The catalytic-dead Pcif1 regulates gene expression and fertility in Drosophila.

Eukaryotic mRNAs are modified at the 5' end with a methylated guanosine (m7G) that is attached to the transcription start site (TSS) nucleotide. The TSS nucleotide is 2'-O-methylated (Nm) by CMTR1 in organisms ranging from insects to human. In mammals, the TSS adenosine can be further N 6 -methylated by RNA polymerase II phosphorylated CTD-interacting factor 1 (PCIF1) to create m6Am. Curiously, the fly ortholog of mammalian PCIF1 is demonstrated to be catalytic-dead, and its functions are not known. Here, we show that Pcif1 mutant flies display a reduced fertility which is particularly marked in females. Deep sequencing analysis of Pcif1 mutant ovaries revealed transcriptome changes with a notable increase in expression of genes belonging to the mitochondrial ATP synthetase complex. Furthermore, the Pcif1 protein is distributed along euchromatic regions of polytene chromosomes, and the Pcif1 mutation behaved as a modifier of position-effect-variegation (PEV) suppressing the heterochromatin-dependent silencing of the white gene. Similar or stronger changes in the transcriptome and PEV phenotype were observed in flies that expressed a cytosolic version of Pcif1. These results point to a nuclear cotranscriptional gene regulatory role for the catalytic-dead fly Pcif1 that is probably based on its conserved ability to interact with the RNA polymerase II carboxy-terminal domain.

PIWI Slicing and EXD1 Drive Biogenesis of Nuclear piRNAs from Cytosolic Targets of the Mouse piRNA Pathway.

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing in the two compartments is coordinated is not known. Here we demonstrate that endonucleolytic slicing of a transcript by the cytosolic mouse PIWI protein MILI acts as a trigger to initiate its further 5'→3' processing into non-overlapping fragments. These fragments accumulate as new piRNAs within both cytosolic MILI and the nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) as a partner of the MIWI2 piRNA biogenesis factor TDRD12. EXD1 homodimers are inactive as a nuclease but function as an RNA adaptor within a PET (PIWI-EXD1-Tdrd12) complex. Loss of Exd1 reduces sequences generated by MILI slicing, impacts biogenesis of MIWI2 piRNAs, and de-represses LINE1 retrotransposons. Thus, piRNA biogenesis triggered by PIWI slicing, and promoted by EXD1, ensures that the same guides instruct PIWI proteins in the nucleus and cytoplasm.

Hyd ubiquitinates the NF-κB co-factor Akirin to operate an effective immune response in Drosophila.

The Immune Deficiency (IMD) pathway in Drosophila melanogaster is activated upon microbial challenge with Gram-negative bacteria to trigger the innate immune response. In order to decipher this nuclear factor κB (NF-κB) signaling pathway, we undertook an in vitro RNAi screen targeting E3 ubiquitin ligases specifically and identified the HECT-type E3 ubiquitin ligase Hyperplastic discs (Hyd) as a new actor in the IMD pathway. Hyd mediated Lys63 (K63)-linked polyubiquitination of the NF-κB cofactor Akirin was required for efficient binding of Akirin to the NF-κB transcription factor Relish. We showed that this Hyd-dependent interaction was required for the transcription of immunity-related genes that are activated by both Relish and Akirin but was dispensable for the transcription of genes that depend solely on Relish. Therefore Hyd is key in NF-κB transcriptional selectivity downstream of the IMD pathway. Drosophila depleted of Akirin or Hyd failed to express the full set of genes encoding immune-induced anti-microbial peptides and succumbed to immune challenges. We showed further that UBR5, the mammalian homolog of Hyd, was also required downstream of the NF-κB pathway for the activation of Interleukin 6 (IL6) transcription by LPS or IL-1ß in cultured human cells. Our findings link the action of an E3 ubiquitin ligase to the activation of immune effector genes, deepening our understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases.

Weevil pgrp-lb prevents endosymbiont TCT dissemination and chronic host systemic immune activation.

