Functions of the Dictyostelium LIMP-2/CD36 homologues in bacteria uptake, phagolysosome biogenesis and host cell defence. Check out our recent paper in Journal of Cell Science:
Phagocytic cells take up, kill and digest microbes by a process called phagocytosis. To this end these cells bind the particle, rearrange their actin cytoskeleton, and orchestrate transport of digestive factors to the particle-containing phagosome. The mammalian lysosomal membrane protein LIMP-2 and CD36, members of the class B of scavenger receptors, play a crucial role in lysosomal enzyme trafficking and uptake of mycobacteria, respectively, and generally in host cell defences against intracellular pathogens. Here, we show that the Dictyostelium discoideum LIMP-2 homologue LmpA regulates phagocytosis and phagolysosome biogenesis. The lmpA knockdown mutant is highly affected in actin-dependent processes such as particle uptake, cellular spreading and motility. Additionally, the cells are severely impaired in phagosomal acidification and proteolysis, likely explaining the higher susceptibility to infection with the pathogenic bacterium Mycobacterium marinum, a close cousin of the human pathogen Mycobacterium tuberculosis. Furthermore, we bring evidence that LmpB is a functional homologue of CD36 and specifically mediates uptake of mycobacteria. Altogether, these data indicate a role for LmpA and LmpB, ancestors of the LIMP-2/CD36 family, in lysosome biogenesis and host cell defence.
This paper can be read here:, , , , , , , , ,
New preprint from the lab:
Professional phagocytes have developed an extensive repertoire of autonomous immunity strategies to ensure killing of bacteria. Besides phagosome acidification and the generation of reactive oxygen species, deprivation of nutrients and the lumenal accumulation of toxic metals are essential to kill ingested bacteria or inhibit growth of intracellular pathogens. We use the soil amoeba Dictyostelium discoideum, a professional phagocyte that digests bacteria for nutritional purposes, to decipher the role of zinc poisoning during phagocytosis of non-pathogenic bacteria and visualize the temporal and spatial dynamics of compartmentalized, free zinc using fluorescent probes. Immediately after particle uptake, zinc is delivered to phagosomes by fusion with 'zincosomes' of endosomal origin, but also by the action of one or more zinc transporters. We localize the four Dictyostelium ZnT transporters to endosomes, the contractile vacuole and the Golgi apparatus, and study the impact of zntknockouts on zinc homeostasis. Finally, we show that zinc is delivered into the lumen of Mycobacterium smegmatis-containing vacuoles, and that Escherichia coli deficient in the zinc efflux P1B-type ATPase ZntA is killed faster than wild type bacteria.
We are very happy to share our bioRxiv preprint "ESCRT and autophagy cooperate to repair ESX-1-dependent damage to the Mycobacterium-containing vacuole".
We believe this is a landmark manuscript, hopefully published soon! It can be read here https://doi.org/10.1101/334755.
Thierry Soldati has been elected as EMBO (European Molecular Biology Organisation) Member
Joining a group of more than 1800 of the best researchers in Europe and around the world.
March brings us another publication, this time in collaboration with P. Cosson (University of Geneva) and M. Leippe (University of Kiel). We propose the Apl protein family of Dictyostelium discoideum as antimicrobial effectors.
Due to their archaic life style and microbivor behavior, amoebae may represent a source of antimicrobial peptides and proteins. The amoebic protozoon Dictyostelium discoideum has been a model organism in cell biology for decades and has recently also been used for research on host-pathogen interactions and the evolution of innate immunity. In the genome of D. discoideum, genes can be identified that potentially allow the synthesis of a variety of antimicrobial proteins. However, at the protein level only very few antimicrobial proteins have been characterized that may interact directly with bacteria and help in fighting infection of D. discoideum with potential pathogens. Here, we focus on a large group of gene products that structurally belong to the saposin-like protein (SAPLIP) family and which members we named provisionally Apls (amoebapore-like peptides) according to their similarity to a comprehensively studied antimicrobial and cytotoxic pore-forming protein of the protozoan parasite Entamoeba histolytica. We focused on AplD because it is the only Apl gene that is reported to be primarily transcribed further during the multicellular stages such as the mobile slug stage. Upon knock-out (KO) of the gene, aplD− slugs became highly vulnerable to virulent Klebsiella pneumoniae. AplD− slugs harbored bacterial clumps in their interior and were unable to slough off the pathogen in their slime sheath. Re-expression of AplD in aplD− slugs rescued the susceptibility toward K. pneumoniae. The purified recombinant protein rAplD formed pores in liposomes and was also capable of permeabilizing the membrane of live Bacillus megaterium. We propose that the multifarious Apl family of D. discoideum comprises antimicrobial effector polypeptides that are instrumental to interact with bacteria and their phospholipid membranes. The variety of its members would allow a complementary and synergistic action against a variety of microbes, which the amoeba encounters in its environment.
