Publications by authors named "Emmanuel Lemichez"

94 Publications

Characterization of a highly neutralizing single monoclonal antibody to botulinum neurotoxin type A.

FASEB J 2021 May;35(5):e21540

Institut Pasteur, Unité des Toxines Bactériennes, UMR CNRS 2001, Paris, France.

Compared to conventional antisera strategies, monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage to be produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. In this study, we characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) with site-directed mutagenesis and functional studies. We found that TA12 recognizes a conformational epitope located at the interface between the H and H subdomains of the BoNT/A1 receptor-binding domain (H ). The TA12-binding interface shares common structural features with the ciA-C2 VHH epitope and lies on the face opposite recognized by ciA-C2- and the CR1/CR2-neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to H confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/A1-binding site for both the neuronal cell surface receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and to a lower extent GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes on the potential of using single mAbs for the treatment of botulism type A.
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http://dx.doi.org/10.1096/fj.202002492RDOI Listing
May 2021

Adaptation of in a Medium Mimicking a Diabetic Foot Environment.

Toxins (Basel) 2021 03 22;13(3). Epub 2021 Mar 22.

Virulence Bactérienne et Infections Chroniques, INSERM U1047, Université de Montpellier, Department of Microbiology and Hospital Hygiene, CHU Nîmes, Univ Montpellier, 30908 Nîmes, France.

is the most prevalent pathogen isolated from diabetic foot infections (DFIs). The purpose of this study was to evaluate its behavior in an in vitro model mimicking the conditions encountered in DFI. Four clinical strains were cultivated for 16 weeks in a specific environment based on the wound-like medium biofilm model. The adaptation of isolates was evaluated as follows: by model (to evaluate virulence); by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) (to evaluate expression of the main virulence genes); and by Biofilm Ring test (to assess the biofilm formation). After 16 weeks, the four had adapted their metabolism, with the development of small colony variants and the loss of β-hemolysin expression. The in vivo nematode model suggested a decrease of virulence, confirmed by qRT-PCRs, showing a significant decrease of expression of the main staphylococcal virulence genes tested, notably the toxin-encoding genes. An increased expression of genes involved in adhesion and biofilm was noted. Our data based on an in vitro model confirm the impact of environment on the adaptation switch of to prolonged stress environmental conditions. These results contribute to explore and characterize the virulence of in chronic wounds.
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http://dx.doi.org/10.3390/toxins13030230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005162PMC
March 2021

Toxemia in Human Naturally Acquired Botulism.

Toxins (Basel) 2020 11 13;12(11). Epub 2020 Nov 13.

Unité des Toxines Bactériennes, UMR CNRS 2001, Institut Pasteur, 75015 Paris, France.

Human botulism is a severe disease characterized by flaccid paralysis and inhibition of certain gland secretions, notably salivary secretions, caused by inhibition of neurotransmitter release. Naturally acquired botulism occurs in three main forms: food-borne botulism by ingestion of preformed botulinum neurotoxin (BoNT) in food, botulism by intestinal colonization (infant botulism and intestinal toxemia botulism in infants above one year and adults), and wound botulism. A rapid laboratory confirmation of botulism is required for the appropriate management of patients. Detection of BoNT in the patient's sera is the most direct way to address the diagnosis of botulism. Based on previous published reports, botulinum toxemia was identified in about 70% of food-borne and wound botulism cases, and only in about 28% of infant botulism cases, in which the diagnosis is mainly confirmed from stool sample investigation. The presence of BoNT in serum depends on the BoNT amount ingested with contaminated food or produced locally in the intestine or wound, and the timeframe between serum sampling and disease onset. BoNT levels in patient's sera are most frequently low, requiring a highly sensitive method of detection. Mouse bioassay is still the most used method of botulism identification from serum samples. However, in vitro methods based on BoNT endopeptidase activity with detection by mass spectrometry or immunoassay have been developed and depending on BoNT type, are more sensitive than the mouse bioassay. These new assays show high specificity for individual BoNT types and allow more accurate differentiation between positive toxin sera from botulism and autoimmune neuropathy patients.
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http://dx.doi.org/10.3390/toxins12110716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697460PMC
November 2020

Dewetting: From Physics to the Biology of Intoxicated Cells.

Adv Exp Med Biol 2020 ;1267:101-115

Unité des Toxines Bactériennes, Institut Pasteur, UMR CNRS 2001, Paris, France.

