Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.     

Humoral immune response upon mild heat-shock conditions in Galleria mellonella larvae

Larvae of Galleria mellonella exposed to mild heat-shock (38 degrees C) showed an enhanced humoral immune response after microbial infection in comparison to infected animals grown at 28 degrees C. This enhanced response was manifested by increased expression of antimicrobial peptide (AMP) genes leading to enhanced antimicrobial activity in the hemolymph. We found an increased level of Hsp90 and changes in the level of a 55kDa protein recognized by anti-Hsp90 antibodies in fat bodies of infected animals reared at 28 degrees C as well as in uninfected animals exposed to elevated temperature. Pre-treatment of animals with an inhibitor of Hsp90, 17-DMAG, prior to immunization resulted in increased expression of AMP genes encoding gallerimycin and cecropin at 38 degrees C. This observation was correlated with the changes in Hsp90 protein and increased level of 55kDa protein. Also G. mellonella larvae pre-treated with 17-DMAG and exposed to mild heat-shock for 30min showed an increased survival rate after infection with entomopathogenic bacteria Pseudomonas aeruginosa. We also show the effect of 17-DMAG on the phosphorylation state of ERK MAP kinase. We postulate that Hsp90 may play a significant role in converging pathways involved in the insect immune response and heat-shock.     

Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.     

青蒿琥酯抗蜡螟烟曲霉感染自噬机制初步研究

目的 探究青蒿琥酯在蜡螟感染烟曲霉后,对蜡螟的自噬相关蛋白的表达影响。方法 用一定量的烟曲霉的活化孢子感染蜡螟,经过1 h,用青蒿琥酯注射一组蜡螟,两性霉素B注射一组蜡螟,剩余的作为感染组。12 h后,取各组蜡螟进行病理切片染色,观察各组的病理情况;取各组的蜡螟的淋巴液,收集各组的孢子,用真菌活性检测试剂盒检测孢子活性并将淋巴细胞分离、裂解,离心取上清,用Western-blot法检测上清液中的Dectin-1、ROS、LC3Ⅱ的表达水平。结果 青蒿琥酯注射组的蜡螟病理切片中的孢子比感染组数量少且聚集在一起,未长出菌丝,而体外分离的真菌孢子,青蒿琥酯组的活性明显受到抑制。经Western-blot显示青蒿琥酯能增强淋巴细胞Dectin-1、ROS、LC3Ⅱ的表达。结论 青蒿琥酯可通过抑制烟曲霉的活性和增强蜡螟淋巴细胞的自噬水平,对抗蜡螟烟曲霉感染。     

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires'' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae''s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.     

Improvement of trypanocidal metabolites production by Aspergillus fumigatus using neural networks

An optimization procedure using artificial neural networks was developed to determine the optimal combination of parameters, such as medium culture, initial pH, temperature and time of fermentation for maximal trypanocidal metabolites production by Aspergillus fumigatus. A data set of 81 experiments was carried out and an artificial neural network was trained to identify the optimal conditions for this process. Good correlation was obtained between the experimental and predicted values of lysis of the trypomastigote forms of Trypanosoma cruzi (r2 = 0.9990). The simulations of fermentation performance were undertaken on combinations of input variables and the highest level of activity against T. cruzi was obtained from the chloroform extract of the modified Jackson medium culture, initial pH of 6.0, incubated at 40 degrees C for 144 h. It displayed lysis of 95% of the trypomastigote forms of T. cruzi and the red blood cells remained normal.     

Electron microscopical-immunocytochemical evidence of ecdysteroids in the prothoracic gland of Galleria mellonella

Summary Fixation of prothoracic glands of Galleria mellonella with a solution containing saponin permits immunocytochemical staining of the entire gland. By this means ecdysteroids were demonstrated electron microscopically to be present in the hyaloplasm and microtubules.Supported by Sächsische Akademie der Wissenschaften zu Leipzig     

A new cell line from the wax moth Galleria mellonella Linne (lepidoptera: Pyralididae)

Summary A cell line derived from the larval-fat body tissues of the wax moth, Galleria mellonella Linne, was established in MGM-450 medium. The cells grew in suspension and were mainly spherical in shape. Population doubling time was between 1.4 and 1.7 d over a range of 15 to 35°C, and the maximum growth rate was at 25°C. The chromosome number ranged from 70–239, with a mode of 170. The cells were sensitive to 20-hydroxyecdysone, which stimulated their growth and induced morphological changes. The cell line was designated GaMe-LF1.     

