Characterization of GEXP15 as a Potential Regulator of Protein Phosphatase 1 in Plasmodium falciparum.

The Protein Phosphatase type 1 catalytic subunit (PP1c) (PF3D7_1414400) operates in combination with various regulatory proteins to specifically direct and control its phosphatase activity. However, there is little information about this phosphatase and its regulators in the human malaria parasite, Plasmodium falciparum. To address this knowledge gap, we conducted a comprehensive investigation into the structural and functional characteristics of a conserved Plasmodium-specific regulator called Gametocyte EXported Protein 15, GEXP15 (PF3D7_1031600). Through in silico analysis, we identified three significant regions of interest in GEXP15: an N-terminal region housing a PP1-interacting RVxF motif, a conserved domain whose function is unknown, and a GYF-like domain that potentially facilitates specific protein-protein interactions. To further elucidate the role of GEXP15, we conducted in vitro interaction studies that demonstrated a direct interaction between GEXP15 and PP1 via the RVxF-binding motif. This interaction was found to enhance the phosphatase activity of PP1. Additionally, utilizing a transgenic GEXP15-tagged line and live microscopy, we observed high expression of GEXP15 in late asexual stages of the parasite, with localization predominantly in the nucleus. Immunoprecipitation assays followed by mass spectrometry analyses revealed the interaction of GEXP15 with ribosomal- and RNA-binding proteins. Furthermore, through pull-down analyses of recombinant functional domains of His-tagged GEXP15, we confirmed its binding to the ribosomal complex via the GYF domain. Collectively, our study sheds light on the PfGEXP15-PP1-ribosome interaction, which plays a crucial role in protein translation. These findings suggest that PfGEXP15 could serve as a potential target for the development of malaria drugs.

Dactylosporolides: Glycosylated Macrolides from Dactylosporangium fulvum.

A series of novel macrolides were discovered from the culture supernatant of the rare soil actinobacteria Dactylosporangium fulvum and named dactylosporolides A-C. The structure and absolute configuration of these dactylosporolides were defined using a combination of NMR structural elucidation and analysis of the dactylosporolide biosynthetic gene cluster. Together these data revealed dactylosporolides to be composed of a central 22-membered macrolactone with an internal hemiketal ring and a protruding ketide tail that were (poly)glycosylated at two distal parts. While bearing no antibiotic activity, these dactylosporolides displayed activity against Plasmodium falciparum 3D7.

Identification of New Potential Biotherapeutics from Human Gut Microbiota-Derived Bacteria.

The role of the gut microbiota in health and disease is well recognized and the microbiota dysbiosis observed in many chronic diseases became a new therapeutic target. The challenge is to get a better insight into the functionality of commensal bacteria and to use this knowledge to select live biotherapeutics as new preventive or therapeutic products. In this study, we set up a screening approach to evaluate the functional capacities of a set of 21 strains isolated from the gut microbiota of neonates and adults. For this purpose, we selected key biological processes involved in the microbiome-host symbiosis and known to impact the host physiology i.e., the production of short-chain fatty acids and the ability to strengthen an epithelial barrier (Caco-2), to induce the release of the anti-inflammatory IL-10 cytokine after co-culture with human immune cells (PBMC) or to increase GLP-1 production from STC-1 endocrine cell line. This strategy highlighted fifteen strains exhibiting beneficial activities among which seven strains combined several of them. Interestingly, this work revealed for the first time a high prevalence of potential health-promoting functions among intestinal commensal strains and identified several appealing novel candidates for the management of chronic diseases, notably obesity and inflammatory bowel diseases.

Multiple Selection Criteria for Probiotic Strains with High Potential for Obesity Management.

Since alterations of the gut microbiota have been shown to play a major role in obesity, probiotics have attracted attention. Our aim was to identify probiotic candidates for the management of obesity using a combination of in vitro and in vivo approaches. We evaluated in vitro the ability of 23 strains to limit lipid accumulation in adipocytes and to enhance the secretion of satiety-promoting gut peptide in enteroendocrine cells. Following the in vitro screening, selected strains were further investigated in vivo, single, or as mixtures, using a murine model of diet-induced obesity. Strain Bifidobacterium longum PI10 administrated alone and the mixture of B. animalis subsp. lactis LA804 and Lactobacillus gasseri LA806 limited body weight gain and reduced obesity-associated metabolic dysfunction and inflammation. These protective effects were associated with changes in the hypothalamic gene expression of leptin and leptin receptor as well as with changes in the composition of gut microbiota and the profile of bile acids. This study provides crucial clues to identify new potential probiotics as effective therapeutic approaches in the management of obesity, while also providing some insights into their mechanisms of action.

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.

The UPR sensor IRE1α promotes dendritic cell responses to control Toxoplasma gondii infection.

