New collaborative paper: 5-ethyl-2’-deoxyuridine fragilizes Klebsiella pneumoniae outer wall and facilitates intracellular killing by phagocytic cells
Our team collaborated with the team of Prof. Pierre Cosson on this work that allowed the identification of a new antibacterial compound targeting K. pneumoniae and facilitating its killing by Dicty. You can learn a bit more about this story here, or directly read the paper published in PLOS One here.
Klebsiella pneumoniae is the causative agent of a variety of severe infections. Many K. pneumoniae strains are resistant to multiple antibiotics, and this situation creates a need for new antibacterial molecules. K. pneumoniae pathogenicity relies largely on its ability to escape phagocytosis and intracellular killing by phagocytic cells. Interfering with these escape mechanisms may allow to decrease bacterial virulence and to combat infections. In this study, we used Dictyostelium discoideum as a model phagocyte to screen a collection of 1,099 chemical compounds. Phg1A KO D. discoideum cells cannot feed upon K. pneumoniae bacteria, unless bacteria bear mutations decreasing their virulence. We identified 3 non-antibiotic compounds that restored growth of phg1A KO cells on K. pneumoniae, and we characterized the mode of action of one of them, 5-ethyl-2’-deoxyuridine (K2). K2-treated bacteria were more rapidly killed in D. discoideum phagosomes than non-treated bacteria. They were more sensitive to polymyxin and their outer membrane was more accessible to a hydrophobic fluorescent probe. These results suggest that K2 acts by rendering the membrane of K. pneumoniae accessible to antibacterial effectors. K2 was effective on three different K. pneumoniae strains, and acted at concentrations as low as 3 μM. K2 has previously been used to treat viral infections but its precise molecular mechanism of action in K. pneumoniae remains to be determined.
A new collaborative work with Dr. Cristina Alvarez-Martinez from the Universidade Estadual de Campinas (Brazil) supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and the Swiss National Science Foundation (SNSF)
Thanks to a research grant, the FAPESP and the SNSF will support a new collaborative work with Dr. Cristina Alvarez-Martinez. This project will aim at deciphering the molecular mechanisms of the Xanthomonas citri resistance to Dictyostelium discoideum predation and the role of the Type VI Secretion System (T6SS) (A) of X. citri in this resistance. Overall, this project will expand our understanding of the role of anti-eukaryotic T6SS and possibly reveal new effector functions and anti-host strategies. This will help to understand better the mechanisms used by X. citri to survive and disseminate in the environment (B).
We have a new postdoctoral fellow in the lab!
Sandra Guallar-Garrido join us for a project on the machineries involved in damage sensing and repairing in Dictyostelium discoideum. Let's give her a warm welcome!
A new comer in the team for a few months!
Lucas Ceseti, a phD student from the group of Cristina Martinez (https://www.ib.unicamp.br/node/99), joined us for three months in order to work on the relationship between our favorite beast, Dictyostelium discoideum, and the phytopathogen Xanthomonas citri.
"As part of my PhD research performed at Unicamp (Brazil), I went to the Soldati Lab for a 3-month internship to evaluate the phenotypes of Dicty expressing putative T6SS effectors that Xanthomonas citri (Xac) uses to resist against the amoeba. Also during this period, I worked together with the Senior postdoc Céline Michard to establish a potential tool for monitoring translocation from bacteria to Dicty, and much more aiming to understand what happens when Xac meets Dicty." - Lucas Ceseti
New preprint from the lab: Disruption of vacuolin microdomains in the host Dictyostelium discoideum increases resistance to Mycobacterium marinum-induced membrane damage and infection
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manipulates the host phagosome maturation pathway to replicate intracellularly. Mycobacterium marinum, a closely-related species, and Dictyostelium discoideum, a social amoeba and alternative phagocytic host, have been used as models to study host-pathogen interactions occurring during mycobacterial infections. Vacuolins, functional homologues of the mammalian flotillins, organize membrane microdomains and play a role in vesicular trafficking. Various pathogens have been reported to manipulate their membrane association and function. During infection of D. discoideum with M. marinum, Vacuolin C was specifically and highly induced and all three vacuolin isoforms were enriched at the mycobacteria-containing-vacuole (MCV). In addition, absence of vacuolins reduced escape from the MCV and conferred resistance to M. marinum infection. Moreover, ESAT-6, the membrane-disrupting virulence factor of M. marinum, was less associated with membranes when vacuolins were absent. Together, these results suggest that vacuolins are important host factors that are manipulated by mycobacteria to inflict membrane damage and escape from their compartment.