This paper can be read here: Dhakshinamoorthy R, Bitzhenner M, Cosson P, Soldati T, Leippe M. The saponin-like protein AplD displays pore-forming activity and participates in defense against bacterial infection during a multicellular stage of Dictyostelium discoideum. Front Cell Infect Microbiol 2018 | DOI:10.3389/fcimb.2018.00073
Glad to announce that our new paper in collaboration with L. Scapozza (University of Geneva), GS. Besra (University of Birmingham), JAG. Cox (Aston University) and L. Ballell (GSK) is now online in Scientific Reports. We identified anti-infective compounds and new mycobacterial targets for treatment.
Tuberculosis remains a serious threat to human health world-wide, and improved efficiency of medical treatment requires a better understanding of the pathogenesis and the discovery of new drugs. In the present study, we performed a whole-cell based screen in order to complete the characterization of 168 compounds from the GlaxoSmithKline TB-set. We have established and utilized novel previously unexplored host-model systems to characterize the GSK compounds, i.e. the amoeboid organisms D. discoideum and A. castellanii, as well as a microglial phagocytic cell line, BV2. We infected these host cells with Mycobacterium marinum to monitor and characterize the anti-infective activity of the compounds with quantitative fluorescence measurements and high-content microscopy. In summary, 88.1% of the compounds were confirmed as antibiotics against M. marinum, 11.3% and 4.8% displayed strong anti-infective activity in, respectively, the mammalian and protozoan infection models. Additionally, in the two systems, 13–14% of the compounds displayed pro-infective activity. Our studies underline the relevance of using evolutionarily distant pathogen and host models in order to reveal conserved mechanisms of virulence and defence, respectively, which are potential “universal” targets for intervention. Subsequent mechanism of action studies based on generation of over-expresser M. bovis BCG strains, generation of spontaneous resistant mutants and whole genome sequencing revealed four new molecular targets, including FbpA, MurC, MmpL3 and GlpK.
This paper can be read here: Trofimov V, Kicka S, Mucaria S, Hanna N, Ramon-Olayo F, Vela-Gonzalez Del Peral L, Lelièvre J, Ballell L, Scapozza L, Besra GS, Cox JAG, Soldati T. Antimycobacterial drug discovery using Mycobacteria-infected amoebae identifies anti-infectives and new molecular targets. Sci Rep 2018, 8:3939 | DOI:10.1038/s41598-018-22228-6
We say "until the next time" to Joe Dan, who has been a maître assistant in our laboratory, and a very good friend, for the last 3 years and a half. We will miss your scientific brainstormings during the lab meetings.
The very best for your foreseeable future, Mr. Dunn!
Our second review of the year was just published! "When Dicty Met Myco, a (Not So) Romantic Story about One Amoeba and Its Intracellular Pathogen".
In this review, we focus on the use of Dictyostelium discoideum as a phagocyte model for the study of mycobacterial infections, in particular Mycobacterium marinum. We look in detail at the intracellular cycle of M. marinum, from its uptake by D. discoideum to its active or passive egress into the extracellular medium. In addition, we introduce the methods and specific tools that have been used so far to monitor the D. discoideum—M. marinum interaction.
Find here our F1000 recommendation of the article "Intracellular Salmonella induces aggrephagy of host endomembranes in persistent infections", published in Autophagy by the laboratory of F García-Del Portillo.
