Previous work demonstrated that PA103 uses type III secretion to kill macrophages and epithelial cells in vitro (31). Biomedical communities and journals need to standardize nomenclature of gene products to enhance accuracy in scientific and public communication. 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). 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. P. aeruginosa utilizes virulence factors that are injected into the host cell through a “type III secretion apparatus” (for review, see ref. Cultures were incubated at 22°C for 30 min, at which time gentamicin was added to kill all extracellular bacteria. It is used to study cell differentiation, chemotaxis, and apoptosis, which are all normal cellular processes. Host for bacterial infections The first study where D. discoideum served as the host for a pathogen was with the … The low phagocytosis rate of strain 12A1 is reflected in reduced plaque size. He had no history of ocular injury or surgery. D. discoideum is able to form plaques in thin lawns of virulent P. aeruginosa strains. The Centers for Disease Control and Prevention (CDC) cannot attest to the accuracy of a non-federal website. 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. Knowing that a type III secretion apparatus was involved, we asked which virulence factors were responsible for the killing of Dictyostelium amoebae. Four toxins that are injected in this way, ExoS, ExoT, ExoU, and ExoY, have been identified (15, 29, 30). Bacteria and amoebae were plated on agar plates with decreasing nutrient content to control the thickness of the bacterial layer, which is proportional to the nutrient concentration in the agar. 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. 28). Free-living amoebae feed on bacteria by a phagocytic mechanism that resembles that of mammalian macrophages (for review, see ref. 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. Comment submitted successfully, thank you for your feedback. Data represent the means of duplicate experiments, and the number of internalized bacteria per well is expressed as cfu/ml +/− SDn−1. After 4 days at 22°C, D. discoideum formed plaques in lawns of 12A1 on SM/25 and plates with lower nutrient content, 8C12 and PA14 allowed plaque formation only on SM/100 plates. The lysates were diluted and plated on Luria broth agar plates. The myxamoebae were seen after 24 hours, and the amebae had transformed into microcysts after 48 hours of incubation. Uptake and digestion rates are comparable to those of PA14. Therefore, the ability of D. discoideum to form plaques in lawns of 12A1 is because of the loss of a virulence mechanism that does not involve the inhibition of phagocytosis. Dictyostelium polycephalum Infection of Human Cornea. Therefore, Dictyostelium opens the exciting possibility of investigating and … Many pathogens, however, start expressing their key virulence factors only at 37°C (temperature of human body). 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). D. discoideum has already been shown to be useful in studying the virulence mechanisms of the intracellular pathogen Legionella pneumophilia (6). 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 … 2 Top, D. discoideum is unable to form plaques in lawns of parental strain PA14 but forms plaques in lawns of the isogenic lasR-mutant 12A1. The identification of free living environmental isolates of amoebae from Bulgaria. 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. The mutation in pscJ prohibits export of the four known cytotoxins by P. aeruginosa strain PA103 (15, 33). 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). 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. D. discoideum cells (AX3) were plated with different bacterial associates on nutrient agar plates (SM/5) at a density of approximately 40 amoebae/plate. The left eye visual acuity was now expressed as the ability to see hand movements near the face. The axenic strains can grow in rich medium and are easily maintained (Sussman, 1987a). Organisms were reidentified as D. polycephalum by sequencing. When D. discoideum is plated on nutrient agar plates with different P. aeruginosa strains, the bacteria form lawns on these plates with amoebae embedded in them. The experimentally versatile Dictyostelium discoideum-Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity … After a variable period, chronic lung disease develops in which the bacterial population consists almost exclusively of mucoid P. aeruginosa in the form of biofilms (2). 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. 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). Because we were not aware of any drug treatment recommendations for infection by this organism, and the disease was advanced, surgical treatment was advised. We directly examined the contribution of rhamnolipids and phenazines to D. discoideum killing but found that these factors were not involved. We constructed a plasmid, pLasR-PUH, which carries lasR fused to the lacZ promoter. Basic processes of host-pathogen interactions, such as phagocytosis, phagosomal maturation, and autophagy, are evolu- However, careful attention to cyst morphology in clinical samples and culture enabled us to identify this organism. 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. Dictyostelium cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. About 95% of the affected individuals have a (Gram stain; original magnification ×100). 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). The influence of methylamine and infectious doses on the results of infection of Dictyostelium discoideum and Acanthamoeba castellanii with Francisella novicida . 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. As shown in Fig. In some of the experiments, low-nutrient plates were used to limit bacterial growth. 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. 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 … 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. Pyocyanin is a phenazine that applies oxidative stress to eukaryotic cells. Infection by tubercular mycobacteria is … Francisella tularensis is a Gram-negative, highly infectious bacterium that causes the zoonotic disease tularemia. We propose that D. discoideum is a genetically tractable system that can be used to study the host biology of P. aeruginosa. To the Editor: Although Dictyostelium spp. 5). We asked whether the same is true on plates when bacteria were plated at low titers. www.ncbi.nlm.nih.gov/blast/megablast.shtml, Dictyostelium polycephalum Infection of Human Cornea, U.S. Department of Health & Human Services, Reddy AK, Balne PK, Garg P, Sangwan VS, Das M, Krishna PV, et al. The plaques formed in lawns of PA14 and 8C12 are smaller in size compared with those in lawns of 12A1. 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. See below ) into lasR-mutant 12A1, we show that the introduction of pLasR-PUH restores lasR function, tested! He had no history of ocular injury or surgery no history of ocular injury or surgery,! 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