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EST analysis of genes expressed by the zygomycete pathogen Conidiobolus coronatus during growth on insect cuticle
Abstract
Conidiobolus coronatus (Zygomycota) is a facultative saprobe that is a pathogen of many insect species. Almost 2000 expressed sequence tag (EST) cDNA clones were sequenced to analyse gene expression during growth on insect cuticle. Sixty percent of the ESTs that could be clustered into functional groups (E<or=10(-5)) had their best BLAST hits among fungal sequences. These included chitinases and multiple subtilisins, trypsin, metalloprotease and aspartyl protease activities with the potential to degrade host tissues and disable anti-microbial peptides. Otherwise, compared to the ascomycete entomopathogen Metarhizium anisopliae, Con. coronatus produced many fewer types of hydrolases (e.g. no phospholipases), antimicrobial agents, toxic secondary metabolites and no ESTs with putative roles in the generation of antibiotics. Instead, Con. coronatus produced a much higher proportion of ESTs encoding ribosomal proteins and enzymes of intermediate metabolism that facilitate its rapid growth. These results are consistent with Con. coronatus having adapted a modification of the saprophytic ruderal-selected strategy, using rapid growth to overwhelm the host and exploit the cadaver before competitors overrun it. This strategy does not preclude specialization to pathogenicity, as Con. coronatus produces the greatest complexity of proteases on insect cuticle, indicating an ability to respond to conditions in the cuticle.
... Probably during evolution, they adapted these weapons to invade the tissues of animals, including arthropods, insects, and, later, mammals (23). For instance, the release of some metabolites allows insect pathogenic entomophthoralean species to secrete several chitinases, successfully penetrating the exoskeleton of their prey (see below) (11,23,76,(99)(100)(101). The release of powerful enzymes in culture and during infection has been reported for Basidiobolus and Conidiobolus species (73,99,102). ...
... For instance, the release of some metabolites allows insect pathogenic entomophthoralean species to secrete several chitinases, successfully penetrating the exoskeleton of their prey (see below) (11,23,76,(99)(100)(101). The release of powerful enzymes in culture and during infection has been reported for Basidiobolus and Conidiobolus species (73,99,102). ...
... The expressions of extracellular cellulases, lipases, and proteases in these fungi have been widely reported (73,99,(102)(103)(104)(105). Likely, Basidiobolus and Conidiobolus took advantage of this ancestral trait to survive in nature and adapted their life cycles to growth inside the environment provided by infected host species (23). ...
The pathogenic entomophthoralean fungi cause infection in insects and mammalian hosts. Basidiobolus and Conidiobolus species can be found in soil and insect, reptile, and amphibian droppings in tropical and subtropical areas. The life cycles of these fungi occur in these environments where infecting sticky conidia are developed. The infection is acquired by insect bite or contact with contaminated environments through open skin. Conidiobolus coronatus typically causes chronic rhinofacial disease in immunocompetent hosts, whereas some Conidiobolus species can be found in immunocompromised patients. Basidiobolus ranarum infection is restricted to subcutaneous tissues but may be involved in intestinal and disseminated infections. Its early diagnosis remains challenging due to clinical similarities to other intestinal diseases. Infected tissues characteristically display eosinophilic granulomas with the Splendore-Höeppli phenomenon. However, in immunocompromised patients, the above-mentioned inflammatory reaction is absent. Laboratory diagnosis includes wet mount, culture serological assays, and molecular methodologies. The management of entomophthoralean fungi relies on traditional antifungal therapies, such as potassium iodide (KI), amphotericin B, itraconazole, and ketoconazole, and surgery. These species are intrinsically resistant to some antifungals, prompting physicians to experiment with combinations of therapies. Research is needed to investigate the immunology of entomophthoralean fungi in infected hosts. The absence of an animal model and lack of funding severely limit research on these fungi.
... Endophytic fungi play an important role in adaption and morphogenesis of host plant by defending it against insects, predators, microbial pathogens and latent pathogens. Similarly, order Mucorales of Zygomycota is considered a sister group of Entomophthorales (insect pathogens) [9]. The pathogenicity of Mucorales is largely believed to be due to endocellular excretions and production of subtilisins, chitinases, proteinases and antioxidant proteins (e.g., superoxide dismutase, catalase and peroxidase) [9]. ...
... Similarly, order Mucorales of Zygomycota is considered a sister group of Entomophthorales (insect pathogens) [9]. The pathogenicity of Mucorales is largely believed to be due to endocellular excretions and production of subtilisins, chitinases, proteinases and antioxidant proteins (e.g., superoxide dismutase, catalase and peroxidase) [9]. ...
... (3.677 ± 0.45) showed highest quenching ability, followed by MC108.16 (4.183 ± 0.16) and WJ11 (7.6 ± 0.44) on Day 5 under stress conditions ( Table 3). The stressful conditions are believed to be responsible for the productions of various extra storage compounds, enzymatic systems and secondary metabolites in filamentous fungi [9,29,36], which partially why all the nutritional deficient media grown filamentous fungi showed higher scavenging ability than normal media. Freimoser and his colleagues [9] also deduced that a change in the stress type and kind of input nutrients also leads to change in the extent of expression of genes responsible for different secondary metabolite production and virulence in Zygomycetes. ...
Three important strains of Mucor circinelloides grown in complete and minimal media for specified period (72 h, 120 h and 168 h) under submerged fermentation conditions were investigated for their potential antioxidants/secondary metabolite production. All mycelial extracts demonstrated effective antioxidant activities in terms of β-carotene/linoleic acid bleaching, radical scavenging, reduction of metal ions and chelating abilities against ferrous ions. Different extraction methods and solvent systems affected the recovery yield and antioxidant activities of the extracts significantly (p ≤ 0.05). Ethanolic extracts were found to be rich source of antioxidant components and subsequently more effective in antioxidant properties. Fermentation period and media used also significantly affected (p ≤ 0.05) the antioxidant production and the resulting antioxidant properties. The (ethanolic) extracts of all the strains from late exponential growth phase (120 h) showed highest antioxidant production with topmost reducing, chelating and radical scavenging capabilities. Strain MC277.49 was found to be the highest producer of antioxidants followed by MC108.16 and WJ11. Phenolic compounds were detected significantly in higher (p ≤ 0.05) amount succeeded by the condensed tannins and flavonoids. Total phenol content of each extract was attributed to overall antioxidant capacity. Submerged fermentation with nutritional stress conditions were found to be excellent way of producing surplus amount of natural antioxidants/secondary metabolites with their vast potential commercial application in food and pharmaceutical industries.
... (3.677 ± 0.45) showed highest quenching ability, followed by MC108.16 (4.183 ± 0.16) and WJ11 (7.6 ± 0.44) on Day 5 under stress conditions ( Table 3). The stressful conditions are believed to be responsible for the productions of various extra storage compounds, enzymatic systems and secondary metabolites in filamentous fungi [9,29,36], which partially why all the nutritional deficient media grown filamentous fungi showed higher scavenging ability than normal media. Freimoser and his colleagues [9] also deduced that a change in the stress type and kind of input nutrients also leads to change in the extent of expression of genes responsible for different secondary metabolite production and virulence in Zygomycetes. ...
... The stressful conditions are believed to be responsible for the productions of various extra storage compounds, enzymatic systems and secondary metabolites in filamentous fungi [9,29,36], which partially why all the nutritional deficient media grown filamentous fungi showed higher scavenging ability than normal media. Freimoser and his colleagues [9] also deduced that a change in the stress type and kind of input nutrients also leads to change in the extent of expression of genes responsible for different secondary metabolite production and virulence in Zygomycetes. ...
... (3.677 ± 0.45) showed highest quenching ability, followed by MC108.16 (4.183 ± 0.16) and WJ11 (7.6 ± 0.44) on Day 5 under stress conditions ( Table 3). The stressful conditions are believed to be responsible for the productions of various extra storage compounds, enzymatic systems and secondary metabolites in filamentous fungi [9,29,36], which partially why all the nutritional deficient media grown filamentous fungi showed higher scavenging ability than normal media. Freimoser and his colleagues [9] also deduced that a change in the stress type and kind of input nutrients also leads to change in the extent of expression of genes responsible for different secondary metabolite production and virulence in Zygomycetes. ...
... The stressful conditions are believed to be responsible for the productions of various extra storage compounds, enzymatic systems and secondary metabolites in filamentous fungi [9,29,36], which partially why all the nutritional deficient media grown filamentous fungi showed higher scavenging ability than normal media. Freimoser and his colleagues [9] also deduced that a change in the stress type and kind of input nutrients also leads to change in the extent of expression of genes responsible for different secondary metabolite production and virulence in Zygomycetes. ...
... 5 A previous study based on expressed sequence tag (EST) analyses of Conidiobolus coronatus and Zoophthora radicans cultures in vitro showed that many transcripts activated by specific insect cuticular components in nutritive media are involved in intermediate metabolism and cuticle-degrading processes. 6,7 Transposon-assisted signal sequence trapping was performed to investigate enzymes secreted by Pandora neoaphidis, Entomophthora planchoniana and Conidiobolus obscurus in naturally infected grain aphids, and an array of secreted hydrolytic enzymes for breeching host cuticles were found. 8 Advanced RNA-sequencing (RNA-seq) based on the high-throughput sequencing Illumina platform has greatly facilitated comparative analyses of transcriptomes generated from biomaterials in vitro and in vivo. ...
... Investigation of the virulence factors of entomopathogenic fungi has largely focused on the complex groups of secreted enzymes involved in host cuticle degradation and fungal proliferation within the hosts, including Pr1-and Pr2-like proteases, carboxypeptidases, chitinases, lipases, glycosyltransferases and carbohydrate esterases. [6][7][8][9]21,22 The characterization of diverse subtilisins (Pr1s) in M. anisopliae has revealed a pathogen fitness associated with the host specificity, as fungal isolates from diverse hosts are capable of secreting diverse Pr1s with different secondary substrate specificities, properties of adsorption to the cuticle and alkaline stability. 23 This study has identified 22 Pr1-like protease unigenes in the aphid-obligate pathogen C. obscurus, approaching the 26 Pr1 transcripts reported in the obligate ant pathogen P. formicae. ...
BACKGROUND: Conidiobolus obscurus is a wide spread fungal entomopathogen holding aphid biocontrol potential. This study focuses on a de novo transcriptomic analysis of C. obscurus.
