Study of the persistence and dynamics of recombinant mCherry-producing Yarrowia lipolytica strains in the mouse intestine using fluorescence imaging.

Yarrowia lipolytica is a dimorphic oleaginous non-conventional yeast widely used as a powerful host for expressing heterologous proteins, as well as a promising source of engineered cell factories for various applications. This microorganism has a documented use in Feed and Food and a GRAS (generally recognized as safe) status. Moreover, in vivo studies demonstrated a beneficial effect of this yeast on animal health. However, despite the focus on Y. lipolytica for the industrial manufacturing of heterologous proteins and for probiotic effects, its potential for oral delivery of recombinant therapeutic proteins has seldom been evaluated in mammals. As the first steps towards this aim, we engineered two Y. lipolytica strains, a dairy strain and a laboratory strain, to produce the model fluorescent protein mCherry. We demonstrated that both Y. lipolytica strains transiently persisted for at least 1 week after four daily oral administrations and they maintained the active expression of mCherry in the mouse intestine. We used confocal microscopy to image individual Y. lipolytica cells of freshly collected intestinal tissues. They were found essentially in the lumen and they were rarely in contact with epithelial cells while transiting through the ileum, caecum and colon of mice. Taken as a whole, our results have shown that fluorescent Y. lipolytica strains constitute novel tools to study the persistence and dynamics of orally administered yeasts which could be used in the future as oral delivery vectors for the secretion of active therapeutic proteins in the gut.

Persistence and dynamics of fluorescent Lactobacillus plantarum in the healthy versus inflamed gut.

The gastrointestinal tract is the main ecological niche in which Lactobacillus strains may provide health benefits in mammals. There is currently a need to characterize host-microbe interactions in space and time by tracking these bacteria in vivo. We combined noninvasive whole-body imaging with ex vivo fluorescence confocal microscopy imaging to monitor the impact of intestinal inflammation on the persistence of orally administered Lactobacillus plantarum NCIMB8826 in healthy and inflamed mouse colons. We developed fluorescent L. plantarum strains and demonstrated that mCherry is the best system for in vivo imaging and ex vivo fluorescence confocal microscopy of these bacteria. We also used whole-body imaging to show that this anti-inflammatory, orally administered strain persists for longer and at higher counts in the inflamed colon than in the healthy colon. We confirmed these results by the ex vivo confocal imaging of colons from mice with experimental colitis for 3 days after induction. Moreover, extended orthogonal view projections enabled us to localize individual L. plantarum in sites that differed for healthy versus inflamed guts. In healthy colons, orally administered bacteria were localized in the lumen (in close contact with commensal bacteria) and sometimes in the crypts (albeit very rarely in contact with intestinal cells). The bacteria were observed within and outside the mucus layer. In contrast, L. plantarum bacteria in the inflamed colon were mostly located in the lumen and (in less inflamed areas) within the mucus layer. In more intensely inflamed areas (i.e., where the colon had undergone structural damage), the L. plantarum were in direct contact with damaged epithelial cells. Taken as a whole, our results show that fluorescently labeled L. plantarum can be used to study the persistence of these bacteria in inflamed guts using both noninvasive whole-body imaging and ex vivo fluorescence confocal microscopy.

High-dose dietary supplementation with zinc prevents gut inflammation: Investigation of the role of metallothioneins and beyond by transcriptomic and metagenomic studies.

Although it is known that zinc has several beneficial roles in the context of gut inflammation, the underlying mechanisms have not been extensively characterized. Zinc (Zn) is known to be the primary physiological inducer of the expression of the metallothionein (MT) superfamily of small stress-responsive proteins. The expression of MTs in various tissues is induced or enhanced (including the gastrointestinal tract (GIT)) by a variety of stimuli, including infection and inflammation. However, the MTs' exact role in inflammation is still subject to debate. In order to establish whether or not MTs are the sole vectors in the Zn-based modulation of intestinal inflammation, we used transcriptomic and metagenomic approaches to assess the potential effect of dietary Zn, the mechanisms underlying the MTs' beneficial effects, and the induction of previously unidentified mediators. We found that the expression of endogenous MTs in the mouse GIT was stimulated by an optimized dietary supplementation with Zn. The protective effects of dietary supplementation with Zn were then evaluated in mouse models of chemically induced colitis. The potential contribution of MTs and other pathways was explored via transcriptomic analyses of the ileum and colon in Zn-treated mice. The microbiota's role was also assessed via fecal 16S rRNA sequencing. We found that high-dose dietary supplementation with Zn induced the expression of MT-encoding genes in the colon of healthy mice. We next demonstrated that the Zn diet significantly protected mice in the two models of induced colitis. When comparing Zn-treated and control mice, various genes were found to be differentially expressed in the colon and the ileum. Finally, we found that Zn supplementation did not modify the overall structure of the fecal microbiota, with the exception of (i) a significant increase in endogenous Clostridiaceae, and (ii) some subtle but specific changes at the family and genus levels. Our results emphasize the beneficial effects of excess dietary Zn on the prevention of colitis and inflammatory events in mouse models. The main underlying mechanisms were driven by the multifaceted roles of MTs and the other potential molecular mediators highlighted by our transcriptomic analyses although we cannot rule out contributions by other factors from the host and/or the microbiota.

