Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8+ T lymphocytes

CD8⁺ cytotoxic T lymphocytes (CTLs) are critical mediators of anti‐tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen‐presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c‐Met (c‐Met⁺ CTLs). Phenotypic and functional analysis of c‐Met⁺ CTLs reveals that they display enhanced cytolytic capacities compared to their c‐Met^? CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c‐Met⁺ CTLs in cell‐mediated cytotoxicity reactions in vitro and in vivo and abrogates T‐cell responses against metastatic melanoma in vivo. Finally, we establish in three murine tumor settings and in human melanoma tissues that c‐Met⁺ CTLs are a naturally occurring CD8⁺ T‐cell population. Together, our findings suggest that the HGF/c‐Met pathway could be exploited to control CD8⁺ T‐cell‐mediated anti‐tumor immunity.     

Synthesis and in vitro antikinetoplastid activity of polyamine–hydroxybenzotriazole conjugates

Thirteen new polyamine derivatives coupled to hydroxybenzotriazole have been synthesized and evaluated for their in vitro antikinetoplastid activity. Trypanosoma Trypanothione reductase (TryR) was envisioned as a potential target. Among all tested molecules, only one compound, a N³-spermidine–benzotriazole derivative, displayed relevant inhibitory activity on this enzyme but was not active on parasites. The corresponding Boc-protected spermidine–benzotriazole was however trypanocidal against Trypanosoma brucei gambiense with an IC₅₀ value of 1 μM and was completely devoid of cytotoxicity. On the intramacrophage amastigotes of Leishmania donovani, a N²-spermidine conjugate of this series, exhibited an interesting IC₅₀ value of 3 μM associated with both low cytotoxicity against axenic Leishmania donovani. These new compounds are promising leads for the development of antikinetoplastid agents and their targets have to be deciphered.     

Initiation of Batrachochytrium dendrobatidis infection in the absence of physical contact with infected hosts – a field study in a high altitude lake

Understanding transmission is a critical prerequisite for predicting disease dynamics and impacts on host populations. It is well established that Batrachochytrium dendrobatidis (Bd), the amphibian fungal pathogen responsible for chytridiomycosis, can be transmitted directly, through physical contact with an infected host. However, indirect pathways of transmission remain poorly investigated. We conducted a five‐week long field infection experiment at a high altitude mountain lake in the French Pyrenees to investigate Bd transmission pathways in larval midwife toads Alytes obstetricans. Uninfected naïve tadpoles were co‐housed either with infected tadpoles (direct and indirect transmission) or with uninfected ones (indirect transmission only). We found that physical contact with an infected host is not necessary for initial infection with Bd and that all tadpoles became infected after only four weeks. However, physical contact with infected tadpoles led to a faster spread within a tadpole group and resulted in higher Bd loads and subsequently higher mortality. Our findings clearly demonstrate that in A. obstetricans, Bd can quickly spread in a population even without physical contact. Our experiment therefore stresses the importance of indirect transmission of Bd zoospores in infected lakes for disease dynamics, especially when a reservoir species such as A. obstetricans is present.     

Protein social behavior makes a stronger signal for partner identification than surface geometry

Cells are interactive living systems where proteins movements, interactions and regulation are substantially free from centralized management. How protein physico‐chemical and geometrical properties determine who interact with whom remains far from fully understood. We show that characterizing how a protein behaves with many potential interactors in a complete cross‐docking study leads to a sharp identification of its cellular/true/native partner(s). We define a sociability index, or S‐index, reflecting whether a protein likes or not to pair with other proteins. Formally, we propose a suitable normalization function that accounts for protein sociability and we combine it with a simple interface‐based (ranking) score to discriminate partners from non‐interactors. We show that sociability is an important factor and that the normalization permits to reach a much higher discriminative power than shape complementarity docking scores. The social effect is also observed with more sophisticated docking algorithms. Docking conformations are evaluated using experimental binding sites. These latter approximate in the best possible way binding sites predictions, which have reached high accuracy in recent years. This makes our analysis helpful for a global understanding of partner identification and for suggesting discriminating strategies. These results contradict previous findings claiming the partner identification problem being solvable solely with geometrical docking. Proteins 2016; 85:137–154. © 2016 Wiley Periodicals, Inc.     

Evaluation of Placental Toxicity of Five Essential Oils and Their Potential Endocrine-Disrupting Effects

Pregnant women may use EOs in case of morning sickness, nausea, stress management, etc. Little is known about the potential danger that EOs represent for the placenta and therefore for the pregnancy. Our aim was to explore and compare the placental toxicity and potential endocrine disrupting effects of niaouli, orange, tea tree, wintergreen and ylang-ylang EOs, and their key compounds: 4-terpineol, 1,8-cineol, limonene, methyl salicylate and benzyl salicylate. We studied the release of four hormones and the activation of P2X7 receptor in JEG-Tox human placental cells as key biomarkers for endocrine toxicity. We observed that niaouli, orange, tea tree, wintergreen and ylang-ylang EOs and their key components disrupted at least one of the studied hormones but none of them activated the P2X7 cell death receptor. The tested EOs appear then to be more hormonal modulators rather than EDCs in human placental cells. The hormonal effects observed with the key components were very different from those observed with the EOs. EOs are very complex mixtures, and it is essential to study whole EOs rather than their components individually in safety assessment.     