RESULTS: A number of pathogenicity-associated factors were annotated for the first time from the assembled 17,231 fungal unigenes, including those encoding subtilisin-like proteolytic enzymes (Pr1s), trypsin-like proteases, metalloproteases, carboxypeptidases and endochitinases. Many of these genes were transcriptionally up-regulated by at least twofold in mycotized cadavers compared to the in vitro fungal cultures. The resultant transcriptomic database was validated by the transcript levels of three selected pathogenicity-related genes quantified from different in vivo and in vitro materials in real-time quantitative PCR. It was also suggested the involvements of multiple Pr1 proteases in the first stage of fungal infection. Interestingly, a unique cytolytic (Cyt)-like -endotoxin gene was highly expressed in both mycotized cadavers and fungal cultures, and was more or less distinct from its homologues in bacteria and other fungi.
CONCLUSION: Our findings provide the first global insight into various pathogenicity-related genes in the obligate aphid pathogen and may help to develop novel biocontrol strategy against aphid pests.
... Expressed sequence tag (EST) analysis of C. coronatus revealed chitinases, multiple subtilisins, trypsin, metalloprotease and aspartyl protease activities (Freimoser et al., 2003). The diversity of proteinases produced by C. coronatus evidences a plasticity of this fungus in exploiting a broad range of proteins of various origins (Freimoser et al., 2003). ...
... Expressed sequence tag (EST) analysis of C. coronatus revealed chitinases, multiple subtilisins, trypsin, metalloprotease and aspartyl protease activities (Freimoser et al., 2003). The diversity of proteinases produced by C. coronatus evidences a plasticity of this fungus in exploiting a broad range of proteins of various origins (Freimoser et al., 2003). The cocktail of enzymes released by C. coronatus into the culture medium efficiently degraded fly cuticles, which resulted in the accumulation of amino acids, N-glucosamine and FFAs as products of the hydrolysis of proteins, chitin and lipids, respectively. ...
Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus–cuticle interactions related to substrate utilization and resistance to host-specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell-free filtrate, for which activity of elastase, N-acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N-glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.
... Only a few studies have examined the gene expression of entomopathogenic fungi in natural conditions or on host-mimicking media. Examples of gene expression studies of entomophthoralean fungi include an expressed sequence tag (EST) library constructed for Conidiobolus coronatus grown on insect cuticle (Freimoser et al., 2003), and EST libraries constructed for two strains of Zoophthora radicans grown on culture media and then screened for pathogenicity related genes (Xu et al., 2009). The final stages of infection and transmission were the focus of a secretome study of a fungusaphid system in which genes from Pandora neoaphidis and Entomophthora planchoniana were analyzed by transposonassisted signal trapping (Grell et al., 2011). ...
... Subtilisin gene family expansion has been shown in fungi that utilize animal tissue as food, either as pathogens or saprobionts (Muszewska et al., 2011), and is hypothesized to be an important evolutionary adaptation in these fungi. In Entomophthoromycota, subtilisin secretion during host tissue utilization has been shown for C. coronatus, P. neoaphidis, and E. planchoniana (Freimoser et al., 2003;Grell et al., 2011). The ability to quickly respond to substrate related changes with production of different types of subtilisins in different stages of the infection process has been shown for M. anisopliae, an entomopathogenic fungus with a very high number of subtilisin genes in its genome compared to other fungi (Gao et al., 2011). ...
Pandora formicae is an obligate entomopathogenic fungus from the phylum Entomophthoromycota, known to infect only ants from the genus Formica. In the final stages of infection, the fungus induces the so-called summit disease syndrome, manipulating the host to climb up vegetation prior to death and fixing the dead cadaver to the surface, all to increase efficient spore dispersal. To investigate this fascinating pathogen-host interaction, we constructed interaction transcriptome libraries from two final infection stages from the material sampled in the field: (1) when the cadavers were fixed, but the fungus had not grown out through the cuticle and (2) when the fungus was growing out from host cadaver and producing spores. These phases mark the switch from within-host growth to reproduction on the host surface, after fungus outgrowth through host integument. In this first de novo transcriptome of an entomophthoralean fungus, we detected expression of many pathogenicity-related genes, including secreted hydrolytic enzymes and genes related to morphological reorganization and nutrition uptake. Differences in expression of genes in these two infection phases were compared and showed a switch in enzyme expression related to either cuticle breakdown or cell proliferation and cell wall remodeling, particularly in subtilisin-like serine protease and trypsin-like protease transcripts.
Copyright © 2015. Published by Elsevier Inc.
... C. coronatus infects susceptible insect hosts via direct cuticle penetration by invasive hyphae formed after the germination of the spores on the cuticle. Penetration of the host integument is achieved by the mechanical pressure imposed by growing hyphae and the enzymatic degradation of major cuticle components (proteins, chitin and lipids) by proteases, chitinases and lipases produced by the fungus 31,32,40 . Upon invasion of the host hemocoel, hyphae expand but do not infest internal organs due to rapid host death caused by toxic metabolites of the fungus which disorganize functioning of Malpighian tubules, incapacitate immune system and affect serotonin-regulating enzymes 8,[10][11][12][14][15][16]38 . ...
Mycoses are a global problem that affects humans and animals. In the present study, the entomopathogenic soil fungus Conidiobolus coronatus (Entomophthorales), infecting in tropics also humans, sheep and horses, was cultivated with the addition of insect cuticular compounds (CCs) previously detected in the cuticle of C. coronatus —resistant fly species (C10–C30 fatty alcohols, butyl oleate, butyl stearate, glycerol oleate, squalene, tocopherol acetate). Our findings indicate that CCs have diversified and complex effects on the growth and sporulation of C. coronatus and its ability to infect the larvae of Galleria mellonella (Lepidoptera). The CCs affected protein content and cuticle-degrading enzymes (CDEs) activity in the conidia. Some CCs inhibited fungal growth (0.1% C10), decreased sporulation (C12, C16, C24, C28, C30, butyl stearate, squalene), virulence (C12, C14, butyl oleate, butyl stearate) and protein content (C18). They also reduced conidial CDE activity: elastase (C24, butyl oleate, butyl stearate, squalene, tocopherol acetate), chitobiosidase (C12, C14, C20) and lipase (C12, C18, C26, squalene, tocopherol acetate). Several CCs enhanced sporulation (C14, C18, C22, C26, C30), virulence (C18, C26, squalene), conidial protein content (C16, C24, C30, squalene) and CDE activity: elastase (C10, C16, C18), NAGase (C16, C20), chitobiosidase (C16) and lipase (C10, C14, C16, C20, butyl oleate). Our findings indicate that C. coronatus colonies grown on media supplemented with CCs employ various compensation strategies: colonies grown with C16 alcohol demonstrated reduced sporulation but greater conidial protein accumulation and increased elastase, NAGase, chitobiosidase and lipase activity, thus preserving high virulence. Also, colonies supplemented with C18 alcohol demonstrated high virulence and enhanced sporulation and elastase activity but slightly decreased conidial protein content. CCs that inhibit the activity of lipases and proteases show promise in the fight against conidiobolomycosis.
... It has been reported that other fungi used for food production, such as Aspergillus oryzae, may produce antibacterial metabolites [38]. However, Zygomycetes are not capable of antibiotic production according to literature [39]. Results of the genome screening support this statement and antibiotics production is therefore not considered a safety concern for Fermotein. ...
Aim: Single-cell proteins (SCPs) are considered as innovative and sustainable alternatives to animal-based products. Fermotein is an innovative SCP obtained from fermentation of the filamentous fungus Rhizomucor pusillus. The toxicity, capability to produce secondary metabolites and allergenic potential of this fungus has never been assessed before. Like other filamentous fungi, there is a lack of information on this species to assess its safety for human consumption. The objective of the current study was to investigate the safety of Fermotein and its source Rhizomucor pusillus regarding toxicity, capability to produce secondary metabolites and allergenicity. In addition, possible contaminants were also examined. Methodology: The genome of Rhizomucor pusillus was sequenced and annotated in order to screen for production of common mycotoxins, antibiotic synthesis pathways, mucormycosis-related virulence factors and in silico potential cross-reactivity with known food allergens. The presence of mycotoxins and allergens were validated by laboratory analysis. The level of RNA, heavy metals and microbiological contaminants were also determined. Results: No mycotoxin production-related genes were identified in the genome of Rhizomucor pusillus nor were mycotoxins found in Fermotein. Six proteins present in Fermotein showed high homology with five known food allergens. No gene clusters were found that corresponded with antibiotic synthesis pathways. Although 10 proteins in the genome of Rhizomucor pusillus may represent mucormycosis-related virulence factors, no cases of mucormycosis after oral intake are reported. The level of heavy metals and microbiological contaminants were below legislative limits, whereas RNA content was 4.9 ± 0.2% of dry matter. Conclusion: No safety concerns were identified for Fermotein or its source Rhizomucor pusillus, except the potential for cross-reactivity with five known food allergens. This should be taken into account for communication with consumers. Information from the current study contributes to the body of evidence for determination of Qualified Presumption of Safety status of Rhizomucor pusillus.
... fungi, such as Metarhizium anisopliae, Beauveria bassiana and Purpureocillium lilacinum [35,[40][41][42] The mycelia of C. coronatus cultivated in vitro secrete a plethora of enzymes, however, the activities of fungal enzymes measured in vitro are not necessarily correlated with their importance in the infection process occurring in nature [15,[43][44][45][46]. The enzymatic cocktail released by C. coronatus mycelia degrades cuticle samples from susceptible insects far more effectively than those from resistant species and/or developmental stages [11,28,47]. ...
The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence.
... Kingdom Fungi provided all phyla with a predominance of endophytic Ascomycota and Basidiomycota occupying wheat seed followed by Incertae sedis, Chytridiomycota, and Zygomycota. Chytridiomycota and Zygomycota represent the earliest terrestrial divergences from ancestral aquatic fungi 48 . Typical environmental conditions, optimal for plant growth, dictate normal or Gaussian fungal distribution in the seed. ...
Illumina-MiSeq next-generation sequencing of ITS 5.8S rRNA gene demonstrated the transgenerational transmission of fungal seed-endophytes (mycobiome) across three consecutive wheat host generations under standard-control and drought conditions in the greenhouse. Drought-stressed plants experienced a positive shift in the seed mycobiome’s composition, moderated by the external acquisition of endophytic Penicillium (E+) at the seed level. Untreated (E−) and unstressed plants harbor a maximal fungal diversity of non-equilibrium ecological communities. While fungal composition in drought-stressed E− plants experienced important fluctuation, E+ plants maintained fungal ecological communities in phase equilibrium across generations. E+ plants hosted a relatively higher abundance of Ascomycota in the 2nd and 3rd seed generations of wheat, whereas higher abundance of Basidiomycota was detected in 1st generation seeds. The dynamic response of ecological communities to environmental stress is conducive to E+ plants’ active recruitment of endosymbiotic consortia in seeds, benefiting host stress resilience and phenotype. In contrast, E− plants showed an erratic distribution of detected OTUs with an increased occurrence of phytopathogens and diminished plant performance under stress. The present study gives insight into the understanding of the seed-mycobiome composition and dynamics with the potential to improve plant host traits in an adverse environment.