Pathogenic bacteria colonize or disseminate into cells and tissues by inducing large-scale remodeling of host membranes. The physical phenomena underpinning these massive membrane extension and deformation are poorly understood. Invasive strategies of pathogens have been recently enriched by the description of a spectacular mode of opening of large transendothelial cell macroaperture (TEM) tunnels correlated to the dissemination of EDIN-producing strains of Staphylococcus aureus via a hematogenous route or to the induction of gelatinous edema triggered by the edema toxin from Bacillus anthracis. Remarkably, these highly dynamic tunnels close rapidly after they reach a maximal size. Opening and closure of TEMs in cells lasts for hours without inducing endothelial cell death. Multidisciplinary studies have started to provide a broader perspective of both the molecular determinants controlling cytoskeleton organization at newly curved membranes generated by the opening of TEMs and the physical processes controlling the dynamics of these tunnels. Here we discuss the analogy between the opening of TEM tunnels and the physical principles of dewetting, stemming from a parallel between membrane tension and surface tension. This analogy provides a broad framework to investigate biophysical constraints in cell membrane dynamics and their diversion by certain invasive microbial agents.
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http://dx.doi.org/10.1007/978-3-030-46886-6_6DOI Listing
September 2020

Human Botulism in France, 1875-2016.

Toxins (Basel) 2020 05 21;12(5). Epub 2020 May 21.

Bacterial Toxins, Institut Pasteur, 75015 Paris, France.

Botulism is a rare but severe disease which is characterized by paralysis and inhibition of secretions. Only a few cases had been reported at the end of the 19th century in France. The disease was frequent during the second world war, and then the incidence decreased progressively. However, human botulism is still present in France with 10-25 cases every year. Food-borne botulism was the main form of botulism in France, whereas infant botulism (17 cases between 2004 and 2016) was rare, and wound and inhalational botulism were exceptional. Type B was the prevalent botulism type and was mainly due to consumption of home-made or small-scale preparations of cured ham and to a lesser extent other pork meat products. In the recent period (2000-2016), a wider diversity of botulism types from various food origin including industrial foods was reported. Severe cases of type A and F botulism as well as type E botulism were more frequent. Albeit rare, the severity of botulism justifies its continued surveillance and recommendations to food industry and consumers regarding food hygiene and preservation practices.
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http://dx.doi.org/10.3390/toxins12050338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291226PMC
May 2020

Cellular microbiology: Bacterial toxin interference drives understanding of eukaryotic cell function.

Cell Microbiol 2020 04;22(4):e13178

Department of Microbiology-Immunology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois.

Intimate interactions between the armament of pathogens and their host dictate tissue and host susceptibility to infection also forging specific pathophysiological outcomes. Studying these interactions at the molecular level has provided an invaluable source of knowledge on cellular processes, as ambitioned by the Cellular Microbiology discipline when it emerged in early 90s. Bacterial toxins act on key cell regulators or membranes to produce major diseases and therefore constitute a remarkable toolbox for dissecting basic biological processes. Here, we review selected examples of recent studies on bacterial toxins illustrating how fruitful the discipline of cellular microbiology is in shaping our understanding of eukaryote processes. This ever-renewing discipline unveils new virulence factor biochemical activities shared by eukaryotic enzymes and hidden rules of cell proteome homeostasis, a particularly promising field to interrogate the impact of proteostasis breaching in late onset human diseases. It is integrating new concepts from the physics of soft matter to capture biomechanical determinants forging cells and tissues architecture. The success of this discipline is also grounded by the development of therapeutic tools and new strategies to treat both infectious and noncommunicable human diseases.
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http://dx.doi.org/10.1111/cmi.13178DOI Listing
April 2020

Neuronal selectivity of botulinum neurotoxins.

Toxicon 2020 Apr 11;178:20-32. Epub 2020 Feb 11.

Bacterial Toxins, Institut Pasteur, Paris, France. Electronic address:

Botulinum neurotoxins (BoNTs) are highly potent toxins responsible for a severe disease, called botulism. They are also efficient therapeutic tools with an increasing number of indications ranging from neuromuscular dysfunction to hypersecretion syndrome, pain release, depression as well as cosmetic application. BoNTs are known to mainly target the motor-neurons terminals and to induce flaccid paralysis. BoNTs recognize a specific double receptor on neuronal cells consisting of gangliosides and synaptic vesicle protein, SV2 or synaptotagmin. Using cultured neuronal cells, BoNTs have been established blocking the release of a wide variety of neurotransmitters. However, BoNTs are more potent in motor-neurons than in the other neuronal cell types. In in vivo models, BoNT/A impairs the cholinergic neuronal transmission at the motor-neurons but also at neurons controlling secretions and smooth muscle neurons, and blocks several neuronal pathways including excitatory, inhibitory, and sensitive neurons. However, only a few reports investigated the neuronal selectivity of BoNTs in vivo. In the intestinal wall, BoNT/A and BoNT/B target mainly the cholinergic neurons and to a lower extent the other non-cholinergic neurons including serotonergic, glutamatergic, GABAergic, and VIP-neurons. The in vivo effects induced by BoNTs on the non-cholinergic neurons remain to be precisely investigated. We report here a literature review of the neuronal selectivity of BoNTs.
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http://dx.doi.org/10.1016/j.toxicon.2020.02.006DOI Listing
April 2020

Revisiting Old Ionophore Lasalocid as a Novel Inhibitor of Multiple Toxins.

