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Background Akkermansia muciniphila is a human gut microbe with a key role in the physiology of the intestinal mucus layer and reported associations with decreased body mass and increased gut barrier function and health. Despite its biomedical relevance, the genomic diversity of A. muciniphila remains understudied and that of closely related species, except for A. glycaniphila, unexplored. Results We present a large-scale population genomics analysis of the Akkermansia genus using 188 isolate genomes and 2226 genomes assembled from 18,600 metagenomes from humans and other animals. While we do not detect A. glycaniphila, the Akkermansia strains in the human gut can be grouped into five distinct candidate species, including A. muciniphila, that show remarkable whole-genome divergence despite surprisingly similar 16S rRNA gene sequences. These candidate species are likely human-specific, as they are detected in mice and non-human primates almost exclusively when kept in captivity. In humans, Akkermansia candidate species display ecological co-exclusion, diversified functional capabilities, and distinct patterns of associations with host body mass. Analysis of CRISPR-Cas loci reveals new variants and spacers targeting newly discovered putative bacteriophages. Remarkably, we observe an increased relative abundance of Akkermansia when cognate predicted bacteriophages are present, suggesting ecological interactions. A. muciniphila further exhibits subspecies-level genetic stratification with associated functional differences such as a putative exo/lipopolysaccharide operon. Conclusions We uncover a large phylogenetic and functional diversity of the Akkermansia genus in humans. This variability should be considered in the ongoing experimental and metagenomic efforts to characterize the health-associated properties of A. muciniphila and related bacteria.

Background Akkermansia muciniphila is a human gut microbe with a key role in the physiology of the intestinal mucus layer and reported associations with decreased body mass and increased gut barrier function and health. Despite its biomedical relevance, the genomic diversity of A. muciniphila remains understudied and that of closely related species, except for A. glycaniphila, unexplored. Results We present a large-scale population genomics analysis of the Akkermansia genus using 188 isolate genomes and 2226 genomes assembled from 18,600 metagenomes from humans and other animals. While we do not detect A. glycaniphila, the Akkermansia strains in the human gut can be grouped into five distinct candidate species, including A. muciniphila, that show remarkable whole-genome divergence despite surprisingly similar 16S rRNA gene sequences. These candidate species are likely human-specific, as they are detected in mice and non-human primates almost exclusively when kept in captivity. In humans, Akkermansia candidate species display ecological co-exclusion, diversified functional capabilities, and distinct patterns of associations with host body mass. Analysis of CRISPR-Cas loci reveals new variants and spacers targeting newly discovered putative bacteriophages. Remarkably, we observe an increased relative abundance of Akkermansia when cognate predicted bacteriophages are present, suggesting ecological interactions. A. muciniphila further exhibits subspecies-level genetic stratification with associated functional differences such as a putative exo/lipopolysaccharide operon. Conclusions We uncover a large phylogenetic and functional diversity of the Akkermansia genus in humans. This variability should be considered in the ongoing experimental and metagenomic efforts to characterize the health-associated properties of A. muciniphila and related bacteria.

Background: Akkermansia muciniphila is a human gut microbe with a key role in the physiology of the intestinal mucus layer and reported associations with decreased body mass and increased gut barrier function and health. Despite its biomedical relevance, the genomic diversity of A. muciniphila remains understudied and that of closely related species, except for A. glycaniphila, unexplored. Results: We present a large-scale population genomics analysis of the Akkermansia genus using 188 isolate genomes and 2226 genomes assembled from 18,600 metagenomes from humans and other animals. While we do not detect A. glycaniphila, the Akkermansia strains in the human gut can be grouped into five distinct candidate species, including A. muciniphila, that show remarkable whole-genome divergence despite surprisingly similar 16S rRNA gene sequences. These candidate species are likely human-specific, as they are detected in mice and non-human primates almost exclusively when kept in captivity. In humans, Akkermansia candidate species display ecological co-exclusion, diversified functional capabilities, and distinct patterns of associations with host body mass. Analysis of CRISPR-Cas loci reveals new variants and spacers targeting newly discovered putative bacteriophages. Remarkably, we observe an increased relative abundance of Akkermansia when cognate predicted bacteriophages are present, suggesting ecological interactions. A. muciniphila further exhibits subspecies-level genetic stratification with associated functional differences such as a putative exo/lipopolysaccharide operon. Conclusions: We uncover a large phylogenetic and functional diversity of the Akkermansia genus in humans. This variability should be considered in the ongoing experimental and metagenomic efforts to characterize the health-associated properties of A. muciniphila and related bacteria.

