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Abstract Glutamine synthetases (GS) catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be under tight control to fit cellular needs. While their catalytic mechanisms and regulations are well-characterized in bacteria and eukaryotes, only limited knowledge exists in archaea. Here, we solved two archaeal GS structures and unveiled unexpected differences in their regulatory mechanisms. GS from Methanothermococcus thermolithotrophicus is inactive in its resting state and switched on by 2-oxoglutarate, a sensor of cellular nitrogen deficiency. The enzyme activation overlays remarkably well with the reported cellular concentration for 2-oxoglutarate. Its binding to an allosteric pocket reconfigures the active site through long-range conformational changes. The homolog from Methermicoccus shengliensis does not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited through glutamine recognition by the catalytic Asp50ʹ-loop, a mechanism common to bacterial homologs, but absent in M. thermolithotrophicus due to residue substitution. Analyses of residue conservation in archaeal GS suggest that both regulations are widespread and not mutually exclusive. While the effectors and their binding sites are surprisingly different, the molecular mechanisms underlying their mode of action on GS activity operate on the same molecular determinants in the active site.

Abstract Background Transcription bridges genetic information and phenotypes. Here, we evaluated how changes in transcriptional regulation enable maize (Zea mays), a crop originally domesticated in the tropics, to adapt to temperate environments. Result We generated 572 unique RNA-seq datasets from the roots of 340 maize genotypes. Genes involved in core processes such as cell division, chromosome organization and cytoskeleton organization showed lower heritability of gene expression, while genes involved in anti-oxidation activity exhibited higher expression heritability. An expression genome-wide association study (eGWAS) identified 19,602 expression quantitative trait loci (eQTLs) associated with the expression of 11,444 genes. A GWAS for alternative splicing identified 49,897 splicing QTLs (sQTLs) for 7614 genes. Genes harboring both cis-eQTLs and cis-sQTLs in linkage disequilibrium were disproportionately likely to encode transcription factors or were annotated as responding to one or more stresses. Independent component analysis of gene expression data identified loci regulating co-expression modules involved in oxidation reduction, response to water deprivation, plastid biogenesis, protein biogenesis, and plant-pathogen interaction. Several genes involved in cell proliferation, flower development, DNA replication, and gene silencing showed lower gene expression variation explained by genetic factors between temperate and tropical maize lines. A GWAS of 27 previously published phenotypes identified several candidate genes overlapping with genomic intervals showing signatures of selection during adaptation to temperate environments. Conclusion Our results illustrate how maize transcriptional regulatory networks enable changes in transcriptional regulation to adapt to temperate regions.

Abstract Background Pervasive translation is a widespread phenomenon that plays a critical role in the emergence of novel microproteins, but the diversity of translation patterns contributing to their generation remains unclear. Based on 54 ribosome profiling (Ribo-Seq) datasets, we investigated the yeast Ribo-Seq landscape using a representation framework that allows the comprehensive inventory and classification of the entire diversity of Ribo-Seq signals, including non-canonical ones. Results We show that if coding regions occupy specific areas of the Ribo-Seq landscape, noncoding regions encompass a wide diversity of Ribo-Seq signals and, conversely, populate the entire landscape. Our results show that pervasive translation can, nevertheless, be associated with high specificity, with 1055 noncoding ORFs exhibiting canonical Ribo-Seq signals. Using mass spectrometry under standard conditions or proteasome inhibition with an in-house analysis protocol, we report 239 microproteins originating from noncoding ORFs that display canonical but also non-canonical Ribo-Seq signals. Each condition yields dozens of additional microprotein candidates with comparable translation properties, suggesting a larger population of volatile microproteins that are challenging to detect. Our findings suggest that non-canonical translation signals may harbor valuable information and underscore the significance of considering them in proteogenomic studies. Finally, we show that the translation outcome of a noncoding ORF is primarily determined by the initiating codon and the codon distribution in its two alternative frames, rather than features indicative of functionality. Conclusion Our results enable us to propose a topology of a species’ Ribo-Seq landscape, opening the way to comparative analyses of this translation landscape under different conditions.