Toxins (Basel) 2020 01 1;12(1). Epub 2020 Jan 1.

Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, F-91191 Gif-sur-Yvette, France.

The ionophore lasalocid is widely used as a veterinary drug against coccidiosis. We found recently that lasalocid protects cells from two unrelated bacterial toxins, the cytotoxic necrotizing factor-1 (CNF1) from and diphtheria toxin. We evaluated lasalocid's capacity to protect cells against other toxins of medical interest comprising toxin B from , Shiga-like toxin 1 from enterohemorrhagic and exotoxin A from . We further characterized the impact of lasalocid on the endolysosomal and the retrograde pathways and organelle integrity, especially the Golgi apparatus. We found that lasalocid protects cells from all toxins tested and impairs the drop of vesicular pH along the trafficking pathways that are required for toxin sorting and translocation to the cytoplasm. Lasalocid also has an impact on the cellular distribution of GOLPH4 and GOLPH2 Golgi markers. Other intracellular trafficking compartments positive for EEA1 and Rab9A display a modified cellular pattern. In conclusion, lasalocid protects cells from multiple deadly bacterial toxins by corrupting vesicular trafficking and Golgi stack homeostasis.
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http://dx.doi.org/10.3390/toxins12010026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020423PMC
January 2020

Public Health Risk Associated with Botulism as Foodborne Zoonoses.

Toxins (Basel) 2019 12 30;12(1). Epub 2019 Dec 30.

Institut Pasteur, Département de Microbiologie, Unité des Toxines Bactériennes, 25 rue du Dr Roux, 75015 Paris, France.

Botulism is a rare but severe neurological disease in man and animals that is caused by botulinum neurotoxins (BoNTs) produced by and atypical strains from other and non- species. BoNTs are divided into more than seven toxinotypes based on neutralization with specific corresponding antisera, and each toxinotype is subdivided into subtypes according to amino acid sequence variations. Animal species show variable sensitivity to the different BoNT toxinotypes. Thereby, naturally acquired animal botulism is mainly due to BoNT/C, D and the mosaic variants CD and DC, BoNT/CD being more prevalent in birds and BoNT/DC in cattle, whereas human botulism is more frequently in the types A, B and E, and to a lower extent, F. Botulism is not a contagious disease, since there is no direct transmission from diseased animals or man to a healthy subject. Botulism occurs via the environment, notably from food contaminated with spores and preserved in conditions favorable for growth and toxin production. The high prevalence of botulism types C, D and variants DC and CD in farmed and wild birds, and to a lower extent in cattle, raises the risk of transmission to human beings. However, human botulism is much rarer than animal botulism, and botulism types C and D are exceptional in humans. Only 15 cases or suspected cases of botulism type C and one outbreak of botulism type D have been reported in humans to date. In contrast, animal healthy carriers of group II, such as type E in fish of the northern hemisphere, and B4 in pigs, represent a more prevalent risk of botulism transmission to human subjects. Less common botulism types in animals but at risk of transmission to humans, can sporadically be observed, such as botulism type E in farmed chickens in France (1998-2002), botulism type B in cattle in The Netherlands (1977-1979), botulism types A and B in horses, or botulism type A in dairy cows (Egypt, 1976). In most cases, human and animal botulisms have distinct origins, and cross transmissions between animals and human beings are rather rare, accidental events. But, due to the severity of this disease, human and animal botulism requires a careful surveillance.
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http://dx.doi.org/10.3390/toxins12010017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020394PMC
December 2019

UBTD1 is a mechano-regulator controlling cancer aggressiveness.

EMBO Rep 2019 04 25;20(4). Epub 2019 Feb 25.

Centre Méditerranéen de Médecine Moléculaire (C3M), Inserm U1065, Nice Cedex 3, France

Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with β-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase β-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.
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http://dx.doi.org/10.15252/embr.201846570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446246PMC
April 2019

Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner.

Elife 2018 09 20;7. Epub 2018 Sep 20.

Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, Paris, France.

