D. polycephalum is ancestral and show different characteristics than other species of Dictyostelium (5,7,8). The patient sought treatment 4 months after his initial visit. SM/25, SM/50, and SM/100-plates contained 1/25, 1/50, or 1/100 of a SM-stock solution [1% glucose/1% Bacto peptone (Difco)/0.1% Bacto yeast extract (Difco)/4.2 mM MgSO4,], respectively. Furthermore, addition of purified pyocyanin did not affect the viability of axenically growing amoebae. Complementation of the lasR mutation restores LasR function and virulence but does not affect synthesis of the pigment pyocyanin. The lasR gene was recovered from plasmid pKDT17 (18) as a 0.8-kb EcoRI/HindIII fragment and subcloned in front of the lac promoter of EcoRI/HindIII-digested pUCP18 (12). P. aeruginosa formed lawns on these plates with amoebae embedded in them. There are a number of ecological changes that offer opportunities for existing nonpathogenic organisms to become pathogenic and adapt to the human body. When amoebae were plated on nutrient agar plates with lawns of PA103, no Dictyostelium plaques emerged (see Figs. discoideum genome harbors several homologs of human genes responsible for a variety of diseases, ­including Chediak-Higashi syndrome, lissencephaly, mucolipidosis, Huntington disease, IBMPFD, and Shwachman-Diamond syndrome. After 48 hours’ of incubation, a nonnutrient agar plate showed growth of double-walled, spherical cysts ≈6–7 µm in diameter that had different morphologic features than those of Acanthamoeba spp. infection. A number of different genetic manipulations, including insertional mutagenesis, chemical mutagenesis, complementation, and cloning, are available to identify mutations that confer resistance to Pseudomonas infections (for a review of available methods, see ref. The small size of the genome (≈34 Mb) and the small amount of noncoding sequence allow us to readily saturate the genome with mutations. Introduction. The bacterium is able to replicate in different phagocytic and nonphagocytic cells including mammalian, amoebae, and arthropod cells. Emerging D. discoideum plaques on each plate were scored after 5 days. Expression of ExoU in P. aeruginosa PA103 prohibits plaque formation by D. discoideum. ), as well as by a grant from the Cystic Fibrosis Foundation (to S.U.P.). 4). Overexpression of lasR from plasmid pLasR-PUH did not restore pyocyanin production in strain SUP17. 5). Furthermore, a PA14 mutant that is unable to secrete pyocyanin because of a deletion in the phenA and phenB genes is still virulent in our plaque assay (see Fig. To survive this situation, it has evolved multiple mechanisms to generate a microbicidal environment within phagosomes and thus, phagocytosis, its means of nutrient acquisition, is simultaneously a major component of its defenses against infection. D. discoideum amoebae could not form plaques in the complemented exoU mutant (see Figs. The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum. The addition of purified pyocyanin to axenic cultures of D. discoideum at a concentration of 7 μg/ml, the highest amount of pyocyanin we ever found in P. aeruginosa supernatants did not affect the viability of growing amoebae (data not shown). consists of an ameboid vegetative phase, a cyst phase, and a plantlike fruiting phase (6). However, careful attention to cyst morphology in clinical samples and culture enabled us to identify this organism. It can be observed at organismic, cellular, and molecular levels primarily because of their restricted number of cell types and behaviors, and their rapid growth. Saving Lives, Protecting People, Author affiliation: L.V. Microscopic examination showed double-walled spherical cysts in potassium hydroxide with calcofluor white stain, Gram stain (Figure, panels A, B), and Giemsa stain. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δ atg1 ), F. noatunensis subsp. The research areas presently pursued include the use of D. discoideum to identify genetic host factors determining the outcome of infections and the use as screening system for identifying bacterial virulence factors. The inositol polyphosphate 5-phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole … Dictyostelium cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. 