral unmixing. The difference in spectra of light emission by the red and green click beetle luciferase mutants and dual bioluminescence detection allowed in vitro and in vivo quantification of the red and green emitted signals; thus, it allowed us to monitor the dynamics and fate of the two bacterial populations simultaneously. Persistence and viability of both strains simultaneously administered to mice in different ratios was studied in vivo in anesthetized mice and ex vivo in mouse feces. The application of dual-luciferase-labeled bacteria has considerable potential to simultaneously study the interactions and potential competitions of different targeted bacteria and their hosts.

Role of VAMP3 and VAMP7 in the commitment of Yersinia pseudotuberculosis to LC3-associated pathways involving single- or double-membrane vacuoles.

Yersinia pseudotuberculosis can replicate inside macrophages by hijacking autophagy and blocking autophagosome acidification. In bone marrow-derived macrophages, the bacteria are mainly observed inside double-membrane vacuoles positive for LC3, a hallmark of autophagy. Here, we address the question of the membrane traffic during internalization of Yersinia investigating the role of vesicle- associated membrane proteins (VAMPs). First, we show that as in epithelial cells, Yersinia pseudotuberculosis replicates mainly in nonacidic LC3-positive vacuoles. Second, in these cells, we unexpectedly found that VAMP3 localizes preferentially to Yersinia-containing vacuoles (YCVs) with single membranes using correlative light-electron microscopy. Third, we reveal the precise kinetics of VAMP3 and VAMP7 association with YCVs positive for LC3. Fourth, we show that VAMP7 knockdown alters LC3's association with single-and multimembrane-YCVs. Finally, in uninfected epithelial cells stimulated for autophagy, VAMP3 overexpression and knockdown led respectively to a lower and higher number of double-membrane, LC3-positive vesicles. Hence, our results highlight the role that VAMPs play in selection of the pathways leading to generation of ultrastructurally different LC3 compartments and pave the way for determining the full set of docking and fusion proteins involved in Yersinia pseudotuberculosis' intravesicular life cycle.

IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo.

Recent studies indicate that IL-1alpha functions intracellularly in pathways independent of its cell surface receptors by translocating to the nucleus and regulating transcription. Similarly, the chromatin-associated protein HMGB1 acts as both a nuclear factor and a secreted proinflammatory cytokine. Here, we show that IL-33, an IL-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines, is an endothelium-derived, chromatin-associated nuclear factor with transcriptional repressor properties. We found that IL-33 is identical to NF-HEV, a nuclear factor preferentially expressed in high endothelial venules (HEV), that we previously characterized. Accordingly, in situ hybridization demonstrated that endothelial cells constitute a major source of IL-33 mRNA in chronically inflamed tissues from patients with rheumatoid arthritis and Crohn's disease. Immunostaining with three distinct antisera, directed against the N-terminal part and IL-1-like C-terminal domain, revealed that IL-33 is a heterochromatin-associated nuclear factor in HEV endothelial cells in vivo. Association of IL-33 with heterochromatin was also observed in human and mouse cells under living conditions. In addition, colocalization of IL-33 with mitotic chromatin was noted. Nuclear localization, heterochromatin-association, and targeting to mitotic chromosomes were all found to be mediated by an evolutionarily conserved homeodomain-like helix-turn-helix motif within the IL-33 N-terminal part. Finally, IL-33 was found to possess transcriptional repressor properties, associated with the homeodomain-like helix-turn-helix motif. Together, these data suggest that, similarly to IL1alpha and HMGB1, IL-33 is a dual function protein that may function as both a proinflammatory cytokine and an intracellular nuclear factor with transcriptional regulatory properties.

Plasticity of endothelial cells: rapid dedifferentiation of freshly isolated high endothelial venule endothelial cells outside the lymphoid tissue microenvironment.

Endothelial cells display remarkable heterogeneity in different organs and vascular beds. Although many studies suggest that tissues "speak" to endothelial cells, endothelial cell diversity remains poorly characterized at the molecular level. Here, we describe a novel strategy to characterize tissue-specific endothelial cell phenotypes and to identify endothelial cell genes that are under the control of the local microenvironment. By comparing post-capillary high endothelial venule endothelial cells (HEVECs), freshly isolated from human tonsils without any cell culture step, with HEVECs cultured for 2 days, we found that HEVECs rapidly lost their specialized characteristics when isolated from the lymphoid tissue microenvironment. Striking changes occurred as early as after 48 hours, with complete loss of the postcapillary venule-specific Duffy antigen receptor for chemokines (DARCs) and the HEV-specific fucosyltransferase Fuc-TVII. DNA microarray analysis identified several other candidate HEV genes that were rapidly down-regulated ex vivo, including type XV collagen, which we characterized as a novel, abundant HEV transcript in situ. Together, our results demonstrate that blood vessel type-specific and tissue-specific characteristics of endothelial cells are under the control of their microenvironment. Therefore, even short-term primary cultures of human endothelial cells may not adequately mimic the differentiated endothelial cell phenotypes existing in vivo.