... The insecticidal activity of C. coronatus is attributed mainly to the presence of toxic metabolites, although it produces a broad range of enzymes, including proteinases, chitinases and lipases, which also play a crucial role in its pathogenicity (Prasertphon & Tanada, 1969;Boguś & Scheller, 2002;Freimoser et al., 2003;Boguś et al., 2007Boguś et al., , 2017bWieloch et al., 2011). Histopathological examination of G. mellonella larvae infected with C. coronatus does not reveal the presence of any nutrient depletion in dying insects (fat bodies were well preserved) or any damage to the vital internal organs of the host by fungal structures. ...
The most effective and important strategy in the insect immune response is based on cellular reactions incorporating haemocytes. The present study uses Galleria mellonella (Lepidoptera: Pyralidae) as a host to study the pathogenesis caused by the entomopthoralean fungus Conidiobolus coronatus (Entomophthorales). Five types of haemocytes with different morphologies and behaviour are observed in the haemolymph of G. mellonella: granulocytes (GRs), plasmatocytes (PLs), spherulocytes (SPs), oenocytes (OEs) and prohaemocytes (PRs). During in vitro cultivation, three morphological subtypes of PLs are distinguished: flattened PLs, sun‐like PLs and oval PLs. In fresh smears of haemolymph observed under phase‐contrast microscopy, only flattened PLs are identified. No morphological changes are observed between fresh smears and in vitro cultures for GR, OE, SP and PR. Haemocytes cultured in vitro form a cellular network composed of PLs and GRs. Changes in the numbers, morphology and behaviour of haemocytes induced by fungal infection are compared with those observed in normally‐developing untreated larvae. Infection results in a significant drop in the number of haemocyte types. Fresh smears of haemocytes from mycosed larvae reveal malformed OEs, vacuolized PLs and GRs, as well as PLs with apoptotic blebs. Haemocytes from mycosed larvae incubated in vitro look similar, with degranulated GRs and vacuolized PLs forming microaggregations, as well as deformed OEs; only the SPs remain unharmed. Fungal infection impairs the ability of haemocytes to attach and spread on the culture dish. The actin cytoskeleton of haemocytes from mycosed larvae appear disorganized.
... EST (expressed sequence tags) analysis confirms our finding that C. coronatus produces a chitinolytic complex to degrade cuticular components [27,28]. Lipolytic activity in C. coronatus cultures has also been reported [20,29]. St. Leger et al. [30] demonstrated that M. anisopliae produces proteases which hydrolyze the cuticles of Caliphora vomitoria and Manduca sexta. ...
The entomopathogenic fungus Conidiobolus coronatus produces enzymes that may hydrolyze the cuticle of Galleria mellonella. Of these enzymes, elastase activity was the highest: this figure being 24 times higher than NAGase activity 553 times higher than chitinase activity and 1844 times higher than lipase activity. The present work examines the differences in the hydrolysis of cuticles taken from larvae, pupae and adults (thorax and wings), by C. coronatus enzymes. The cuticles of the larvae and adult thorax were the most susceptible to digestion by proteases and lipases. Moreover, the maximum concentration of free N-glucosamine was in the hydrolysis of G. mellonella thorax. These differences in the digestion of the various types of cuticle may result from differences in their composition. GC-MS analysis of the cuticular fatty acids isolated from pupae of G. mellonella confirmed the presence of C 8:0, C 9:0, C 12:0, C 14:0, C 15:0, C 16:1, C 16:0, C 17:0, C 18:1, C 18:0, with C 16:0 and C 18:0 being present in the highest concentrations. Additional fatty acids were found in extracts from G. mellonella imagines: C 10:0, C 13:0, C 20:0 and C 20:1, with a considerable dominance of C 16:0 and C 18:1. In larvae, C 16:0 and C 18:1 predominated. Statistically significant differences in concentration (p≤0.05) were found between the larvae, pupae and imago for each fatty acid. The qualitative and quantitative differences in the fatty acid composition of G. mellonella cuticle occurring throughout normal development might be responsible for the varied efficiency of fungal enzymes in degrading larval, pupal and adult cuticles.
... Kingdom Fungi provided all phyla with a predominance of endophytic Ascomycota and Basidiomycota occupying wheat seed followed by Incertae sedis, Chytridiomycota, and Zygomycota. Chytridiomycota and Zygomycota represent the earliest terrestrial divergences from ancestral aquatic fungi 48 . Typical environmental conditions, optimal for plant growth, dictate normal or Gaussian fungal distribution in the seed. ...
... These molecules might promote taxis behaviors (e.g., in relation to temperature, humidity, sunlight, or gravity) or specific muscle-related functions such as wing or proboscis extension. Recent studies have shed some light on the molecular mechanisms of entomophthoralean pathogenesis [18,[21][22][23][24]. Two such mechanisms are (1) penetration of the host cuticle by subtilisin-and trypsin-like proteases and (2) protein and lipid uptake using lipases, serine proteases, and zinc-dependent metalloproteases. ...
... Once inside the host, these fungi grow while utilizing the host resources and ultimately kill and penetrate the host from within to produce new infective conidia for transmission (Roy et al. 2006;Vega et al. 2012; Fig. 1). The molecular mechanisms of these stages of host-pathogen interactions are in many cases not known, although several extracellular secreted peptidases including subtilisins and trypsin-like proteases, chitinases, glycoside hydrolases, lipases, glycosyltransferases and carbohydrate esterases involved in penetration of the insect cuticle and disruption of host membranes have been identified from several species within Entomophthoraceae (Freimoser et al. 2003;Hu & St. Leger 2004;Xu et al. 2006Xu et al. , 2009Grell et al. 2011;Małagocka et al. 2015). It is thus evident that hydrolytic enzymes contribute to fungal pathogenicity, but host-specific variation in enzyme-coding genes has so far not been observed within Entomophthoraceae (Xu et al. 2006). ...
Obligate parasites are under strong selection to increase exploitation of their host to survive while evading detection by host immune defenses. This has often led to elaborate pathogen adaptations and extreme host specificity. Specialization on one host, however, often incurs a trade-off influencing the capacity to infect alternate hosts. Here we investigate host adaptation in two morphologically indistinguishable and closely related obligate specialist insect-pathogenic fungi from the Phylum Entomophthoromycota, Entomophthora muscae sensu stricto and E. muscae sensu lato, pathogens of houseflies (Musca domestica) and cabbage flies (Delia radicum), respectively. We compared single nucleotide polymorphisms within and between these two E. muscae species using 12 RNAseq transcriptomes from five biological samples. All five isolates contained intra-isolate polymorphisms that segregate in 50:50 ratios, indicative of genetic duplication events or functional diploidy. Comparative analysis of dN/dS ratios between the multinucleate E. muscae s.str. and E. muscae s.l. revealed molecular signatures of positive selection in transcripts related to utilization of host lipids and the potential secretion of toxins that interfere with the host immune response. Phylogenetic comparison with the non-obligate generalist insect-pathogenic fungus Conidiobolus coronatus revealed a gene-family expansion of trehalase enzymes in E. muscae. The main sugar in insect hemolymph is trehalose, and efficient sugar utilization was probably important for the evolutionary transition to obligate insect pathogenicity in E. muscae. These results support the hypothesis that genetically based host specialisation in specialist pathogens evolves in response to the challenge of using resources and dealing with the immune system of different hosts.
... Conidiobolus coronatus is a member of the fungal order Entomophthorales (phylum Zygomyces), commonly known as insect destroyers, and is a potent entomopathogen (Boguś & Scheller, 2002;Domsch et al., 2007). Conidiobolus coronatus can probably attack any stressed insect and it is also an opportunistic pathogen of vertebrates (Freimoser et al., 2003). Histological examination of C. vicina larvae exposed to sporulating C. coronatus colonies confirms that the fungal conidia are unable to germinate on the fly cuticle, thus suggesting the presence of compounds inhibiting spore germination (Boguś et al., 2007). ...
The composition of cuticular and internal lipids in females of the cockroach Blatta orientalis L. exposed to the entomopathogenic fungus Conidiobolus coronatus is investigated. The compositions of the fatty acids, n-alkanes, alcohol, sterols and methyl esters in the lipids are chemically characterized. Although contact with virulent colonies of the fungus does not induce insect mortality, significant changes in the lipid profiles, both cuticular and internal, are found. The cuticular extracts of a control group of B. orientalis females contain 24 compounds varying in carbon chain length from C6 to C22. The main cuticular fatty acids identified are: C16:1, C16:0, C18:1 and C18:0. The cuticular lipids of B. orientalis females after exposure to C. coronatus contain only 14 free fatty acids from C8 to C20. The highest concentrations identified are C16:0, C18:2 and C18:1. Analysis by gas chromatography-mass spectrometry identifies the presence of a homologous series of n-alkanes containing from 25 to 31 carbon atoms. In the case of the insects after fungal exposure, the content of the n-alkanes in the cuticular lipid is two-fold higher compared with the controls. Of the cuticular lipids, 11 alcohols are found, ranging from C12:0 to C20:0. There is no presence of alcohols in the internal lipids of the control B. orientalis females and in all of the extracts from the B. orientalis females after fungal exposure. In the samples analyzed, the most common sterol is cholesterol. This is present in the cuticular lipids and the internal lipids of all of the insects sampled. The cuticular and internal lipids of females contain five fatty acid methyl esters, ranging in size from C15 to C19.
... A patogenia da conidiobolomicose não está esclarecida. Genes associados à virulência foram estudados na infecção do fungo em insetos (Freimoser et al. 2003) e a sua capacidade de termo resistência em diferentes temperaturas (Silva et al. 2012, Godoy et al. 2014. ...
The study of sheep conidiobolomycosis has been carried out in its clinical, epidemiological, pathological and molecular aspects. Information, however, about the host immune response in infection Conidiobolus lamprauges is absent. This study aimed to identify immunoreactive proteins that may play an important role in the immune response of sheep naturally infected by C. lamprauges. For protein and immunological characterization, C. lamprauges (strain FIOCRUZ-INCQS 40316) isolated from a sheep with clinical signs of conidiobolomycosis in the MT state and five sera samples of naturally infected sheep were used. The presence of IgG antibody was observed in all patients with reagent titers in dilutions up to 1:1600. In immunoblot technique, the antigenic profile against infected sheep sera showed twelve reactive bands with molecular weights ranging from 35 to 198 kDa. Among them, the 198 kDa protein was reactive against sera from three sheep and the 53 kDa showed increased intensity compared to other bands probably being immunodominant. Healthy animal serum samples showed no reactivity demonstrating the specificity of the technique. The presence of antigenic proteins of C. lamprauges and specific IgG in sheep sera observed in this study may assist in the development of early diagnostic methods and the use of protein as candidate vaccines for the control and prevention of infection in animals and human.
