dictyostelium discoideum human infection

Dictyostelium discoideum is a unicellular, free-living amoebae that lives in soil and feeds on bacteria (Bonner, 1944). D. discoideum cells from midlogarithmic cultures were collected by centrifugation (1,000 × g; 4 min), washed once with SorC (8), and added to the bacterial suspensions at a final concentration of 5 × 102 cells/ml suspension; 0.2 ml of this mixture was plated out on SM/5 plates and allowed to dry under a sterile flow of air. PA14 is a multihost pathogen that has been used widely for studying the molecular basis of biofilm formation (9–11, 23). At the time of his medical visit, he was using ophthalmic solutions of 5% natamycin sulfate, 0.5% moxifloxacin hydrochloride, and 0.3% gentamicin sulfate, each instilled every hour, and 1% atropine sulfate, 3×/d. Pyocyanin is a phenazine that applies oxidative stress to eukaryotic cells. D. discoideum formed plaques in lawns of K. aerogenes but was unable to do so in lawns of PA14 and PA14ΔrhlA. Centers for Disease Control and Prevention. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. The soil-dwelling social amoeba Dictyostelium discoideum feeds on bacteria. D. polycephalum is ancestral and show different characteristics than other species of Dictyostelium (5,7,8). The social amoeba Dictyostelium discoideum is increasingly being used as a simple model for the investigation of problems that are relevant to human health. It is assumed that these procedures simply enhance bacterial attachment to … D. discoideum is well suited for studying the host biology of pathogens, because it offers a variety of different genetic manipulations. It is a genetically close cousin of Mycobacterium tuberculosis, and thereby the infection process remarkably shares many of the hallmarks of M. tuberculosis infection in human, at both the cellular and organism levels. A 35-year-old man sought treatment for redness, pain, and watering in the left eye of 11 days’ duration. One of these cytotoxins is ExoU, and exoU mutants are avirulent toward D. discoideum. Although the evidence is still limited, a growing body of research suggests music may have beneficial effects for diseases such as Parkinson’s. D. discoideum has already been shown to be useful in studying the virulence mechanisms of the intracellular pathogen Legionella pneumophilia (6). Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella. noatunensis, which has an optimal growth temperature similar to that of the Dictyostelium host, as a pathogen. infection. This finding suggests that P. aeruginosa strains that use ExoU as a virulence factor are readily cleared from the lungs of cystic fibrosis patients. Data represent the means of duplicate elastase assays, and activity is expressed as the activity relative to wild-type PA14 (=100%) +/−SDn−1. The axenic strains can grow in rich medium and are easily maintained (Sussman, 1987a). As a reference for plating efficiency, AX3 was also cultured with K. aerogenes. At the last follow-up (3 months after surgery), the corneal graft was clear with no evidence of infection. Saving Lives, Protecting People, Author affiliation: L.V. For example, insertional mutagenesis by restriction-enzyme-mediated integration (REMI) may introduce null mutations that result in the genetic removal of proteins from cells (41). 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 complemented lasR strain SUP17 shows elastase levels comparable to PA14, indicating that the function of LasR is restored in this strain. Overexpression of lasR from plasmid pLasR-PUH did not restore pyocyanin production in strain SUP17. D. discoideum is a complex eukaryote that lives part of its life cycle as a unicellular amoeba. 5). It is also used to study other aspects of development, including cell sorting, pattern formation, phagocytosis, mot… We then tested the virulence of this strain in our plaque assay. A mutant in the lasR gene, which codes for the transcriptional activator of the lasR quorum-sensing mechanism, is less virulent toward D. discoideum and allows plaque formation. The experimentally versatile Dictyostelium discoideum-Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity … To determine whether this is the case, we applied a modified gentamicin protection protocol to measure the uptake rates of various P. aeruginosa strains, including strains 12A1 and PA14 (21). Cell growth and development. Because D. discoideum is haploid, mutations are not masked by additional alleles, so that mutations, including recessive ones, are readily identified. This experiment does not distinguish between killing and growth inhibition, but we have shown that the bacteria kill the amoebae (see below). Autophagy, a pathway of nutrient reallocation, has also been incorporated into its defenses and is activated when microbes disrupt the ph… 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. Therefore, Dictyostelium opens the exciting possibility of investigating and … Shapeshifting designs could have wide-ranging pharmaceutical and biomedical applications in coming years. Thus, P. aeruginosa predation of D. discoideum depends on LasR, which likely regulates expression of a downstream effector gene through the quorum-sensing pathway. To the Editor: Although Dictyostelium spp. The soil amoeba Dictyostelium discoideum is a haploid eukaryote that, upon starvation, aggregates and enters a developmental cycle to produce fruiting bodies. major areas using D. discoideum as a model organism in biomedicine. The influence of methylamine