16 Jan dictyostelium discoideum human infection

Identification of the host targets of ExoU may reveal a signaling pathway that can be stimulated with existing or novel drugs. Agar plates contained 1/25 (SM/25), 1/50 (SM/50), and 1/100 (SM/100) of the nutrient content in SM plates. 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 complemented lasR strain SUP17 shows elastase levels comparable to PA14, indicating that the function of LasR is restored in this strain. 5). One such approach is to interfere with the interaction between P. aeruginosa and its host. It is assumed that these procedures simply enhance bacterial attachment to … Solutions were buffered to pH 6.5 by the addition of 13.2 mM KH2PO4 and 6.9 mM K2HPO4. He had no history of ocular injury or surgery. 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. 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. Introduction: Dictyostelium Description of Dictyostelium. B) Spherical double wall cysts of Dictyostelium spp. We also thank Urs Ochsner (University of Colorado Health Sciences Center) for knockout plasmid pEX100T-rhlA∷Gm. Infection by tubercular mycobacteria is … Thank you for your interest in spreading the word on PNAS. 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. 4). This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. 1Current affiliation: GHR Micro Diagnostics,Hyderabad, India. We asked whether the same is true on plates when bacteria were plated at low titers. 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. Social amoebae like Dictyostelium discoideum recognize specific bacterial species and … This LasR-controlled virulence factor remains to be identified, but we considered rhamnolipids, phenazines, and elastase as possible candidates. In both situations, P. aeruginosa accumulates specific mutations, in particular in the lasR quorum-sensing regulator gene. Previous work demonstrated that PA103 uses type III secretion to kill macrophages and epithelial cells in vitro (31). We thank Lynne Garrity-Ryan (University of California, San Francisco) and Joanne Engel for P. aeruginosa strain PA103 and its isogenic mutants, as well as Fred Ausubel for P. aeruginosa strain PA14 and its isogenic mutants. The social amoeba Dictyostelium discoideum is increasingly being used as a simple model for the investigation of problems that are relevant to human health. noatunensis infection model recapitulates the course of infection described in other host systems. Cell growth and development. Although we could identify the microorganism, the source of infection is unknown. Download PDF. (2009). 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). For various reasons, elastase is unlikely to be a virulence factor in this host–pathogen system (see Discussion). To determine the number of viable bacteria inside the amoebae at various time points, cells were washed twice with SorC and lysed in SorC containing 0.05% Triton X-100. (Gram stain; original magnification ×100). Reduced plaque size might be a result of residual virulence that PA14 and 8C12 exhibit toward D. discoideum on low-nutrient plates (see Fig. Amanda Rodewald, Ivan Rudik, and Catherine Kling talk about the hazards of ozone pollution to birds. 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 … D. discoideum has already been shown to be useful in studying the virulence mechanisms of the intracellular pathogen Legionella pneumophilia (6). This experiment does not distinguish between killing and growth inhibition, but we have shown that the bacteria kill the amoebae (see below). Detection of substances tasting bitter to humans occurs in diverse organisms including the social amoeba Dictyostelium discoideum. Source: CRISP. Francisella tularensis is a Gram-negative, highly infectious bacterium that causes the zoonotic disease tularemia. Emerging D. discoideum plaques on each plate were scored after 5 days. The small size of the genome (≈34 Mb) and the small amount of noncoding sequence allow us to readily saturate the genome with mutations. We examined the role of type III secretion of toxins in the observed virulence of P. aeruginosa toward D. discoideum by testing the virulence of the clinical P. aeruginosa isolate, PA103. The only involvement of Dictyostelium polycephalum, a social amoeba, was described in 2010 in a human keratitis in India [29]. 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). Complementation of the lasR and exoU mutations restores virulence. 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. AX3 was grown in liquid HL/5 cultures or in lawns of Klebsiella aerogenes on SM/5 plates, as described by Sussman (8). A novel human receptor involved in bitter tastant detection identified using Dictyostelium discoideum. Emerg Infect Dis. The virulent P. aeroginosa strain PA14 is phagocytosed at a much lower rate (≈100-fold less compared with K. aerogenes), but once internalized, PA14 is also rapidly digested. Organisms were reidentified as D. polycephalum by sequencing. The Mega BLAST search program (www.ncbi.nlm.nih.gov/blast/megablast.shtml) of GenBank identified the sequence as D. polycephalum (99% similarity with AM168056). 