... A patogenia da conidiobolomicose não está esclarecida. Genes associados à virulência foram estudados na infecção do fungo em insetos (Freimoser et al. 2003) e a sua capacidade de termo resistência em diferentes temperaturas (Silva et al. 2012, Godoy et al. 2014. ...
O estudo de conidiobolomicose ovina tem sido realizado nos seus aspectos clínicos, epidemiológicos, patológicos e moleculares. Informações, entretanto, sobre a resposta imune do hospedeiro na infecção por Conidiobolus lamprauges são inexistentes. Este estudo teve por objetivo a identificação de proteínas imunorreativas que possam desempenhar papel importante na resposta imune de ovinos naturalmente infectados por C. lamprauges. Para a caracterização protéica e imunológica foi utilizada a cepa de C. lamprauges (FIOCRUZ-INCQS 40316) isolada de ovino com sinais clínicos de conidiobolomicose no Estado do MT e cinco amostras de soro de ovinos infectados naturalmente pelo fungo. A presença de anticorpos IgG foi observada em todos os animais doentes com títulos reagentes em diluições de até 1:1.600. Na técnica do immunoblot, o perfil antigênico frente aos soros ovinos com a doença apresentou doze bandas reativas, com massas moleculares variando de 35 a 198 kDa. Dentre estas, a proteína de 198 kDa foi reativa em 3 soros de ovinos e a de 53 kDa apresentou a maior intensidade comparativamente com outras bandas, sendo provavelmente imunodominante. Amostras de soro de animais sadios não apresentaram reatividade demostrando a especificidade da técnica. A presença de proteínas antigênicas de C. lamprauges e IgG específicos em soros de ovinos observados no presente trabalho poderá auxiliar no desenvolvimento de métodos de diagnóstico precoces e na utilização de proteínas candidatas a vacinas para o controle e prevenção da infecção em animais e humanos.
... However, when M. anisopliae was transformed to overexpress its native chitinase, the pathogenicity towards the tobacco hornworm was unaltered, suggesting that wild type levels of chitinase are not limiting for cuticle penetration (Screen et al., 2001). Another fungal species, Conidiobolus coronatus, also produces both endo-and exo-acting chitinolytic enzymes during growth on insect cuticle (Freimoser et al., 2003b). Apparently, both M. anisopliae and C. coronatus produce a chitinolytic enzyme system to degrade cuticular components. ...
This chapter highlights some of the recent and important findings obtained from studies conducted on the synthesis, structure, physical state, modification, organization, and degradation of chitin in insect tissues, as well as the interplay of chitin with chitin-binding proteins, the regulation of genes responsible for chitin metabolism, and, finally, the targeting of chitin metabolism for insect-control purposes. Chitin is the major polysaccharide present in insects and many other invertebrates as well as in several microbes, including fungi. It serves as the skeletal polysaccharide of several animal phyla, such as the Arthropoda, Annelida, Molluska, and Coelenterata. In several groups of fungi, chitin replaces cellulose as the structural polysaccharide. In insects, it is found in the body wall or cuticle, gut lining or peritrophic matrix (PM), salivary gland, trachea, eggshells, and muscle attachment points. In the course of evolution, insects have made excellent use of the rigidity and chemical stability of the polymeric chitin to assemble both hard and soft extracellular structures such as the cuticle (exoskeleton) and PM respectively, both of which enable insects to be protected from the environment while allowing for growth, mobility, respiration, and communication. All of these structures are primarily composites of chitin fibers and proteins with varying degrees of hydration and trace materials distributed along the structures.
... Expression of such genes involved in general metabolic pathways coupled to antioxidants and host-targeting enzymes appeared similar among B. bassiana and C. coronatus. Among the genes expressed in C. coronatus are the antioxidants c-glutamylcysteine synthetase and gluthatione reductase, subtilisin type hydrolases including metalloprotease, trypsin protease and aspartyl protease and antimicrobials similar to cytochrome p450 of tobacco and cephalosporin (Freimoser et al., 2003b). As indicated from our EST analysis some similar metabolic pathways are also present in B. bassiana. ...
The coffee berry borer (CBB; Hypothenemus hampei) is a major pest of coffee responsible for significant crop losses worldwide. The entomopathogenic fungus Beauveria bassiana represents a natural means of controlling this insect pest; however, little is known concerning the molecular determinants that contribute to the virulence of the fungus towards the CBB. In order to examine genes involved in insect virulence, two expressed sequence tag (EST) libraries, representing germinating conidia and growing hyphae/mycelia of B. bassiana cells grown on cuticular extracts of the CBB were constructed and analysed. In total, 4186 cDNA transcripts were obtained, which included 2141 from the cuticle-germinated conidia and 2045 from the cuticle-grown mycelium libraries, respectively. The average sequence length obtained was 470 bp and transcript assembly resulted in a set of 1271 and 1305 unique gene sequences for the conidial and mycelia libraries, respectively. Around 50 % of the sequences in each library could be annotated by gene ontology terms. An analysis of the two generated libraries as well as a previously reported EST library of B. bassiana grown on chitin was performed. Between the cuticle-germinated conidia and the cuticle-grown mycelia libraries, 322 unique gene sequences were shared, of which 90 % could be annotated, leaving 949 unique cuticle-germinated conidial genes and 983 unique growing hyphae/mycelia genes of which around 65 % were annotated. ESTs shared between the libraries indicated a basic response pattern for B. bassiana against H. hampei, which included genes implicated in pathogenicity. The expression profiles of four genes were evaluated with a cyclophilin, an alkaline-like serine protease and a mitogen-activated protein kinase (MAPK), showing elevated expression during initial phases of infection, i.e. conidia germinating on insect extracts. These data provide clues and gene candidates for further exploration concerning the biology and molecular mechanisms of entomopathogenicity by this fungus.
... PR1A and CDEP-1 (96% identical to bassiasin I) have been shown to be pathogenicity determinants of M. anisopliae and B. bassiana, respectively (Charnley, 2003; St. Leger et al., 1996a; Zhang et al., 2008). A number of clones deduced to encode cuticle-degrading enzymes were identified in two EST studies with cultures of the entomophthoralean pathogens Conidiobolus coronatus and Zoophthora radicans, grown on insect cuticle and isolated from lepidopteran larvae, respectively (Freimoser et al., 2003; Xu et al., 2009). Our findings were substantiated by the identification of homologous clones in these libraries (Table 2, EST hits). ...
The discovery of novel secretome proteins can add to our understanding of host-pathogen interactions. Here we report a rich diversity of secreted proteins from the interaction between grain aphids (host, insect order Hemiptera) and fungi of the order Entomophthorales (insect pathogens). The proteins were identified using a unique method unbiased by known sequences or functions to screen a cDNA library constructed directly from field-sampled material. We show for the first time that fungi from the genera Pandora and Entomophthora are armed with a battery of hydrolytic enzymes for penetrating the host cuticle. This enables both access to the hemolymph and exit for sporulation. Further, they secrete enzymes, most notably a number of lipases, for digestion of easily accessible high-energy compounds in the hemolymph. In contrast, we identified only few host genes potentially involved in the interaction, indicating that aphids respond only weakly to the pathogens. These results support recent findings that aphids have a reduced immune repertoire.
... The secretome has been defined as being the combination of native secreted proteins and the cell machinery involved in their secretion [285]. A defining characteristic of plant pathogenic fungi is the secretion of a large number of degradative enzymes and other proteins, which have diverse functions in nutrient acquisition, substrate colonization, and ecological interactions286287288. Several extracellular fungal enzymes, such as polygalacturonase, pectate lyase, xylanase, and lipase, have been shown or postulated to be required for virulence in at least one host-pathogen interaction289290291292293294295. ...
Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection.
... In other basal members of the kingdom (Fig. 3), wc-1 homologs are evident in the genomes of two chytrids A. macrogynus (phylum Blastocladiomycota) and Spizellomyces punctatus (phylum Chytridiomycota). A potential homolog is also present in Conidiobolus conatus (subphylum Entomophthoromycotina) represented by an expressed sequence tag (Freimoser et al., 2003), thus implicating the gene in a lineage in addition to the Mucormycotina of what was previously the zygomycetes. ...
Virtually all organisms exposed to light are capable of sensing this environmental signal. In recent years the photoreceptors that mediate the ability of fungi to "see" have been identified in diverse species, and increasingly characterized. The small sizes of fungal genomes and ease in genetic and molecular biology manipulations make this kingdom ideal amongst the eukaryotes for understanding photosensing. The most widespread and conserved photosensory protein in the fungi is White collar 1 (WC-1), a flavin-binding photoreceptor that functions with WC-2 as a transcription factor complex. Other photosensory proteins in fungi include opsins, phytochromes and cryptochromes whose roles in fungal photobiology are not fully resolved and their distribution in the fungi requires further taxon sampling. Additional unknown photoreceptors await discovery. This review discusses the effects of light on fungi and the evolutionary processes that may have shaped the ability of species to sense and respond to this signal.
Fungi have emerged as a significant reservoir of potent antioxidant compounds among numerous natural alternative sources of bioactive metabolites. Such scarcely studied microorganisms are prolific producers of novel metabolites and are capable of producing compounds, which are isolated exclusively from Plantae. A broad variety of compounds were known as natural antioxidants, including nucleobases, polyketides, terpenoids, flavonoids, coumarins, xanthones, semiquinones, peptides, and phenolic acids. Some of these free radical scavengers have shown wide use as potential food preservatives, nutraceuticals, antibacterial, anticancer, and antifungal agents in the food and pharmaceutical industries. This chapter aims to spot light fungal antioxidant jewelry and discuss it through different biological assays.
Background
Insect-killing fungal species, Beauveria bassiana, is as an environment-friendly pest management tool, and many isolates are on the track of industrialization. However, some of B. bassiana isolates show similar morphology and virulence against insect pests, and so it is hard to differentiate them. Herein we used two patented isolates, ERL836 and JEF-007, and investigated their virulence against western flower thrips, Frankliniella occidentalis, and further analyzed genome structures and transcriptional responses when interacting with cuticles of thrips to see possible differences on the initial step of fungal infection.
