scholarly journals Eca1, a Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase, Is Involved in Stress Tolerance and Virulence in Cryptococcus neoformans

2007 ◽  
Vol 75 (7) ◽  
pp. 3394-3405 ◽  
Author(s):  
Weihua Fan ◽  
Alexander Idnurm ◽  
Julia Breger ◽  
Eleftherios Mylonakis ◽  
Joseph Heitman
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Tolerance ◽  
Cryptococcus Neoformans ◽  
Galleria Mellonella ◽  
Acanthamoeba Castellanii ◽  
Calcineurin Inhibitors ◽  
Normal Growth ◽  
Mammalian Host ◽  
Wax Moth

ABSTRACT The basidiomycetous fungal pathogen Cryptococcus neoformans is adapted to survive challenges in the soil and environment and within the unique setting of the mammalian host. A C. neoformans mutant was isolated with enhanced virulence in a soil amoeba model that nevertheless exhibits dramatically reduced growth at mammalian body temperature (37°C). This mutant phenotype results from an insertion in the ECA1 gene, which encodes a sarcoplasmic/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA)-type calcium pump. Infection in murine macrophages, amoebae (Acanthamoeba castellanii), nematodes (Caenorhabditis elegans), and wax moth (Galleria mellonella) larvae revealed that the eca1 mutants are virulent or hypervirulent at permissive growth temperatures but attenuated at 37°C. Deletion mutants lacking the entire ECA1 gene were also hypersensitive to the calcineurin inhibitors cyclosporin and FK506 and to ER and osmotic stresses. An eca1Δ cna1Δ mutant lacking both Eca1 and the calcineurin catalytic subunit was more sensitive to high temperature and ER stresses than the single mutants and exhibited reduced survival in C. elegans and attenuated virulence towards wax moth larvae at temperatures that permit normal growth in vitro. Eca1 is likely involved in maintaining ER function, thus contributing to stress tolerance and virulence acting in parallel with Ca2+-calcineurin signaling.

Transgenic expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco

2006 ◽  
Vol 387 (5) ◽  
pp. 549-557 ◽  
Author(s):  
Gregor Langen ◽  
Jafargholi Imani ◽  
Boran Altincicek ◽  
Gernot Kieseritzky ◽  
Karl-Heinz Kogel ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Fungal Pathogens ◽  
Galleria Mellonella ◽  
Ectopic Expression ◽  
Pathogenic Fungi ◽  
Transgenic Expression ◽  
Greater Wax Moth ◽  
Insect Defensin ◽  
Wax Moth

Abstract A cDNA encoding gallerimycin, a novel antifungal peptide from the greater wax moth Galleria mellonella, was isolated from a cDNA library of genes expressed during innate immune response in the caterpillars. Upon ectopic expression of gallerimycin in tobacco, using Agrobacterium tumefaciens as a vector, gallerimycin conferred resistance to the fungal pathogens Erysiphe cichoracearum and Sclerotinia minor. Quantification of gallerimycin mRNA in transgenic tobacco by real-time PCR confirmed transgenic expression under control of the inducible mannopine synthase promoter. Leaf sap and intercellular washing fluid from transgenic tobacco inhibited in vitro germination and growth of the fungal pathogens, demonstrating that gallerimycin is secreted into intercellular spaces. The feasibility of the use of gallerimycin to counteract fungal diseases in crop plants is discussed.

scholarly journals Natural Products can Efficiently Control the Greater Wax Moth (Lepidoptera: Pyralidae), but are Harmless to Honey Bees

Sociobiology ◽  
2020 ◽  
Vol 67 (1) ◽  
pp. 89
Author(s):  
Daniele Maria Telles ◽  
Gabriel Moreno Martineli ◽  
Maurice Fabian Scaloppi ◽  
Marina Pagliai Ferreira Luz ◽  
Samir Moura Kadri ◽  
...  
Keyword(s):  
Natural Products ◽  
Population Growth ◽  
Honey Bees ◽  
Galleria Mellonella ◽  
Greater Wax Moth ◽  
Lepidoptera Pyralidae ◽  
Tobacco Extract ◽  
Bee Colonies ◽  
Wax Moth