Long-term intracellular symbiosis (or endosymbiosis) is widely distributed across invertebrates and is recognized as a major driving force in evolution. However, the maintenance of immune homeostasis in organisms chronically infected with mutualistic bacteria is a challenging task, and little is known about the molecular processes that limit endosymbiont immunogenicity and host inflammation. Here, we investigated peptidoglycan recognition protein (PGRP)-encoding genes in the cereal weevil Sitophilus zeamais's association with Sodalis pierantonius endosymbiont. We discovered that weevil pgrp-lb generates three transcripts via alternative splicing and differential regulation. A secreted isoform is expressed in insect tissues under pathogenic conditions through activation of the PGRP-LC receptor of the immune deficiency pathway. In addition, cytosolic and transmembrane isoforms are permanently produced within endosymbiont-bearing organ, the bacteriome, in a PGRP-LC-independent manner. Bacteriome isoforms specifically cleave the tracheal cytotoxin (TCT), a peptidoglycan monomer released by endosymbionts. pgrp-lb silencing by RNAi results in TCT escape from the bacteriome to other insect tissues, where it chronically activates the host systemic immunity through PGRP-LC. While such immune deregulations did not impact endosymbiont load, they did negatively affect host physiology, as attested by a diminished sexual maturation of adult weevils. Whereas pgrp-lb was first described in pathogenic interactions, this work shows that, in an endosymbiosis context, specific bacteriome isoforms have evolved, allowing endosymbiont TCT scavenging and preventing chronic endosymbiont-induced immune responses, thus promoting host homeostasis.

CHMP1B is a target of USP8/UBPY regulated by ubiquitin during endocytosis.

Integration and down-regulation of cell growth and differentiation signals rely on plasma membrane receptor endocytosis and sorting towards either recycling vesicles or degradative lysosomes via multivesicular bodies (MVB). In this process, the endosomal sorting complex-III required for transport (ESCRT-III) controls membrane deformation and scission triggering intraluminal vesicle (ILV) formation at early endosomes. Here, we show that the ESCRT-III member CHMP1B can be ubiquitinated within a flexible loop known to undergo conformational changes during polymerization. We demonstrate further that CHMP1B is deubiquitinated by the ubiquitin specific protease USP8 (syn. UBPY) and found fully devoid of ubiquitin in a ~500 kDa large complex that also contains its ESCRT-III partner IST1. Moreover, EGF stimulation induces the rapid and transient accumulation of ubiquitinated forms of CHMP1B on cell membranes. Accordingly, CHMP1B ubiquitination is necessary for CHMP1B function in both EGF receptor trafficking in human cells and wing development in Drosophila. Based on these observations, we propose that CHMP1B is dynamically regulated by ubiquitination in response to EGF and that USP8 triggers CHMP1B deubiquitination possibly favoring its subsequent assembly into a membrane-associated ESCRT-III polymer.

Isoconazole and Clemizole Hydrochloride Partially Reverse the Xeroderma Pigmentosum C Phenotype.

Xeroderma Pigmentosum protein C (XPC) is involved in recognition and repair of bulky DNA damage such as lesions induced by Ultra Violet (UV) radiation. XPC-mutated cells are, therefore, photosensitive and accumulate UVB-induced pyrimidine dimers leading to increased cancer incidence. Here, we performed a high-throughput screen to identify chemicals capable of normalizing the XP-C phenotype (hyper-photosensitivity and accumulation of photoproducts). Fibroblasts from XP-C patients were treated with a library of approved chemical drugs. Out of 1280 tested chemicals, 16 showed ≥25% photo-resistance with RZscore above 2.6 and two drugs were able to favor repair of 6-4 pyrimidine pyrimidone photoproducts (6-4PP). Among these two compounds, Isoconazole could partially inhibit apoptosis of the irradiated cells especially when cells were post-treated directly after UV irradiation while Clemizole Hydrochloride-mediated increase in viability was dependent on both pre and post treatment. No synergistic effect was recorded following combined drug treatment and the compounds exerted no effect on the proliferative capacity of the cells post UV exposure. Amelioration of XP-C phenotype is a pave way towards understanding the accelerated skin cancer initiation in XP-C patients. Further examination is required to decipher the molecular mechanisms targeted by these two chemicals.

Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries.