2 Top) (10). This model system of host-pathogen interaction has a critical distinction: because Dictyostelium discoideum cannot survive in temperatures higher than 27°C, the incubation temperature of the model has to be about 25.5°C. Cells were infected with bacteria at a multiplicity of infection of approximately 100:1. (Middle) Elastase activity of supernatants from overnight cultures were assayed by using elastin Congo red (ECR) as the substrate. However, on further incubation for 3 weeks at 36°C, no sporocarp formed. 5 and 6B). Previous work demonstrated that PA103 uses type III secretion to kill macrophages and epithelial cells in vitro (31). The entire genomic sequence will be known soon, and this will accelerate the identification of genes that emerge from any genetic screen for resistance to P. aeruginosa. We report a case of keratitis caused by Dictyostelium polycephalum in an immunocompetent person. To survive such a hostile environment, D. discoideum has in turn evolved efficient antimicrobial responses that are intertwined with phagocytosis and autophagy, its nutrient acquisition pathways. P. aeruginosa therefore has the capacity to kill D. discoideum under these conditions. To study the interaction of P. aeruginosa and a host cell, we suggest the genetically tractable organism Dictyostelium discoideum. We constructed a plasmid, pLasR-PUH, which carries lasR fused to the lacZ promoter. Reduced pyocyanin production is expected in a lasR mutant because LasR directly controls the expression of RhlR, a transcription factor that is involved in pyocyanin production (24). D. discoideum forms plaques in lawns of 12A1 that are approximately one-tenth the size of those formed in lawns of K. aerogenes (data not shown). Infection was initiated by centrifugation (750 × g; 10 min). Iris and lens details could not be distinguished because of corneal haze. Virulence of the complemented exoU strain is not because of an increased growth rate. It is also used to study other aspects of development, including cell sorting, pattern formation, phagocytosis, mot… Because strain PA103 does not produce cytotoxins ExoY and ExoS, we determined the virulence of a PA103 mutant that carries a Tn5-transposon insertion in the exoU gene (15, 30, 34). The influence of methylamine and infectious doses on the results of infection of Dictyostelium discoideum and Acanthamoeba castellanii with Francisella novicida . Dictyostelium discoideum has long been used as a model organism to study many different biological processes. We deposited the sequence of our isolate in GenBank (accession no. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. Amanda Rodewald, Ivan Rudik, and Catherine Kling talk about the hazards of ozone pollution to birds. Kenneth Bryan Raper (1908-1987) This Special Issue of The International Journal of Developmental Biology is dedicated to Kenneth Bryan Raper (1908- 1987), who discovered Dictyostelium discoideum and described the remarkable features of this organism, setting up the basis for its use as a model system. Dictyostelium: genus of protozoa, formerly also considered a fungus; its natural habitat is decaying forest leaves, where it feeds on bacteria; often called cellular slime mold; D. discoideum is the best known species and is widely used in biomedical research. Reddy AK, Balne PK, Garg P, et al. in potassium hydroxide (calcoflour white stain; original magnification ×40) preparation. The identification of free living environmental isolates of amoebae from Bulgaria. 5). As shown in Fig. To the Editor: Although Dictyostelium spp. D. discoideum amoebae were plated on nutrient agar plates in combination with K. aerogenes or P. aeruginosa strains PA14 and PA14ΔrhlA. D. discoideum is well suited for studying the host biology of pathogens, because it offers a variety of different genetic manipulations. Dictyostelium amoebae formed plaques in lawns of the pscJ mutant, suggesting that during infections with wild-type strain PA103, cytotoxins are injected into the D. discoideum host cells (see Fig. Immunocompromised individuals are at high risk of becoming infected in a hospital setting where P. aeruginosa causes a variety of nosocomial infections, including pneumonia, urinary tract infections, surgical wound infections, and blood stream infections (for review, see ref. The cornea showed a ring-shaped infiltrate, central thinning, surrounding corneal edema, and pigments on the endothelium (Figure, panel C); these findings were identical to the clinical picture of Acanthamoeba keratitis. major areas using D. discoideum as a model organism in biomedicine. After an incubation at 37°C for 16 h, the colony-forming units were counted. (Top) D. discoideum cells (AX3) were plated on nutrient agar (SM/5) with P. aeruginosa