Honey bees (Apis mellifera L.) have great global socioeconomic and environmental importance. However, the greater wax moth (Galleria mellonella L.) is a pest that causes serious worldwide damage to honey bee colonies. Good beekeeping practices and physical, chemical, or natural methods can be used to control wax moths. The use of natural products is a more sustainable option because of their lower toxicity to the environment and the colony. Therefore, we evaluated the efficiency of four natural products for greater wax moth control: neem oil (Azadirachta indica), eucalyptus oil (Eucalyptus spp.), tobacco extract (Nicotiana tabacum), and malagueta pepper extract (Capsicum frutescens). We also evaluated their effects on adult bees and on the population growth of colonies. The 4th instar wax moths and adult bees were subjected to in vitro bioassays of different concentrations of the products. The results allowed usto establish a concentration for each product that was safe for the bees and effectively controlled the moth. Then, we sprayed them on bee colonies to evaluate their effects on population growth. The neem and eucalyptus oils caused wax moth mortality at low concentrations, but did not affect colony population growth. However, they did have a toxic effect on adult bees. The tobacco and pepper extracts efficiently controlled the moth, but did not cause adult bee mortality or interfered with the population growth of the colonies. Therefore, the tobacco and pepper extracts could efficiently control the greater wax moth, without damaging honey bees.

scholarly journals Leishmania mexicana Mutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

2000 ◽  
Vol 11 (4) ◽  
pp. 1183-1195 ◽  
Author(s):  
James D. Hilley ◽  
Jody L. Zawadzki ◽  
Malcolm J. McConville ◽  
Graham H. Coombs ◽  
Jeremy C. Mottram
Keyword(s):  
Normal Growth ◽  
Surface Proteins ◽  
Host Cells ◽  
Mammalian Host ◽  
Putative Protein ◽  
Major Class ◽  
Major Surface Proteins ◽  
Major Surface

The major surface proteins of the parasitic protozoonLeishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (ΔGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein–anchor precursors. However, the synthesis and cellular levels of other non–protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the ΔGPI8 mutant. Significantly, the ΔGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.

scholarly journals Cryptococcus neoformans Phosphoinositide-Dependent Kinase 1 (PDK1) Ortholog Is Required for Stress Tolerance and Survival in Murine Phagocytes

2012 ◽  
Vol 12 (1) ◽  
pp. 12-22 ◽  
Author(s):  
Yeissa Chabrier-Roselló ◽  
Kimberly J. Gerik ◽  
Kristy Koselny ◽  
Louis DiDone ◽  
Jennifer K. Lodge ◽  
...  
Keyword(s):  
Cell Wall ◽  
Stress Tolerance ◽  
Cryptococcus Neoformans ◽  
Nitrosative Stress ◽  
Galleria Mellonella ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Oxidative And Nitrosative Stress ◽  
Independent Manner ◽  
Content Type

ABSTRACTCryptococcus neoformansPKH2-01andPKH2-02are orthologous to mammalian PDK1 kinase genes. Although orthologs of these kinases have been extensively studied inS. cerevisiae, little is known about their function in pathogenic fungi. In this study, we show thatPKH2-02but notPKH2-01is required forC. neoformansto tolerate cell wall, oxidative, nitrosative, and antifungal drug stress. Deletion ofPKH2-02leads to decreased basal levels of Pkc1 activity and, consequently, reduced activation of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway in response to cell wall, oxidative, and nitrosative stress.PKH2-02function also is required for tolerance of fluconazole and amphotericin B, two important drugs for the treatment of cryptococcosis. Furthermore, OSU-03012, an inhibitor of human PDK1, is synergistic and fungicidal in combination with fluconazole. Using aGalleria mellonellamodel of low-temperature cryptococcosis, we found thatPKH2-02is also required for virulence in a temperature-independent manner. Consistent with the hypersensitivity of thepkh2-02Δ mutant to oxidative and nitrosative stress, this mutant shows decreased survival in murine phagocytes compared to that of wild-type (WT) cells. In addition, we show that deletion ofPKH2-02affects the interaction betweenC. neoformansand phagocytes by decreasing its ability to suppress production of tumor necrosis factor alpha (TNF-α) and reactive oxygen species. Taken together, our studies demonstrate that Pkh2-02-mediated signaling inC. neoformansis crucial for stress tolerance, host-pathogen interactions, and both temperature-dependent and -independent virulence.

scholarly journals Identification of ENA1 as a Virulence Gene of the Human Pathogenic Fungus Cryptococcus neoformans through Signature-Tagged Insertional Mutagenesis

2009 ◽  
Vol 8 (3) ◽  
pp. 315-326 ◽  
Author(s):  
Alexander Idnurm ◽  
Felicia J. Walton ◽  
Anna Floyd ◽  
Jennifer L. Reedy ◽  
Joseph Heitman
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Insertional Mutagenesis ◽  
Pathogenic Fungus ◽  
Mammalian Host ◽  
New Genes ◽  
Strain Collection ◽  
Mutant Strains ◽  
Human Pathogenic Fungus

