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Phylogenetic tree of the ZnT family. The sequence of human ZNT4 was used to search for zinc transporters of the ZnT family among non-redundant sequences in selected organisms using NCBI PSI-BLAST. A score of 2 × 10⁻⁵ was used as a threshold. Sequences were aligned with the software AliView [(218); http://ormbunkar.se/aliview/] and curated manually, to remove divergent N- and C-terminus. Trees were built using MAFFT and the E-INS-i strategy (219).
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The soil-dwelling social amoeba Dictyostelium discoideum feeds on bacteria. Each meal is a potential infection because some bacteria have evolved mechanisms to resist predation. To survive such a hostile environment, D. discoideum has in turn evolved efficient antimicrobial responses that are intertwined with phagocytosis and autophagy, its nutrien...
Citations
... Dictyostelium discoideum, a soil-dwelling social amoeba, has emerged as a valuable model for host-pathogen interactions and drug discovery (Froquet et al. 2009). D. discoideum is amenable to high-throughput screening of natural product extracts and has been used as a host model system for several human pathogens, including Mycobacterium tuberculosis (Dunn et al. 2017), Legionella pneumophila (Escoll et al. 2013), Pseudomonas aeruginosa (Cosson et al. 2002;Pukatzki et al. 2002;Bravo-Toncio et al. 2016) and K. pneumoniae (Marcoleta et al. 2018). Some reports have used this invertebrate model system to screen the cytotoxicity of natural extracts (Hwang and Kim 2002;McQuade et al. 2013;Ilacqua et al. 2017); however, few have simultaneously used the social amoeba to screen potential anti-virulence drugs without cytotoxic side effects (Bravo-Toncio et al. 2016). ...
Abstract
The rise of antibiotic-resistant bacterial strains represents an important challenge for global health, underscoring the critical need for innovative strategies to confront this threat. Natural products and their derivatives have emerged as a promising reservoir for drug discovery. The social amoeba Dictyostelium discoideum is a potent model organism in this effort. Employing this invertebrate model, we introduce a novel perspective to investigate natural plant extracts in search of molecules with potential antivirulence activity. Our work established an easy-scalable developmental assay targeting a virulent strain of Klebsiella pneumoniae, with Helenium aromaticum as the representative plant. The main objective was to identify tentative compounds from the Helenium aromaticum extract that attenuate the virulence of K. pneumoniae virulence without inducing cytotoxic effects on amoeba cells. Notably, the methanolic root extract of H. aromaticum fulfilled these prerequisites compared to the dichloromethane extract. Using UHPLC Q/Orbitrap/ESI/MS/MS, 63 compounds were tentatively identified in both extracts, 47 in the methanolic and 29 in the dichloromethane, with 13 compounds in common. This research underscores the potential of employing D. discoideum-assisted pharmacognosy to discover new antivirulence agents against multidrug-resistant pathogens.
... In this paper, we develop theory for concentration sensing in a fluctuating environment inspired by Dictyostelium discoideum (Dicty), a common model organism for chemotaxis (16)(17)(18). Dicty consumes bacteria and can detect these bacteria by sensing folic acid given off as a byproduct of bacterial metabolism (3). We develop theory and simulation describing Dictyostelium in a relatively simple environment-a cell surrounded by bacteria in 3D liquid. ...
... whereT ¼ T=t r . Equation 17 shows that the optimal averaging behavior depends on two key parameters: the ratio of the environmental variation to the snapshot detection error, s 2 env =s 2 det , and ut r . Here, ut r is the ratio of the correlation time for the receptors t r to the correlation time for the environment, which is 1=u. ...
When cells measure concentrations of chemical signals, they may average multiple measurements over time in order to reduce noise in their measurements. However, when cells are in an environment that changes over time, past measurements may not reflect current conditions—creating a new source of error that trades off against noise in chemical sensing. What statistics in the cell’s environment control this trade-off? What properties of the environment make it variable enough that this trade-off is relevant? We model a single eukaryotic cell sensing a chemical secreted from bacteria (e.g., folic acid). In this case, the environment changes because the bacteria swim—leading to changes in the true concentration at the cell. We develop analytical calculations and stochastic simulations of sensing in this environment. We find that cells can have a huge variety of optimal sensing strategies ranging from not time averaging at all to averaging over an arbitrarily long time or having a finite optimal averaging time. The factors that primarily control the ideal averaging are the ratio of sensing noise to environmental variation and the ratio of timescales of sensing to the timescale of environmental variation. Sensing noise depends on the receptor-ligand kinetics, while environmental variation depends on the density of bacteria and the degradation and diffusion properties of the secreted chemoattractant. Our results suggest that fluctuating environmental concentrations may be a relevant source of noise even in a relatively static environment.