Understanding the viral-host cell interface during HIV-1 infection is a prerequisite for the development of innovative antiviral therapies. Here we show that the suppressor of G2 allele of skp1 (SUGT1) is a permissive factor for human immunodeficiency virus (HIV)-1 infection. Expression of SUGT1 increases in infected cells on human brain sections and in permissive host cells. We found that SUGT1 determines the permissiveness to infection of lymphocytes and macrophages by modulating the nuclear import of the viral genome. More importantly, SUGT1 stabilizes the microtubule plus-ends (+MTs) of host cells (through the modulation of microtubule acetylation and the formation of end-binding protein 1 (EB1) comets). This effect on microtubules favors HIV-1 retrograde trafficking and replication. SUGT1 depletion impairs the replication of HIV-1 patient primary isolates and mutant virus that is resistant to raltegravir antiretroviral agent. Altogether our results identify SUGT1 as a cellular factor involved in the post-entry steps of HIV-1 infection that may be targeted for new therapeutic approaches.

Therapy resistance is a major roadblock in oncology. Exacerbation of molecular dysfunctions typical of cancer cells have proven effective in twisting oncogenic mechanisms to lethal conditions, thus offering new therapeutic avenues for cancer treatment. Here, we demonstrate that selective agonists of Transient Receptor Potential cation channel subfamily M member 8 (TRPM8), a cation channel characteristic of the prostate epithelium frequently overexpressed in advanced stage III/IV prostate cancers (PCa), sensitize therapy refractory models of PCa to radio, chemo or hormonal treatment. Overall, our study demonstrates that pharmacological-induced Ca2+ cytotoxicity is an actionable strategy to sensitize cancer cells to standard therapies.

The acquired immune deficiency syndrome (AIDS) has caused over 60 million deaths since the etiological agent, human immunodeficiency virus type 1 (HIV-1), was first discovered in 1981. Over 6000 new HIV-1 infections are reported every day, predominantly in economically deprived regions of sub-Saharan Africa. Despite impressive developments in antiretroviral therapy, current medical intervention is unable to prevent or cure HIV-1 infection, necessitating expensive life-long treatment. Difficulties in establishing a vaccine or cure, arise from its capacity to cause life-long latent infection, and its extraordinary ability to evolve resistance to therapeutic intervention. Capsid uncoating is the process by which p24CA proteins (CA) disassemble from the viral ribonucleoprotein during the early phase of the HIV-1 lifecycle. Despite intensive investigation, much is yet unknown about the spatial and temporal occurrence of this process within the cell, and the viral or host cellular factors involved. However, studies investigating p24CA mutations which alter the stability, and consequently the kinetics of capsid uncoating, have shown that timely capsid uncoating is crucial for efficient HIV-1 infection. Recent advancements in microscopic techniques have enabled high resolution analysis of cellular protein interactions, including the in situ localisation and dynamics of these events. We have developed three imaging techniques for the analysis of different aspects of HIV-1 capsid uncoating: 1) a dual-fluorescently labelled virus 2) a fluorescently labelled antibody targeting a capsid internalised repeat peptide array, and 3) a split-luciferase system tagging an internalised component of the capsid core. Fluorescent labelling of both the CA and IN proteins enabled the sensitive and specific analysis of uncoating in response to both restriction factors and CA mutations, and the visualisation of CA colocalised pre-integration complexes (PICs) within the nucleus. This system is i

CRISPR nucleases are efficient tools to edit cellular genomes in a variety of organisms. However, the in vivo application of this technology is still severely limited by unwanted genomic cleavages, that are further increased by long-term expression of the nuclease and can lead to unpredictable results. To address this limitation, we developed a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets in order to select optimized Streptococcus pyogenes Cas9 (SpCas9) variants. The screening of SpCas9 variants obtained by random mutagenesis of the Rec1-II domain allowed the identification of hits with increased on/off ratios. Through the combination of the identified mutations within a single variant we isolated the best performing nuclease, that we named evoCas9 (evolved Cas9). Side by side analyses with recently reported rationally designed variants demonstrated a significant improvement in fidelity of our evoCas9. In addition, to control Cas9 persistence into cells over time, we developed a Self-Limiting Cas9 circuitry for Enhanced Safety (SLiCES) which consists of an expression unit for SpCas9, a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. This self-limiting circuit, by controlling Cas9 levels, results in increased genome editing specificity. For its in vivo utilization, we integrated SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Following its delivery into target cells, the lentiSLiCES circuit is switched on to edit the intended genomic locus while simultaneously promoting its own neutralization through SpCas9 inactivation. The two strategies here developed represent complementary approaches to address a major issue in the genome editing field. On one hand, by preserving target cells from residual nuclease activity, our hit and go SLiCES system increases the safety margins for genome engineering

