scholarly journals Comparison of different extraction kits to isolate microRNA fromGalleria mellonella(wax moth) larvae infected withMetarhizium brunneum(ARSEF4556)

2019 ◽  
Author(s):  
Muneefah A. Alenezi ◽  
Tariq M. Butt ◽  
Daniel C. Eastwood
Keyword(s):  
Fungal Pathogens ◽  
High Throughput Sequencing ◽  
Galleria Mellonella ◽  
Host Tissue ◽  
Tissue Cultures ◽  
Complex Environments ◽  
Infected Insect ◽  
Sequencing And Expression ◽  
Wax Moth

ABSTRACTMicroRNAs (miRNAs) play an important role in regulating gene expression and are involved in developmental processes in animals, plants and fungi. To understand the role of miRNAs in a biological system, it is important to optimise the extraction procedures to obtain high quality and quantity nucleic acid that enable high throughput sequencing and expression analysis. Numerous kit-based miRNA extraction protocols have been optimised generally to single cell or tissue cultures. Fungi, however, often occupy physically and chemically complex environments which miRNA make extraction challenging, such as fungal pathogens interacting within plant or animal host tissue. We used aGalleria mellonella(wax moth) larvae and entomopathogenic fungusMetarhizium brunneum ARSEF 4556host/pathogen model to compare commercially available miRNA extraction kits (Invitrogen PureLink™ miRNA Isolation Kit, Ambion mirVana™miRNA Isolation Kit and Norgen microRNA purification Kit). Our results showed reproducible and significant differences in miRNAs extraction between the kits, with the Invitrogen PureLink™ miRNA Isolation protocol demonstrating the best performance in terms of miRNA quantity, quality and integrity isolated from fungus-infected insect tissue.

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 Use of the Galleria mellonella Caterpillar as a Model Host To Study the Role of the Type III Secretion System in Pseudomonas aeruginosa Pathogenesis

2003 ◽  
Vol 71 (5) ◽  
pp. 2404-2413 ◽  
Author(s):  
Sachiko Miyata ◽  
Monika Casey ◽  
Dara W. Frank ◽  
Frederick M. Ausubel ◽  
Eliana Drenkard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Galleria Mellonella ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Triple Mutant ◽  
Type Iii ◽  
Wax Moth

ABSTRACT Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ΔexoU ΔexoY, ΔexoT ΔexoY, and ΔexoT ΔexoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a ΔexoT ΔexoU ΔexoY triple mutant and a ΔpscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.

scholarly journals Role of gliotoxin in the symbiotic and pathogenic interactions of Trichoderma virens

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2319-2330 ◽  
Author(s):  
Walter A. Vargas ◽  
Prasun K. Mukherjee ◽  
David Laughlin ◽  
Aric Wiest ◽  
Maria E. Moran-Diez ◽  
...  
Keyword(s):  
Fungal Pathogen ◽  
Gene Disruption ◽  
Galleria Mellonella ◽  
Pythium Ultimum ◽  
Trichoderma Virens ◽  
Human Pathogen ◽  
Opportunistic Human Pathogen ◽  
Confrontation Assay ◽  
Wax Moth

Using a gene disruption strategy, we generated mutants in the gliP locus of the plant-beneficial fungus Trichoderma virens that were no longer capable of producing gliotoxin. Phenotypic assays demonstrated that the gliP-disrupted mutants grew faster, were more sensitive to oxidative stress and exhibited a sparse colony edge compared with the WT strain. In a plate confrontation assay, the mutants deficient in gliotoxin production were ineffective as mycoparasites against the oomycete, Pythium ultimum, and the necrotrophic fungal pathogen, Sclerotinia sclerotiorum, but retained mycoparasitic ability against Rhizoctonia solani. Biocontrol assays in soil showed that the mutants were incapable of protecting cotton seedlings from attack by P. ultimum, against which the WT strain was highly effective. The mutants, however, were as effective as the WT strain in protecting cotton seedlings against R. solani. Loss of gliotoxin production also resulted in a reduced ability of the mutants to attack the sclerotia of S. sclerotiorum compared with the WT. The addition of exogenous gliotoxin to the sclerotia colonized by the mutants partially restored their degradative abilities. Interestingly, as in Aspergillus fumigatus, an opportunistic human pathogen, gliotoxin was found to be involved in pathogenicity of T. virens against larvae of the wax moth, Galleria mellonella. The loss of gliotoxin production in T. virens was restored by complementation with the gliP gene from A. fumigatus. We have, thus, demonstrated that the putative gliP cluster of T. virens is responsible for the biosynthesis of gliotoxin, and gliotoxin is involved in mycoparasitism and biocontrol properties of this plant-beneficial fungus.

scholarly journals Galleria mellonella as an alternative infection model for Yersinia pseudotuberculosis

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1516-1522 ◽  
Author(s):  
Olivia L. Champion ◽  
Ian A. M. Cooper ◽  
Sarah L. James ◽  
Donna Ford ◽  
Andrey Karlyshev ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Yersinia Pseudotuberculosis ◽  
Galleria Mellonella ◽  
Infection Model ◽  
Phenotypic Properties ◽  
Infection Models ◽  
Insect Haemocytes ◽  
Increased Susceptibility ◽  
Wax Moth