Congratulations to our new PhD Lyudmil Raykov!
On September 6th, Lyudmil Raykov successfully defended his phD, entilted "Identification and Characterization of Dictyostelium discoideum Conserved Response Factors Involved in Pathogen Detection and Stress Signal Transduction". A big thank you to the jury members: Prof. Aurélien Roux, Prof. Pierre Cosson and Prof. Félix Randow
Our new Methods chapter is out! If you want to learn how to study infection at different scale, you have to read it!
The Dictyostelium discoideum–Mycobacterium marinum host–pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three microscopy methods that allow the precise identification and quantification of very diverse phenotypes arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was established to study the infection dynamics with high-throughput imaging of a large number of cells at the different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning disc confocal microscope.
We are very happy to share our bioRxiv preprint: The Dictyostelium discoideum E3 ubiquitin ligase TrafE coordinates endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum.
Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage. Endolysosomal perforations are recognised as severe stress, however the mechanisms of the cellular response that ensure quality control, repair and endolysosomal homeostasis are just beginning to be unravelled. The endosomal sorting complex required for transport (ESCRT) and the autophagy machinery are recruited to damaged membranes to either repair or to remove membrane remnants. Crucial element of the endolysosomal damage response (ELDR) are factors that sense damage, paralleled by extensive tagging of the damaged organelles with signals, such as ubiquitin, required for the recruitment of ELDR components. Unattended membrane damage leads to leakage of harmful components including protons and reactive oxygen species that cause cell death. To explore ELDR key factors responsible for detection and marking of damaged compartments we use the professional phagocyte Dictyostelium discoideum. We found an evolutionary conserved E3-ligase TrafE that is robustly recruited to intracellular compartments disrupted after infection with Mycobacterium marinum or after sterile damage caused by chemical components. Importantly, we show that the absence of TrafE severely compromises the xenophagy restriction of bacteria as well as autophagy-mediated and ESCRT-mediated ELDR, resulting in early cell death.
Our latest paper is out! Have a look at the effect of Zinc during infection!
Macrophages use diverse strategies to restrict intracellular pathogens, including either depriving the bacteria of (micro)nutrients such as transition metals or intoxicating them via metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum, a close relative of Mycobacterium tuberculosis (Mtb), and its hosts. We use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn2+ during M. marinum infection. We show that M. marinum senses toxic levels of Zn2+ and responds by upregulating one of its isoforms of the Zn2+ efflux transporter CtpC. Deletion of ctpC (MMAR_1271) leads to growth inhibition in broth supplemented with Zn2+ as well as reduced intracellular growth. Both phenotypes were fully rescued by constitutive ectopic expression of the Mtb CtpC orthologue demonstrating that MMAR_1271 is the functional CtpC Zn2+ efflux transporter in M. marinum. Infection leads to the accumulation of Zn2+ inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn2+ efflux pumps ZntA and ZntB. In cells lacking ZntA, there is further attenuation of M. marinum growth, presumably due to a compensatory efflux of Zn2+ into the MCV, carried out by ZntB, the main Zn2+ transporter in endosomes and phagosomes. Counterintuitively, bacterial growth is also impaired in zntB KO cells, in which MCVs appear to accumulate less Zn2+ than in wild-type cells, suggesting restriction by other Zn2+-mediated mechanisms. Absence of CtpC further epistatically attenuates the intracellular proliferation of M. marinum in zntA and zntB KO cells, confirming that mycobacteria face noxious levels of Zn2+.
We have a new postdoctoral fellow in the lab!
Mélanie Foulon joined us in January for a project on the host-derived lipids: transport and utilization by mycobacteria during their intracellular life! Let's give her a warm welcome!