strain PA14, the isogenic lasR-mutant 12A1, complemented strains SUP16 (ΔlasR with pUCP18) and SUP17 (ΔlasR with plasR-PUH), and the pyocyanin-deficient strain ΔphenA/ΔphenB. Most myxamoebae aggregate to form sporocarps; however, some may round up in individual cells to form microcysts (8).The D. polycephalum isolated from our patient grew at 36°C on nonnutrient agar with an Escherichia coli overlay. Dictyostelium discoideum is a natural predator of bacteria and must contend with the fact that every meal is a potential infection. In some of the experiments, low-nutrient plates were used to limit bacterial growth. Deep anterior lamellar keratoplasty was performed after 2 days. The cellular slime mold Dictyostelium discoideum occupies a unique position in phylogeny, branching from the phylogenic trunk after plants but before the fungal lineages . Initially, P. aeruginosa colonizes the airways with other pathogens, like Haemophilis influenzae and Staphylococcus aureus. The experimentally versatile Dictyostelium discoideum – Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity and host response. Work with C. elegans as a model host showed that P. aeruginosa secretes a pigment, pyocyanin, that exerts its toxic effect on eukaryotic cells through reactive oxygen species (10). The myxamoebae were seen after 24 hours, and the amebae had transformed into microcysts after 48 hours of incubation. Strain 8C12 is identical to 12A1, except that the TnphoA transposon is in another gene, which does not affect virulence toward amoebae (10). D. discoideum is a complex eukaryote that lives part of its life cycle as a unicellular amoeba. Furthermore, several genes in D. discoideum have shown to be homologous with human genes. INTRODUCTION Tuberculosis (TB) is an old but re-emerging global health threat caused by mycobacteria belonging to the Mycobacterium tuberculosis (Mtb) complex (WHO, 2018). Identification of the host targets of ExoU may reveal a signaling pathway that can be stimulated with existing or novel drugs. Reddy, A. K., Balne, P. K., Garg, P., Sangwan, V. S., Das, M., Krishna, P. V....Vemuganti, G. K. (2010). 4). We speculate that this is because high levels of intracellular LasR inhibit the activity of the transcription factor RhlR at a posttranslational level (25). The soil-dwelling social amoeba Dictyostelium discoideum feeds on bacteria. Because pyocyanin synthesis is controlled by quorum sensing, we investigated whether P. aeruginosa-induced killing of Dictyostelium was mediated by pyocyanin. Starvation triggers a change in life style, forcing cells to gather into aggregates to form multicellular organisms capable of cell differentiation and morphogenesis. Agar plates contained 1/25 (SM/25), 1/50 (SM/50), and 1/100 (SM/100) of the nutrient content in SM plates. D. discoideum cells were placed in tissue culture wells at 106 cells/ml. D. discoideum cells do not form plaques in lawns of P. aeruginosa strain PA14. Using D. discoideum as a genetically tractable model host takes advantage of the organism's ability to integrate complicated activities in a single cell, which includes responses to pathogens. The lysates were diluted and plated on Luria broth agar plates. Increasing the host response in such a manner may help cystic fibrosis patients to clear infections with P. aeruginosa that lack ExoU. The gentamicin protection experiment demonstrates that D. discoideum has the capacity to phagocytose all P. aeruginosa strains tested. Chagas disease (human American trypanosomiasis), which is caused by the protozoan parasite Trypanosoma cruzi, is responsible for numerous deaths each year; however, established treatments for the disease are limited.Differentiation-inducing factor-1 (DIF-1) and DIF-3 are chlorinated alkylphenones originally found in the cellular slime mold Dictyostelium discoideum that have been … Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. The similarities between the Dictyostelium and mammalian cells extend to membrane trafficking, endocytic transit and sorting events. Amoebae were collected at indicated time points, lysed, and plated on nutrient agar plates to determine the colony-forming units (cfu) in these lysates. Pyocyanin was extracted from supernatants and assayed as described in (20). Dictyostelium as host model for pathogenesis Dictyostelium as host model for pathogenesis Steinert, Michael; Heuner, Klaus 2005-03-01 00:00:00 Introduction Whether or not host organisms become infected by pathogens is the result of a complex interplay