One challenge in cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and associated with cellular membranes that are flat, positively or negatively curved. Using and cell biology approaches, we assess mechanisms of ezrin's enrichment on curved membranes. We evidence that wild-type ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTD's specific conformation reduces intermolecular interactions, allows binding to actin filaments, which reduces membrane tethering, and promotes ezrin binding to positively-curved membranes. While neither ezrinTD nor ezrinWT senses negative curvature alone, we demonstrate that interacting with curvature-sensing I-BAR-domain proteins facilitates ezrin enrichment in negatively-curved membrane protrusions. Overall, our work demonstrates that ezrin can tether membranes, or be targeted to curved membranes, depending on conformations and interactions with actin and curvature-sensing binding partners.
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http://dx.doi.org/10.7554/eLife.37262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167055PMC
September 2018

Variability of Botulinum Toxins: Challenges and Opportunities for the Future.

Toxins (Basel) 2018 09 13;10(9). Epub 2018 Sep 13.

Institut Pasteur, Département de Microbiologie, Unité des Toxines Bactériennes, 25 Rue du Docteur Roux, 75015 Paris, France.

Botulinum neurotoxins (BoNTs) are the most potent known toxins, and are therefore classified as extremely harmful biological weapons. However, BoNTs are therapeutic drugs that are widely used and have an increasing number of applications. BoNTs show a high diversity and are divided into multiple types and subtypes. Better understanding of the activity at the molecular and clinical levels of the natural BoNT variants as well as the development of BoNT-based chimeric molecules opens the door to novel medical applications such as silencing the sensory neurons at targeted areas and dermal restoration. This short review is focused on BoNTs' variability and the opportunities or challenges posed for future clinical applications.
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http://dx.doi.org/10.3390/toxins10090374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162648PMC
September 2018

In silico analysis of Schmidtea mediterranea TIR domain-containing proteins.

Dev Comp Immunol 2018 09 7;86:214-218. Epub 2018 May 7.

CNRS, 31 Chemin Joseph Aiguier, 13402, Marseille, France; CNRS UMR 7278, IRD198, INSERM U1095, APHM, Institut Hospitalier Universitaire Méditerranée-Infection, Aix-Marseille Université, 19-21 Bd Jean Moulin, 13385, Marseille Cedex 05, France. Electronic address:

While genetic evidence points towards an absence of Toll-Like Receptors (TLRs) in Platyhelminthes, the Toll/IL-1 Receptor (TIR)-domains that drive the assembly of signalling complexes downstream TLR are present in these organisms. Here, we undertook the characterisation of the repertoire of TIR-domain containing proteins in Schmidtea mediterranea in order to gain valuable information on TLR evolution in metazoan. We report the presence of twenty proteins containing between one and two TIR domains. In addition, our phylogenetic-based reconstruction approach identified Smed-SARM and Smed-MyD88 as conserved TLR adaptors.
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http://dx.doi.org/10.1016/j.dci.2018.05.004DOI Listing
September 2018

CNF1-like deamidase domains: common Lego bricks among cancer-promoting immunomodulatory bacterial virulence factors.

Pathog Dis 2018 07;76(5)

Bacterial Toxins Unit, Department of Microbiology, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris, France.

Alterations of the cellular proteome over time due to spontaneous or toxin-mediated enzymatic deamidation of glutamine (Gln) and asparagine (Asn) residues contribute to bacterial infection and might represent a source of aging-related diseases. Here, we put into perspective what is known about the mode of action of the CNF1 toxin from pathogenic Escherichia coli, a paradigm of bacterial deamidases that activate Rho GTPases, to illustrate the importance of determining whether exposure to these factors are risk factors in the etiology age-related diseases, such as cancer. In particular, through in silico analysis of the distribution of the CNF1-like deamidase active site Gly-Cys-(Xaa)n-His sequence motif in bacterial genomes, we unveil the wide distribution of the super-family of CNF-like toxins and CNF-like deamidase domains among members of the Enterobacteriacae and in association with a large variety of toxin delivery systems. We extent our discussion with recent findings concerning cellular systems that control activated Rac1 GTPase stability and provide protection against cancer. These findings point to the urgency for developing holistic approaches toward personalized medicine that include monitoring for asymptomatic carriage of pathogenic toxin-producing bacteria and that ultimately might lead to improved public health and increased lifespans.
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http://dx.doi.org/10.1093/femspd/fty045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693396PMC
July 2018

Group-I PAKs-mediated phosphorylation of HACE1 at serine 385 regulates its oligomerization state and Rac1 ubiquitination.

Sci Rep 2018 01 23;8(1):1410. Epub 2018 Jan 23.

C3M, Inserm, Equipe labellisée Ligue contre le Cancer, University of Côte d'Azur, Nice, F-06204, France.