New review by our lab: "Eat Prey, Live: Dictyostelium discoideum As a Model for Cell-Autonomous Defenses".
Here, we discuss the advantages and relevance of Dictyostelium discoideum as a model phagocyte to study cell-autonomous defenses. We cover the antimicrobial functions of phagocytosis and autophagy and describe the processes that create a microbicidal phagosome. We also describe microbial interference with these defenses and highlight observations made first in D. discoideum. Finally, we discuss galectins, TNF receptor-associated factors, tripartite motif-containing proteins, and signal transducers and activators of transcription, microbial restriction factors initially characterized in mammalian phagocytes that have either homologs or functional analogs in D. discoideum.
Our new paper "Survey on medicinal plants traditionally used in Senegal for the treatment of tuberculosis (TB) and assessment of their antimycobacterial activity", in collaboration with Prof. Wolfender, is out!
In West Africa, populations are used to taking traditional medicine as a first aid against common health problems. In this aspect, many plants are claimed to be effective in the treatment of Tuberculosis (TB), which according to the World Health Organization (WHO) remains one of the world’s deadliest communicable diseases.The main aim of this study was to identify plants used to treat TB-symptoms by the population of Senegal and to evaluate their possible concomitant use with clinically approved TB-drugs. This approach allowed the selection of plants effectively used in traditional medicine. In order to verify if the usage of some of these plants can be rationalized, the activity of their traditional preparations was assessed with both an intracellular and extracellular antimycobacterial host-pathogen assays.
This paper can be read here: Diop EA, Queiroz EF, Kicka S, Rudaz S, Diop T, Soldati T, Wolfender JL. Survey on medicinal plants traditionally used in Senegal for the treatment of tuberculosis (TB) and assessment of their antimycobacterial activity. J Ethnopharmacol 2017, doi: 10.1016/j.jep.2017.12.037.
We published the biochemical characterization and the first crystal structure of DdPoxA, a secreted heme peroxidase from Dictyostelium discoideum. Find all the information in our new paper in JBC.
Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals. Interestingly, phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum. This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages. Here we demonstrate that peroxidase A from D. discoideum (DdPoxA) is a stable, monomeric, glycosylated and secreted heme peroxidase with homology to mammalian peroxidases. The first crystal structure (2.5 Å resolution) of a mycetozoan peroxidase of this superfamily shows the presence of a posttranslationally-modified heme with one single covalent ester bond between the 1-methyl heme substituent and E236. The metalloprotein follows the halogenation cycle, whereby Compound I oxidizes iodide and thiocyanate at high (> 108 M-1 s-1) and bromide at very low rates. It is demonstrated that DdPoxA is upregulated and likely secreted at late multicellular development stages of D. discoideum when migrating slugs differentiate into fruiting bodies that contain persistent spores on top of a cellular stalk. Expression of DdPoxA is shown to restrict bacterial contamination of fruiting bodies. Structure and function of DdPoxA are compared to evolutionary related mammalian peroxidases in the context of non specific immune defense.
This paper can be read here: A Nicolussi, JD Dunn, G Mlynek, M Bellei, M Zamocky, G Battistuzzi, K Djinović-Carugo, PG Furtmüller, T Soldati, and C Obinger. Secreted Heme Peroxidase from Dictyostelium discoideum: Insights into Catalysis, Structure and Biological Role. JBC 2017, doi: 10.1074/jbc.RA117.000463. http://m.jbc.org/content/early/2017/12/14/jbc.RA117.000463.full.pdf
We have a new PhD in the lab!
Congratulations, Dr. Ana Teresa López-Jiménez!
Ethel Bayer Santos is visiting us!
Ethel, from Universidade de Sao Paulo, will be in our lab for 2 months to study the role of the type VI secretion system of Xanthomonas citri in the interaction with Dictyostelium discoideum. For more info, click here.
Welcome to our new PhD student Manon Mottet!
She will study the single-cell dynamics of mycobacterial infection. Click here to learn more about her previous studies.
The Dicty 2017 conference, organised this year by Prof. Cosson’s lab and our lab, has been a success!
Exciting science hold in an excellent venue. For more info, click here.