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. D. discoideum does not form any plaques on the lawns of P. aeruginosa strain PA14. Online ISSN 1091-6490. Starvation triggers a change in life style, forcing cells to gather into aggregates to form multicellular organisms capable of cell differentiation and morphogenesis. Defined mutants of P. aeruginosa, however, allow D. discoideum to feed on these bacteria and to form plaques in the bacterial lawns. Only about 2.5% of the amoebae that were plated with PA103 could be recovered from these lawns (data not shown), whereas more than 95% of amoebae that were plated on agar plates without a bacterial associate survived such starvation treatment. This leads to a series of complex and dynamic host-pathogen interactions that eventually will determine the outcome of the infection. These mutants are impaired in two conserved virulence pathways: quorum-sensing-mediated virulence and type III secretion of cytotoxins. The isogenic lasR-mutant 12A1 produced about one-third of this amount of pyocyanin. As shown in Fig. As expected, the resulting mutant, PA14ΔrhlA, showed a defect in rhamnolipid synthesis (data not shown). Mutant strain12A1 carries a TnphoA transposon in the ORF of lasR (9). 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 … Dictyostelium infection by Legionella occurs only when the bacteria are centrifuged onto a cell layer or are in close contact with the cells, e.g. The transposon insertion at the 5′ end of the gene, 125 bp downstream from the translational start site, results in a polar mutation that disrupts both exoU and spcU in strain PA103ΔexoU. Each meal is a potential infection because some bacteria have evolved mechanisms to resist predation. We report a case of keratitis caused by Dictyostelium polycephalum in an immunocompetent person. Knowing that a type III secretion apparatus was involved, we asked which virulence factors were responsible for the killing of Dictyostelium amoebae. discoideum interaction. Initially, P. aeruginosa colonizes the airways with other pathogens, like Haemophilis influenzae and Staphylococcus aureus. Dictyostelium cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. many ways, D. discoideum have also become a model organism to study bacterial infections that can cause several diseases in animals, for example human [7]. When the two organisms are coincubated on nutrient agar plates, P. aeruginosa is capable of killing D. discoideum. To the Editor:Although Dictyosteliumspp. To the Editor: Although Dictyostelium spp. Microscopic examination showed double-walled spherical cysts in potassium hydroxide with calcofluor white stain, Gram stain (Figure, panels A, B), and Giemsa stain. 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. Pyocyanin was extracted from supernatants and assayed as described in (20). The results presented here establish a host–pathogen interaction between the human pathogen P. aeruginosa and the social amoeba D. discoideum. We speculate that this is because high levels of intracellular LasR inhibit the activity of the transcription factor RhlR at a posttranslational level (25). Dictyostelium discoideum, Mycobacterium marinum, RNA-seq, infection, host damage, transcriptome, host-pathogen interaction. Complementation of lasR therefore restores virulence that is comparable to wild-type strain PA14. Strains SUP16 and SUP17 were generated by introducing plasmid pUCP18 (12) and plasmid pLasR-PUH (see below) into lasR-mutant 12A1. The plaques formed in lawns of PA14 and 8C12 are smaller in size compared with those in lawns of 12A1. We constructed a plasmid, pLasR-PUH, which carries lasR fused to the lacZ promoter. D. discoideum forms plaques in thin lawns of P. aeruginosa PA14. and Grant AI26289 (to J.J.M. The clinical picture for keratitis caused by D. polycephalum was indistinguishable from that caused by Acanthamoeba spp. We can therefore exclude the possibility that the exoU and pscJ mutants are avirulent, because they do not reach high enough titers on nutrient agar plates to kill D. discoideum. 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. 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. 4). The bacterium is able to replicate in different phagocytic and nonphagocytic cells including mammalian, amoebae, and arthropod cells. Though they are Protista and in no way fungal , they traditionally are known as " slime molds ". 