Polymorphism in the Yersinia LcrV Antigen Enables Immune Escape From the Protection Conferred by an LcrV-Secreting Lactococcus Lactis in a Pseudotuberculosis Mouse Model.

Yersinioses caused by Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are significant concerns in human and veterinary health. The link between virulence and the potent LcrV antigen has prompted the latter's selection as a major component of anti-Yersinia vaccines. Here, we report that (i) the group of Yersinia species encompassing Y. pestis and Y. pseudotuberculosis produces at least five different clades of LcrV and (ii) vaccination of mice with an LcrV-secreting Lactococcus lactis only protected against Yersinia strains producing the same LcrV clade as that of used for vaccination. By vaccinating with engineered LcrVs and challenging mice with strains producing either type of LcrV or a LcrV mutated for regions of interest, we highlight key polymorphic residues responsible for the absence of cross-protection. Our results show that an anti-LcrV-based vaccine should contain multiple LcrV clades if protection against the widest possible array of Yersinia strains is sought.

Characterization of the protective immune response to Yersinia pseudotuberculosis infection in mice vaccinated with an LcrV-secreting strain of Lactococcus lactis.

BACKGROUND: Pseudotuberculosis is an infection caused by the bacterial enteropathogen Yersinia pseudotuberculosis and is considered to be a significant problem in veterinary medicine. We previously found that intranasal administration of a recombinant Lactococcus lactis strain that secretes the low-calcium response V (LcrV) antigen from Y. pseudotuberculosis (Ll-LcrV) confers protection against a lethal Y. pseudotuberculosis infection. Here, we aimed at characterizing the immunological basis of this LcrV-elicited protective response and at determining the duration of vaccine-induced immunity. METHODS: Splenocytes from BALB/c mice intranasally immunized with Ll-LcrV or Ll as control were immunostained then analyzed by flow cytometry. Protection against a lethal intravenous injection of Y. pseudotuberculosis was also determined (i) in immunized BALB/c mice depleted or not of CD4+, CD8+ or CD25+ cells and (ii) in naïve BALB/c mice receiving serum from immunized mice by counting the number of bacteria in liver and spleen. Lastly, survival rate of immunized BALB/c mice following a lethal intravenous injection of Y. pseudotuberculosis was followed up to 9-months. RESULTS: We found that T and B lymphocytes but not non-conventional lymphoid cells were affected by Ll-LcrV immunization. We also observed that depletion of CD4+ and CD25+ but not CD8+ cells in immunized mice eradicated protection against a lethal systemic Y. pseudotuberculosis infection, suggesting that activated CD4+ T lymphocytes are required for vaccine-induced protection. Adoptive transfer of LcrV-specific antibodies from Ll-LcrV-immunized animals significantly reduced the bacterial counts in the liver compared to non-vaccinated mice. Lastly, the protective immunity conferred by Ll-LcrV decreased slightly over time; nevertheless almost 60% of the mice survived a lethal bacterial challenge at 9months post-vaccination. CONCLUSION: Mucosal vaccination of mice with Ll-LcrV induced cell- and antibody-mediated protective immunity against Y. pseudotuberculosis infection in the mouse and the protection is long-lasting.

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.

Bioluminescence imaging study of spatial and temporal persistence of Lactobacillus plantarum and Lactococcus lactis in living mice.

Lactic acid bacteria, especially lactobacilli, are common inhabitants of the gastrointestinal tract of mammals, for which they have received considerable attention due to their putative health-promoting properties. In this study, we describe the development and application of luciferase-expressing Lactobacillus plantarum and Lactococcus lactis strains for noninvasive in vivo monitoring in the digestive tract of mice. We report for the first time the functional in vitro expression in Lactobacillus plantarum NCIMB8826 and in Lactococcus lactis MG1363 of the click beetle luciferase (CBluc), as well as Gaussia and bacterial luciferases, using a combination of vectors, promoters, and codon-optimized genes. We demonstrate that a CBluc construction is the best-performing luciferase system for the noninvasive in vivo detection of lactic acid bacteria after oral administration. The persistence and viability of both strains was studied by bioluminescence imaging in anesthetized mice and in mouse feces. In vivo bioluminescence imaging confirmed that after a single or multiple oral administrations, L. lactis has shorter survival times in the mouse gastrointestinal tract than L. plantarum, and it also revealed the precise gut compartments where both strains persisted. The application of luciferase-labeled bacteria has significant potential to allow the in vivo and ex vivo study of the interactions of lactic acid bacteria with their mammalian host.

Protection against Yersinia pseudotuberculosis infection conferred by a Lactococcus lactis mucosal delivery vector secreting LcrV.