We identified new antimycobacterial compounds. Find all the information in our new paper published in PLoS One.
Tuberculosis remains one of the major threats to public health worldwide. Given the prevalence of multi drug resistance (MDR) in Mycobacterium tuberculosis strains, there is a strong need to develop new anti-mycobacterial drugs with modes of action distinct from classical antibiotics. Inhibitors of mycobacterial virulence might target new molecular processes and may represent a potential new therapeutic alternative. In this study, we used a Dictyostelium discoideum host model to assess virulence of Mycobacterium marinum and to identify compounds inhibiting mycobacterial virulence. Among 9995 chemical compounds, we selected 12 inhibitors of mycobacterial virulence that do not inhibit mycobacterial growth in synthetic medium. Further analyses revealed that 8 of them perturbed functions requiring an intact mycobacterial cell wall such as sliding motility, bacterial aggregation or cell wall permeability. Chemical analogs of two compounds were analyzed. Chemical modifications altered concomitantly their effect on sliding motility and on mycobacterial virulence, suggesting that the alteration of the mycobacterial cell wall caused the loss of virulence. We characterized further one of the selected compounds and found that it inhibited the ability of mycobacteria to replicate in infected cells. Together these results identify new antimycobacterial compounds that represent new tools to unravel the molecular mechanisms controlling mycobacterial pathogenicity. The isolation of compounds with anti-virulence activity is the first step towards developing new antibacterial treatments.
This paper can be read here: Ouertatani-Sakouhi H, Kicka S, Chiriano G, Harrison CF, Hilbi H, Scapozza L, Soldati T, Cosson P. Inhibitors of Mycobacterium marinum virulence identified in a Dictyostelium discoideum host model. PLoS One 2017, 12(7):e0181121. journals.plos.org/plosone/article?id=10.1371/journal.pone.0181121
Find here our F1000 recommendation of the article "A Rab20-Dependent Membrane Trafficking Pathway Controls M. tuberculosis Replication by Regulating Phagosome Spaciousness and Integrity", recently published in Cell Host & Microbe by the laboratory of MG Gutierrez.
This review describes recent evidence about the dual interaction of mycobacteria with host lipid droplets and membrane phospholipids, and integrates them in a broader view of the underlying cellular processes manipulated by various intracellular pathogens to gain access to host lipids.
Iuliia Viediernikova, from the Leibniz Institute for Natural Product Research and Infection Biology (Hans-Knöll-Institute, Jena, Germany) got a short-term EMBO fellowship to work with us for two months on the project:
An investigation of the evolutionary origin and defensive role of extracellular traps by social amoeba against filamentous fungi
Our new article on how Mycobacterium marinum induces the formation of autophagosomes in its host while repressing the autophagic killing capacity has been published in PLoS Pathogens
One of the cell-autonomous defence pathways against intracellular pathogens is autophagy, an ancestral eukaryotic process surprisingly conserved throughout evolution. Recent studies have highlighted contradictory roles for autophagy during mycobacterial infection. Whereas some studies revealed a role for autophagy to control intracellular bacterial growth, others brought evidence that mycobacteria somehow inhibit autophagic killing. Here, we demonstrate for the first time that Mycobacterium marinum induces both an early autophagic response and its simultaneous repression by blocking the autophagic digestion. This antagonistic manipulation of autophagy is dependent on a functional ESX-1 secretion system, which secretes the membrane-damaging factor ESAT-6, proposed to participate in the perforation of the M. marinum-containing vacuole (MCV). We show here that these membrane damages activate the formation of autophagosomes and their recruitment to the MCV. However, M. marinum also utilizes its ESX-1 secretion system to avoid killing inside autolysosomes by blocking the autophagic flux. In addition, we bring evidence that this manipulation of autophagy is orchestrated via the regulation of TORC1, the major eukaryotic kinase complex controlling nutrient-sensing and cell metabolism.