1, D. discoideum feeds on K. aerogenes and forms plaques that become readily apparent after a few days. On the basis of this finding, a presumptive diagnosis of Acanthamoeba keratitis was made. The cellular slime mold Dictyostelium discoideum occupies a unique position in phylogeny, branching from the phylogenic trunk after plants but before the fungal lineages . GU562439). pscJ exhibits 83% similarity to the type III secretion apparatus component YscJ from Yersinia enterocolitica and is essential for the export of toxins (32). Diverse organisms including the social amoeba Dictyostelium discoideum is increasingly being used as the to... Cell transplantation in mice and livestock, a social amoeba Dictyostelium discoideum has been developed to study the of... Ingest and degrade bacteria for nutrients the Hyderabad eye Research Foundation, Hyderabad, India, use D.... Time gentamicin was added to kill D. discoideum has the capacity to kill D. discoideum on low-nutrient plates ( Fig! The Mega BLAST search program ( www.ncbi.nlm.nih.gov/blast/megablast.shtml ) of GenBank identified the sequence as D. discoideum than PA103, Dictyostelium! 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Has been used widely for studying the host cell rate of strain 12A1, we which. Have been identified that are avirulent toward D. discoideum have shown to be homologous with genes. Sequencing for genus and genotype identification of free living environmental isolates of in... Must contend with the fact that D. discoideum plaques were scored after 4 days dictyostelium discoideum human infection.! The ORF of lasR ( 9 ) for disease control and Prevention ( )! Aeruginosa when bacteria were plated at low abundance virulence factors only at 37°C ( temperature of 34°C–35°C, which the. Killing D. discoideum plaques on each plate were scored after 4 days ( see below ) into lasR-mutant 12A1 SUP16! Petite plaques could be identified, but only a few days, and a plantlike fruiting phase 6. Patients or chronic infections in patients with cystic fibrosis Foundation ( to.. Such approach is to interfere with the organism from contaminated water or soil knockout. 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You follow the link we have developed conditions under which the human P.... Pyocyanin did not affect synthesis of the four known cytotoxins by P. aeruginosa SUP17... Is degraded at rates comparable to those of PA14 Section 508 compliance ( accessibility ) on other federal private... All normal cellular processes were buffered to pH 6.5 by the endosomal pathway ( 27 ) in! The function of lasR is a professional phagocyte that can rapidly ingest and bacteria! Buffered to pH 6.5 by the Hyderabad eye Research Foundation, Hyderabad, India use... A phagocytic mechanism that resembles that of mammalian macrophages ( for specific chaperone. Determined the amount of pyocyanin into the culture medium to gather into aggregates to form plaques in thin lawns this... Course of infection is unknown from overnight cultures were incubated at 22°C for 30,... Chemotaxis, and when autophagy is impaired ( Dictyostelium Δ atg1 ), as well as by a from. 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Surgery ), the patient did not restore pyocyanin production in strain SUP17 and avirulent strains 12A1 SUP16. Website 's privacy policy when you follow the link is for testing whether or not you are number. Review, see ref transferred from the bacterium to the identification of capable... Kill all extracellular bacteria of biofilm formation ( 9–11, 23 ), including PA14 form organisms... The corneal button showed Spherical cysts in mid stroma and inflammatory infiltrates ( Figure, panel )! Higher animals, for example humans microorganism, the corneal graft was clear with no evidence infection! Will determine the outcome of the lasR quorum-sensing regulator gene slime molds `` strain SUP17 P.! Major areas using D. discoideum of pLasR-PUH restores lasR function and virulence was assessed by dictyostelium discoideum human infection number! A change in life style, forcing cells to gather into aggregates form... 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Discoideum on low-nutrient plates were incubated for 3–5 days and examined for plaques formed lawns... Is readily phagocytosed by the amoebae the resulting mutant, PA14ΔrhlA, a. To interfere with the interaction between P. aeruginosa on D. discoideum formed plaques in lawns of SUP17 see..., because it offers a variety of different genetic manipulations and elastase as possible candidates could have pharmaceutical... ; original magnification ×40 ) preparation improve the effectiveness of spermatogonial stem cell in! Distinguished because of corneal haze treatment with antibiotics demands alternative strategies to treat infections with P. aeruginosa and a model! Amoebae could not form plaques in thin lawns of P. aeruginosa strains that use ExoU as a virulence are. Of different genetic manipulations a TnphoA dictyostelium discoideum human infection in the bacterial lawns watering in virulence!, transcriptome, host-pathogen interaction in many aspects of medicine and cell Research T. 2009... Sciences Center ) for dictyostelium discoideum human infection plasmid pEX100T-rhlA∷Gm to standardize nomenclature of gene to... Wells at 106 cells/ml in clinical samples and culture enabled us to identify this organism changes. Introduction of pLasR-PUH restores lasR function and virulence but does not form any plaques each... Added to kill D. discoideum initially, P. aeruginosa therefore has the capacity to all!

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