Herein, we sought to evaluate the potential of a recombinant Lactococcus lactis strain secreting the Yersinia pseudotuberculosis low-calcium response V (LcrV) antigen for mucosal vaccination against Yersinia infections. We showed that the recombinant strain induced specific systemic and mucosal antibody and cellular immune responses after intranasal immunization and protected mice against both oral and systemic Y. pseudotuberculosis infections. This constitutes the first proof of principle for a novel anti-Yersinia mucosal vaccination strategy using recombinant lactic acid bacteria.

Therapeutic potential of Yersinia anti-inflammatory components.

Microbial pathogens have developed various stratagems for modulating and/or circumventing the host's innate and adaptive immunity. Hence, certain virulence factors can be viewed as potential therapeutic agents for human immunopathological diseases. This is the case for virulence plasmid-encoded proteins from pathogenic Yersiniae that inhibit the host's inflammatory response by interfering with various cellular signaling pathways.

Prevention and treatment of colitis with Lactococcus lactis secreting the immunomodulatory Yersinia LcrV protein.

BACKGROUND & AIMS: The low calcium response V (LcrV) protein synthesized by gram-negative, pathogenic yersiniae participates in bacterial evasion of the host's innate immune response by stimulating synthesis of the anti-inflammatory interleukin (IL)-10 and preventing the activation of proinflammatory cytokines. METHODS: We genetically engineered the food-grade bacterium Lactococcus lactis to secrete the LcrV protein from the enteropathogenic species Yersinia pseudotuberculosis. The protective and therapeutic potential of orally administered LcrV-secreting L lactis was evaluated in 2 models of acute experimental colitis (induced by trinitrobenzene sulfonic acid [TNBS] and dextran sodium sulfate [DSS], respectively) in wild-type and knockout mice. RESULTS: Oral administration of LcrV-secreting L lactis led to active delivery of LcrV and induction of IL-10 (via a Toll-like receptor 2-dependent pathway) in the colon and prevented TNBS-induced colitis, in contrast to the L lactis control not producing LcrV. Down-regulation of tissue inflammatory markers correlated well with the reduction in damage to the colonic mucosa. In contrast, TNBS-induced colitis was not prevented in IL-10(-/-) mice pretreated with LcrV-secreting L lactis, thus showing that IL-10 is required for LcrV protection. Administration of LcrV-secreting L lactis also proved to be very effective in preventing and treating acute DSS-induced colitis. CONCLUSIONS: LcrV-secreting L lactis decreased experimentally induced intestinal inflammation in 2 murine models of colitis. This novel approach highlights the potential of using pathogen-derived immunomodulating molecules in vivo as novel therapeutics for inflammatory bowel diseases.

Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria.

AIM: To investigate the correlation between in vitro and in vivo immunomodulation potential of the probiotic strain and its ability to prevent experimental colitis in mice. METHODS: In vitro immunomodulation was assessed by measuring interleukin (IL)-12p70, IL-10, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) release by human peripheral blood mononuclear cells (PBMCs) after 24 h stimulation with 13 live bacterial strains. A murine model of acute TNBS-colitis was next used to evaluate the prophylactic protective capacity of the same set of strains. RESULTS: A strain-specific in vivo protection was observed. The strains displaying an in vitro potential to induce higher levels of the anti-inflammatory cytokine IL-10 and lower levels of the inflammatory cytokine IL-12, offered the best protection in the in vivo colitis model. In contrast, strains leading to a low IL-10/IL-12 cytokine ratio could not significantly attenuate colitis symptoms. CONCLUSION: These results show that we could predict the in vivo protective capacity of the studied lactic acid bacteria (LAB) based on the cytokine profile we established in vitro. The PBMC-based assay we used may thus serve as a useful primary indicator to narrow down the number of candidate strains to be tested in murine models for their anti-inflammatory potential.

Selecting lactic acid bacteria for their safety and functionality by use of a mouse colitis model.

Studies showed that specific probiotics might provide therapeutic benefits in inflammatory bowel disease. However, a rigorous screening of new probiotics is needed to study possible adverse interactions with the host, particularly when intended for administration to individuals with certain health risks. In this context, the objective of this study was to investigate the role of three lactobacilli (LAB) on intestinal inflammation and bacterial translocation using variations of the mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute colitis. We first compared the in vitro ability of LAB to survive gastrointestinal tract (GIT) conditions and their ability to persist in the GIT of mice following daily oral administration. As a control, we included a nonprobiotic Lactobacillus paracasei strain, previously isolated from an endocarditis patient. Feeding high doses of LAB strains to healthy and to TNBS-treated mice did not induce any detrimental effect or abnormal translocation of the bacteria. Oral administration of Lactobacillus salivarius Ls-33 had a significant preventive effect on colitis in mice, while Lactobacillus plantarum Lp-115 and Lactobacillus acidophilus NCFM did not. None of the three selected LAB strains translocated to extraintestinal organs of TNBS-treated mice. In contrast, L. paracasei exacerbated colitis under severe inflammatory conditions and translocated to extraintestinal organs. This study showed that evaluations of the safety and functionality of new probiotics are recommended. We conclude that not all lactobacilli have similar effects on intestinal inflammation and that selected probiotics such as L. salivarius Ls-33 may be considered in the prevention or treatment of intestinal inflammation.