Small RNAs called PIWI -interacting RNAs (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded RNA precursors to generate millions of individual piRNAs, but the molecular mechanisms that identify a transcript as a precursor are poorly understood. Here we demonstrate that artificial tethering of the piRNA biogenesis factor, Armi, to a transcript is sufficient to direct it into primary processing in Drosophila ovaries and in an ovarian cell culture model. In the fly ovarian somatic follicle cells, the transcript becomes cleaved in a stepwise manner, with a 5'→3' directionality, liberating U1-containing ~24 nt piRNAs that are loaded into Piwi. Although uridines are preferred for generation of piRNA 5' ends, processing takes place even in their absence, albeit at a lower efficiency. We show that recombinant Armi has 5'→3' helicase activity, and mutations that abolish this activity also reduce piRNA processing in vivo. Another somatic piRNA pathway factor Yb, an interactor of Armi, is also able to trigger piRNA biogenesis when tethered to a transcript. Tethering-mediated primary piRNA biogenesis is also functional in the fly ovarian germline and loads all the three PIWI proteins present in this environment. Our study finds a broad correlation between piRNA processing and localization of the tethered factors to the cytoplasmic perinuclear ribonucleoprotein granules called germline nuage or somatic Yb bodies. We conclude that transcripts bound by Armi and Yb are identified as piRNA precursors, resulting in localization to cytoplasmic processing granules and their subsequent engagement by the resident piRNA biogenesis machinery.

A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila.

BACKGROUND: Lysosomes are the major catabolic compartment within eukaryotic cells, and their biogenesis requires the integration of the biosynthetic and endosomal pathways. Endocytosis and autophagy are the primary inputs of the lysosomal degradation pathway. Endocytosis is specifically needed for the degradation of membrane proteins whereas autophagy is responsible for the degradation of cytoplasmic components. We previously identified the deubiquitinating enzyme UBPY/USP8 as being necessary for lysosomal biogenesis and productive autophagy in Drosophila. Because UBPY/USP8 has been widely described for its function in the endosomal system, we hypothesized that disrupting the endosomal pathway itself may affect the biogenesis of the lysosomes. RESULTS: In the present study, we blocked the progression of the endosomal pathway at different levels of maturation of the endosomes by expressing in fat body cells either dsRNAs or dominant negative mutants targeting components of the endosomal machinery: Shibire, Rab4, Rab5, Chmp1 and Rab7. We observed that inhibition of endosomal trafficking at different steps in vivo is systematically associated with defects in lysosome biogenesis, resulting in autophagy flux blockade. CONCLUSION: Our results show that the integrity of the endosomal system is required for lysosome biogenesis and productive autophagy in vivo.

Synthesis and biological evaluation of thiophene and benzo[b]thiophene analogs of combretastatin A-4 and isocombretastatin A-4: A comparison between the linkage positions of the 3,4,5-trimethoxystyrene unit.

Combretastatin A-4 and isocombretastatin A-4 derivatives having thiophenes or benzo[b]thiophenes instead of the B ring were prepared and evaluated for their in cellulo tubulin polymerization inhibition (TPI) and antiproliferative activities. The presence of the benzo[b]thiophene ring proved to have a crucial effect as most of the thiophene derivatives, except those having one methoxy group, were inactive to inhibit tubulin polymerization into microtubules. The influence of the attachment position was also studied: benzo[b]thiophenes having iso or cis 3,4,5-trimethoxystyrenes at position 2 were 12-30-fold more active than the 3-regioisomers for the TPI activity. Some of the novel designed compounds exhibited interesting anti-proliferative effects on two different cell lines.

Small flies to tackle big questions: assaying complex bacterial virulence mechanisms using Drosophila melanogaster.

A successful raid on a fortress requires ingenious strategies in addition to a large number of soldiers. When a microorganism faces a potential host many factors are important, including not only the capacity to proliferate but also the ability to hide, escape or subvert the defence arsenal of the infected organism. This ability confers microbial pathogenicity and relies on complex virulence mechanisms, which are tightly regulated during the course of the infection. The amazing versatility of some microbes that can infect a wide broad of hosts undoubtedly relies on virulence factors intent on fighting evolutionarily conserved innate immune mechanisms. This makes the use of alternative invertebrate models, which are of outstanding interest because they demand less ethical consideration and lower experimental costs, extremely relevant. These simpler organisms are used to analyse genes and mechanisms involved in resistance or tolerance to microorganisms. They can also be used to study bacterial virulence factors that allow proliferation or persistence in the host. In particular, the Drosophila fruit fly has a complex immune response (similar to the mammalian innate immune response) and is particularly appropriate for deciphering many events underlying bacterial pathogenicity from acute virulence to biofilm formation. As highlighted in this review, Drosophila has been notably extensively used to study virulence traits of the opportunistic bacteria Pseudomonas aeruginosa, such as proliferation or persistence, translocation through an epithelial barrier, subversion of the phagocytic machinery, in vivo biofilm formation and enhanced virulence provided by commensal flora or a polymicrobial community. Moreover, these small flies now appear to be a useful system for assaying chemicals with therapeutic potential.

