Biology subjects

Soldati, T.

Publications and source records attributed to Soldati, T..

28 records · Page 2Linked to original sources

bioRxiv2019

Lowe Syndrome-linked endocytic adaptors direct membrane cycling kinetics with OCRL in Dictyostelium discoideum.

Mutations of the inositol 5-phosphatase OCRL cause Lowe Syndrome (LS), characterized by congenital cataract, low IQ and defective kidney proximal tubule resorption. A key subset of LS mutants abolishes OCRLs interactions with endocytic adaptors containing F&H peptide motifs. Converging unbiased methods examining human peptides and the unicellular phagocytic organism Dictyostelium discoideum, reveal that, like OCRL, the Dictyostelium OCRL orthologue Dd5P4 binds two proteins closely related to the F&H proteins APPL1 and Ses1/2 (also referred to as IPIP27A/B). In addition, a novel conserved F&H interactor was identified, GxcU (in Dictyostelium) and the Cdc42-GEF Frabin (in human cells). Examining these proteins in Dictyostelium discoideum, we find that, like OCRL, Dd5P4 acts at well-conserved and physically distinct endocytic stations. Dd5P4 functions in coordination with F&H proteins to control membrane deformation at multiple stages of endocytosis, and suppresses GxcU-mediated activity during fluid-phase micropinocytosis. We also reveal that OCRL/Dd5P4 acts at the contractile vacuole, an exocytic osmoregulatory organelle. We propose F&H peptide-containing proteins may be key modifiers of LS phenotypes.

cell biology↗

bioRxiv2019

Proteomic characterization of the Mycobacterium marinum-containing vacuole in Dictyostelium discoideum

Mycobacterium tuberculosis, the causative agent of tuberculosis, is able to manipulate the phagosome compartment in which it resides in order to establish a permissive replicative compartment called the Mycobacterium-containing vacuole (MCV). Mycobacterium marinum, a fish pathogen and a close relative of the tuberculosis group, is able to infect the free-living amoeba and professional phagocyte Dictyostelium discoideum and to manipulate its phagosome maturation. By using this host-pathogen model system, we have established an innovative process to isolate MCVs. This procedure allowed us to isolate the M. marinum-MCV at 1, 3 and 6 hours post infection to study the early M. marinum-MCV proteome. By using isobaric labelling and mass spectrometry, we quantitatively compared the proteomic composition of those MCVs isolated at different stages of the early infection phase to understand how M. marinum impacts on this compartment to divert it from the normal phagosomal pathway. Furthermore, we also compared the manipulated compartment M. marinum-MCV to non- or less manipulated compartments containing different mycobacteria strains: the non-pathogenic M. smegmatis, the avirulent M. marinum-L1D or the attenuated M. marinum-RD1.

systems biology↗

bioRxiv2019

Time-resolved RNA-seq profiling of the infection of Dictyostelium discoideum by Mycobacterium marinum reveals an integrated host response to damage and stress

Tuberculosis remains the most pervasive infectious disease and the recent emergence of multiple drug-resistant strains emphasizes the need for more efficient drug treatments. The experimentally versatile Dictyostelium discoideum - Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity and host response. In this study, a time-resolved transcriptomic analysis of the amoeba D. discoideum was performed to decipher the different host pathways impacted during infection. We investigated how D. discoideum fine-tunes its gene expression in response to M. marinum infection by assessing the transcriptomic profile covering the critical stages of entry, establishment of a permissive niche, proliferation and dissemination (1, 3, 6, 12, 24 and 48 hours post infection). Differential gene expression provided a fingerprint of the transcriptome of the host cell in the presence of mycobacteria, and helped identify specific markers and molecular signatures of infection. Enrichment pathway analysis showed that most of the Biological Processes (BP) of upregulated genes at early time point of infection hinted towards damage response and cellular defence, especially in specific pathways involved in membrane repair (ESCRT) and bacteria elimination (autophagy). Whereas at late time points of infection, BP related to starvation were upregulated. Some other signatures were more unexpected, such as cell cycle (downregulation of cytosolic large & small ribosomal subunits) and upregulation of metabolic adaptations (lipids transport).