and infectious doses on the results of infection of Dictyostelium discoideum and Acanthamoeba castellanii with Francisella novicida . Amoebae feed on these bacteria and form plaques in their bacterial lawns. D. discoideum and P. aeruginosa were coincubated for 30 min, and any bacteria that were not taken up by the amoebae were killed by the addition of gentamicin. No plaques were formed on plates with the virulent P. aeruginosa strain. are used for studying signal transduction, cytoskeletal functions, endocytosis, and molecular pathogenesis of infectious and other diseases (), human or animal infections caused by this organism have not been reported.We report a case of keratitis caused by Dictyostelium polycephalum in an immunocompetent person. This LasR-controlled virulence factor remains to be identified, but we considered rhamnolipids, phenazines, and elastase as possible candidates. 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. 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. noatunensis infection model recapitulates the course of infection described in other host systems. Dictyostelium discoideum is a cooperative, genetically tractable phagocyte The soil amoeba D. discoideum can internalize a remarkably wide range of particles, including latex beads, yeast, benign bacteria and different pathogenic bacteria (Cardelli, 2001; Maniak, 2003; Steinert et al., 2003). Because pyocyanin synthesis is controlled by quorum sensing, we investigated whether P. aeruginosa-induced killing of Dictyostelium was mediated by pyocyanin. The exoU-mutant PA103∷exoU (mut 8) (15), the pscJ-mutant PA103∷pscJ (mut N) (15), and their isogenic parent PA103 (16) were gifts from Joanne Engel (University of California, San Francisco). The similarities between the Dictyostelium and mammalian cells extend to membrane trafficking, endocytic transit and sorting events. Introduction: Dictyostelium Description of Dictyostelium. The mechanism by which ExoU induces rapid lysis is not yet known. We have developed conditions under which the human pathogen Pseudomonas aeruginosa infects Dictyostelium discoideum , a genetically tractable eukaryotic organism. Francisella tularensis is a Gram-negative, highly infectious bacterium that causes the zoonotic disease tularemia. 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. This study was supported by the Hyderabad Eye Research Foundation, Hyderabad, India. We investigated whether the avirulent phenotype of the lasR-mutant 12A1 was due to a reduction in rhamnolipid secretion. The exoU operon contains the exoU and spcU genes. Mycobacterium marinum is the causative agent of fish and amphibian tuberculosis in the wild. D. discoideum cells (AX3) were plated with different bacterial associates on nutrient agar plates (SM/5) at a density of approximately 40 amoebae/plate. 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). Infection by bacterial pathogens Due to the similarities between D. discoideum and human cells, Dictyostelium is a good model for the study of microbial infection since the damage made by the pathogen is mimicked (Annesley and Fisher, 2009). Due to these facts, D. discoideum have also been used to 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. To establish a molecular mechanism for bitter tastant detection in Dictyostelium, we screened a mutant library for resistance to a commonly used bitter standard, phenylt … The virulence of PA103, as well as isogenic mutants PA103∷pscJ (ΔpscJ), PA103∷exoU (ΔexoU), PA103∷exoU+pLAFRSKI (ΔexoU with plasmid control pLAFRSKI), and PA103∷exoU+pAH807 [ΔexoU with plasmid pAH807 (exoU+/spcU+)] was tested in this assay. We obtained corneal scrapings, and the material was subjected to a detailed microbiologic analysis (2). Organisms were reidentified as D. polycephalum by sequencing. First, we determined the amount of pyocyanin that was secreted by PA14 and its mutants during stationary phase. Dictyostelium cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. The bacterium is able to replicate in different phagocytic and nonphagocytic cells including mammalian, amoebae, and arthropod cells. The cornea showed a large central epithelial defect with underlying stromal infiltrate and Descemet folds. Using Dictyostelium as a genetic model host takes advantage of the organism's ability to integrate complicated activities in a single cell, which includes responses to pathogens. A few genes have already been studied, providing new insights on the mechanism of action of the encoded proteins and in some cases on the defect … Table of Contents – Volume 16, Number 10—October 2010. 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. As expected, the resulting mutant, PA14ΔrhlA, showed a defect in rhamnolipid synthesis (data not shown). We deposited the sequence of our isolate in GenBank (accession no. Dictyostelium cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. Quorum sensing is thought to be an important determinant of P. aeruginosa virulence, because it guarantees that the organism expresses virulence factors only when it is present at high numbers. Get PDF (1 MB) Abstract. ↵† To whom reprint requests should be addressed. and Grant AI26289 (to J.J.M. 