The article can be read here: Cardenal-Muñoz E, Arafah S, López-Jiménez AT, Kicka S, Falaise A, Bach F, et al. (2017) Mycobacterium marinum antagonistically induces an autophagic response while repressing the autophagic flux in a TORC1- and ESX-1-dependent manner. PLoS Pathog 13(4): e1006344. https://doi.org/10.1371/journal.ppat.1006344
Mycobacterium marinum not only acquire fatty acids from triacylglycerols stored in host lipid droplets, but also from host phospholipids. Our article has been published in PLoS Pathogens
Mycobacterium tuberculosis (Mtb) survives the human immune defence mechanisms leading to latent tuberculosis in one third of the world population. The ability to persist latently in human macrophages is due to a remarkable physiological change that is accompanied by a slowdown in replication, low metabolism, and phenotypic tolerance to antibiotics. It was proposed that fatty acids released from bacterial intracytosolic lipid inclusions (ILIs), a characteristic of dormant Mtb, serve as carbon source during reactivation from dormancy. In the article “Mycobacterium marinum degrades both triacylglycerols and phospholipids from its Dictyostelium host to synthesise its own triacylglycerols and generate lipid inclusions” we show that the bacteria accumulate ILIs even in a Dictyostelium mutant that is deficient in triacylglycerol synthesis and therefore incapable to build up lipid droplets. In addition, the accumulation of ILIs is not sufficient to induce a dormancy-like phenotype in M. marinum inside its host Dictyostelium. Moreover, we propose an alternative lipid transfer route from the host to the pathogen via degradation and recycling of host phospholipids.
The article can be read here: Barisch C, Soldati T (2017) Mycobacterium marinum Degrades Both Triacylglycerols and Phospholipids from Its Dictyostelium Host to Synthesise Its Own Triacylglycerols and Generate Lipid Inclusions. PLoS Pathog 13(1): e1006095. https://doi.org/10.1371/journal.ppat.1006095
The study has been highlighted in the most various places:
- Docu on Swiss TV, RTS (French)
- Press Release UNIGE (French)
- Swiss newspaper (German)
- Swiss website (German)
- Spanish newspaper (Spanish)
Andrea Nicolussi is visiting us!
Andrea is a PhD student in the Protein Biochemistry Group of Prof. Christian Obinger at the University of Natural Resources and Life Sciences, Vienna. She will be in our lab for 4 months to analyze the function, structure and physiological role of ancestral heme peroxidases.
She tell us more about her PhD project Secreted heme peroxidase from Dictyostelium discoideum – Insights into Catalysis, Structure and Biological Role:
"After producing the metalloprotein recombinantly, we characterized it by various biochemical and biophysical methods, e.g. UV-vis, ECD or EPR spectroscopy, pre-steady state kinetics or differential scanning calorimetry. Solving the X-ray structure of the heme enzyme increased our understanding of the structure-function relationships within the active site. My work on this peroxidase will be complemented by my research stay in the group of Prof. Thierry Soldati in Geneva, where we will investigate the in-vivo role of the peroxidase in the unspecific immune defense of Dictyostelium discoideum.”
New lab member!
Welcome to the MSc Lyudmil Raykov, from the Department of Molecular Biology of the University of Geneva.
Our article on the invention of DNA-based extracellular traps by Dictyostelium Sentinel slug cells has been published in Nature Communications
Our innate immune system, made up mainly of phagocytes, protects our body by exterminating bacteria. To do this, it uses two mechanisms. The first kills foreign bodies within the phagocyte itself. The second kills them outside the cell. These two strategies were already known to researchers, but only in humans and other higher animals. In collaboration with our colleagues from the Baylor College of Medicine in Huston (USA) we discovered that the social amoeba Dictyostelium, a unicellular microorganism living in the soils of temperate forests, also uses both these mechanisms, and has done so for over a billion years. Since this amoeba possesses an innate defense system similar to that of humans, while being genetically modifiable, the researchers can therefore carry out experiments on it in order to understand and fight genetic diseases of the immune system. This discovery can be read in the journal Nature Communications.
To defend themselves, our immune cells have two mechanisms. The first, called phagocytosis, kills bacteria within the phagocytic cell itself. The cell envelops the foreign body and exterminates it specifically by using reactive oxygen species (ozone, hydrogen peroxide, bleach), generated thanks to the enzyme NOX2. However, when the invader is too large to be taken up, cells use a second defense mechanism which consists of expelling their genetic material, that is to say their DNA. This DNA transforms into sticky and poisoned nets called «neutrophil extracellular traps» (NETs). These DNA nets then capture bacteria outside of the cell and kill them.