During tuberculosis (TB), migration of dendritic cells (DCs) from the site of infection to the draining lymph nodes is known to be impaired, hindering the rapid development of protective T-cell-mediated immunity. However, the mechanisms involved in the delayed migration of DCs during TB are still poorly defined. Here, we found that infection of DCs with Mycobacterium tuberculosis (Mtb) triggers HIF1A-mediated aerobic glycolysis in a TLR2-dependent manner, and that this metabolic profile is essential for DC migration. In particular, the lactate dehydrogenase inhibitor oxamate and the HIF1A inhibitor PX-478 abrogated Mtb-induced DC migration in vitro to the lymphoid tissue-specific chemokine CCL21, and in vivo to lymph nodes in mice. Strikingly, we found that although monocytes from TB patients are inherently biased toward glycolysis metabolism, they differentiate into poorly glycolytic and poorly migratory DCs compared with healthy subjects. Taken together, these data suggest that because of their preexisting glycolytic state, circulating monocytes from TB patients are refractory to differentiation into migratory DCs, which may explain the delayed migration of these cells during the disease and opens avenues for host-directed therapies for TB.

The general objective of this work was to isolate from yoghourts cultured strains of lactobacilli with potential for use as probiotics in poultry farming. Three yoghourts were cultured to see the presence of lactobacilli in the Rogosa agar base culture medium. It was found that only one yoghourt (number 1) showed the growth of lactobacilli. This yoghourt was immediately selected for further cultivation. Afterwards, the Lactobacillus strains were isolated and fortified under CO2 and then inoculated into a solution of peptone water, which constituted inoculums to be administered to an experimental group of poultry. Another group served as controls. Pathogenic strains of Escherichia coli were also administered to both groups (experimental and control, each comprising five hens). The results showed significant weight gain from the experimental group (positive effect on immunity) and freedom from disease after an incubation period (inhibitory effect of the lactobacillus strains on the Escherichia coli strains), whereas the control group showed less weight gain than the experimental group and development of colibacillosis after an incubation period. The positive effects of the Lactobacillus strains observed on the poultry of the experimental group proved the ecological role of these microorganisms in improving the health of the poultry by inhibiting the effects of pathogenic Escherichia coli strains. This suggests reassuring prospects for the reduction of antibiotic use in both human and animal care.

Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

Abstract Background Internal tandem duplications in the FLT3 gene, termed FLT3-ITDs, are useful molecular markers in acute myeloid leukemia (AML) for patient risk stratification and follow-up. FLT3-ITDs are increasingly screened through high-throughput sequencing (HTS) raising the need for robust and efficient algorithms. We developed a new algorithm, which performs no alignment and uses little resources, to identify and quantify FLT3-ITDs in HTS data. Results Our algorithm (FiLT3r) focuses on the k-mers from reads covering FLT3 exons 14 and 15. We show that those k-mers bring enough information to accurately detect, determine the length and quantify FLT3-ITD duplications. We compare the performances of FiLT3r to state-of-the-art alternatives and to fragment analysis, the gold standard method, on a cohort of 185 AML patients sequenced with capture-based HTS. On this dataset FiLT3r is more precise (no false positive nor false negative) than the other software evaluated. We also assess the software on public RNA-Seq data, which confirms the previous results and shows that FiLT3r requires little resources compared to other software. Conclusion FiLT3r is a free software available at https://gitlab.univ-lille.fr/filt3r/filt3r . The repository also contains a Snakefile to reproduce our experiments. We show that FiLT3r detects FLT3-ITDs better than other software while using less memory and time.

A cell’s ability to secrete extracellular vesicles (EVs) for communication is present in all three domains of life. Notably, Gram-negative bacteria produce a specific type of EVs called outer membrane vesicles (OMVs). We previously observed the presence of OMVs in human blood, which could represent a means of communication from the microbiota to the host. Here, in order to investigate the possible translocation of OMVs from the intestine to other organs, the mouse was used as an animal model after OMVs administration. To achieve this, we first optimized the signal of OMVs containing the fluorescent protein miRFP713 associated with the outer membrane anchoring peptide OmpA by adding biliverdin, a fluorescence cofactor, to the cultures. The miRFP713-expressing OMVs produced in E. coli REL606 strain were then characterized according to their diameter and protein composition. Native- and miRFP713-expressing OMVs were found to produce homogenous populations of vesicles. Finally, in vivo and ex vivo fluorescence imaging was used to monitor the distribution of miRFP713-OMVs in mice in various organs whether by intravenous injection or oral gavage. The relative stability of the fluorescence signals up to 3 days post-injection/gavage paves the way to future studies investigating the OMV-based communication established between the different microbiotas and their host.