Results
The two isolates showed no significant differences in fungal growth, conidial production, and virulence against thrips, and they were structurally similar in genome. But, in transcription level, ERL836 appeared to infect thrips easily, while JEF-007 appeared to have more difficulty. In the GO analysis of ERL836 DEGs (differentially expressed genes), the number of up-regulated genes was much larger than that of down-regulated genes, when compared to JEF-007 DEGs (more genes down-regulated). Interestingly, in the enrichment analysis using shared DEGs between two infecting isolates, plasma membrane-mediated transporter activity and fatty acid degradation pathway including cytochrome P450 were more active in infecting ERL836.
Conclusion
The two B. bassiana isolates had similar morphology and virulence as well as genome structure, but in transcription level they differently interacted with the cuticle of western flower thrips. This comparative approach using shared DEG analysis could be easily applied to characterize the difference of the two B. bassiana isolates, JEF-007 and ERL836.
Broad host range insect pathogenic fungi penetrate through the host cuticle, necessitating an ability to confront and overcome surface lipids and other molecules that often include antimicrobial compounds. In this context, induction of lipid assimilatory pathways by exogenous substrates is crucial for successful infection to occur, and lipid growth substrates can have significant effects on the virulence of fungal infectious propagules, e.g. conidia. The production of lipases is a critical part of the cuticle-degrading repertoire and pathways involved in triglyceride metabolism and phospholipid homeostasis have been shown to contribute to host invasion. Mobilization of endogenous lipid stores via the activities of the caleosin and perilipin lipid storage-turnover proteins, have been linked to diverse processes including formation of penetration structures, e.g. germ tubes and appressoria, spore properties and dispersal, and the ability to respond to lipid growth substrates and virulence. Here, we summarize recent advances in our understanding of lipid assimilation and mobilization pathways in the ability of entomogenous fungi to infect and use host substrates. Host surface and internal lipids can alternatively act as antifungal barriers, inducers of pathogenesis-related pathways, and/or as fungal growth substrates. Lipids and lipid assimilation can be considered as forming a co-evolutionary web between in insect host and entomogenous fungi.
Coronatin-2, a 14.5 kDa protein, was isolated from culture filtrates of the entomopathogenic fungus Conidiobolus coronatus (Costantin) Batko (Entomophthoramycota: Entomophthorales). After LC–MS/MS (liquid chromatography tandem mass spectrometry) analysis of the tryptic peptide digest of coronatin-2 and a mass spectra database search no orthologs of this protein could be found in fungi. The highest homology was observed to the partial translation elongation factor 1a from Sphaerosporium equinum (protein sequence coverage, 21%), with only one peptide sequence, suggesting that coronatin-2 is a novel fungal protein that has not yet been described. In contrast to coronatin-1, an insecticidal 36 kDa protein, which shows both elastolytic and chitinolytic activity, coronatin-2 showed no enzymatic activity. Addition of coronatin-2 into cultures of hemocytes taken from larvae of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), resulted in progressive disintegration of nets formed by granulocytes and plasmatocytes due to rapid degranulation of granulocytes, extensive vacuolization of plasmatocytes accompanied by cytoplasm expulsion, and cell disintegration. Spherulocytes remained intact, while oenocytes rapidly disintegrated. Coronatin-2 produced 80% mortality when injected into G. mellonella at 5 µg larva −1 . Further study is warranted to determine the relevance of the acute toxicity of coronatin-2 and its effects on hemocytes in vitro to virulence of C. coronatus against its hosts.
The order Entomophthorales, which formerly contained c.280 species, has recently been recognized as a separate phylum, Entomophthoromycota, consisting of three recognized classes and six families. Many genera in this group contain obligate insect-pathogenic species with narrow host ranges, capable of producing epizootics in natural insect populations. Available sequence information from the phylum Entomophthoromycota can be classified into three main categories: first, partial gene regions (exons+introns) used for phylogenetic inference; second, protein coding gene regions obtained using degenerate primers, expressed sequence tag methodology or de novo transcriptome sequencing with molecular function inferred by homology analysis; and third, primarily forthcoming whole-genome sequencing data sets. Here we summarize the current genetic resources for Entomophthoromycota and identify research areas that are likely to be significantly advanced from the availability of new whole-genome resources.
Topics not Covered, or Receiving Secondary Emphasis Biosafety Considerations: Before You Begin Work with Pathogenic Fungi… Fungi Defined: Their Ecologic Niche Medical Mycology A Brief History of Medical Mycology Rationale for Fungal Identification Sporulation Dimorphism Sex in Fungi Classification of Mycoses Based on the Primary Site of Pathology Taxonomy/Classification: Kingdom Fungi General Composition of the Fungal Cell Primary Pathogens Endemic Versus Worldwide Presence Opportunistic Fungal Pathogens Determinants of Pathogenicity General References in Medical Mycology Selected References for Introduction to Fundamental Medical Mycology Websites Cited Questions
Sustainable agricultural systems must be adopted to increase the food and fiber production keeping in view of human health and increase in population; the number of undernourished has increased to almost 20% (The state of food insecurity in the world economic crises – impacts and lessons learned. In Food and Agriculture Organization of the United Nations, Rome). Insect pests have been causing serious damage in the fields and stored grains and their products. Interventions is required to limit the losses, therefore, synthetic insecticides have played a significant role in their management for more than 60 years. Indiscriminate use of insecticides have left undesirable residues in the environment, which are toxic to human beings and non target organisms as well as insects have developed resistance against them and resurgence of pests that lead to find a suitable, sustainable and efficient method of management. Microorganisms: bacteria, viruses, fungi and protozoa form the most abundant and diverse groups, which offer a vast resource for exploitation to use in the management program. Bacillus thuringiensis is a gram positive, occurs in soil and ubiquitous in distribution. It produces parasporal crystalline body which contains one or more cry proteins that can be toxic to a number of insects. cry proteins are encoded by cry genes and 200 of them are identified. Similarly, a number of insect pests are also vulnerable to viral diseases. Nuclear Polyhedrosis and Granulosis Viruses are commonly used against the Lepidoptera. They are highly species specific and safer to human beings. Fungi, often act as important natural control agents that limit the insect population. Promising results are obtained by Beauveria bassiana and Metarhizium anisopliae against many insect pests. B. bassiana grows naturally in soil throughout the world and causes white muscardine disease. Therefore, intensive work is required to improve the efficacy of microbial insecticides through molecular biology and genetic engineering to enhance their role in the insect management for better food security.
Al igual que la genómica y la transcriptómica, la proteómica ha evolucionado para incorporar
técnicas de alto rendimiento y protocolos que permiten un análisis más rápido de un gran número
de proteinas [24, 25]. Así la proteómica constituye una herramienta eficaz para proporcionar
información importante sobre la patogenicidad y los factores de virulencia relacionados con
ésta, a fin de abrir nuevas posibilidades para el diagnóstico de las enfermedades, y desarrollar
estrategias de protección de cultivos eficientes y que sean además respetuosas con el medio
ambiente [1921]. A diferencia de otros sistemas biológicos, como en las levaduras [26] y en
humanos [27], el potencial de la proteómica en investigación de hongos patógenos de plantas
está lejos de ser plenamente explotado, como pone de manifiesto el bajo número de dichas
especies bajo investigación con dicha técnica, la baja cobertura del proteoma de las especies
investigadas, y el uso casi exclusivo de las técnicas clásicas de primera generación que se
basan en electroforesis bidimensional (2-DE) acoplada a la espectrometría de masas (MS) (revisado
recientemente en [21]). Aún así, todo esto, junto con la posibilidad de obtener mutantes
‘knockout’ o de silenciar genes [18], está contribuyendo a la compresión de la patogenicidad,
virulencia y agresividad de los hongos fitopatógenos.
The insect pathogenic plant root symbiont Metarhizium experiences many situations that restrict its growth whether living in host insects or on plant roots. These include a range of physical, chemical and biological effects involving UV and extremes of temperature, pH, nutrient availability, toxic metals and other pollutants, and insect host defenses such as production of reactive oxygen species. Aside virulence, the major impediment to reliable pest control with Metarhizium is its sensitivity to UV and temperature extremes. However, increased levels of stress tolerance can be engineered into Metarhizium quite simply by reprogramming the expression of single downstream endogenous genes. For example, overexpression of RNA-binding proteins resulted in Metarhizium with increased tolerance to cold stress, overexpression of photolyase increased tolerance to UV, and increased expression of heat shock protein 25 improved tolerance to several stress conditions, including heat, and osmotic pressure. Conversely, disruption of these genes greatly reduced persistence, and could provide genetic containment for genetically engineered hypervirulent strains.
Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption
by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence of serinocyclins,
cyclic heptapeptides, in the extracts of conidia of control strains, whereas the compounds were undetectable in ΔManps1 mutants treated identically or in other developmental stages, suggesting that MaNPS1 encodes a serinocyclin synthetase. Production of the cyclic depsipeptide destruxins, M. anisopliae metabolites also predicted to be synthesized by an NPS, was similar in ΔManps1 mutant and control strains, indicating that MaNPS1 does not contribute to destruxin biosynthesis. Surprisingly, a MaNPS1 fragment detected DNA polymorphisms that correlated with relative destruxin levels produced in vitro, and MaNPS1 was expressed concurrently with in vitro destruxin production. ΔManps1 mutants exhibited in vitro development and responses to external stresses comparable to control strains. No detectable differences
in pathogenicity of the ΔManps1 mutants were observed in bioassays against beet armyworm and Colorado potato beetle in comparison to control strains. This
is the first report of targeted disruption of a secondary metabolite gene in M. anisopliae, which revealed a novel cyclic peptide spore factor.
Among the major microbial taxa, fungi are surpassed only by actinomycetes as a source of therapeutic agents, especially antibiotics. It has been argued that there is a higher likelihood of discovering producers of novel antibiotics among parasitic microorganisms because of the unique adaptive roles adduced to secondary metabolites during the infection, both in overcoming host defenses and in competing with other microorganisms for host resources. The extensive polyketide synthase sequence data available for fungi now makes molecular screening for novel bioactive polyketides a viable alternative to traditional screening approaches. The development, application, and utility of one such screening approach to predict chemistries in insect pathogenic fungi are described herein.