The regulation of Rac1 by HACE1-mediated ubiquitination and proteasomal degradation is emerging as an essential element in the maintenance of cell homeostasis. However, how the E3 ubiquitin ligase activity of HACE1 is regulated remains undetermined. Using a proteomic approach, we identified serine 385 as a target of group-I PAK kinases downstream Rac1 activation by CNF1 toxin from pathogenic E. coli. Moreover, cell treatment with VEGF also promotes Ser-385 phosphorylation of HACE1. We have established in vitro that HACE1 is a direct target of PAK1 kinase activity. Mechanistically, we found that the phospho-mimetic mutant HACE1(S385E), as opposed to HACE1(S385A), displays a lower capacity to ubiquitinate Rac1 in cells. Concomitantly, phosphorylation of Ser-385 plays a pivotal role in controlling the oligomerization state of HACE1. Finally, Ser-385 phosphorylated form of HACE1 localizes in the cytosol away from its target Rac1. Together, our data point to a feedback inhibition of HACE1 ubiquitination activity on Rac1 by group-I PAK kinases.
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http://dx.doi.org/10.1038/s41598-018-19471-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780496PMC
January 2018

Screening of a Drug Library Identifies Inhibitors of Cell Intoxication by CNF1.

ChemMedChem 2018 04 27;13(7):754-761. Epub 2018 Feb 27.

Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, 91191, Gif/Yvette, France.

Cytotoxic necrotizing factor 1 (CNF1) is a toxin produced by pathogenic strains of Escherichia coli responsible for extra-intestinal infections. CNF1 deamidates Rac1, thereby triggering its permanent activation and worsening inflammatory reactions. Activated Rac1 is prone to proteasomal degradation. There is no targeted therapy against CNF1, despite its clinical relevance. In this work we developed a fluorescent cell-based immunoassay to screen for inhibitors of CNF1-induced Rac1 degradation among 1120 mostly approved drugs. Eleven compounds were found to prevent CNF1-induced Rac1 degradation, and five also showed a protective effect against CNF1-induced multinucleation. Finally, lasalocid, monensin, bepridil, and amodiaquine protected cells from both diphtheria toxin and CNF1 challenges. These data highlight the potential for drug repurposing to fight several bacterial infections and Rac1-based diseases.
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http://dx.doi.org/10.1002/cmdc.201700631DOI Listing
April 2018

Decoding glycan recognition by bacterial toxins.

Nat Microbiol 2018 02;3(2):124-126

Institut Pasteur, Département de Microbiologie, Unité des Toxines Bactériennes, Université Paris Descartes, Paris, France.

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http://dx.doi.org/10.1038/s41564-018-0107-9DOI Listing
February 2018

ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

Sci Rep 2017 Nov 14;7(1):15567. Epub 2017 Nov 14.

Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, LabEx LERMIT, 91191, Gif-sur-Yvette, France.

Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.
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http://dx.doi.org/10.1038/s41598-017-15466-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686106PMC
November 2017

Force-induced transcellular tunnel formation in endothelial cells.

Mol Biol Cell 2017 Aug 9. Epub 2017 Aug 9.

UC Berkeley/UC San Francisco Graduate Group in Bioengineering, Berkeley, California 94720

The endothelium serves as a protective semipermeable barrier in blood vessels and lymphatic vessels. Leukocytes and pathogens can pass directly through the endothelium by opening holes in endothelial cells, known as transcellular tunnels, which are formed by contact and self-fusion of the apical and basal plasma membranes. Here we test the hypothesis that the actin cytoskeleton is the primary barrier to transcellular tunnel formation using a combination of atomic force microscopy and fluorescence microscopy of live cells. We find that localized mechanical forces are sufficient to induce the formation of transcellular tunnels in HUVECs. When HUVECs are exposed to the bacterial toxin EDIN, which can induce spontaneous transcellular tunnels, less mechanical work is required to form tunnels due to the reduced cytoskeletal stiffness and thickness of these cells, similar to the effects of a ROCK inhibitor. We also observe actin enrichment in response to mechanical indentation that is reduced in cells exposed to the bacterial toxin. Our study shows that the actin cytoskeleton of endothelial cells provides both passive and active resistance against transcellular tunnel formation, serving as a mechanical barrier that can be overcome by mechanical force as well as disruption of the cytoskeleton.
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http://dx.doi.org/10.1091/mbc.E17-01-0080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620373PMC
August 2017

Ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation.

Nat Commun 2017 06 23;8:15839. Epub 2017 Jun 23.

INSERM, U1065, Université de Nice-Sophia Antipolis, Centre Méditerranéen de Médecine Moléculaire (C3M), Department of microbial toxins in host-pathogen interactions, 151 Route St Antoine de Ginestière, BP 2 3194, 06204 Nice, France.