For forty years, there has been growing uncertainty about whether Hill's horseshoe bat (Rhinolophus hilli) still persists in Nyungwe National Park, Rwanda. Only known from one small area within the National Park, R. hilli is listed as Critically Endangered by the International Union for the Conservation of Nature (IUCN), based on its extremely small geographic range and presumed low number of mature individuals. Here, we present and describe bat species occurrence data contributed to the Global Biodiversity Information Facility (GBIF) that we collected as part of a long-term collaborative project to rediscover this lost species. This data paper describes the survey methods and findings resulting from cave roost surveys, capture surveys, and acoustic sampling of bat echolocation activity in Nyungwe National Park and surrounding areas in south-western Rwanda from 2013-2020 and their conservation relevance.We report the discovery of an extant population of Hill's horseshoe bat (Rhinolophus hilli) in Nyungwe National Park, Rwanda, 40 years since the last reported observation of the species in 1981. We also report the first record of Lander's horseshoe bat (Rhinolophus landeri) in Nyungwe National Park and the first record of the Damara woolly bat (Kerivoula argentata) in Rwanda. The dataset contributed to GBIF and described in this paper includes 278 occurrence records from 10 bat species of five families detected at 71 locations in or near Nyungwe National Park, Rwanda. We include a description of the morphological descriptions of R. hilli and present the first acoustic echolocation signatures and phylogenetic information for this species.

Xer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild-type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.

The coronavirus disease 2019 (COVID-19) pandemic continues to cause significant morbidity and mortality worldwide. Since a large portion of the world’s population is currently unvaccinated or incompletely vaccinated and has limited access to approved treatments against COVID-19, there is an urgent need to continue research on treatment options, especially those at low cost and which are immediately available to patients, particularly in low- and middle-income countries. Prior in vitro and observational studies have shown that fluoxetine, possibly through its inhibitory effect on the acid sphingomyelinase/ceramide system, could be a promising antiviral and anti-inflammatory treatment against COVID-19. In this report, we evaluated the potential antiviral and anti-inflammatory activities of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and against variants of concern in vitro, i.e., SARS-CoV-2 ancestral strain, Alpha B.1.1.7, Gamma P1, Delta B1.617 and Omicron BA.5. Fluoxetine, administrated after SARS-CoV-2 infection, significantly reduced lung tissue viral titres and expression of several inflammatory markers (i.e., IL-6, TNFα, CCL2 and CXCL10). It also inhibited the replication of all variants of concern in vitro. A modulation of the ceramide system in the lung tissues, as reflected by the increase in the ratio HexCer 16:0/Cer 16:0 in fluoxetine-treated mice, may contribute to explain these effects. Our findings demonstrate the antiviral and anti-inflammatory properties of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and its in vitro antiviral activity against variants of concern, establishing fluoxetine as a very promising candidate for the prevention and treatment of SARS-CoV-2 infection and disease pathogenesis.

The ability of Mycobacterium tuberculosis (Mtb) to persist inside host cells relies on metabolic adaptation, like the accumulation of lipid bodies (LBs) in the so-called foamy macrophages (FM), which are favorable to Mtb. The activation state of macrophages is tightly associated to different metabolic pathways, such as lipid metabolism, but whether differentiation towards FM differs between the macrophage activation profiles remains unclear. Here, we aimed to elucidate whether distinct macrophage activation states exposed to a tuberculosis-associated microenvironment or directly infected with Mtb can form FM. We showed that the triggering of signal transducer and activator of transcription 6 (STAT6) in interleukin (IL)-4-activated human macrophages (M(IL-4)) prevents FM formation induced by pleural effusion from patients with tuberculosis. In these cells, LBs are disrupted by lipolysis, and the released fatty acids enter the β-oxidation (FAO) pathway fueling the generation of ATP in mitochondria. Accordingly, murine alveolar macrophages, which exhibit a predominant FAO metabolism, are less prone to become FM than bone marrow derived-macrophages. Interestingly, direct infection of M(IL-4) macrophages with Mtb results in the establishment of aerobic glycolytic pathway and FM formation, which could be prevented by FAO activation or inhibition of the hypoxia-inducible factor 1-alpha (HIF-1α)-induced glycolytic pathway. In conclusion, our results demonstrate that Mtb has a remarkable capacity to induce FM formation through the rewiring of metabolic pathways in human macrophages, including the STAT6-driven alternatively activated program. This study provides key insights into macrophage metabolism and pathogen subversion strategies.