[Strength and specificity of the CMBA screening platform for bioactive molecules discovery]. / Force et spécificité du criblage pour des molécules bioactives au CMBA-Grenoble - Une plate-forme dédiée à la découverte et à l'analyse de molécules bioactives et candidats médicaments.

Used as powerful chemical probes in Life science fundamental research, the application potential of new bioactive molecular entities includes but extends beyond their development as therapeutic drugs in pharmacology. In this review, we wish to point out the methodology of chemical libraries screening on living cells or purified proteins at the CMBA academic platform of Grenoble Alpes University, and strategies employed to further characterize the selected bioactive molecules by phenotypic profiling on human cells. Multiple application fields are concerned by the screening activity developed at CMBA with bioactive molecules previously selected for their potential as tools for fundamental research purpose, therapeutic candidates to treat cancer or infection, or promising compounds for production of bioenergy.

Unique biofilm signature, drug susceptibility and decreased virulence in Drosophila through the Pseudomonas aeruginosa two-component system PprAB.

Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections.

Identifying USPs regulating immune signals in Drosophila: USP2 deubiquitinates Imd and promotes its degradation by interacting with the proteasome.

BACKGROUND: Rapid activation of innate immune defences upon microbial infection depends on the evolutionary conserved NF-κB dependent signals which deregulation is frequently associated with chronic inflammation and oncogenesis. These signals are tightly regulated by the linkage of different kinds of ubiquitin moieties on proteins that modify either their activity or their stability. To investigate how ubiquitin specific proteases (USPs) orchestrate immune signal regulation, we created and screened a focused RNA interference library on Drosophila NF-κB-like pathways Toll and Imd in cultured S2 cells, and further analysed the function of selected genes in vivo. RESULTS: We report here that USP2 and USP34/Puf, in addition to the previously described USP36/Scny, prevent inappropriate activation of Imd-dependent immune signal in unchallenged conditions. Moreover, USP34 is also necessary to prevent constitutive activation of the Toll pathway. However, while USP2 also prevents excessive Imd-dependent signalling in vivo, USP34 shows differential requirement depending on NF-κB target genes, in response to fly infection by either Gram-positive or Gram-negative bacteria. We further show that USP2 prevents the constitutive activation of signalling by promoting Imd proteasomal degradation. Indeed, the homeostasis of the Imd scaffolding molecule is tightly regulated by the linkage of lysine 48-linked ubiquitin chains (K48) acting as a tag for its proteasomal degradation. This process is necessary to prevent constitutive activation of Imd pathway in vivo and is inhibited in response to infection. The control of Imd homeostasis by USP2 is associated with the hydrolysis of Imd linked K48-ubiquitin chains and the synergistic binding of USP2 and Imd to the proteasome, as evidenced by both mass-spectrometry analysis of USP2 partners and by co-immunoprecipitation experiments. CONCLUSION: Our work identified one known (USP36) and two new (USP2, USP34) ubiquitin specific proteases regulating Imd or Toll dependent immune signalling in Drosophila. It further highlights the ubiquitin dependent control of Imd homeostasis and shows a new activity for USP2 at the proteasome allowing for Imd degradation. This study provides original information for the better understanding of the strong implication of USP2 in pathological processes in humans, including cancerogenesis.

Drosophila cellular immunity: a story of migration and adhesion.

Research during the past 15 years has led to significant breakthroughs, providing evidence of a high degree of similarity between insect and mammalian innate immune responses, both humoural and cellular, and highlighting Drosophila melanogaster as a model system for studying the evolution of innate immunity. In a manner similar to cells of the mammalian monocyte and macrophage lineage, Drosophila immunosurveillance cells (haemocytes) have a number of roles. For example, they respond to wound signals, are involved in wound healing and contribute to the coagulation response. Moreover, they participate in the phagocytosis and encapsulation of invading pathogens, are involved in the removal of apoptotic bodies and produce components of the extracellular matrix. There are several reasons for using the Drosophila cellular immune response as a model to understand cell signalling during adhesion and migration in vivo: many genes involved in the regulation of Drosophila haematopoiesis and cellular immunity have been maintained across taxonomic groups ranging from flies to humans, many aspects of Drosophila and mammalian innate immunity seem to be conserved, and Drosophila is a simplified and well-studied genetic model system. In the present Commentary, we will discuss what is known about cellular adhesion and migration in the Drosophila cellular immune response, during both embryonic and larval development, and where possible compare it with related mechanisms in vertebrates.