systems biology↗

bioRxiv2019

The developmental cycle of Dictyostelium discoideum ensures curing of a mycobacterial infection at both cell autonomous level and by collaborative exclusion

During its life cycle, the social amoeba Dictyostelium discoideum alternates between a predatory amoeba and a facultative multicellular form. The single-celled amoeba is a well-established model system to study cell-autonomous mechanisms of phagocytosis and defence against intracellular bacterial pathogens, whereas the multicellular forms are arising as models to study the emergence of innate immune defence strategies. Importantly, during evolution, prokaryotes have also evolved their own strategies to resist predation. Considering these complex ecological relationships, we wondered whether D. discoideum cells infected with intracellular pathogenic mycobacteria would be able to undergo their developmental cycle and what would be the fate of the infection. We show that the combination of cell-autonomous mechanisms and the organisation into a multicellular organism leads to the efficient multistep-curing of a mycobacteria-infected population, thereby ensuring germ-free spores and progeny. Specifically, using a microfluidic device to trap single infected cells, we revealed that in the first curing phase, individual cells rely on three mechanisms to release intracellular bacteria: exocytic release, ejection and lytic release. The second phase occurs at the collective level, when remaining infected cells are excluded from the forming cell aggregates.

microbiology↗

bioRxiv2019

Dictyostelium discoideum flotillin homologues are essential for phagocytosis and participate in plasma membrane recycling and lysosome biogenesis

The metazoan flotillins are lipid rafts residents involved in membrane trafficking and recycling of plasma membrane proteins. Dictyostelium discoideum, a social soil amoeba, uses phagocytosis to digest, kill and feed on bacteria. D. discoideum possesses three flotillin-like proteins, termed VacA, VacB and the recently identified VacC. All three vacuolins gradually accumulate on postlysosomes and, like flotillins, are strongly associated with membranes and partly with lipid rafts. Vacuolins are absolutely required for uptake of various particles. Their absence impairs particle recognition possibly because of defective recycling of plasma membrane or cortex-associated proteins. In addition, vacuolins are involved in phagolysosome biogenesis, although this does not impact digestion and killing of a wide range of bacteria. Furthermore, vacuolin knockout affects early recruitment of the WASH complex on phagosomes, suggesting that vacuolins may be involved in the WASH-dependent plasma membrane recycling. Altogether, these results indicate that vacuolins act as the functional homologues of flotillins in D. discoideum.

cell biology↗

bioRxiv2019

Zinc intoxication of Mycobacterium marinum during Dictyostelium discoideum infection is counteracted by induction of the pathogen Zn2+ exporter CtpC

Macrophages use diverse strategies to kill or restrict intracellular pathogens. Some of these strategies involve the deprivation of bacteria from (micro)nutrients such as transition metals, and the bacteria intoxication through metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum, a close relative of the human pathogen M. tuberculosis, and its hosts. Here we use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn2+ during M. marinum infection. We show that M. marinum infection induces 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 the ZntA detoxifying transporter there is further attenuation of M. marinum growth, possibly due to a compensatory efflux of Zn2+ into the MCV. This efflux is presumably carried out by ZntB, the main Zn2+ transporter in endosomes and phagosomes. Counterintuitively, M. marinum growth is also impaired in zntB KO cells, where MCVs accumulate less Zn2+. We also demonstrate that M. marinum senses toxic levels of Zn2+ and responds by upregulating its Zn2+ exporter CtpC, which supports bacteria survival under these restrictive conditions. Attenuation of M. marinum intracellular proliferation in zntA and zntB KO cells is accentuated in the absence of CtpC, confirming that mycobacteria face noxious levels of Zn2+. Altogether, we show for the first time that M. marinum infection induces a deleterious Zn2+ elevation in D. discoideum, which is counteracted by the bacteria with the induction of its Zn2+ exporter CtpC.

cell biology↗

bioRxiv2018

Identification of novel anti-bacterial compounds from a chemically highly diverse pathways-based library using phenotypic screens in amoebae host models