... The stages of infection of M. tuberculosis and M. marinum in a variety of evolutionary distant phagocytes are extremely well conserved, and a single cycle of infection lasts about 48 hours (illustrated in Fig. 1A). After entry by phagocytosis in animal phagocytes of the innate immune system (8) or in amoebae (3,9), secretion of virulence factors, and especially of EsxA, via the ESX-1 secretion system inflicts membrane damage to the Mycobacteria-containing Vacuole (MCV) (10)(11)(12), which are repaired by a combination of cytosolic machinery. After cycles of damage and repair, the mycobacteria reach the cytosol where they continue to proliferate until they disseminate via lytic and non-lytic mechanisms (13)(14)(15)(16). ...
... The social amoeba, Dictyostelium discoideum is an alternative phagocytic host model to study interactions with environmental and pathogenic bacteria (9,22). The phagocytic pathway is highly conserved and represents a major restriction point for bacteria in both macrophages and amoebae. ...
... D. discoideum has emerged as a powerful genetically and biochemically tractable model to study processes of M. marinum infection, and in particular has recently been used to demonstrate the role of the ESCRT and autophagy machinery in MCV membrane repair (24,25), the role of metal poisoning in bacterial growth restriction (26,27) as well as to identify anti-infective compounds (28,29). Therefore, we use D. discoideum and M. marinum as a model system to study cell-autonomous defense mechanisms that are relevant to the pathogenesis of tuberculosis (3,8,9). ...
The emergence of biochemically and genetically tractable host model organisms for infection studies holds the promise to accelerate the pace of discoveries related to the evolution of innate immunity and the dissection of conserved mechanisms of cell-autonomous defenses. Here, we have used the genetically and biochemically tractable infection model system Dictyostelium discoideum / Mycobacterium marinum to apply a genome-wide transposon-sequencing experimental strategy to reveal comprehensively which mutations confer a fitness advantage or disadvantage during infection and compare these to a similar experiment performed using the murine microglial BV2 cells as host for M. marinum to identify conservation of virulence pathways between hosts.
... Dictyostelium is equipped with a broad array of surface receptors that exhibit substantial homology to mammalian PRRs, such as scavenger receptors (LIMP-2), TLRs (tirA, tirB), leucine-rich repeat receptors (LrrA), and C-type lectin receptors (Bajgar and Krejcǒva, 2023). Activation of such receptors triggers intracellular signaling cascades initiating phagocytosis, phagosome maturation, and bacterial death, as well as cascades related to stress and detoxification responses (Dunn et al., 2018). Metchnikoff's seminal observations shed light on the intimate connection between Biology (physiology) and Pathology (defense) (Metchnikoff, 1893). ...
The formation of the innate immune system of animals can only be envisioned after the development of the first metazoan embryo. The decisive role of Embryology in understanding the evolution of the immune system has been inexplicably disregarded in the history of science. Some characteristics of our holozoan ancestors, including macrophage-like movement and enteric phagocytosis, were suppressed by the formation of chains of physically attached cells in the context of embryo multicellularity. The formation of the archenteron during morphogenesis of the first embryo resulted in a meta-organism whose survival was dependent on the ability to perform enteric phagocytosis (nutrition on bacteria). By recognizing the neoplastic basis of embryo formation, it is possible to venture a glimpse at its other face, a process that becomes evident when the extracellular matrix and cadherin junctions are destroyed. What ensues is metastasis (in the case of cancer) or an alternative version controlled by cell differentiation (during embryogenesis). In the context of innate immunity, the development of mesogleal cells by epithelial–mesenchymal transition and differentiation into cells specialized in bacterial recognition allowed the newly formed animal to preserve homeostasis, an innovation that has been maintained throughout evolution. In this article, I will share my first reflections on the embryonic origin of innate immunity and its close relationship with cancer. Innate immunity arises naturally during embryogenesis, which explains why the immune system typically does not react against cancer cells. In its essence, the immune system was created from them. Here, I argue that the first embryo can be understood as a benign tumor nourished and protected by the innate immune system.
... Following acidification of the phagosome, a pH of about 4.5-5 is attained in macrophages and 3.5-4 in Dictyostelium discoideum [32,33]. In these acidic conditions, a variety of distinct vesicular enzymes, including proteases, hydrolases, and lysozymes, are sequentially recruited to the phagosome to break down different bacterial components [34]. ...