While tuberculosis (TB) is a risk factor in HIV-1-infected individuals, the mechanisms by which Mycobacterium tuberculosis (Mtb) worsens HIV-1 pathogenesis remain scarce. We showed that HIV-1 infection is exacerbated in macrophages exposed to TB-associated microenvironments due to tunneling nanotube (TNT) formation. To identify molecular factors associated with TNT function, we performed a transcriptomic analysis in these macrophages, and revealed the up-regulation of Siglec-1 receptor. Siglec-1 expression depends on Mtb-induced production of type I interferon (IFN-I). In co-infected non-human primates, Siglec-1 is highly expressed by alveolar macrophages, whose abundance correlates with pathology and activation of IFN-I/STAT1 pathway. Siglec-1 localizes mainly on microtubule-containing TNT that are long and carry HIV-1 cargo. Siglec-1 depletion decreases TNT length, diminishes HIV-1 capture and cell-to-cell transfer, and abrogates the exacerbation of HIV-1 infection induced by Mtb. Altogether, we uncover a deleterious role for Siglec-1 in TB-HIV-1 co-infection and open new avenues to understand TNT biology.

The recruitment of pericytes and vascular smooth muscle cells (SMCs) that enwrap endothelial cells (ECs) is a crucial process for vascular maturation and stabilization. Communication between these two cell types is crucial during vascular development and in maintaining vessel homeostasis. Extracellular vesicles (EVs) have emerged as a new communication tool involving the exchange of microRNAs between cells. In the present study, we searched for microRNAs that could be transferred via EVs from ECs to SMCs and vice versa. Thanks to a microRNA profiling experiment, we found that two microRNAs are more exported in each cell type in coculture experiments: while miR-539 is more secreted by ECs, miR-582 is more present in EVs from SMCs. Functional assays revealed that both microRNAs can modulate both cell-type phenotypes. We further identified miR-539 and miR-582 targets, in agreement with their respective cell functions. The results obtained in vivo in the neovascularization model suggest that miR-539 and miR-582 might cooperate to trigger the process of blood vessel coverage by smooth muscle cells in a mature plexus. Taken together, these results are the first to highlight the role of miR-539 and miR-582 in angiogenesis and communication between ECs and SMCs.

Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning. In this review, we present the different modifications and how they are deposited on the rRNA. We also describe the most recent results showing that the modified positions are not 100% modified, which creates a heterogeneous population of ribosomes. This gave rise to the concept of specialized ribosomes that we discuss. The knowledge accumulated in the yeast Saccharomyces cerevisiae is very helpful to better understand the role of rRNA modifications in humans, especially in ribosomopathies.

Summary: The tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), and HIV-1 act synergistically; however, the mechanisms by which Mtb exacerbates HIV-1 pathogenesis are not well known. Using in vitro and ex vivo cell culture systems, we show that human M(IL-10) anti-inflammatory macrophages, present in TB-associated microenvironment, produce high levels of HIV-1. In vivo, M(IL-10) macrophages are expanded in lungs of co-infected non-human primates, which correlates with disease severity. Furthermore, HIV-1/Mtb co-infected patients display an accumulation of M(IL-10) macrophage markers (soluble CD163 and MerTK). These M(IL-10) macrophages form direct cell-to-cell bridges, which we identified as tunneling nanotubes (TNTs) involved in viral transfer. TNT formation requires the IL-10/STAT3 signaling pathway, and targeted inhibition of TNTs substantially reduces the enhancement of HIV-1 cell-to-cell transfer and overproduction in M(IL-10) macrophages. Our study reveals that TNTs facilitate viral transfer and amplification, thereby promoting TNT formation as a mechanism to be explored in TB/AIDS potential therapeutics. : Tuberculosis is a clear, yet confounding, risk factor for HIV-1-induced morbidity and mortality. In this issue, Souriant et al. reveal that a tuberculosis-associated microenvironment triggers IL-10/STAT3-dependent tunneling nanotube formation in M(IL-10) macrophages, which promotes HIV-1 exacerbation during co-infection. M(IL-10) macrophage accumulation is also observed in vivo in co-infected subjects. Keywords: AIDS, HIV-1, tuberculosis, Mycobacterium tuberculosis, co-infection, macrophage, monocyte, IL-10, STAT3, viral spread, tunneling nanotubes, biomarker

The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t6A and mcm5s2U are not determinants for each other’s formation. Our results suggest that t6A deficiency, like mcm5s2U deficiency, leads to protein folding defects, and show that the absence of t6A led to stress sensitivities (heat, ethanol, salt) and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t6A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs) were increased in the mutants. The global consequences on translation caused by t6A absence were examined by ribosome profiling. Interestingly, the absence of t6A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t6A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t6A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.