Transendothelial cell macroaperture (TEM) tunnels control endothelium barrier function and are triggered by several toxins from pathogenic bacteria that provoke vascular leakage. Cellular dewetting theory predicted that a line tension of uncharacterized origin works at TEM boundaries to limit their widening. Here, by conducting high-resolution microscopy approaches we unveil the presence of an actomyosin cable encircling TEMs. We develop a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This establishes the critical role of ezrin and non-muscle myosin II (NMII) in the progressive implementation of line tension. Mechanistically, fluorescence-recovery-after-photobleaching experiments point for the upstream role of ezrin in stabilizing actin filaments at the edges of TEMs, thereby favouring their crosslinking by NMIIa. Collectively, our findings ascribe to ezrin and NMIIa a critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting the formation of an actomyosin ring.
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http://dx.doi.org/10.1038/ncomms15839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490010PMC
June 2017

Pseudomonas aeruginosa Exolysin promotes bacterial growth in lungs, alveolar damage and bacterial dissemination.

Sci Rep 2017 05 18;7(1):2120. Epub 2017 May 18.

Université Grenoble Alpes, F-38000, Grenoble, France.

Exolysin (ExlA) is a recently-identified pore-forming toxin secreted by a subset of Pseudomonas aeruginosa strains identified worldwide and devoid of Type III secretion system (T3SS), a major virulence factor. Here, we characterized at the ultrastructural level the lesions caused by an ExlA-secreting strain, CLJ1, in mouse infected lungs. CLJ1 induced necrotic lesions in pneumocytes and endothelial cells, resulting in alveolo-vascular barrier breakdown. Ectopic expression of ExlA in an exlA-negative strain induced similar tissue injuries. In addition, ExlA conferred on bacteria the capacity to proliferate in lungs and to disseminate in secondary organs, similar to bacteria possessing a functional T3SS. CLJ1 did not promote a strong neutrophil infiltration in the alveoli, owing to the weak pro-inflammatory cytokine reaction engendered by the strain. However, CLJ1 was rapidly eliminated from the blood in a bacteremia model, suggesting that it can be promptly phagocytosed by immune cells. Together, our study ascribes to ExlA-secreting bacteria the capacity to proliferate in the lung and to damage pulmonary tissues, thereby promoting metastatic infections, in absence of substantial immune response exacerbation.
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http://dx.doi.org/10.1038/s41598-017-02349-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437091PMC
May 2017

Staphylococcus aureus Promotes Smed-PGRP-2/Smed-setd8-1 Methyltransferase Signalling in Planarian Neoblasts to Sensitize Anti-bacterial Gene Responses During Re-infection.

EBioMedicine 2017 Jun 24;20:150-160. Epub 2017 Apr 24.

CNRS UMR 7278, IRD198, INSERM U1095, APHM, Institut Hospitalier Universitaire Méditerranée-Infection, Aix-Marseille Université, 19-21 Bd Jean Moulin 13385, Marseille, Cedex 05, France. Electronic address:

Little is known about how organisms exposed to recurrent infections adapt their innate immune responses. Here, we report that planarians display a form of instructed immunity to primo-infection by Staphylococcus aureus that consists of a transient state of heightened resistance to re-infection that persists for approximately 30days after primo-infection. We established the involvement of stem cell-like neoblasts in this instructed immunity using the complementary approaches of RNA-interference-mediated cell depletion and tissue grafting-mediated gain of function. Mechanistically, primo-infection leads to expression of the peptidoglycan receptor Smed-PGRP-2, which in turn promotes Smed-setd8-1 histone methyltransferase expression and increases levels of lysine methylation in neoblasts. Depletion of neoblasts did not affect S. aureus clearance in primo-infection but, in re-infection, abrogated the heightened elimination of bacteria and reduced Smed-PGRP-2 and Smed-setd8-1 expression. Smed-PGRP-2 and Smed-setd8-1 sensitize animals to heightened expression of Smed-p38 MAPK and Smed-morn2, which are downstream components of anti-bacterial responses. Our study reveals a central role of neoblasts in innate immunity against S. aureus to establish a resistance state facilitating Smed-sted8-1-dependent expression of anti-bacterial genes during re-infection.
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http://dx.doi.org/10.1016/j.ebiom.2017.04.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478204PMC
June 2017

Decrease of Virulence by in a Model.

Front Cell Infect Microbiol 2017 16;7:77. Epub 2017 Mar 16.

Institut National de la Santé et de la Recherche Médicale, U1047, UFR de Médecine, Université de MontpellierNîmes, France; Service de Microbiologie, CHU CarémeauNîmes, France.