Advances in the application of microbial-based technology in insect pest management assist us to counter problems created by the application of chemical pesticides. These are mainly strong environmental effects, resistance development and high costs. Among the microbial pesticides, fungal pesticides are now preferred as they are target specific, ecofriendly, lacking in toxic residue and are economical. Being numerous with great diversification, entomopathogenic fungi therefore have great potential to control a large variety of insect pests. Fungi are applied directly in form of spores, mycelia or blastospores or by their metabolites (mycotoxins). Both approaches have very promising roles in insect pest management. However, there are three main obstacles in the development of fungal pesticides: (i) scant production of mycotoxins; (ii) carcinogenic mycotoxicosis in non-target organisms; and (iii) slow effectiveness. Therefore, to eliminate these problems, attention has recently been paid to a synergistic approach to combating insecticide resistance. Next to synergism, genetic manipulation is also used to enhance the pathogenicity and virulence of fungal insecticides. However, the key risk associated with the release of recombinant microorganisms into the environment is that the novel organism may have unforeseen undesirable characteristics. Therefore, the introduction of synergists in pest control could have great benefit both economically and ecologically. An ideal synergistic approach is the mixing of more than two accelerating components together, i.e. tripartite or multiple synergism to enhance effectiveness. Thus, synergistic approaches have a bright future and require further research and financial support.
EST (expressed sequence tag) technology has long been used for gene discovery. As more and more EST data have become publicly available, the usage of ESTs has expanded to other areas, such as in silico genetic marker discovery, in silico gene discovery, construction of gene models, alternative splicing prediction, genome annotation, expression profiling, and comparative genomics. In comparison with whole genome sequencing, EST technology is simpler and less costly, especially in the case of large genomes. Moreover, since ESTs represent “the expressed parts” of genomes, they are more immediately informative about the transcriptomes. On the other hand, ESTs are not suitable for the studies related to “the control parts” of genomes, such as promoters and transcription enhancing/inhibiting elements. In addition, information for rarely expressed genes is also difficult to mine from EST data. EST data mining requires bioinformatics resources such as databases, data retrieving tools and analysis algorithms. Bioinformatics tools are also required to deal with EST errors and contaminations. Many of these tools are freely available to the academic community. In this chapter, EST data resources, tools used for processing and mining EST data, and applications of ESTs in genomics, particularly, fungal genomics, are reviewed.
Entomopathogenic fungi (EPF) have become a significant force in shaping the larger context of insecticides within contemporary
insect pest management schemes (Lord 2005; Roy et al., 2006; Khachatourians 2008). Needless to say, as mycologists we need
the perspective and understanding to explain the diversity of EPF and their spatial and temporal distribution within the insect
ecosystem. In the past decade, the accelerated focus of research and scholarly studies has generated two perspectives: (a)
the molecular biology, genomics and proteomics of EPF, and (b) the practical use of EPF in insect pest management schemes.
Their value therefore is two-fold, first in the study of microbial pathogenicity and second in their application to the microbial
control of phytophagous insects as much as biting and hematophagous insect pest populations. Altogether some 90 genera and
700 species are involved with entomopathogenicity, only a few members of the Entomophthorales and Hyphomycetes have been well
studied. In the past decade, major new developments in the realm of application of the knowledge of EPF to insect pest management
have been realized (Khachatourians 1996). New developments in genomic and molecular research and serious interest in commercialization
of EPF for pest control have become the new drivers of understanding in the field, challenges that were forecasted to meet
the promise of new biotechnology (Khachatourians 1986). With such knowledge, physiological manipulations, isolation of mutants
with enhanced virulence, and construction of environmentally safe strains with limited persistence should be possible within
the near future. This chapter primarily reviews the literature since 1995 on the biochemistry and molecular biology of EPF
and their involvement in the disease of insects. Additional sources of information can be followed from Table 3.1.
Pathogenic fungi are important natural regulators of insect populations. However, many attempts to harness their potential for pest control have met with comparatively minor commercial success. Studies on mechanisms of pathogenesis have yet to contribute to the development of more efficient mycoinsecticides, but new insights into the pathogenic process are laying the groundwork. Significant progress has been made in particular in understanding enzymes involved with the penetration of host cuticle and the role of insecticidal toxins. Insect cuticle comprises up to 70% protein and it is not surprising that extracellular fungal proteases appear to be particularly important in the penetration process. Subtilisins, chymotrypsins, trypsins and metalloproteases, usually with multiple isoforms of each, provide an impressive backed-up arsenal. Pathogenic fungi produce a wide variety of toxic metabolites, which vary from low molecular weight products of secondary metabolism to complex cyclic peptides and proteolytic enzymes. Comparatively few compounds have been found in diseased insects, in quantities sufficient to account for symptoms of mycosis. An exception, a family of cyclic peptides called the destruxins, are dealt with in detail. The potential for synergy between toxins is explored also.
Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic metalloproteases and serine proteases have also been examined. The sites of protease toxic activity range from the insect midgut to the hemocoel (body cavity) to the cuticle. This review discusses these insecticidal proteases along with their evaluation and use as potential pesticides.
Entomopathogenic fungi are important natural regulatory factors of insect populations and have potential as biological control agents of insect pests. The cosmopolitan soil fungus Conidiobolus coronatus (Entomopthorales) easily attacks Galleria mellonella (Lepidoptera) larvae. Prompt death of invaded insects is attributed to the action of toxic metabolites released by the invader. Effect of fungal metabolites on hemocytes, insect blood cells involved in innate defense response, remains underexplored to date.
An enduring theme in pathogenic microbiology is poor understanding of the mechanisms of host specificity. Metarhizium is a cosmopolitan genus of invertebrate pathogens that contains generalist species with broad host ranges such as M. robertsii (formerly known as M. anisopliae var. anisopliae) as well as specialists such as the acridid-specific grasshopper pathogen M. acridum. During growth on caterpillar (Manduca sexta) cuticle, M. robertsii up-regulates a gene (Mest1) that is absent in M. acridum and most other fungi. Disrupting M. robertsii Mest1 reduced virulence and overexpression increased virulence to caterpillars (Galleria mellonella and M. sexta), while virulence to grasshoppers (Melanoplus femurrubrum) was unaffected. When Mest1 was transferred to M. acridum under control of its native M. robertsii promoter, the transformants killed and colonized caterpillars in a similar fashion to M. robertsii. MEST1 localized exclusively to lipid droplets in M. robertsii conidia and infection structures was up-regulated during nutrient deprivation and had esterase activity against lipids with short chain fatty acids. The mobilization of stored lipids was delayed in the Mest1 disruptant mutant. Overall, our results suggest that expression of Mest1 allows rapid hydrolysis of stored lipids, and promotes germination and infection structure formation by M. robertsii during nutrient deprivation and invasion, while Mest1 expression in M. acridum broadens its host range by bypassing the regulatory signals found on natural hosts that trigger the mobilization of endogenous nutrient reserves. This study suggests that speciation in an insect pathogen could potentially be driven by host shifts resulting from changes in a single gene.
Metarhizium anisopliae and Beauveria bassiana are ubiquitous insect pathogens and possible plant symbionts, as some strains are endophytic or colonize the rhizosphere. We evaluated 11 strains of M. anisopliae and B. bassiana, and two soil saprophytes (the non-rhizospheric Aspergillus niger and the rhizosphere-competent Trichoderma harzianum) for their ability to germinate in bean root exudates (REs). Our results showed that some generalist strains of M. anisopliae were as good at germinating in RE as T. harzianum, although germination rates of the specialized acridid pathogen Metarhizium acridum and the B. bassiana strains were significantly lower. At RE concentrations of <1 mg ml(-1), M. anisopliae strain ARSEF 2575 showed higher germination rates than T. harzianum. Microarrays showed that strain 2575 upregulated 29 genes over a 12 h period in RE. A similar number of genes (21) were downregulated. Upregulated genes were involved in carbohydrate metabolism, lipid metabolism, cofactors and vitamins, energy metabolism, proteolysis, extracellular matrix/cell wall proteins, transport proteins, DNA synthesis, the sexual cycle and stress response. However, 41.3% of the upregulated genes were hypothetical or orphan sequences, indicating that many previously uncharacterized genes have functions related to saprophytic survival. Genes upregulated in response to RE included the subtilisin Pr1A, which is also involved in pathogenicity to insects. However, the upregulated Mad2 adhesin specifically mediates adhesion to plant surfaces, demonstrating that M. anisopliae has genes for rhizosphere competence that are induced by RE.
Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.
Covering: up to the end of February 2010
Eighteen fatty acids identified in the cuticle of three insect species representing differing susceptibilities to C. coronatus infection, were tested for effects on the in vitro growth and pathogenicity of the parasitic fungus. At all applied concentrations (0.1-0.0001% w/v) growth was inhibited by C(16:0), C(16:1), C(18:0), C(18:1), C(18:2), C(18:3), C(20:0) and C(20:1). At high concentrations spore germination was inhibited by C(7:0), C(8:0), C(9:0), C(10:0), C(12:0), C(18:2) and C(18:3) and hyphal growth was merely retarded by C(5:0), C(6:0), C(6:2), C(14:0), C(16:0), C(16:1), C(18:0,) C(18:1), C(20:0) and C(20:1). The presence of C(15:0) at the 0.1% concentration stimulated growth of C. coronatus. Sporulation was inhibited by all concentrations of C(16:0) and C(18-20) fatty acids. Low concentrations of C(5:0), C(6:0), C(6:2) and C(7:0) enhanced sporulation. Fatty acids C(5-12) as well as C(18:3), C(20:0) and C(20:1) decreased the ability of fungal colonies to infect G. mellonella while C(16:1) elevated it thus suggesting that C(16:1) may stimulate production of enzymes involved in the host invasion. Toxicity of metabolites released into incubation medium decreased with varying degrees in the presence of C(6:0), C(6:2,) C(7:0), C(9:0), C(12:0), C(16:1), C(18:2), C(18:3), C(20:0) and C(20:1); other fatty acids had no effect. Further work is needed to analyse the effects of exogenous fatty acids on the C. coronatus enzymes implicated in fungal pathogenicity as well as on the production of insecticidal metabolites.
Catalases and peroxidases are the most important enzymes that degrade hydrogen peroxide into water and oxygen. These enzymes and superoxide dismutase are the first lines of cell defense against reactive oxygen species. Metarhizium anisopliae displays an increase in catalase-peroxidase activity during germination and growth. To determine the importance of catalase during the invasion process of M. anisopliae, we isolated the cat1 gene. cat1 cDNA expression in Escherichia coli and the subsequent purification of the protein confirmed that the cat1 gene codes for a monofunctional catalase. Expression analysis of this gene by RT-PCR from RNA isolated from fungus grown in liquid cultures showed a decrease in the expression level of the cat1 gene during germination and an increase during mycelium growth. The expression of this gene in the fungus during the infection process of the larvae of Plutella xylostella also showed a significant increase during invasive growth. Transgenic strains overexpressing the cat1 gene had twice the catalase activity of the wild-type strain. This increase in catalase activity was accompanied by a higher level of resistance to exogenous hydrogen peroxide and a reduction in the germination time. This improvement was also observed during the infection of P. xylostella larvae. M. anisopliae transgenic strains overexpressing the cat1 gene grew and spread faster in the soft tissue of the insect, reducing the time to death of the insect by 25% and the dose required to kill 50% of the population 14-fold.