We report that larvae of the wax moth (Galleria mellonella) are susceptible to infection with the human enteropathogen Yersinia pseudotuberculosis at 37 °C. Confocal microscopy demonstrated that in the initial stages of infection the bacteria were taken up into haemocytes. To evaluate the utility of this model for screening Y. pseudotuberculosis mutants we constructed and tested a superoxide dismutase C (sodC) mutant. This mutant showed increased susceptibility to superoxide, a key mechanism of killing in insect haemocytes and mammalian phagocytes. It showed reduced virulence in the murine yersiniosis infection model and in contrast to the wild-type strain IP32953 was unable to kill G. mellonella. The complemented mutant regained all phenotypic properties associated with SodC, confirming the important role of this metalloenzyme in two Y. pseudotuberculosis infection models.

scholarly journals Fighting fungi with fungi: the mycobiome contribution to emerging disease in amphibians

2017 ◽  
Author(s):  
Patrick Kearns ◽  
Sarah Fischer ◽  
Saioa Fernández-Beaskoetxea ◽  
Caitlin Gabor ◽  
Jaime Bosch ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
High Throughput Sequencing ◽  
Batrachochytrium Dendrobatidis ◽  
Emerging Infectious Diseases ◽  
Pathogen Defense ◽  
Colonization Potential ◽  
Additional Resistance ◽  
Symbiotic Fungi

Emerging infectious diseases caused by fungal taxa are increasing and are placing a substantial burden on economies and ecosystems worldwide. Of the emerging fungal diseases, chytridomycosis caused by the fungus Batrachochytrium dendrobatidis (hereafter Bd) is causing a global amphibian extinction. The host frog does have come internal innate immunity, as well as additional resistance through cutaneous microbial communities, leading to the development of probiotic bacterial therapies with mixed results. Unknown is the role of fungi in the protection against Bd infection, and as such, we examined the overlapping roles of bacterial and fungal microbiota in pathogen defense with a combination of high-throughput sequencing and culturing of symbiotic fungi from poison arrow frogs (Dendrobates sp.). Our analyses revealed that abundance of cutaneous fungi contributed more to pathogen defense (~45%), than bacteria (~10%) and these differed from environmental microbiota. Further, we demonstrated that a fungal probiotic therapy did not induce an endocrine-immune reaction in contrast to bacterial probiotics that stressed amphibian hosts and suppressed antimicrobial peptide responses, limiting their long-term colonization potential. Our results suggest that probiotic strategies against amphibian fungal pathogens should refocus on host-associated and environmental fungi such as Penicillium and member of the families Chaetomiaceae and Lasiosphaeriaceae.

scholarly journals Culture Degeneration Reduces Sex-Related Gene Expression, Alters Metabolite Production and Reduces Insect Pathogenic Response in Cordyceps militaris

2021 ◽  
Vol 9 (8) ◽  
pp. 1559
Author(s):  
Peter A. D. Wellham ◽  
Abdul Hafeez ◽  
Andrej Gregori ◽  
Matthias Brock ◽  
Dong-Hyun Kim ◽  
...  
Keyword(s):  
Secondary Metabolite ◽  
Galleria Mellonella ◽  
Cordyceps Militaris ◽  
Insect Immunity ◽  
Pharmacological Activities ◽  
Metabolite Production ◽  
Expression Of Genes ◽  
Greater Wax Moth ◽  
Wax Moth

Cordyceps militaris is an entomopathogenic ascomycete, known primarily for infecting lepidopteran larval (caterpillars) and pupal hosts. Cordycepin, a secondary metabolite produced by this fungus has anti-inflammatory properties and other pharmacological activities. However, little is known about the biological role of this adenosine derivate and its stabilising compound pentostatin in the context of insect infection the life cycle of C. militaris. During repeated subcultivation under laboratory conditions a degeneration of C. militaris marked by decreasing levels of cordycepin production can occur. Here, using degenerated and parental control strains of an isolate of C. militaris, we found that lower cordycepin production coincides with the decline in the production of various other metabolites as well as the reduced expression of genes related to sexual development. Additionally, infection of Galleria mellonella (greater wax moth) caterpillars indicated that cordycepin inhibits the immune response in host haemocytes. Accordingly, the pathogenic response to the degenerated strain was reduced. These data indicate that there are simultaneous changes in sexual reproduction, secondary metabolite production, insect immunity and infection by C. militaris. This study may have implications for biological control of insect crop pests by fungi.

scholarly journals Standardization of G. mellonella Larvae to Provide Reliable and Reproducible Results in the Study of Fungal Pathogens

2018 ◽  
Vol 4 (3) ◽  
pp. 108 ◽  
Author(s):  
Olivia Champion ◽  
Richard Titball ◽  
Steven Bates
Keyword(s):  
Fungal Pathogens ◽  
Galleria Mellonella ◽  
Infection Model ◽  
Full Potential ◽  
Development Cycle ◽  
Mammalian Infection ◽  
High Throughput Screens ◽  
Wax Moth

In the past decade, Galleria mellonella (wax moth) larvae have become widely used as a non-mammalian infection model. However, the full potential of this infection model has yet to be realised, limited by the variable quality of larvae used and the lack of standardised procedures. Here, we review larvae suitable for research, protocols for dosing larvae, and methods for scoring illness in larvae infected with fungal pathogens. The development of standardised protocols for carrying out our experimental work will allow high throughput screens to be developed, changing the way in which we evaluate panels of mutants and strains. It will also enable the in vivo screening of potential antimicrobials at an earlier stage in the research and development cycle.

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