between host and pathogen genotypes, as well as the physiological condition of both species. Dictyostelium formed plaques in lawns of SUP16, but only a few petite plaques could be identified in lawns of SUP17 (see Fig. Cytotoxin ExoU contributes to the pathogenicity of P. aeruginosa PA103 toward D. discoideum, because PA103 mutants that are defective in type III secretion, or production of cytotoxin ExoU are avirulent toward D. discoideum. Dictyostelium polycephalum Infection of Human Cornea. Although we could identify the microorganism, the source of infection is unknown. Dictyostelium species are found in arctic, tropical, desert, and rain forest soils . The vision in his right eye and results of a clinical examination were within normal limits. For example, strain 12A1 might be taken up at an increased rate if the loss of LasR had affected a virulence mechanism that inhibits phagocytosis by the host cell. To identify the organism, we extracted DNA from the growth on nonnutrient agar and subjected it to PCR specific for Acanthamoeba spp (3); results were negative. Introduction: Dictyostelium Description of Dictyostelium. The experimentally versatile Dictyostelium discoideum – Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity and host response. Download PDF. The complemented lasR strain SUP17 shows elastase levels comparable to PA14, indicating that the function of LasR is restored in this strain. A number of P. aeruginosa mutants have been identified that are avirulent in this assay. Genetically accessible host models are useful for studying microbial pathogenesis because they offer the means to identify novel strategies that pathogens use to evade immune mechanisms, cause cellular injury, and induce disease. Because we were not aware of any drug treatment recommendations for infection by this organism, and the disease was advanced, surgical treatment was advised. Bacteria were grown in Luria broth for 16 h, pelleted by centrifugation (1,600 × g; 15 min), and resuspended in HL/5. 1, D. discoideum feeds on K. aerogenes and forms plaques that become readily apparent after a few days. ... DOI: 10.1016/S0140- human or animal infections caused by To identify the organism, we ex- 6736(85)90740-8 this organism have not been reported. The mutation in pscJ prohibits export of the four known cytotoxins by P. aeruginosa strain PA103 (15, 33). Dictyostelium discoideum is a species of soil-dwelling amoeba belonging to the phylum Amoebozoa, infraphylum Mycetozoa.Commonly referred to as slime mold, D. discoideum is a eukaryote that transitions from a collection of unicellular amoebae into a multicellular slug and then into a fruiting body within its lifetime. Rhamnolipids are biosurfactants that lyse host cells by disrupting their cell membranes (26). We measured the amounts of extracellular rhamnolipids from P. aeruginosa cultures in stationary phase and found no significant differences among the lasR-mutant 12A1, the plasmid-complemented strain SUP17, and wild-type PA14 (data not shown). It is used to study cell differentiation, chemotaxis, and apoptosis, which are all normal cellular processes. Altogether, the Dictyostelium-F. noatunensis subsp. To test whether the ability of Dictyostelium to form plaques on strain 12A1 is due to the removal of LasR, we carried out a plasmid complementation experiment. In culture, it grows at a temperature of 34°C–35°C, which is higher than that for other species of Dictyostelium (8). RhlA encodes a subunit of rhamnosyltransferase 1, an essential enzyme in the synthesis of rhamnolipids, and mutations in rhlA result in the inability to secrete any rhamnolipids (13). Because the patient was a manual laborer, he could have become infected with the organism from contaminated water or soil. We then tested the virulence of this strain in our plaque assay. Shapeshifting designs could have wide-ranging pharmaceutical and biomedical applications in coming years. Therefore, rhamnolipids do not play a role in the virulence of P. aeruginosa on D. discoideum (data not shown). the importance of the microbiome in human development, health, and well being, drives the study of host effectors that control its bacterial composition (Peterson and Artis 2014, Singh et al., 2017, Dominguez-Bello et al., 2019). Dictyostelium is a genus of single- and multi-celled eukaryotic, phagotrophic bacterivores. Enter multiple addresses on separate lines or separate them with commas. Abstract. Photo reproduced with permission from the University Archives