The article can be read here: Zhang, X., Zhuchenko, O., Kuspa, A., and Soldati, T. (2016) Social amoebae trap and kill bacteria by casting DNA nets Nature Communications 1;7:10938. doi: 10.1038/ncomms10938.
The study has been highlighted in the most various places:
New lab member!
Welcome to the PhD Louise Lefrancois, from the Research institue of the McGill University Health Centre (Montreal, Canada).
We have a new PhD in the lab!
Congratulations, Dr. Valentin Trofimov!
We have a new PhD in the lab!
Congratulations, Dr. Xuezhi Zhang!
We have been awarded a COST grant from the Swiss State Secretariat for Education, Research and Innovation
This project aims to study cellular and molecular functions of ROS and NOX in a social amoeba model system.
During this project supported by the COST / SEFRI, we propose to use our simple and powerful experimental model system Dictyostelium infected with Mycobacterium marinum to dissect functions of ROS/NOX in host pathogen interactions, phagosomal bactericidal activities, including metal poisoning, but also in chemotaxis and morphogenesis.
We have become a member of iGE3
The mission of the Institute of Genetics and Genomics of Geneva (iGE3) is to promote internationally competitive biomedical research and high quality teaching by using primarily genetic and genomic scientific analysis.
"iGE3 is a dynamic structure of the University of Geneva to promote research and teaching related to the human and other genomes. Our ambition is to understand life in the light of the structure, variation and function of genomes, and to contribute to the promotion of health based on the studies of the various human genomes." The major aims of the Soldati group are to understand the integration, the cooperation of signalling, cytoskeleton and membrane trafficking in phagocytosis and its relevance to host-pathogen interactions.
We have been awarded a SystemsX grant
The HostPathX project aims to study the host-pathogen interface, using modelling and chemical genetics perturbation of the phagocyte – mycobacteria interface.
This collaborative grant is a partnership with the labs of:
- Pierre Cosson at the Department of Cell Physiology and Metabolism of the university of Geneva
- Hubert Hilbi at Institute of Medical Microbiology of the University of Zurich
- Marco Pagni at the Swiss Institute of Bioinformatic in Lausanne
- Heinz Koeppel at the BISON Group | D-ITET | ETH Zurich
During this 4 year SystemsX.ch RTD project, we propose to identify the mode of action of anti-infective compounds by innovative methodologies that probe the perturbations of the host-pathogen interaction landscape. We will exploit the technological developments in high throughput RNA-sequencing to determine transcriptional signatures triggered by compounds that decrease bacterial pathogenicity. Notably, we will analyze the transcriptional response of host cells to infection with bacteria, and its perturbation by anti-infective compounds. Through developing novel computational methods and leveraging state-of-the art network analysis and modelling tools, we will predict a putative mode of action for each compound, and test the hypotheses directly using appropriate biological assays. To this end, we will exploit the genetic tractability of both Dictyostelium and Mycobacterium marinum.
We have a new PhD in the lab!
Congratulations, Dr. Aurélie Guého!
The Soldati Group had a productive retreat
The Soldati lab has come back from their scientific retreat in Hotel Bellevue Le Rocheray, in the Vallée de Joux. It took place on the 30th and 31th May.
We have become a member of the BMBS COST Action BM1203
The EU-ROS COST Action aims to study the functions of ROS and NOX at all levels, from bench to bed side.
Unravelling the fine balance between Reactive Oxygen Species (ROS) acting as a friend or a foe is fundamental to understand aerobic life. To advance this important area of biology and medicine, highly synergistic approaches combining diverse and scattered disciplines are needed. For this, COST provides an ideal framework. EU-ROS brings together multi-disciplinary experts to enhance the competitiveness of European research. Collectively, EU-ROS will overcome the fragmentation of European R&D on oxygen/ROS research while its translational components will contribute to European societies’ economic growth and wellbeing.