[Phenotypic screens or one stone to kill two birds: discover the target and its pharmacological regulator]. / Les criblages phénotypiques ou comment faire d'une pierre deux coups: découvrir la cible et la molécule pharmacologique capable de la réguler.

Phenotypic screens, in which chemical libraries are assayed on cells with the aim to isolate compounds that interfere with a given cell function, are a risky but powerful strategy to discover, in the same approach, new therapeutic targets and the compounds able to regulate them. With a strong experience of nearly 10 years in the field, we present the advantages of such an approach, the possible troubles and technical solutions. We also present in this paper a french network which has been recently built and that gather all the competencies needed for screening approaches.

Investigation of Chemical Composition and Biological Activities of Ajuga pyramidalis-Isolation of Iridoids and Phenylethanoid Glycosides.

Despite several studies on the Ajuga L. genus, the chemical composition of Ajuga pyramidalis, an alpine endemic species, is still largely unknown. The purpose of this study was to therefore deeper describe it, particularly from the phytochemistry and bioactivity perspectives. In that respect, A. pyramidalis was investigated and 95% of the extracted mass of the plant was characterized by chromatography and mass spectrometry. Apart from the already determined chemical compounds, namely, harpagide and 8-O-acetylharpagide, two iridoids, and neoajugapyrin A, a neo-clerodane diterpene, and three polyphenols (echinacoside, verbascoside and teupoloside) were identified for the first time in A. pyramidalis. Incidentally, the first RX structure of a harpagoside derivative is also described in this paper. The extracts and isolated compounds were then evaluated for various biochemical or biological activities; notably a targeted action on the renewal of the epidermis was highlighted with potential applications in the cosmetic field for anti-aging.

NLRP7 Enhances Choriocarcinoma Cell Survival and Camouflage in an Inflammasome Independent Pathway.

BACKGROUND: Gestational choriocarcinoma (GC) is a highly malignant trophoblastic tumor that often develops from a complete hydatidiform mole (HM). NLRP7 is the major gene responsible for recurrent HM and is involved in the innate immune response, inflammation and apoptosis. NLRP7 can function in an inflammasome-dependent or -independent pathway. Recently, we have demonstrated that NLRP7 is highly expressed in GC tumor cells and contributes to their tumorigenesis. However, the underlying mechanisms are still unknown. Here, we investigated the mechanism by which NLRP7 controls these processes in malignant (JEG-3) and non-tumor (HTR8/SVneo) trophoblastic cells. Cell survival, dedifferentiation, camouflage, and aggressiveness were compared between normal JEG-3 cells or knockdown for NLRP7, JEG-3 Sh NLRP7. In addition, HTR8/SVneo cells overexpressing NLRP7 were used to determine the impact of NLRP7 overexpression on non-tumor cells. NLRP7 involvement in tumor cell growth and tolerance was further characterized in vivo using the metastatic mouse model of GC. RESULTS: We demonstrate that NLRP7 (i) functions in an inflammasome-dependent and -independent manners in HTR8/SVneo and JEG-3 cells, respectively; (ii) differentially regulates the activity of NF-κB in tumor and non-tumor cells; (iii) increases malignant cell survival, dedifferentiation, and camouflage; and (iv) facilitates tumor cells colonization of the lungs in the preclinical model of GC. CONCLUSIONS: This study demonstrates for the first time the mechanism by which NLRP7, independently of its inflammasome machinery, contributes to GC growth and tumorigenesis. The clinical relevance of NLRP7 in this rare cancer highlights its potential therapeutic promise as a molecular target to treat resistant GC patients.

Genome-wide analysis of genes encoding core components of the ubiquitin system during cerebral cortex development.