Histopathology of experimental Aspergillus fumigatus keratitis

Histopathological studies in rabbit's eyes, 7 and 14 days after intracorneal inoculation with 1×10⁵ Aspergillus fumigatus conidia have been performed.Similar lesions were found in both periods with fungal hyphae in the anterior third of corneal stroma, round cell infiltration from the sclero-corneal edge and in the anterior chamber and, neovascularization.No lesions were found in the Descemet's membrane.Gomori silver-methenamine stain with hematoxiline-eosine counter-stain was found to be the most reliable stain to detect fungal presence in corneal stroma, and Masson's trichromic stain in the study of pathological changes in ocular elements.     

Phagocytic activity and encapsulation rate of Galleria mellonella larval haemocytes during bacterial infection by Bacillus thuringiensis

The bacterium Bacillus thuringiensis (Bt) is a pathogen of many insect species and is actively used in biocontrol. After the peroral inoculation of Galleria mellonella by the Bt in 5% sublethal concentration (LC₅), a 1.5-fold increase in the phagocytic activity of infected larvae has been registered on the second and third days after the inoculation. With the increase of Bt-inoculum amount to 15% of sublethal concentration (LC₁₅), a further increase of the phagocytic activity and enhanced encapsulation rates in the haemolymph of infected larvae has been observed. The enhanced cellular immunity during the bacteriosis seems to have resulted from the destruction of midgut epithelium cells followed by the subsequent exposure of gut content to lymph factors activating the immune system of haemocoel.     

Immunocompetence of Galleria mellonella: sex- and stage-specific differences and the physiological cost of mounting an immune response during metamorphosis

The antibacterial immune response of the wax moth, Galleria mellonella, was analysed by use of an inhibition zone plate assay. We demonstrated significant stage-specific differences as the immune response was most effective in the pupal, next the larval and then the adult stage. In addition, we demonstrated that an immune challenge at the onset of, or during metamorphosis does not increase nor decrease the strength of the antibacterial immune response in the subsequent developmental stage(s). These findings illustrate that induced immunity is not preserved during metamorphosis but also deny any cost to the immune system itself. However, an immune challenge does induce a significant shortening of the direct development time and affects the mass loss during metamorphosis in a sex-dependent manner: males emerged smaller whereas the mass of females was not significantly affected. These observations indicate that there are sex-specific costs to mounting an immune response during metamorphosis which affect physiological traits, implicating a trade-off between immunity and development.     

The structures of polysaccharides and glycolipids of Aspergillus fumigatus grown in the presence of human serum

A study was made of polysaccharides and glycosphingolipids isolated from Aspergillus fumigatus grown in media supplemented with human serum from healthy donors. Fractionation of Cetavlon-precipitated polysaccharides on Sephacryl S-400 gave rise to an excluded fraction (Fraction I) with molecular weight of >400 kDa and an included peak (Fraction II) with an average molecular weight of 30–80 kDa. Fraction I comprises about 5% of total polysaccharide and was identified as a glycogen-like molecule. Its structure was deduced from methylation data, treatment with amyloglucosidase, a red-brown coloration produced with an iodine solution and by ¹H and ¹³C-NMR spectroscopy. It was previously suggested that higher amounts of glycogen-like polysaccharide (20%) were present in A. fumigatus grown in serum-free medium. Fraction II was identified as a galactomannan and was the main polysaccharide of A. fumigatus grown in serum-supplemented medium. Its structure was elucidated mainly by ¹³C-NMR spectroscopy combined with partial acetolysis and methylation analysis. The ¹³C-NMR spectrum of the galactomannan showed a much greater complexity in the -d-galf and -d-manp C-1 regions, than was evident for galactomannan from serum-free cultures previously described, reflecting differences in the glycosylation pattern, stimulated in serum-supplemented medium.No differences in A. fumigatus glycosphingolipid could be detected between serum-containing and serum-free growth conditions.Our results demonstrate that the change in polysaccharide structure is a more specific response to the altered growth conditions and not merely a symptom of more general changes.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effect of pre-incubation temperature on susceptibility of Galleria mellonella larvae to infection by Candida albicans

The use of insects for evaluating the virulence of microbial pathogens and for determining the efficacy of antimicrobial drugs is increasing. When larvae of the greater wax moth Galleria mellonella were incubated at 4 or 37°C for 24 h. prior to infection, they manifested increased resistance to infection by the yeast Candida albicans compared to larvae that had been pre-incubated for 24 h at 30°C. Incubation at 4 or 37°C led to an increase in haemocyte density and the expression of genes coding for gallerimycin, transferrin, an inducible metalloproteinase inhibitor (IMPI) and galiomicin. Peak expression of these genes was recorded at approximately 24 h after the commencement of the 4 or 37°C incubation. These results indicate that exposure of larvae to mild thermal shock conditions induces a protective cellular and humoral immune response mediated by increased numbers of haemocytes and elevated expression of antimicrobial peptides.     