Metals and metalloids are used as weapons for predatory feeding by unicellular eukaryotes on prokaryotes. This review emphasizes the role of metal(loid) bioavailability over the course of Earth’s history, coupled with eukaryogenesis and the evolution of the mitochondrion to trace the emergence and use of the metal(loid) prey-killing phagosome as a feeding strategy. Members of the genera Acanthamoeba and Dictyostelium use metals such as zinc (Zn) and copper (Cu), and possibly metalloids, to kill their bacterial prey after phagocytosis. We provide a potential timeline on when these capacities first evolved and how they correlate with perceived changes in metal(loid) bioavailability through Earth’s history. The origin of phagotrophic eukaryotes must have post-dated the Great Oxidation Event (GOE) in agreement with redox-dependent modification of metal(loid) bioavailability for phagotrophic poisoning. However, this predatory mechanism is predicted to have evolved much later – closer to the origin of the multicellular metazoans and the evolutionary development of the immune systems.
... D. discoideum is a soil amoeba feeding on microorganisms. Its small haploid genome allows relatively easy genetic manipulation and has made it a convenient model system to study many biological processes, in particular the complex interactions between phagocytic cells and bacteria (Cosson & Soldati, 2008), and the largely conserved mechanisms ensuring killing of bacteria (Dunn et al., 2017). P. aeruginosa is an opportunistic pathogen, ubiquitous in the soil and water (Hardalo & Edberg, 1997). ...
Ingestion and killing of bacteria by phagocytic cells are critical processes to protect the human body from bacterial infections. In addition, some immune cells (neutrophils, NK cells) can release microbicidal molecules in the extracellular medium to eliminate non‐ingested microorganism. Molecular mechanisms involved in the resulting intracellular and extracellular killing are still poorly understood. In this study, we used the amoeba Dictyostelium discoideum as a model phagocyte to investigate the mechanisms allowing intracellular and extracellular killing of Pseudomonas aeruginosa. When a D. discoideum cell establishes a close contact with a P. aeruginosa bacterium, it can either ingest it and kill it in phagosomes, or kill it extracellularly, allowing a direct side‐by‐side comparison of these two killing modalities. Efficient intracellular destruction of P. aeruginosa requires the presence of the Kil2 pump in the phagosomal membrane. On the contrary, extracellular lysis is independent on Kil2 but requires the expression of the superoxide‐producing protein NoxA, and the extracellular release of the AplA bacteriolytic protein. These results shed new light on the molecular mechanisms allowing elimination of P. aeruginosa bacteria by phagocytic cells.
... Dictyostelium discoideum, a soil-dwelling social amoeba, has emerged as a valuable model for host-pathogen interactions and drug discovery 5 . D. discoideum is amenable to high-throughput screening of natural product extracts and has been used as a host model system for several human pathogens, including Mycobacterium tuberculosis 6 , Legionella pneumophila 7 , Pseudomonas aeruginosa [8][9][10] and Klebsiella pneumoniae 11 . Some reports have used this ...
The rise of antibiotic-resistant bacterial strains poses a significant global health challenge, underscoring the critical need for innovative strategies to address this threat. Natural products and their derivatives have emerged as a promising reservoir for drug discovery. The social amoeba Dictyostelium discoideum is an advantageous model organism in this effort. Using this invertebrate model, we introduce a novel perspective to screen natural plant extracts for molecules with potential antivirulence activity. As a proof of concept, we established a simple high-throughput assay to screen for antivirulence molecules targeting Klebsiella pneumoniae among extracts of Helenium aromaticum . Thus, we aimed to identify compounds attenuating K. pneumoniae virulence without inducing cytotoxic effects on amoeba cells. Notably, the methanolic root extract of H. aromaticum but not other extracts fulfilled these prerequisites. Further analysis via UHPLC-ESI-MS/MS led to the identification of 24 chemical compounds boasting potential antivirulence attributes. This research underscores the potential of employing D. discoideum -assisted pharmacognosy for unearthing novel antivirulence agents against multidrug-resistant pathogens.
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... As a result, infection by Paraburkholderia allows D. discoideum spores to co-disperse with prey bacteria that can seed new populations, which is beneficial to spores that disperse to areas with limited prey [13,15]. D. discoideum amoebae can also be infected by numerous human intracellular pathogens under laboratory conditions, making it a popular model system for studying bacterial pathogenesis [16][17][18][19]. In nature, amoebae sometimes act as environmental reservoirs for pathogenic bacteria such as Bordetella bronchiseptica [20] and Mycobacterium bovis [21]. ...