Social bacterial interactions are considered essential in numerous infectious diseases, particularly in wounds. Foot ulcers are a common complication in diabetic patients and these ulcers become frequently infected. This infection is usually polymicrobial promoting cell-to-cell communications. is the most prevalent pathogen isolated. Its association with , commensal Gram-positive cocci, is frequently described. The aim of this study was to assess the impact of co-infection on virulence of both and strains in a model. To study the host response, qRT-PCRs targeting host defense genes were performed. We observed that strains harbored a very low (LT50: 5.7 days ± 0.4) or an absence of virulence (LT50: 6.9 days ± 0.5). In contrast, strains (LT50: 2.9 days ± 0.4) were significantly more virulent than all ( < 0.001). When and strains were associated, significantly reduced the virulence of the strain in nematodes (LT50 between 4.4 and 5.2 days; < 0.001). To evaluate the impact of these strains on host response, transcriptomic analysis showed that the ingestion of led to a strong induction of defense genes (, and ) while did not. No statistical difference of host response genes expression was observed when were infected with either alone or with + . Moreover, two well-characterized virulence factors ( and ) present in were down-regulated when were co-infected with . This study showed that decreased the virulence of without modifying directly the host defense response. Factor(s) produced by this bacterium modulating the staphylococci virulence must be investigated.
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http://dx.doi.org/10.3389/fcimb.2017.00077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352687PMC
September 2017

Identification of cancer-associated missense mutations in hace1 that impair cell growth control and Rac1 ubiquitylation.

Sci Rep 2017 03 20;7:44779. Epub 2017 Mar 20.

Inserm, U1065, C3M, Team Bacterial Toxins in Host-Pathogens Interactions France, Equipe labellisée la ligue contre le cancer, Nice 06204, France.

The E3 ubiquitin ligase HACE1 is a potent tumor suppressor that controls cell proliferation and ubiquitylates the small GTPase Rac1 to target it to proteasomal degradation. Whether and how the activity of HACE1 is regulated by the N-terminal ankyrin (ANK) and the middle (MID) domains is ill defined. Here, we identified in the version 64 of the Catalogue of Somatic Mutations in Cancer (COSMIC) 13 missense mutations of hace1 located outside the HECT domain, and found that all lead to defective control of cell proliferation. In addition, several mutations located in the ankyrin domain displayed a dramatic reduction in Rac1 ubiquitylation associated with a decrease of colony formation in soft agar. 3D structure modelling of the 7 ankyrin-repeats coupled to functional analysis identified a surface epitope centered on one of the mutated residue, Gly-175, which is critical for controlling Rac1 binding and ubiquitylation. We also identified a role for the MID domain in conferring the specificity of association of HACE1 to the active form of Rac1. Our study of the functional interplay between HACE1 and Rac1 in cancer thus sheds a new light on the molecular mechanism of Rac1 ubiquitylation by HACE1 and the impact of its cancer-associated mutations in cell proliferation.
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http://dx.doi.org/10.1038/srep44779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5357957PMC
March 2017

New Aspects on Bacterial Effectors Targeting Rho GTPases.

Curr Top Microbiol Immunol 2017 ;399:155-174

UCA, Inserm, C3M, U1065, Team Microbial Toxins in Host Pathogen Interactions, Equipe Labellisée la Ligue Contre le Cancer, Nice, 06204, France.

The virulence of highly pathogenic bacteria such as Salmonella, Yersinia, Staphylococci, Clostridia, and pathogenic strains of Escherichia coli involves intimate cross-talks with the host actin cytoskeleton and its upstream regulators. A large number of virulence factors expressed by these pathogens modulate Rho GTPase activities either by mimicking cellular regulators or by catalyzing posttranslational modifications of these small proteins. This impressive convergence of virulence toward Rho GTPases and actin indeed offers pathogens the capacity to breach host defenses and invade their host, while it promotes inflammatory reactions. In return, the study of this targeting of Rho GTPases in infection has been an invaluable source of information in cell signaling, cell biology, and biomechanics, as well as in immunology. Through selected examples, I highlight the importance of recent studies on this crosstalk, which have unveiled new mechanisms of regulation of Rho GTPases; the relationship between cell shape and actin cytoskeleton organization; and the relationship between Rho GTPases and innate immune signaling.
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http://dx.doi.org/10.1007/82_2016_27DOI Listing
June 2017

Immunoadjuvant Properties of the Rho Activating Factor CNF1 in Prophylactic and Curative Vaccination against Leishmania infantum.

PLoS One 2016 3;11(6):e0156363. Epub 2016 Jun 3.

Inserm U1065, Centre Méditerranéen de Médecine Moléculaire, Team "Microbial toxins in host pathogen interactions", Equipe labellisée ligue contre le cancer, Nice, France.