The acquired immune deficiency syndrome (AIDS) has caused over 60 million deaths since the etiological agent, human immunodeficiency virus type 1 (HIV-1), was first discovered in 1981. Over 6000 new HIV-1 infections are reported every day, predominantly in economically deprived regions of sub-Saharan Africa. Despite impressive developments in antiretroviral therapy, current medical intervention is unable to prevent or cure HIV-1 infection, necessitating expensive life-long treatment. Difficulties in establishing a vaccine or cure, arise from its capacity to cause life-long latent infection, and its extraordinary ability to evolve resistance to therapeutic intervention. Capsid uncoating is the process by which p24CA proteins (CA) disassemble from the viral ribonucleoprotein during the early phase of the HIV-1 lifecycle. Despite intensive investigation, much is yet unknown about the spatial and temporal occurrence of this process within the cell, and the viral or host cellular factors involved. However, studies investigating p24CA mutations which alter the stability, and consequently the kinetics of capsid uncoating, have shown that timely capsid uncoating is crucial for efficient HIV-1 infection. Recent advancements in microscopic techniques have enabled high resolution analysis of cellular protein interactions, including the in situ localisation and dynamics of these events. We have developed three imaging techniques for the analysis of different aspects of HIV-1 capsid uncoating: 1) a dual-fluorescently labelled virus 2) a fluorescently labelled antibody targeting a capsid internalised repeat peptide array, and 3) a split-luciferase system tagging an internalised component of the capsid core. Fluorescent labelling of both the CA and IN proteins enabled the sensitive and specific analysis of uncoating in response to both restriction factors and CA mutations, and the visualisation of CA colocalised pre-integration complexes (PICs) within the nucleus. This system is i

CRISPR nucleases are efficient tools to edit cellular genomes in a variety of organisms. However, the in vivo application of this technology is still severely limited by unwanted genomic cleavages, that are further increased by long-term expression of the nuclease and can lead to unpredictable results. To address this limitation, we developed a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets in order to select optimized Streptococcus pyogenes Cas9 (SpCas9) variants. The screening of SpCas9 variants obtained by random mutagenesis of the Rec1-II domain allowed the identification of hits with increased on/off ratios. Through the combination of the identified mutations within a single variant we isolated the best performing nuclease, that we named evoCas9 (evolved Cas9). Side by side analyses with recently reported rationally designed variants demonstrated a significant improvement in fidelity of our evoCas9. In addition, to control Cas9 persistence into cells over time, we developed a Self-Limiting Cas9 circuitry for Enhanced Safety (SLiCES) which consists of an expression unit for SpCas9, a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. This self-limiting circuit, by controlling Cas9 levels, results in increased genome editing specificity. For its in vivo utilization, we integrated SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Following its delivery into target cells, the lentiSLiCES circuit is switched on to edit the intended genomic locus while simultaneously promoting its own neutralization through SpCas9 inactivation. The two strategies here developed represent complementary approaches to address a major issue in the genome editing field. On one hand, by preserving target cells from residual nuclease activity, our hit and go SLiCES system increases the safety margins for genome engineering

Persistence of latent, replication-competent Human Immunodeficiency Virus type 1 (HIV-1) provirus is the main impediment towards a cure for HIV/AIDS (Acquired Immune Deficiency Syndrome). Therefore, different therapeutic strategies to eliminate the viral reservoirs are currently being explored. We here propose a novel strategy to reduce the replicating HIV reservoir during primary HIV infection by means of drug-induced retargeting of HIV integration. A novel class of integration inhibitors, referred to as LEDGINs, inhibit the interaction between HIV integrase and the LEDGF/p75 host cofactor, the main determinant of lentiviral integration site selection. We show for the first time that LEDGF/p75 depletion hampers HIV-1 reactivation in cell culture. Next we demonstrate that LEDGINs relocate and retarget HIV integration resulting in a HIV reservoir that is refractory to reactivation by different latency-reversing agents. Taken together, these results support the potential of integrase inhibitors that modulate integration site targeting to reduce the likeliness of viral rebound.

Persistence of latent, replication-competent Human Immunodeficiency Virus type 1 (HIV-1) provirus is the main impediment towards a cure for HIV/AIDS (Acquired Immune Deficiency Syndrome). Therefore, different therapeutic strategies to eliminate the viral reservoirs are currently being explored. We here propose a novel strategy to reduce the replicating HIV reservoir during primary HIV infection by means of drug-induced retargeting of HIV integration. A novel class of integration inhibitors, referred to as LEDGINs, inhibit the interaction between HIV integrase and the LEDGF/p75 host cofactor, the main determinant of lentiviral integration site selection. We show for the first time that LEDGF/p75 depletion hampers HIV-1 reactivation in cell culture. Next we demonstrate that LEDGINs relocate and retarget HIV integration resulting in a HIV reservoir that is refractory to reactivation by different latency-reversing agents. Taken together, these results support the potential of integrase inhibitors that modulate integration site targeting to reduce the likeliness of viral rebound.