The unfolded protein response (UPR) has emerged as a central regulator of immune cell responses in several pathologic contexts including infections. However, how intracellular residing pathogens modulate the UPR in dendritic cells (DCs) and thereby affect T cell-mediated immunity remains uncharacterized. Here, we demonstrate that infection of DCs with Toxoplasma gondii (T. gondii) triggers a unique UPR signature hallmarked by the MyD88-dependent activation of the IRE1α pathway and the inhibition of the ATF6 pathway. Induction of XBP1s controls pro-inflammatory cytokine secretion in infected DCs, while IRE1α promotes MHCI antigen presentation of secreted parasite antigens. In mice, infection leads to a specific activation of the IRE1α pathway, which is restricted to the cDC1 subset. Mice deficient for IRE1α and XBP1 in DCs display a severe susceptibility to T. gondii and succumb during the acute phase of the infection. This early mortality is correlated with increased parasite burden and a defect in splenic T-cell responses. Thus, we identify the IRE1α/XBP1s branch of the UPR as a key regulator of host defense upon T. gondii infection.

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.

Differential Fractionation of Erythrocytes Infected by Plasmodium berghei.

The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected erythrocyte. The task remains delicate despite the use of enzymes or detergents theoretically capable of degrading specific membranes inside the infected cell. The remit of this protocol is to propose a method to isolate the erythrocyte cytosol and ghosts from the other compartments of the infected cell via a percoll gradient. Also, the lysis of the erythrocyte membrane is done using equinatoxin II, which has proven to be more effective at erythrocyte lysis regardless of the cell infection status, compared to the commonly used streptolysin. The parasitophorous vacuole (PV) content is collected after saponin lysis, before recovering membrane and parasite cytosol proteins by Triton X-100 lysis. The lysates thus obtained are analyzed by Western blot to assess the accuracy of the various extraction steps. This protocol allows the separation of the host compartment from the parasite compartments (PV and parasite), leading to potential studies of host proteins as well as parasite proteins exported to the host cell.

Autophagy: A Novel Mechanism Involved in the Anti-Inflammatory Abilities of Probiotics.

BACKGROUND/AIMS: Deregulation of the complex interaction among host genetics, gut microbiota and environmental factors on one hand and aberrant immune responses on the other hand, are known to be associated with the development of inflammatory bowel disease. Recent studies provided strong evidence that autophagy plays a key role in the etiology of Crohn's disease (CD). Probiotics may exhibit many therapeutic properties, including anti-inflammatory abilities. While successful results have been obtained in ulcerative colitis patients, probiotics remain inefficient in CD for unknown reason. It remains therefore important to better understand their molecular mechanisms of action. METHODS: The activation of autophagy was examined by stimulating bone marrow-derived dendritic cells by the bacteria, followed by confocal microscopy and western blot analysis. The impact of blocking in vitro autophagy was performed in peripheral blood mononuclear cells using 3-methyl adenine or bafilomycin followed by cytokine secretion measurement by ELISA. The role of autophagy in the anti-inflammatory capacities of the bacterial strains was evaluated in vivo using an acute trinitrobenzene sulfonic acid-induced murine model of colitis. The impact of BMDC was evaluated by adoptive transfer, notably using bone marrow cells derived from autophagy-related 16-like 1-deficient mice. RESULTS: We showed that selected lactobacilli and bifidobacteria are able to induce autophagy activation in BMDCs. Blocking in vitro autophagy abolished the capacity of the strains to induce the release of the anti-inflammatory cytokine interleukin-10, while it exacerbated the secretion of the pro-inflammatory cytokine interleukin-1ß. We confirmed in the TNBS-induced mouse model of colitis that autophagy is involved in the protective capacity of these selected strains, and showed that dendritic cells are involved in this process. CONCLUSION: We propose autophagy as a novel mechanism involved in the regulatory capacities of probiotics.

Beneficial metabolic effects of selected probiotics on diet-induced obesity and insulin resistance in mice are associated with improvement of dysbiotic gut microbiota.

Alterations in gut microbiota composition and diversity were suggested to play a role in the development of obesity, a chronic subclinical inflammatory condition. We here evaluated the impact of oral consumption of a monostrain or multi-strain probiotic preparation in high-fat diet-induced obese mice. We observed a strain-specific effect and reported dissociation between the capacity of probiotics to dampen adipose tissue inflammation and to limit body weight gain. A multi-strain mixture was able to improve adiposity, insulin resistance and dyslipidemia through adipose tissue immune cell-remodelling, mainly affecting macrophages. At the gut level, the mixture modified the uptake of fatty acids and restored the expression level of the short-chain fatty acid receptor GPR43. These beneficial effects were associated with changes in the microbiota composition, such as the restoration of the abundance of Akkermansia muciniphila and Rikenellaceae and the decrease of other taxa like Lactobacillaceae. Using an in vitro gut model, we further showed that the probiotic mixture favours the production of butyrate and propionate. Our findings provide crucial clues for the design and use of more efficient probiotic preparations in obesity management and may bring new insights into the mechanisms by which host-microbe interactions govern such protective effects.