Legionella pneumophila and tubercular Mycobacteria are the causative agents of potentially fatal diseases due to their pathogenesis but also to the emergence of antibiotic resistance that limits treatment strategies. The aim of our study is to explore the antimicrobial activity of a small ligand-based chemical library of 1,255 structurally diverse compounds. These compounds were screened in a combination of three assays, two monitoring the intracellular growth of the pathogenic bacteria, Mycobacterium marinum and L. pneumophila, and an additional anti-virulence \"plaque\" assay for M. marinum. We set up these assays using two amoeba strains, the genetically tractable Dictyostelium discoideum and the free-living amoeba Acanthamoeba castellanii. In summary, sixty-four compounds showed anti-infective/anti-virulence activity in at least one of the 3 assays. The intracellular assays hit rate varied between 1.7% (n=22) for M. marinum and 2.8% (n=35) for L pneumophila with 7 compounds in common between both pathogens. In parallel, 1.2 % (n= 15) of the tested compounds were able to restore D. discoideum growth in presence of M. marinum spiked in a lawn of Klebsiella pneumoniae. We also validated the generality of the hit compounds identified using the A. castellanii-M. marinum anti-infective screen in the powerful D. discoideum-M. marinum host-pathogen model. The characterization of anti-infective and antibacterial hits in the latter infection model revealed compounds able to reduce intracellular growth more than 50% at 30 M. Our studies underline the relevance of using a combination of low-cost and low-complexity assays with full 3R compliance associated with a rationalized focused library of compounds to help identifying new chemical scaffolds and dissect some of their properties prior to run further compounds development steps.

microbiology↗

bioRxiv2018

Think zinc: Role of zinc poisoning in the intraphagosomal killing of bacteria by the amoeba Dictyostelium

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 znt knockouts 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.\n\nSummary statementMetal poisoning is one of the bactericidal strategies of macrophages. Here, we describe the dynamics of free Zn and the role of Zn transporters during phagocytosis in Dictyostelium.

cell biology↗

bioRxiv2018

PIKfyve/Fab1 is required for efficient V-ATPase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum.\n\nPIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.\n\nImportanceCells that capture or eat bacteria must swiftly kill them to prevent pathogens from surviving long enough to escape the bactericidal pathway and establish an infection. This is achieved by the rapid delivery of components that produce an antimicrobial environment in the phagosome, the compartment containing the captured microbe. This is essential both for the function of immune cells and for amoebae that feed on bacteria in their environment. Here we identify a central component of the pathway used by cells to deliver antimicrobial components to the phagosome and show that bacteria survive over three times as long within the host if this pathway is disabled. We show that this is of general importance for killing a wide range of pathogenic and non-pathogenic bacteria, and that it is physiologically important if cells are to avoid infection by the opportunistic human pathogen Legionella.

cell biology↗

bioRxiv2018

ESCRT and autophagy cooperate to repair ESX-1-dependent damage to the Mycobacterium-containing vacuole

Phagocytes capture invader microbes within the bactericidal phagosome. Some pathogens subvert killing by damaging and escaping from this compartment. To prevent and fight bacterial escape, cells contain and repair the membrane damage, or finally eliminate the cytosolic escapees. All eukaryotic cells engage highly conserved mechanisms to ensure integrity of membranes in a multitude of physiological and pathological situations, including the Endosomal Sorting Complex Required for Transport (ESCRT) and autophagy machineries. In Dictyostelium discoideum, recruitment of the ESCRT-III protein Snf7/Chmp4/Vps32 and the ATPase Vps4 to sites of membrane repair relies on the ESCRT-I component Tsg101 and occurs in absence of Ca2+. The ESX-1 dependent membrane perforations produced by the pathogen Mycobacterium marinum separately engage both ESCRT and autophagy. In absence of Tsg101, M. marinum escapes earlier to the cytosol, where it is restricted by xenophagy. We propose that ESCRT has an evolutionary conserved function in containing intracellular pathogens in intact compartments.

microbiology↗