ABSTRACT A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals.

scholarly journals Serial Passage of Cryptococcus neoformans in Galleria mellonella Results in Increased Capsule and Intracellular Replication in Hemocytes, but Not Increased Resistance to Hydrogen Peroxide

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 732 ◽  
Author(s):  
Muhammad Fariz Ali ◽  
Stephen M. Tansie ◽  
John R. Shahan ◽  
Rebecca L. Seipelt-Thiemann ◽  
Erin E. McClelland
Keyword(s):  
Hydrogen Peroxide ◽  
Cryptococcus Neoformans ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Histopathological Examination ◽  
Serial Passage ◽  
Infection Model ◽  
Hydrogen Peroxide Production ◽  
Species Response

To gain insight into how pathogens adapt to new hosts, Cryptococcus neoformans (H99W) was serially passaged in Galleria mellonella. The phenotypic characteristics of the passaged strain (P15) and H99W were evaluated. P15 grew faster in hemolymph than H99W, in vitro and in vivo, suggesting that adaptation had occurred. However, P15 was more susceptible to hydrogen peroxide in vitro, killed fewer mouse macrophages, and had less fungal burden in human ex vivo macrophages than H99W. Analysis of gene expression changes during Galleria infection showed only a few different genes involved in the reactive oxygen species response. As P15 sheds more GXM than H99W, P15 may have adapted by downregulating hemocyte hydrogen peroxide production, possibly through increased capsular glucuronoxylomannan (GXM) shedding. Hemocytes infected with P15 produced less hydrogen peroxide, and hydrogen peroxide production in response to GXM-shedding mutants was correlated with shed GXM. Histopathological examination of infected larvae showed increased numbers and sizes of immune nodules for P15 compared to H99W, suggesting an enhanced, but functionally defective, response to P15. These results could explain why this infection model does not always correlate with murine models. Overall, C. neoformans’ serial passage in G. mellonella resulted in a better understanding of how this yeast evolves under selection.

USING OF LARGE WAX MOTH LARVAE PRODUCTS EXCRETED IN CLONAL MICROREPRODUCTION OF GARDEN STRAWBERRY

1970 ◽  
pp. 66-68
Author(s):  
М. G. Markova ◽  
Е. N. Somova
Keyword(s):  
Aqueous Solution ◽  
Survival Rate ◽  
Nutrient Medium ◽  
Galleria Mellonella ◽  
Waste Product ◽  
Biologically Active ◽  
Clonal Micropropagation ◽  
Wax Moth

Work on the clonal micropropagation of strawberries comes down to the search for new growth regulators, which include a biologically active substance - the waste product of the wax moth Galleria mellonella L. The effect of the waste product of the wax moth on the efficiency of clonal micropropagation of strawberries (Fragaria х ananassa duch) in vitro and in vivo conditions in 2018-2020 is shown. The object of research is micro-cuttings, rooted micro-cuttings and adapted micro-plants of garden strawberries of the Korona variety and of the remontant strawberries of the Brighton variety. It was revealed that at the proliferation stage, the propagation coefficient of the Korona variety increased significantly with the introduction of the waste product of the wax moth in doses of 4.0 mg/L and 6.0 mg/L and amounted to 4.2 and 3.8 pcs./explant, respectively; for Brighton variety, the coefficient increased significantly when the dose of the waste product of the wax moth 2.0 mg/L and amounted to 4.6 pcs./explant. The introduction of the waste product of the wax moth in doses of 4.0 mg/L and 6.0 mg/L into the nutrient medium had a significant effect on the yield of Brighton micro-cuttings suitable for rooting: the yield was 95.5 and 94.1%, respectively 87.7% in the control. For the Korona variety, no significant positive effect of the waste product of the wax moth on this indicator was noted. The rooting of micro-cuttings of strawberries of both varieties significantly increased with the introduction of the waste product of the wax moth into the nutrient medium in all studied doses and amounted to 86.4-100% in the Korona variety, and 88.9-100% in the Brighton variety.  The survival rate of adaptable micro-cuttings of Corona variety strawberries when sprayed with an aqueous solution of the waste product of the wax moth at a dose of 4.0 mg/L was 100%; the maximum survival rate of micro-cuttings Brighton variety is 99.8% in the variant with spraying with an aqueous solution of the waste product of the wax moth at a dose of 6.0 mg/L.

Identification of a Novel Secreted Metabolite Cyclo(Phenylalanyl-Prolyl) from Batrachochytrium Dendrobatidis and its effect on Galleria Mellonella.