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). D. discoideum does not form any plaques on the lawns of P. aeruginosa strain PA14. Members of the genus Dictyostelium (social amebas or cellular slime molds) are divided into 4 high-level taxa with several species on the basis of DNA phylogeny (5). Bacteria were grown in Luria broth for 16 h, pelleted by centrifugation (1,600 × g; 15 min), washed once, and resuspended in SorC (16.7 mM Na2H/KH2PO4/50 μM CaCl2, pH 6.0) at a final optical density of 5.5 at 600 nm. Dictyostelium discoideum is a unicellular protozoan that can be grown axenically in its amoebal form (67, 74); it feeds on bacteria through well-characterized phagosomal and endolysosomal mechanisms similar to those of mammalian phagocytes in aspects of cytoskeletal organization, membrane trafficking, endocytic transit, and sorting events (8, 21, 37, 41, 52, 54). 2 Middle, lasR-mutant 12A1 and vector control strain SUP16 show low elastase activity, compared with wild-type PA14. Though they are Protista and in no way fungal , they traditionally are known as " slime molds ". Using a Dictyostelium discoideum amoeba model, we assessed whether these mutations affect bacterial virulence. The inositol polyphosphate 5-phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole … Bacteria were grown in Luria broth for 16 h, pelleted by centrifugation (1,600 × g; 15 min), and resuspended in HL/5. 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. The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum. Another class of virulence factors that is controlled by the lasR quorum-sensing mechanism are the rhamnolipids. Cystic fibrosis patients, who carry mutations in both alleles of the cystic fibrosis transmembrane conductance regulator, are at high risk of developing chronic lung infections at some stage in their lives. 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). No plaques could be seen in lawns of wild-type PA14 and the isogenic mutant 8C12 on SM/25 and SM/50 plates. In this study, we infected single‐cell stages of D. discoideum with different Legionella species. Thus, the loss of amoebae in lawns of PA103 is because of killing rather than starvation. The lmpA knockdown mutant is highly affected in actin-dependent ... an infection model for human pathogens such as Legionella, Klebsiella, Mycobacterium, Pseudomonas (Alibaud et al., 2008; E-mail: stefan_pukatzki{at}hms.harvard.edu. ... the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. In this study, we infected single‐cell... Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella - Hägele - 2000 - Cellular Microbiology - Wiley Online Library. 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. The lasR-mutant 12A1, the phenA/phenB-mutant ΔphenA/phenB, the TnphoA-mutant 8C12, as well as their parent strain UCB-PP PA14, were kind gifts from Fred Ausubel (Massachusetts General Hospital, Boston) (9–11). 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). The cellular slime mold Dictyostelium discoideum occupies a unique position in phylogeny, branching from the phylogenic trunk after plants but before the fungal lineages . Complementation of the lasR and exoU mutations restores virulence. ... the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. When exposed to low titers of bacterial cells in a liquid medium, D. discoideum amoebae ingested all P. aeruginosa strains by phagocytosis in our gentamicin-protection experiment. Dictyostelium discoideum has long been used as a model organism to study many different biological processes. Pyocyanin levels in bacterial cultures were determined as described by Essar et al. By Elena Ivek. A number of P. aeruginosa mutants have been identified that are avirulent in this assay. ... 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. Madhusmita Das. The mutation in pscJ prohibits export of the four known cytotoxins by P. aeruginosa strain PA103 (15, 33). Photo reproduced with permission from the University Archives and Records Mana - To the Editor:Although Dictyosteliumspp. These mutants are impaired in two conserved virulence pathways: quorum-sensing-mediated virulence and type III secretion of cytotoxins. noatunensis accumulates within Dictyostelium cells. The experimentally versatile Dictyostelium discoideum – Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity and host response. INTRODUCTION Tuberculosis (TB) is an old but re-emerging global health threat caused by mycobacteria belonging to the Mycobacterium tuberculosis (Mtb) complex (WHO, 2018). Four toxins that are injected in this way, ExoS, ExoT, ExoU, and ExoY, have been identified (15, 29, 30). 1Current affiliation: GHR Micro Diagnostics,Hyderabad, India. Elastolytic activity was determined as described by Gambello and Iglewski (19). Thus, ExoU is responsible for killing D. discoideum. On SM/25 and SM/50 plates, which contain one-fifth and one-tenth of the nutrient concentration of SM/5, respectively, plaques emerged in lawns of the lasR-mutant 12A1. (Middle) Elastase activity of supernatants from overnight cultures were assayed by using elastin Congo red (ECR) as the substrate. A PCR product ≈800 bp was obtained and subjected to bidirectional sequencing with fluorescent-labeled dideoxy nucleotide terminators by using ABI 3130 XI automated sequencer in accordance with the manufacturer’s instructions (PE Applied Biosystems, Foster City, CA, USA). To study the interaction of P. aeruginosa and a host cell, we suggest the genetically tractable organism Dictyostelium discoideum. The organism showed cytotoxicity after in vitro inoculation of a rabbit corneal epithelial cell line. 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). 4). Because we were not aware of any drug treatment recommendations for infection by this organism, and the disease was advanced, surgical treatment was advised. This cellular slime mold is a major player in many aspects of medicine and cell research. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. 2 Top) (10). Thirty minutes after the initiation of infection, cells were washed twice and resuspended in SorC (16.7 mM Na2H/KH2PO4/50 μM CaCl2, pH 6.0) containing 400 μg/ml of gentamicin to kill all bacteria that were not ingested by the amoebae. To ensure that the introduction of pLasR-PUH restores LasR function, we determined the activity of the LasR-controlled lasB gene product, elastase. The vision in his right eye and results of a clinical examination were within normal limits. Rhamnolipids are biosurfactants that lyse host cells by disrupting their cell membranes (26). Strain 8C12 is identical to 12A1, except that the TnphoA transposon is in another gene, which does not affect virulence toward amoebae (10). Dictyostelium discoideum, a social amoeba, is a profes- sional phagocyte that can rapidly ingest and degrade bacteria for nutrients. 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. About 95% of the affected individuals have a SpcU (for specific Pseudomonas chaperone for ExoU) encodes a chaperone that is necessary for proper translocation of ExoU (35). Message not sent. Pseudomonas aeruginosa is an opportunistic pathogen that causes life-threatening infections in individuals with compromised immune systems, such as cancer patients undergoing chemotherapy or patients with cystic fibrosis. This “quorum-sensing mechanism” is thought to be an important regulator of pathogenesis, because it guarantees that certain virulence factors are made only when P. aeruginosa has reached a crucial density and can launch a successful infection (22). 5). However, careful attention to cyst morphology in clinical samples and culture enabled us to identify this organism. in potassium hydroxide (calcoflour white stain; original magnification ×40) preparation. D. discoideum cells from midlogarithmic cultures were collected by centrifugation (1,000 × g; 4 min), washed once with fresh HL/5 (8), and resuspended in HL/5 at a concentration of 106 cells/ml. 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. Reduced plaque size might be a result of residual virulence that PA14 and 8C12 exhibit toward D. discoideum on low-nutrient plates (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. (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. 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. major areas using D. discoideum as a model organism in biomedicine. 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 … CRISPR-Cas9 gene editing can improve the effectiveness of spermatogonial stem cell transplantation in mice and livestock, a study finds. At the same time, the infected host turns on defense mechanisms to clear the infection. Any genetic manipulation of P. aeruginosa that results in a growth defect could lead to the formation of thin bacterial lawns on rich-medium plates, allowing D. discoideum to form plaques. Therefore, D. discoideum amoebae are able to feed on virulent P. aeruginosa when bacteria are present at low abundance. Furthermore, addition of purified pyocyanin did not affect the viability of axenically growing amoebae. P. aeruginosa utilizes virulence factors that are injected into the host cell through a “type III secretion apparatus” (for review, see ref. One such approach is to interfere with the interaction between P. aeruginosa and its host. We report a case of keratitis caused by Dictyostelium polycephalum in an immunocompetent person. To test whether P. aeruginosa could infect D. discoideum, the pathogenicity of P. aeruginosa toward D. discoideum cells was determined by using a simple plating assay. The amoeba is very similar to macrophages present in higher animals, for example humans. Free-living amoebae feed on bacteria by a phagocytic mechanism that resembles that of mammalian macrophages (for review, see ref. The lysates were diluted and plated on Luria broth agar plates. Most P. aeruginosa isolates from cystic fibrosis patients lack the exoU gene or are unable to secrete the toxin, whereas a higher proportion of strains isolated from corneal infections are reported to contain ExoU (38–40). 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 … The inositol polyphosphate 5-phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole … Plates were incubated for 3–5 days and examined for plaques formed by Dictyostelium amoebae. 5 and 6B). Cells were infected with bacteria at a multiplicity of infection of approximately 100:1. One avirulent mutant strain carries an insertional mutation in the lasR gene. As shown in Fig. 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. Complementation of the lasR mutation restores virulence, as D. discoideum amoebae are not able to form plaques in lawns of this complemented strain. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δ atg1 ), F. noatunensis subsp. D. discoideum amoebae could not form plaques in the complemented exoU mutant (see Figs. In culture, it grows at a temperature of 34°C–35°C, which is higher than that for other species of Dictyostelium (8). the amoeba Dictyostelium discoideum as a surrogate macrophage and F. noatunensis subsp. Complementation of the lasR mutation restores LasR function and virulence but does not affect synthesis of the pigment pyocyanin. We constructed a rhlA mutant of P. aeruginosa PA14 by using the site-specific insertional mutagenesis strategy of Schweizer and Hoang (14).

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