and Records Mana - Social amoebae like Dictyostelium discoideum recognize specific bacterial species and … As shown in Fig. Liquid SM/5 medium, the same medium that was used to prepare nutrient agar plates for the plaque assay, was inoculated with various PA103 mutants, and their growth at 22°C was followed over 30 h. The growth properties of strains defective in the exoU or pscJ genes did not differ from their isogenic parent PA103. Although the evidence is still limited, a growing body of research suggests music may have beneficial effects for diseases such as Parkinson’s. Prasad Eye Institute, Hyderabad, India, Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of. Each meal is a potential infection because some bacteria have evolved mechanisms to resist predation. Therefore, D. discoideum amoebae are able to feed on virulent P. aeruginosa when bacteria are present at low abundance. are used for studying signal transduction, cytoskeletal functions, endocytosis, and molecular pathogenesis of infectious and other diseases (1), human or animal infections caused by this organism have not been reported. This cellular slime mold is a major player in many aspects of medicine and cell research. SpcU (for specific Pseudomonas chaperone for ExoU) encodes a chaperone that is necessary for proper translocation of ExoU (35). Microbiol. E-mail: stefan_pukatzki{at}hms.harvard.edu. A rhlA mutant of PA14 was constructed with knockout-plasmid pEX100T-rhlA∷Gm by using the site-specific insertional mutagenesis strategy of Schweizer and Hoang (13, 14). noatunensis infection model recapitulates the course of infection described in other host systems. Dictyostelium discoideum shares many of the physiological functions seen in mammalian cells as well as other amoebae, such Dictyostelium strain AX3 was used in all experiments (7). To test whether P. aeruginosa is internalized and digested by D. discoideum, a modification of the gentamicin protection assay was used (21). The use of D. discoideum as a host model may lead to the identification of cellular host components that are targeted by ExoU. It is this unicellular lifestyle that enables us to study the interaction between a host and a pathogen directly with only these two organisms present. Social amoebae like Dictyostelium discoideum recognize specific bacterial species and … If such a protein is a host component that is targeted by a particular virulence factor, its removal may confer resistance to the pathogen. Besides quorum-sensing mediated virulence, P. aeruginosa utilizes a type III secretion mechanism to kill D. discoideum. We have identified a number of P. aeruginosa mutants that are avirulent in this assay. After 3 days, D. discoideum plaques appeared on plates with K. aerogenes. In this study, a time-resolved transcriptomic analysis of the amoeba D. discoideum was performed to decipher the different host pathways impacted during infection. by incubating bacteria in a thin liquid layer with cells. P. aeruginosa utilizes virulence factors that are injected into the host cell through a “type III secretion apparatus” (for review, see ref. This structure allows cytotoxins to be transferred from the bacterium to the host cell. These mutants are impaired in two conserved virulence pathways: quorum-sensing-mediated virulence and type III secretion of cytotoxins. The exoU mutation was complemented by introducing plasmid pAH807 (pLAFRSK1 with a 3.8-kb genomic fragment containing exoU) (17); the resulting strain was also a kind gift from Joanne Engel. His left eye visual acuity was expressed as the ability to count fingers at 1 m. The eyelids were edematous and the conjunctivae were congested. Autophagy, a pathway of nutrient reallocation, has also been incorporated into its defenses and is activated when microbes disrupt the ph… D. discoideum forms plaques in thin lawns of P. aeruginosa PA14. Dictyostelium discoideum, Mycobacterium marinum, RNA-seq, infection, host damage, transcriptome, host-pathogen interaction. Comment submitted successfully, thank you for your feedback. INTRODUCTION Tuberculosis (TB) is an old but re-emerging global health threat caused by mycobacteria belonging to the Mycobacterium tuberculosis (Mtb) complex (WHO, 2018). the importance of the microbiome in human development, health, and well being, drives the study of host effectors that control its bacterial composition (Peterson and Artis 2014, Singh et al., 2017, Dominguez-Bello et al., 2019). 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