There is a need to develop new effective immunoadjuvants for prophylactic or therapeutic vaccines against intracellular pathogens. The activation of Rho GTPases by bacterial cytotoxic necrotizing factor 1 (CNF1) elicits humoral protective responses against protein antigens. Here, we set out to investigate whether CNF1 activity initiates humoral immunity against co-administered parasite antigens and anti-microbial immune signaling. We report that co-administration of wild-type (WT) CNF1 with Leishmania (L.) promastigote antigens at the nasal mucosa triggered prophylactic and curative vaccine responses against this parasite. Vaccination of the mucosa with promastigote lysate antigens combined with WT CNF1 conferred protection against high inoculum L. infantum infection, which reached 82% in the spleen. Immune parameter analysis by antigen recall indicated robust T-helper (Th)1 polarization of immune memory cells, with high IL-2 and IFN-γ production combined with decreased IL-4 production. Additionally, we explored the curative effect of WT CNF1 on previously infected animals. We observed that PL combined with WT CNF1, but not the inactive C866S mutant CNF1 (mCNF1), induced a 58% decrease in the parasite burden in the spleen.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156363PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892475PMC
August 2017

System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.

Mol Cell 2016 Apr 3;62(1):121-36. Epub 2016 Mar 3.

Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, ON M5S3E1, Canada; Department of Molecular Genetics, University of Toronto, 1 King's College Cir, Toronto, ON M5S1A8, Canada. Electronic address:

HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.
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http://dx.doi.org/10.1016/j.molcel.2016.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988125PMC
April 2016

Microbial pathogenesis meets biomechanics.

Curr Opin Cell Biol 2016 Feb 2;38:31-7. Epub 2016 Feb 2.

Pathogenesis of vascular infections unit, INSERM, Institut Pasteur, 75015 Paris, France. Electronic address:

Introducing concepts from soft matter physics and mechanics has largely contributed to our understanding of a variety of biological processes. In this review, we argue that this holds true for bacterial pathogenesis. We base this argument on three examples of bacterial pathogens and their interaction with host cells during infection: (i) Shigella flexneri exploits actin-dependent forces to come into close contact with epithelial cells prior to invasion of the epithelium; (ii) Neisseria meningitidis manipulates endothelial cells to resist shear stress during vascular colonization; (iii) bacterial toxins take advantage of the biophysical properties of the host cell plasma membrane to generate transcellular macroapertures in the vascular wall. Together, these examples show that a multidisciplinary approach integrating physics and biology is more necessary than ever to understand complex infectious phenomena. Moreover, this avenue of research will allow the exploration of general processes in cell biology, highlighted by pathogens, in the context of other non-communicable human diseases.
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http://dx.doi.org/10.1016/j.ceb.2016.01.005DOI Listing
February 2016

Genetic Diversity and Population Structure of Leishmania infantum from Southeastern France: Evaluation Using Multi-Locus Microsatellite Typing.

PLoS Negl Trop Dis 2016 Jan 25;10(1):e0004303. Epub 2016 Jan 25.

UMR MIVEGEC IRD 224-CNRS 5290-Université Montpellier, Montpellier, France.

In the south of France, Leishmania infantum is responsible for numerous cases of canine leishmaniasis (CanL), sporadic cases of human visceral leishmaniasis (VL) and rare cases of cutaneous and muco-cutaneous leishmaniasis (CL and MCL, respectively). Several endemic areas have been clearly identified in the south of France including the Pyrénées-Orientales, Cévennes (CE), Provence (P), Alpes-Maritimes (AM) and Corsica (CO). Within these endemic areas, the two cities of Nice (AM) and Marseille (P), which are located 150 km apart, and their surroundings, concentrate the greatest number of French autochthonous leishmaniasis cases. In this study, 270 L. infantum isolates from an extended time period (1978-2011) from four endemic areas, AM, P, CE and CO, were assessed using Multi-Locus Microsatellite Typing (MLMT). MLMT revealed a total of 121 different genotypes with 91 unique genotypes and 30 repeated genotypes. Substantial genetic diversity was found with a strong genetic differentiation between the Leishmania populations from AM and P. However, exchanges were observed between these two endemic areas in which it seems that strains spread from AM to P. The genetic differentiations in these areas suggest strong epidemiological structuring. A model-based analysis using STRUCTURE revealed two main populations: population A (consisting of samples primarily from the P and AM endemic areas with MON-1 and non-MON-1 strains) and population B consisting of only MON-1 strains essentially from the AM endemic area. For four patients, we observed several isolates from different biological samples which provided insight into disease relapse and re-infection. These findings shed light on the transmission dynamics of parasites in humans. However, further data are required to confirm this hypothesis based on a limited sample set. This study represents the most extensive population analysis of L. infantum strains using MLMT conducted in France.
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http://dx.doi.org/10.1371/journal.pntd.0004303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726517PMC
January 2016

The Saccharomyces boulardii CNCM I-745 strain shows protective effects against the B. anthracis LT toxin.

Toxins (Basel) 2015 Oct 30;7(11):4455-67. Epub 2015 Oct 30.

Centre Scientifique de Monaco, Monaco 98000, Monaco.

The probiotic yeast Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.
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http://dx.doi.org/10.3390/toxins7114455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663514PMC
October 2015