Zygomycosis is an important granulomatous disease that affects humans and animals, particularly sheep in tropical regions. Rhinofacial and nasopharyngeal zygomycosis were described in sheep in association with Conidiobolus spp. The present study characterized 5 samples of Conidiobolus isolated from 3 herds with clinical disease in Mato Grosso State, Brazil. The clinical and pathological findings were similar to nasopharyngeal zygomycosis. Based on morphological features, isolates were classified as Conidiobolus spp., and molecular phylogenetic analyses based on 18S ribosomal DNA grouped all isolates in a Conidiobolus lamprauges cluster. The current report describes the molecular characterization of ovine nasopharyngeal zygomycosis associated with C. lamprauges.
We have isolated and characterized cDNA and genomic DNA clones encoding the 70-kDa heat-shock protein (Hsp70) from the aquatic fungus Blastocladiella emersonii (Be). Nucleotide (nt) sequence analysis predicts an acidic protein containing 650 amino acids, with a calculated molecular mass of 70.8 kDa. The Be hsp70 gene is induced by heat shock (HS), as well as during sporulation of the fungus, and its coding region is interrupted by a single intron. All the evidence seems to indicate that this is the only hsp70 in Be. S1 nuclease protection assays revealed that splicing of the hsp70 intron is highly thermoresistant; at the lethal temperature of 42°C, only 30% of the hsp70 mRNAs have not been processed. A single transcription start point (tsp), localized about 30 nt downstream from a putative TATA box, was determined both during HS and at normal temperatures. The promoter region presented several NGAAN repeats (where N is any nucleotide) characteristic of HS elements, as well as putative binding sites for ATF, Spl and two metal-responsive elements.
The pvdA gene, encoding the enzyme L-ornithine N5-oxygenase, catalyzes a key step of the pyoverdin biosynthetic pathway in Pseudomonas aeruginosa. Expression studies with a promoter probe vector made it possible to identify three tightly iron-regulated promoter regions in the 5.9-kb DNA fragment upstream of pvdA. The promoter governing pvdA expression was located within the 154-bp sequence upstream of the pvdA translation start site. RNA analysis showed that expression of PvdA is iron regulated at the transcriptional level. Primer extension and S1 mapping experiments revealed two 5'termini of the pvdA transcript, 68 bp (T1) and 43 bp (T2) 5' of the PvdA initiation. The pvdA transcripts were monocystronic, with T1 accounting for 90% of the pvdA mRNA. Fur box-like sequences were apparently absent in the regions 5' of pvdA transcription start sites. A sequence motif resembling the -10 hexamer of AlgU-dependent promoters and the iron starvation box of pyoverdin genes controlled by the sigmaE -like factor PvdS were identified 5' of the T1 start site. The minimum DNA region required for iron-regulated promoter activity was mapped from bp -41 to -154 relative to the ATG translation start site of pvdA. We used pvdA'::lacZ transcriptional fusions and Northern (RNA) analyses to study the involvement of Fur and PvdS in the iron-regulated expression of pvdA. Two fur mutants of P. aeruginosa were much less responsive than wild-type PAO1 to the iron-dependent regulation of pvdA expression. Transcription from the pvdA promoter did not occur in a heterologous host unless in the presence of the pvdS gene in trans and was abrogated in a pvdS mutant of P. aeruginosa. Interaction of the Fur repressor with a 150-bp fragment encompassing the pvdS promoter was demonstrated in vivo by the Fur titration assay and confirmed in vitro by gel retardation experiments with a partially purified Fur preparation. Conversely, the promoter region of pvdA did not interact with Fur. Our results support the hypothesis that the P. aeruginosa Fur repressor indirectly controls pvdA transcription through the intermediary sigma factor PvdS; in the presence of sufficient iron, Fur blocks the pvdS promoter, thus preventing PvdS expression and consequently transcription of pvdA and other pyoverdin biosynthesis genes.
Mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides. A key aim of much recent work has been to increase the speed of kill and so improve commercial efficacy of these biocontrol agents. This might he achieved by adding insecticidal genes to the fungus, an approach considered to have enormous potential for the improvement of biological pesticides. We report here the development of a genetically improved entomopathogenic fungus. Additional copies of the gene encoding a regulated cuticle-degrading protease (Pr1) from Metarhizium anisopliae were inserted into the genome of M. anisopliae such that Pr1 was constitutively overproduced in the hemolymph of Manduca sexta, activating the prophenoloxidase system. The combined toxic effects of Pr1 and the reaction products of phenoloxidase caused larvae challenged with the engineered fungus to exhibit a 25% reduction in time of death and reduced food consumption by 40% compared to infections by the wild-type fungus. In addition, infected insects were rapidly melanized, and the resulting cadavers were poor substrates for fungal sporulation. Thus, environmental persistence of the genetically engineered fungus is reduced, thereby providing biological containment.
A secreted aspartic proteinase from Glomerella cingulata (GcSAP) was purified to homogeneity by ion exchange chromatography. The enzyme has an M, of 36000 as estimated by SDS-PAGE, optimal activity from pH 3.5 to pH 4.0 and is inhibited by pepstatin. The N-terminal sequence, 23 residues long, was used to design a gene-specific primer. This was used in 3' RACE (rapid amplification of cDNA ends) PCR to amplify a 1.2 kb fragment of the gcsap cDNA. A second gene-specific primer was designed and used in 5' RACE PCR to clone the 5' region. This yielded a 600 bp DNA fragment and completed the open reading frame. The gcsap open reading frame encodes a protein with a 78 residue prepro-sequence typical of other fungal secreted aspartic proteinases. Based on the deduced sequence, the mature enzyme contains 329 amino acids and shows approximately 40% identity to other fungal aspartic proteinases. Subsequent cloning and sequencing of gcsap fragments obtained from PCR with genomic DNA revealed a 73 bp intron beginning at nt 728. Southern analyses at medium and high stringency indicated that G. cingulata possesses one gene for the secreted aspartic proteinase, and Northern blots indicated that gene expression was induced by exogenous protein and repressed by ammonium salts. GcSAP is a putative pathogenicity factor of G. cingulata, and it will now be possible to create SAP-mutants and assess the role GcSAP plays in pathogenicity.
The abilities of isolates of saprophytes (Neurospora crassa, Aspergillus nidulans), an opportunistic human pathogen (Aspergillus fumigatus), an opportunistic insect pathogen (Aspergillus flavus), plant pathogens (Verticillium albo-atrum, Verticillium dahliae, Nectria haematococca), a mushroom pathogen (Verticillium fungicola) and entomopathogens (Verticillium lecanii, Beauveria bassiana, Metarhizium anisopliae) to utilize plant cell walls and insect cuticle components in different nutrient media were compared. The pathogens showed enzymic adaptation to the polymers present in the integuments of their particular hosts. Thus, the plant pathogens produced high levels of enzymes capable of degrading pectic polysaccharides, cellulose and xylan, as well as cutinase substrate, but secreted little or no chitinase and showed no proteolytic activity against elastin and mucin. The entomopathogens and V. fungicola degraded a broad spectrum of proteins (including elastin and mucin) but, except for chitinase, cellulase (V. lecanii and V. fungicola only) and cutinase (B. bassiana only), produced very low levels of polysaccharidases. The saprophytes (Neu. crassa and A. nidulans) and the opportunistic pathogens (A. fumigatus and A. flavus) produced the broadest spectrum of protein and polysaccharide degrading enzymes, indicative of their less specialized nutritional status. V. lecanii and V. albo-atrum were compared in more detail to identity factors that distinguish plant and insect pathogens. V. albo-atrum, but not V. lecanii, grew well on different plant cell wall components. The major class of proteases produced in different media by isolates of V. albo-atrum and V. dahliae were broad spectrum basic (pI > 10) trypsins which degrade Z-AA-AA-Arg-NA substrates (Z, benzoyl; AA, various amino acids; Na, nitroanilide), hide protein azure and insect (Manduca sexta) cuticles. Analogous peptidases were produced by isolates of V. lecanii and V. fungicola but they were specific for Z-Phe-Val-Arg-NA. V. albo-atrum and V. dahliae also produced low levels of neutral (pI ca 7) and basic (pI ca 9.5) subtilisin-like proteases active against a chymotrypsin substrate (Succinyl-Ala2-Pro-Phe-NA) and insect cuticle. In contrast, subtilisins comprised the major protease component secreted by V. lecanii and V. fungicola. Both V. lecanii and V. albo-atrum produced the highest levels of subtilisin and trypsin-like activities during growth on collagen or insect cuticle. Results are discussed in terms of the adaptation of fungi to the requirements of their ecological niches.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
Signaling by Notch family receptors is involved in many cell-fate decisions during development. Several modifiers of Notch activity have been identified, suggesting that regulation of Notch signaling is complex. In a genetic screen for modifiers of Notch activity, we identified a gene encoding a novel WD40-repeat protein. The gene is called Notchless, because loss-of-function mutant alleles dominantly suppress the wing notching caused by certain Notch alleles. Reducing Notchless activity increases Notch activity. Overexpression of Notchless in Xenopus or Drosophila appears to have a dominant-negative effect in that it also increases Notch activity. Biochemical studies show that Notchless binds to the cytoplasmic domain of Notch, suggesting that it serves as a direct regulator of Notch signaling activity.
To date zinc carboxypeptidases have only been found in animals and actinomycete bacteria. A cDNA clone (MeCPA) for a novel fungal (Metarhizium anisopliae) carboxypeptidase (MeCPA) was obtained by using reverse transcription differential display polymerase chain reaction to identify
pathogenicity genes. MeCPA resembles pancreatic carboxypeptidases in being synthesized as a precursor species (418 amino acids)
containing a large amino-terminal fragment (99 amino acids). The mature (secreted) form of MeCPA shows closest amino acid
identity to human carboxypeptidases A1 (35%) and A2 (37%). MeCPA was expressed in an insect cell line yielding an enzyme with
dual A1 + A2 specificity for branched aliphatic and aromatic COOH-terminal amino acids. However, in contrast to the very broad
spectrum A + B-type bacterial enzymes, MeCPA lacks B-type activity against charged amino acids. This is predictable as key
catalytic residues determining the specificity of MeCPA are conserved with those of mammalian A-type carboxypeptidases. Thus,
in evolutionary terms the fungal enzyme is an intermediate between the divergence of A and B forms and the differentiation
of the A form into A1 and A2 isoforms. Ultrastructural immunocytochemistry of infected host (Manduca sexta) cuticle demonstrated that MeCPA participates with the concurrently produced endoproteases in procuring nutrients; an equivalent
function to digestive pancreatic enzymes.