TSAP6 facilitates the secretion of translationally controlled tumor protein/histamine-releasing factor via a nonclassical pathway

Translationally controlled tumor protein (TCTP) is cytoplasmic and structurally related to guanine-nucleotide free chaperones. TCTP (also called histamine-releasing factor) has been described previously as a secreted protein that participates in inflammatory responses by promoting the release of histamine. How TCTP is eventually exported out of the cell to promote such activities is unknown. Here we show that TCTP secretion was insensitive to either brefeldin A or monensin, suggesting that it proceeds via an endoplasmic reticulum/Golgi-independent or nonclassical pathway. Moreover, our analyses also suggest that secreted TCTP originates from pre-existing pools. TSAP6, a p53-inducible 5-6 transmembrane protein, was found to interact with TCTP in a yeast two-hybrid hunt. GST pull down assays confirmed their direct interaction, and immunofluorescence analysis revealed their partial co-distribution to vesicular-like structures at the plasma membrane and around the nucleus. Functionally, the overexpression of TSAP6 consistently leads to enhanced secretion of both endogenously and exogenously expressed TCTP. Finally, we found TCTP in preparations of small secreted vesicles called exosomes, which have been suggested as a possible pathway for nonclassical secretion. Overexpression of TSAP6 also increased TCTP levels in exosome preparations. Altogether, these data identify a novel role for TSAP6 in the export of TCTP and indicate that this multipass membrane protein could have a general role in the regulation of vesicular trafficking and secretion.     

Evolutionary History and Attenuation of Myxoma Virus on Two Continents

The attenuation of myxoma virus (MYXV) following its introduction as a biological control into the European rabbit populations of Australia and Europe is the canonical study of the evolution of virulence. However, the evolutionary genetics of this profound change in host-pathogen relationship is unknown. We describe the genome-scale evolution of MYXV covering a range of virulence grades sampled over 49 years from the parallel Australian and European epidemics, including the high-virulence progenitor strains released in the early 1950s. MYXV evolved rapidly over the sampling period, exhibiting one of the highest nucleotide substitution rates ever reported for a double-stranded DNA virus, and indicative of a relatively high mutation rate and/or a continually changing selective environment. Our comparative sequence data reveal that changes in virulence involved multiple genes, likely losses of gene function due to insertion-deletion events, and no mutations common to specific virulence grades. Hence, despite the similarity in selection pressures there are multiple genetic routes to attain either highly virulent or attenuated phenotypes in MYXV, resulting in convergence for phenotype but not genotype.     

Characterization of the Bubblegum acyl-CoA synthetase of Microchloropsis gaditana

The metabolic pathways of glycerolipids are well described in cells containing chloroplasts limited by a two-membrane envelope but not in cells containing plastids limited by four membranes, including heterokonts. Fatty acids (FAs) produced in the plastid, palmitic and palmitoleic acids (16:0 and 16:1), are used in the cytosol for the synthesis of glycerolipids via various routes, requiring multiple acyl-Coenzyme A (CoA) synthetases (ACS). Here, we characterized an ACS of the Bubblegum subfamily in the photosynthetic eukaryote Microchloropsis gaditana, an oleaginous heterokont used for the production of lipids for multiple applications. Genome engineering with TALE-N allowed the generation of MgACSBG point mutations, but no knockout was obtained. Point mutations triggered an overall decrease of 16:1 in lipids, a specific increase of unsaturated 18-carbon acyls in phosphatidylcholine and decrease of 20-carbon acyls in the betaine lipid diacylglyceryl–trimethyl–homoserine. The profile of acyl-CoAs highlighted a decrease in 16:1-CoA and 18:3-CoA. Structural modeling supported that mutations affect accessibility of FA to the MgACSBG reaction site. Expression in yeast defective in acyl-CoA biosynthesis further confirmed that point mutations affect ACSBG activity. Altogether, this study supports a critical role of heterokont MgACSBG in the production of 16:1-CoA and 18:3-CoA. In M. gaditana mutants, the excess saturated and monounsaturated FAs were diverted to triacylglycerol, thus suggesting strategies to improve the oil content in this microalga.

A heterokont Bubblegum acyl-CoA synthetase (ACSBG), or lipidosin, is essential in Microchloropsis (Nannochloropsis) gaditana and thio-esterifies 16:1 and 18:3 fatty acids to Coenzyme A in vivo.