The soil amoeba Dictyostelium discoideum acts as both a predator and potential host for diverse bacteria. We tested fifteen Pseudomonas strains that were isolated from transiently infected wild D. discoideum for ability to escape predation and infect D. discoideum fruiting bodies. Three predation-resistant strains frequently caused extracellular infections of fruiting bodies but were not found within spores. Furthermore, infection by one of these species induces secondary infections and suppresses predation of otherwise edible bacteria. Another strain can persist inside of amoebae after being phagocytosed but is rarely taken up. We sequenced isolate genomes and discovered that predation-resistant isolates are not monophyletic. Many Pseudomonas isolates encode secretion systems and toxins known to improve resistance to phagocytosis in other species, as well as diverse secondary metabolite biosynthetic gene clusters that may contribute to predation resistance. However, the distribution of these genes alone cannot explain why some strains are edible and others are not. Each lineage may employ a unique mechanism for resistance.
... marinum) hostpathogen system (Tobin and Ramakrishnan, 2008;Habjan et al., 2021). D. discoideum is a social amoeba and a professional phagocyte with conserved innate immune response pathways, such as phagocytosis, ROS production and autophagy (Dunn et al., 2018). Working on Mycobacterium tuberculosis (Mtb) as a model in laboratories entails high operational costs and constitutes a clear obstacle to the development of high-throughput testing methods (World Health Organisation, 2012), as it presents high risks of contaminations for humans and therefore requires biosafety level 3 laboratories. ...
Antibiotics resistance is a clear threat to the future of current tuberculosis treatments like rifampicin, prompting the need for new treatment options in this field. While plants can offer a plethora of chemical diversity in their constitutive natural products to tackle this issue, finding potentially bioactive compounds in them has not always proven to be that simple. Classical bioactivity-guided fractionation approaches are still trendy, but they bear significant shortfalls, like their time-consuming nature as well as the ever-increasing risk of isolating known bioactive compounds. In this regard, we have developed an alternative method to the latter approach that allows for natural derivatives of a known bioactive scaffold to be efficiently targeted and isolated within a large library of plant extracts. Hence our approach allows for the anticipation of bioactive structure independently of preliminary bioassays. By relying on the chemical diversity of a set of 1,600 plant extracts analyzed by HRMS/MS, we were able to isolate and characterize several minor derivatives of a previously reported bioactive aza-anthraquinone compound from Cananga brandisiana , selected within the plant set. Assessment of bioactivity on these derivatives (especially onychine, with an IC 50 value of 39 µM in infection) confirmed their expected activity on Mycobacterium marinum in our anti-infective assay. This proof-of-concept study has established an original path towards bioactive compounds isolation, with the advantage of potentially highlighting minor bioactive compounds, whose activity may not even be detectable at the extract level.
... In turn, innate immune cells may exploit ROS to activate the fungal apoptosis-like pathway, thereby killing ingested conidia of the human pathogen A. fumigatus (Shlezinger et al. 2017). Besides immune cells, the generation of ROS as a key component of cell autonomous defence against various pathogens has also been observed for environmental predators, such as insects (Bergin et al. 2005), nematodes (McCallum and Garsin 2016), or amoebae (Dunn et al. 2018). ...
Predator–prey interactions are obvious drivers in the evolution among higher animals, but until today, there is little known about such relationships in the microverse. Like any other microbes, fungi can be prone to predation by bacteria, amoebae, or nematodes. While the boundaries between parasitism, biotrophy, and predation appear to be less sharp for bacteria, highly sophisticated mechanisms to feed on are obvious for unicellular amoebae and nematodes. Even phagocytic amoebae are not restricted to the ingestion and intracellular killing of single yeast cells or conidia but they can also attack and invade entire hyphae of filamentous fungi. Nematodes, in turn, can specifically open hyphae via injection-needle like stylets. In this chapter, we provide a selected overview of examples from recent years on how fungal preys exploit a variety of strategies to either escape or survive attacks by these specialized environmental predators. At least some of these factors have been shown to fulfil a dual function, serving in predator defence but also against innate immune cells when colonizing higher animals. Whether predatory defence has thus acted as a selection pressure towards virulence determinants in fungi is a matter of ongoing research.KeywordsAmoebaPredationPhagocytosisVirulence