The calcium/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous and central player in Ca2+ signaling that is best known for its functions in the brain. In particular, the α isoform of CaMKII has been the subject of intense research and it has been established as a central regulator of neuronal plasticity. In contrast, little attention has been paid to CaMKIIβ, the other predominant brain isoform that interacts directly with the actin cytoskeleton, and the functions of CaMKIIβ in this organ remain largely unexplored. However, recently, the perturbation of CaMKIIβ expression has been associated with multiple neuropsychiatric and neurodevelopmental diseases, highlighting CAMK2B as a gene of interest. Herein, after highlighting the main structural and expression differences between the α and β isoforms, we will review the specific functions of CaMKIIβ, as described so far, in neuronal development and plasticity, as well as its potential implication in brain diseases.

The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

The range of hosts that parasites can infect is a key determinant of the emergence and spread of disease. Yet, the impact of host range variation on the evolution of parasite genomes remains unknown. Here, we show that codon optimization underlies genome adaptation in broad host range parasites. We found that the longer proteins encoded by broad host range fungi likely increase natural selection on codon optimization in these species. Accordingly, codon optimization correlates with host range across the fungal kingdom. At the species level, biased patterns of synonymous substitutions underpin increased codon optimization in a generalist but not a specialist fungal pathogen. Virulence genes were consistently enriched in highly codon-optimized genes of generalist but not specialist species. We conclude that codon optimization is related to the capacity of parasites to colonize multiple hosts. Our results link genome evolution and translational regulation to the long-term persistence of generalist parasitism.

Prions are infectious proteins that can adopt a structural conformation that is then propagated among other molecules of the same protein. [PSI+] is an aggregated conformation of the translational release factor eRF3. [PSI+] modifies cellular fitness, inducing various phenotypes depending on genetic background. However, the genes displaying [PSI+]-controlled expression remain unknown. We used ribosome profiling in isogenic [PSI+] and [psi−] strains to identify the changes induced by [PSI+]. We found 100 genes with stop codon readthrough events and showed that many stress-response genes were repressed in the presence of [PSI+]. Surprisingly, [PSI+] was also found to affect reading frame selection independently of its effect on translation termination efficiency. These results indicate that [PSI+] has a broader impact than initially anticipated, providing explanations for the phenotypic differences between [psi−] and [PSI+] strains.

Melatonin receptors have been studied for several decades. The low expression of the receptors in tissues led the scientific community to find a substitute for the natural hormone melatonin, the agonist 2-[125I]-iodomelatonin. Using the agonist, several hundreds of studies were conducted, including the discovery of agonists and antagonists for the receptors and minute details about their molecular behavior. Recently, we attempted to expand the panel of radioligands available for studying the melatonin receptors by using the newly discovered compounds SD6, DIV880, and S70254. These compounds were characterized for their affinities to the hMT1 and hMT2 recombinant receptors and their functionality in the classical GTPS system. SD6 is a full agonist, equilibrated between the receptor isoforms, whereas S70254 and DIV880 are only partial MT2 agonists, with Ki in the low nanomolar range while they have no affinity to MT1 receptors. These new tools will hopefully allow for additions to the current body of information on the native localization of the receptor isoforms in tissues.

For many years, it was of interest to identify the sequences encoding the two melatonin receptors (MT1 and MT2) from various species. After publishing the basic molecular characterization of the human, rat, mouse, sheep, and platypus MT1, MT2, or Mel1c receptors, we began cloning the genes from other animals, such as birds, bats, and vipers. The goal was to advance the receptor crystallization, which could greatly contribute the understanding of the sequence/stability relationship. European hamster MT1 receptor was cloned for the first time from this gender, was expressed in stable form in cells, and its binding characterized with a sample of 19 melatonin ligands. Siberian hamster (Phodopus sungorus) expresses a non-functional MT2. We observed that unlike this hamster, the European hamster (Cricetus cricetus) does not have a stop codon in the MT2 sequence. Thus, we undertook the tedious task of cloning the MT2 receptor. We partially succeeded, sequencing the complete exon 2 and a fragment of exon 1 (from putative amino acids 12 to 38 and 77 to 323), after several years of efforts. In order to show that the protein parts we cloned were capable to sustain some binding capacities, we designed a chimeric MT2 receptor using a consensus sequence to replace the unknown amino acids, based on other small rodent MT2 sequences. This chimeric construct could bind melatonin in the nanomolar range. This work is meant to be the basis for attempts from other laboratories of the community to determine the complete natural sequence of the European hamster MT2 receptor. The present work is the first to show that, among the hamsters, if the Siberian is a natural knockout for MT2, the European one is not.

Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s), that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes.

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.