2021 ◽  
Author(s):  
Amanda Michelle Starr ◽  
Masoud Zabet-Moghaddam ◽  
Michael San Francisco
Keyword(s):  
Batrachochytrium Dendrobatidis ◽  
Galleria Mellonella ◽  
Ultra Performance Liquid Chromatography ◽  
Liquid Chromatography Mass Spectrometry ◽  
Loss Of Righting Reflex ◽  
Righting Reflex ◽  
Fragmentation Patterns ◽  
Eventual Death ◽  
Wax Moth

Abstract The fungus, Batrachochytrium dendrobatidis, is the causative agent of chytridiomycosis and a leading cause of global decline in amphibian populations . The first stages of chytridiomycosis include: inflammation, hyperkeratosis, lethargy, loss of righting reflex, and disruption of internal electrolyte levels leading to eventual death of the host. Previous work indicates that B. dendrobatidis can produce immunomodulatory compounds and other secreted molecules that regulate the growth of the fungus. In this study, filtrates of the fungus grown in media and water were subjected to ultra performance liquid chromatography-mass spectrometry and analyzed using Compound Discoverer 3.0. Identification of cyclo(phenylalanyl-prolyl), chitobiose, and S-adenosylmethionine were verified by their retention times and fragmentation patterns from B. dendrobatidis supernatants. Previous studies have analyzed the effects of B. dendrobatidis on amphibian models, in vitro, or in cell culture. We studied the effects of live B. dendrobatidis cells, spent culture filtrates containing secreted metabolites, and cyclo(pheylalanyl-prolyl) on wax moth larvae ( Galleria mellonella) . Concentrated filtrates caused melanization within 24 hours, while live B. dendrobatidis caused melanization within 48 hours. Our results indicate B. dendrobatidis produces secreted metabolites previously unreported. These findings provide another alternative for the use of a non-amphibian model system to study pathogenicity traits in this fungus.

The secretion of collagen by insects: uptake of [3H]proline by collagen-synthesizing cells in Locusta migratoria and Galleria mellonella

1976 ◽  
Vol 20 (2) ◽  
pp. 377-403
Author(s):  
D.E. Ashhurst ◽  
N.M. Costin
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Galleria Mellonella ◽  
Unit Area ◽  
Locusta Migratoria ◽  
Relative Number ◽  
Collagen Secretion ◽  
The Matrix ◽  
Intracellular Pathway ◽  
Wax Moth

The uptake of [H3]proline by collagen-secreting cells of the locust, Locusta migratoria, and wax-moth, Galleria mellonella, has been investigated by electron autoradiography. The locust cells are around the ejaculatory duct and they secrete collagen in the young adult male, while the wax-moth cells are those which produce the dorsal mass of connective tissue on the abdominal nerve cord during the late pupal stage. The cells were exposed to [H3]proline either by injection of the [3H]proline into the insect, or as a pulse while the tissue was maintained in a culture medium. The tissues were fixed at differeing experimental times after exposure to the [3H]proline. The resulting electron autoradiographs were subjected to quantitative analysis, and the silver grain distribution was determined as the relative number of grains per unit area over a series of tissue compartments. When the results of this analysis for the matrix, rough endoplasmic reticulum and Golgi complexes of the two tissues were plotted against experimental time, it was seen that the relative number of grains per unit area over the rough endoplasmic reticulum decreases while that over the matrix increases; statistical analysis has shown that these changes are significant. For the Golgi complexes, however, the theoretical variances are much greater, due to the small relative area occupied by this organelle. There is little evidence for anything other than random sampling fluctuations in the relative numbers of grains per unit area, and hence it is unlikely that the time course of the label over the Golgi complexes follows that over the rough endoplasmic reticulum. The conclusions drawn from these experiments are firstly that a large portion of the labelled protein passes straight from the rough endoplasmic reticulum to the matrix, but that a smaller portion of the labelled material might pass from the rough endoplasmic reticulum to the Golgi complexes and thence to the matrix. It is assumed that collagen comprises most of the protein which passes straight from the rough endoplasmic reticulum to the matrix, and while there is no evidence to exclude collagen from the material passing through the Golgi complexes, it is probable that other proteins and glycosaminoglycans are also present in this labelled material. These conclusions about the intracellular pathway for collagen secretion are similar to those derived from recent studies of some vertebrate fibroblasts. There is, however, conflicting opinion about the intracellular pathway of collagen and it is pointed out that there is diversity in collagen-synthesizing cells, which may account for the differences in the intracellular pathways for collagen secretion which have been proposed.

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