Toxicity of mycotoxins of Fusarium sambucinum for feeding in Galleria mellonella

Fusarium sambucinum Fuckel showed antifeedant activity towards larvae of Galleria mellonella L. when incorporated into insect diet. The activity appeared mostly due to the concentration of trichothecenes present in the fungal extracts. Diacetoxyscirpenol and neosolaniol showed similar levels of activity and were significant potent antifeedants against larvae at 50 and 100 ppm. On the contrary, enniatin B showed no activity up to 100 ppm.     

The effects of dsRNA mycoviruses on growth and murine virulence of Aspergillus fumigatus

Some isolates of the opportunistic human pathogenic fungus Aspergillus fumigatus are known to be infected with mycoviruses. The dsRNA genomes of two of these mycoviruses, which include a chrysovirus and a partitivirus, have been completely sequenced and an RT-PCR assay for the viruses has been developed. Through curing virus-infected A. fumigatus isolates by cycloheximide treatment and transfecting virus-free isolates with purified virus, as checked by RT-PCR, isogenic virus-free and virus-infected lines of the fungus were generated whose phenotypes and growth have been directly compared. Mycovirus infection of A. fumigatus with either the chrysovirus or the partitivirus resulted in significant aberrant phenotypic alterations and attenuation of growth of the fungus but had no effect on susceptibility to common antifungals. Chrysovirus infection of A. fumigatus caused no significant alterations to murine pathogenicity.     

A study for minisatellitic markers of Conidiobolus coronatus' pathogenicity to Galleria mellonella larvae

A selected panel of 13 colonies of entomopathogenic fungus Conidiobolus coronatus representing 6 variants of pathogenicity to Galleria mellonella larvae (ranged from 100 to 10% of efficiency), derived from single spores, were tested for the presence of hypervariable loci in their genomes by hybridization with Jeffreys' human minisatellite probe 33.6. The probe produced informative fingerprints and revealed slight differences among colonies analyzed. Up to 20 variable bands per colony were recognized in the size range of 2-20 kb. The band sharing within groups with the same pathogenicity ranged from 0.966 to 0.800. The genetic distance between different variants ranged from 0.026 to 0.282. A few characteristic bands for high and low pathogenicity to the larvae were found.     

The ultrastructure and ultracytochemistry of the basement membrane of the Galleria mellonella fat body

Summary The fat body lobes of Galleria mellonella are surrounded by basement membrane — a fine granular layer of connective tissue. This membrane has an affinity for ruthenium red. The results obtained after treatment of the fat body with neuraminidase, hyaluronidase, phospholipase C and proteolytic enzymes suggest that glycoproteins and phospholipoproteins are constituents of this basement membrane. The basement membrane also has the ability to bind concanavalin A-peroxidase, which is associated with the presence of mannoside residues.The preliminary results of these studies were presented at the IX Conference of Electron Microscopy, Gdask, Poland (Dutkowski, 1975)I am greatly indebted to Professor A. Przececka for her encouragement to undertake this study. The excellent technical assistance of Mrs. K. Mroziska, Mrs. Z. Kamiska and the engineering staff of the Laboratory is gratefully acknowledged     

Entry into the stationary phase is associated with a rapid loss of viability and an apoptotic-like phenotype in the opportunistic pathogen Aspergillus fumigatus

When the opportunistic pathogen Aspergillus fumigatus entered the stationary phase, there was a rapid loss in cell viability which was associated with the appearance of markers characteristic of apoptosis, namely annexin V-FITC binding to the cytoplasmic membrane, demonstrating exposure of phosphatidylserine to the outer leaflet of the membrane; and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining of the nuclei, indicating DNA fragmentation. This was followed later by a loss of membrane integrity as revealed by propidium iodide staining. The development of the apoptotic phenotype was blocked when the protein synthesis inhibitor cycloheximide was added to the culture 1h prior to the onset of the stationary phase, demonstrating active participation of the cell. In addition, intracellular activity against substrates specific for caspase-1 and -8 also increased on stationary phase entry and the development of the apoptotic phenotype was blocked when the cell permeant caspase inhibitor Z-FAD-fmk was present in the medium. Cell death in A. fumigatus during the stationary phase therefore appears to share similarities to apoptotic cell death in higher eukaryotes and to be dependent on a caspase-like activity.     

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system¹. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides^2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals¹. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis^4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model^8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C^12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD₅₀¹⁴, measurement of bacterial survival^15,16 and examination of the infection process¹⁷. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections^18,19.