Our latest collaboration is out! Dr Mohammad Parhizkar, in the team of Prof Giovanna Di Marzo Serugendo at UNIGE is working on Dictyostelium discoideum as an Inspiration for Higher-Order Emergence in Collective Adaptive Systems and Swarm Robotics.
Collective behaviour in nature provides a source of inspiration to engineer artificial collective adaptive systems, due to their mechanisms favouring adaptation to environmental changes and enabling complex emergent behaviour to arise from a relatively simple behaviour of individual entities. As part of our ongoing research, we study the social amoeba Dictyostelium discoideum to derive agent-based models and mechanisms that we can then exploit in artificial systems, in particular in swarm robotics. In this paper, we present a selection of agent-based models of the aggregation phase of D. discoideum, their corresponding biological illustrations and how we used them as an inspiration for transposing this behaviour into swarms of Kilobots. We focus on the stream-breaking phenomenon occurring during the aggregation phase of the life cycle of D. discoideum. Results show that the breakup of aggregation streams depends on cell density, motility, motive force and the concentration of cAMP and CF. The breakup also comes with the appearance of late centres. Our computational results show similar behaviour to our biological experiments, using Ax2(ka) strain. For swarm robotics experiments, we focus on signalling and aggregation towards a centre.
We are happy to announce that our collaboration with the lab of Jason King at the University of Sheffield is now out !
Engulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialized cup-shaped protrusions. To effectively capture and internalize their targets, these cups are organized into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR, and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac, and phospholipid interactions to localize at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, we propose RGBARG shapes the protrusion by expanding Rac activation at the rim while suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of coordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte.
Read our new article on the role of Vacuolins in phagocytic uptake and phagosomal membrane recycling in Dictyostelium discoideum
Flotillins are lipid rafts residents involved in membrane trafficking and recycling of plasma membrane proteins. Dictyostelium discoideum uses phagocytosis to kill, digest and feed on bacteria. It possesses three flotillin-like vacuolins that are strongly associated with membranes and gradually accumulate on maturing phagosomes. Absence of vacuolins reduced adhesion and particle recognition resulting in a drastic reduction in the uptake of various types of particles. This was caused by a block in the recycling of plasma membrane components and the absence of their specific cortex-associated proteins. In addition, absence of vacuolins also impaired phagolysosome biogenesis, without significantly impacting killing and digestion of a range of bacteria. Strikingly, both absence and overexpression of vacuolins induced a strong down-regulation of myosin VII expression, as well as its partner talin A. Episomal expression of myosin VII fully rescued defects in uptake and adhesion, but not in phagosome maturation. These results suggest a dual role for vacuolins: a novel mechanism involving membrane microdomains and myosin VII/talin A in clustering phagosomal receptors and adhesion molecules at the plasma membrane, and a role in phagolysosomal biogenesis.
We are happy to announce that our collaboration with the lab of Falk Hillmann at the HKI in Jena, and especially with Iuliia Viediernikova who came two month in 2017 with an EMBO fellowship was fruitful and the article is now out !
The human-pathogenic fungus Aspergillus fumigatusis a ubiquitous saprophyte that causes fatal lung infections in immunocompromised individuals. Following inhalation, conidia are ingested by innate immune cells and can arrestphagolysosome maturation. How this virulence trait could have been selected for innatural environments is unknown. Here, we found that surface exposure of thegreen pigment 1,8-dihydroxynaphthalene-(DHN)-melanin can protect conidia from phagocytic uptake and intracellular killing by the fungivorous amoeba Protostelium aurantium and delays its exocytosis from the non fungivorous species Dictyostelium discoideum. To elucidate the antiphagocytic properties of the surface pigment, we followed the antagonistic interactions of A. fumigatus conidia with the amoebae in real time. For both amoebae, conidia covered with DHN-melanin were internalizedat far lower rates than were seen with conidia lacking the pigment, despite high rates of initial attachment to non killing D. discoideum. When ingested by D. discoideum, the formation of nascent phagosomes was followed by transient acidification of phagolysosomes, their subsequent neutralization, and, finally, exocytosis of the conidia. While the cycle was completed in less than 1 h for unpigmented conidia, the process was significantly prolonged for conidia covered with DHN-melanin, leading to an extended intracellular residence time. At later stages of this cellular infection, pigmente