Ubiquitination involves three types of enzymes (E1, E2, and E3) that sequentially attach ubiquitin (Ub) to target proteins. This posttranslational modification controls key cellular processes, such as the degradation, endocytosis, subcellular localization and activity of proteins. Ubiquitination, which can be reversed by deubiquitinating enzymes (DUBs), plays important roles during brain development. Furthermore, deregulation of the Ub system is linked to the pathogenesis of various diseases, including neurodegenerative disorders. We used a publicly available RNA-seq database to perform an extensive genome-wide gene expression analysis of the core components of the ubiquitination machinery, covering Ub genes as well as E1, E2, E3 and DUB genes. The ubiquitination network was governed by only Uba1 and Ube2m, the predominant E1 and E2 genes, respectively; their expression was positively regulated during cortical formation. The principal genes encoding HECT (homologous to the E6-AP carboxyl terminus), RBR (RING-in-between-RING), and RING (really interesting new gene) E3 Ub ligases were also highly regulated. Pja1, Dtx3 (RING ligases) and Stub1 (U-box RING) were the most highly expressed E3 Ub ligase genes and displayed distinct developmental expression patterns. Moreover, more than 80 DUB genes were expressed during corticogenesis, with two prominent genes, Uch-l1 and Usp22, showing highly upregulated expression. Several components of the Ub system overexpressed in cancers were also highly expressed in the cerebral cortex under conditions not related to tumour formation or progression. Altogether, this work provides an in-depth overview of transcriptomic changes during embryonic formation of the cerebral cortex. The data also offer new insight into the characterization of the Ub system and may contribute to a better understanding of its involvement in the pathogenesis of neurodevelopmental disorders.

Identification of chemicals breaking the USP8 interaction with its endocytic substrate CHMP1B.

The ubiquitin-specific protease USP8 plays a major role in controlling the stability and intracellular trafficking of numerous cell surface proteins among which the EGF receptor that regulates cell growth and proliferation in many physio-pathological processes. The function of USP8 at the endocytic pathway level partly relies on binding to and deubiquitination of the Endosomal Sorting Complex Required for Transport (ESCRT) protein CHMP1B. In the aim of finding chemical inhibitors of the USP8::CHMP1B interaction, we performed a high-throughput screening campaign using an HTRF® assay to monitor the interaction directly in lysates of cells co-expressing both partners. The assay was carried out in an automated format to screen the academic Fr-PPIChem library (Bosc N et al., 2020), which includes 10,314 compounds dedicated to the targeting of protein-protein interactions (PPIs). Eleven confirmed hits inhibited the USP8::CHMP1B interaction within a range of 30% to 70% inhibition at 50 µM, while they were inactive on a set of other PPI interfaces demonstrating the feasibility of specifically disrupting this particular interface. In parallel, we adapted this HTRF® assay to compare the USP8 interacting capacity of CHMP1B variants. As anticipated from earlier studies, a deletion of the MIM (Microtubule Interacting and Trafficking domain Interacting Motif) domain or mutation of two conserved leucine residues, L192 and L195, in this domain respectively abolished or strongly impeded the USP8::CHMP1B interaction. By contrast, a CHMP1B mutant that displays a highly decreased ubiquitination level following mutation of four lysine residues in arginine interacted at a similar level as the wild-type form with USP8. Therefore, conserved leucine residues within the MIT domain rather than its ubiquitinated status triggers CHMP1B substrate recognition by USP8.

Genomic erosion and horizontal gene transfer shape functional differences of the ExlA toxin in Pseudomonas spp.

Two-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of Pseudomonas aeruginosa is conserved in pathogenic and environmental Pseudomonas. While P. chlororaphis and P. entomophila displayed ExlA-dependent killing, P. putida did not cause damage to eukaryotic cells. ExlA proteins interacted with epithelial cell membranes; however, only ExlA Pch induced the cleavage of the adhesive molecule E-cadherin. ExlA proteins participated in insecticidal activity toward the larvae of Galleria mellonella and the fly Drosophila melanogaster. Evolutionary analyses demonstrated that the differences in the C-terminal domains are partly due to horizontal movements of the operon within the genus Pseudomonas. Reconstruction of the evolutionary history revealed the complex horizontal acquisitions. Together, our results provide evidence that conserved TPS toxins in environmental Pseudomonas play a role in bacteria-insect interactions and discrete differences in CTDs may determine their specificity and mode of action toward eukaryotic cells.