The cytoplasmic carboxyl-terminal domain of AE1, the plasma membrane chloride/bicarbonate exchanger of erythrocytes, contains a binding site for carbonic anhydrase II (CAII). To examine the physiological role of the AE1/CAII interaction, anion exchange activity of transfected HEK293 cells was monitored by following the changes in intracellular pH associated with AE1-mediated bicarbonate transport. AE1-mediated chloride/bicarbonate exchange was reduced 50-60% by inhibition of endogenous carbonic anhydrase with acetazolamide, which indicates that CAII activity is required for full anion transport activity. AE1 mutants, unable to bind CAII, had significantly lower transport activity than wild-type AE1 (10% of wild-type activity), suggesting that a direct interaction was required. To determine the effect of displacement of endogenous wild-type CAII from its binding site on AE1, AE1-transfected HEK293 cells were co-transfected with cDNA for a functionally inactive CAII mutant, V143Y. AE1 activity was maximally inhibited 61 +/- 4% in the presence of V143Y CAII. A similar effect of V143Y CAII was found for AE2 and AE3cardiac anion exchanger isoforms. We conclude that the binding of CAII to the AE1 carboxyl-terminus potentiates anion transport activity and allows for maximal transport. The interaction of CAII with AE1 forms a transport metabolon, a membrane protein complex involved in regulation of bicarbonate metabolism and transport.
Active oxygen species have been postulated to perform multiple functions in plant defense, but their exact role in plant resistance to diseases is not fully understood. We have recently demonstrated H2O2-mediated disease resistance in transgenic potato (Solanum tuberosum) plants expressing a foreign gene encoding glucose oxidase. In this study we provide further evidence that the H2O2-mediated disease resistance in potato is effective against a broad range of plant pathogens. We have investigated mechanisms underlying the H2O2-mediated disease resistance in transgenic potato plants. The constitutively elevated levels of H2O2 induced the accumulation of total salicylic acid severalfold in the leaf tissue of transgenic plants, although no significant change was detected in the level of free salicylic acid. The mRNAs of two defense-related genes encoding the anionic peroxidase and acidic chitinase were also induced. In addition, an increased accumulation of several isoforms of extracellular peroxidase, including a newly induced one, was observed. This was accompanied by a significant increase in the lignin content of stem and root tissues of the transgenic plants. The results suggest that constitutively elevated sublethal levels of H2O2 are sufficient to activate an array of host defense mechanisms, and these defense mechanisms may be a major contributing factor to the H2O2-mediated disease resistance in transgenic plants.
Expressed sequence tag (EST) libraries for Metarhizium anisopliae, the causative agent of green muscardine disease, were developed from the broad host-range pathogen Metarhizium anisopliae sf. anisopliae and the specific grasshopper pathogen, M. anisopliae sf. acridum. Approximately 1,700 5' end sequences from each subspecies were generated from cDNA libraries representing fungi grown under conditions that maximize secretion of cuticle-degrading enzymes. Both subspecies had ESTs for virtually all pathogenicity-related genes cloned to date from M. anisopliae, but many novel genes encoding potential virulence factors were also tagged. Enzymes with potential targets in the insect host included proteases, chitinases, phospholipases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites. A diverse array of proteases composed 36 % of all M. anisopliae sf. anisopliae ESTs. Eighty percent of the ESTs that could be clustered into functional groups had significant matches (E<10(-5)) in other ascomycete fungi. These included genes reported to have specific roles in pathogens with plant or vertebrate hosts. Many of the remaining ESTs had their best BLAST match among animal, plant and bacterial sequences. These include genes with plant and microbial counterparts that produce potent antimicrobials. The abundance of transcripts discovered for different functional groups varied between the two subspecies of M. anisopliae in a manner consistent with ecological adaptations of the two pathogens. By hastening gene discovery this project has enhanced development of improved mycoinsecticides. In addition, the M. anisopliae ESTs represent a significant contribution to the extensive database of sequences from ascomycetes that are saprophytes or plant and vertebrate pathogens. Comparative analyses of these sequences is providing important information about the biology and evolutionary history of this clade.
This book presents topics on the development, improvement and commercialization of fungi for the biological control of pests, weeds and diseases which are of economic importance. Common themes such as production, formulation and application of technologies, biosafety, risk assessment and registration requirements are all covered. The book attempts to bring together scientists, industry and government agencies involved in all aspects of fungal biological control agents for the first time.
The Zygomycota may be one of the most diverse and the least-studied of the fungal phyla, and as presently classified, appears to be polyphyletic. Zygomycota is one of the four divisions of the Fungi accepted by Barr (1992), and consists of Trichomycetes and Zygomycetes. Zygomycetes are saprobes or haustorial or nonhaustorial parasites of animals, plants, or fungi. Trichomycetes are symbionts in the gut, or less commonly around the anal region, of arthopods, including insects and their larvae; they attach to the host via a cellular or noncellular holdfast (Lichtwardt 1986). Because of their unique characteristics Trichomycetes will be discussed in the following chapter (Benny, Chap. 7, this Vol.).
Fungal phylogenetics has always been based on characters, but technological and intellectual advances are introducing new kinds of characters and new ways of thinking about them. First light microscopy, then electron microscopy, and now DNA sequencing successively upset previous views of fungal relationships. Phenetics, cladistics, and computerized data analysis and phylogenetic tree generation are now changing the intellectual rules for taxonomy and phylogenetics. The combination of new characters and new analytical tools have supported some taxonomic groups, established some new ones, and demolished a few old ones.
Fungal pathogens are capable of causing sensational levels of mortality in insect populations. As early as about 1000 AD, sericulturists in Asia reported Beauveria bassiana infections in silkworms (Steinhaus, 1956). The “germ theory of disease,” the concept that microbes can cause disease, was first experimentally proven by Agostino Bassi in 1834 working with B. bassiana and silkworms. From the late 1800s through 1925, research on the potential use of fungi for insect control was conducted. In recent years, largely due to our present knowledge of the hazards and inefficiencies of dependence on synthetic chemical pesticides for insect control, interest in developing fungal pathogens for control purposes has increased dramatically (Roberts and Hajek, 1992; Vandenberg, 1993). Although abundant research on use of entomopathogenic fungi for control has been conducted, there are major gaps in our understanding of the basic ecology of these fungal species, in part due to the complexity of host/pathogen/environment interactions and the diversity of host/pathogen systems to be studied. Lack of successful control in some systems and difficulties in adapting some species for typical control practices has promoted a shift in research emphasis. At present, some research efforts are directed toward understanding the factors leading to fungal infection in insects in order to investigate the potential for manipulating these systems to enhance levels of infection and promote development of epizootics (disease outbreaks).
Synthesis of chymoelastase and trypsin by the entomopathogenic fungus Metarhizium anisopliae occurs rapidly (<2 h) during carbon and nitrogen derepression in minimal media. Enzyme levels were enhanced when minimal media were supplemented with insect cuticle or other insoluble polymetic nutrients (e.g. cellulose) that were insufficient to produce catabolite repression. Addition of more readily utilized metabolites (e.g. glucose or alanine) repressed protease production confirming that production is constitutive but repressible. Operational control of protease release involves synthesis rather than secretion because catabolite repression reduced endocellular activity (associated with a sedimentable vacuole containing fraction) as well as extracellular enzyme levels. Studies with metabolic inhibitors indicated that production of Pr1 and Pr2 does not require DNA synthesis. However, synthesis is substantially reduced by inhibitors of transcription (actinomycin D and 8-azoguanine) and translation (cyclohexamide and puromycin).
Inhibition by 8-azoguanine is relieved by guanine. These results imply that the operative steps in protease regulation involve de novo synthesis of mRNA. Inhibition of enzyme production by an AMP analogue adenosine 5′-0-thiophosphate implies an involvement for AMP-dependent enzyme systems in derepression. However, neither exogenous cAMP nor an inhibitor of cAMP phosphodiesterase relieved catabolite repression by glucose or NH4Cl. Use of o-vanadate to inhibit plasmalemma ATPase confirmed that secretion of chymoelastase-like protease and trypsin-like protease via the cell membrane is an active process.
Aspergillus fumigatus is saprotrophic with an unusual ability to colonize the respiratory tract. The mechanisms that permit pathogenicity may have evolved to adapt the fungus to life as a saprobe. To define the nature of these adaptations and identify common themes in fungal pathogenesis to vertebrates, insects and plants, we compared A. fumigatus with a plant pathogen (Haematonectria haematococca) and an insect pathogen (Metarhizium anisopliae) in their abilities to degrade and utilize host-derived macromolecules (horse lung polymers, porcine mucin, hyaluronic acid, alfalfa cell walls and cockroach cuticle). Each fungus produced a similar range of proteases on mucin and lung polymers, and high levels of several glycosidic enzymes on mucin and plant cell walls, which contain inductive carbohydrate substrates. Following 18 h of growth by A. fumigatus at pH 4 or pH 8, the degradation of mucin carbohydrates and mucin protein were approximately 40% and 75% respectively, suggesting that the aspartyl proteases (produced at pH 4) and the subtilisin proteases (produced at pH 8) are more important than carbohydrases for degrading mucin. The highly glycosylated mucin residue remaining after 18 h growth resisted further degradation, in part due to bound sialic acid as A. fumigatus secretes a sulphatase but not sialidase. Hyaluronidase activity (an important virulence factor in bacteria) was not produced by A. fumigatus, M. anisopliae or H. haematococca, but each fungus secreted a range of other enzymes (phospholipase A2, phospholipase C, acid phosphatase, alkali phosphatase, phosphodiesterase and esterase) that are common toxic components of bacteria as well as reptilia and invertebrate venoms. Thus thermotolerant opportunists such as A. fumigatus may sustain themselves and cause disease in human hosts using depolymerases that are widely distributed in fungi and that provide them with the versatility to exploit many environments.
The gene encoding the aspartyl protease of the filamentous fungus Mucor miehei has been cloned in Escherichia coli and the DNA sequenced. The deduced primary translation product contains an N-terminal region of 69 amino acid (aa) residues not present in the mature protein. By analogy to the evolutionarily related mammalian gastric aspartyl proteases it is inferred that the primary secreted product is a zymogen containing a 47-aa propeptide. This propeptide is presumably removed in the later steps of the secretion process or upon secretion into the medium. To study the effects of modifications of the protease structure on its maturation by enzyme-engineering methods, an efficient expression system was sought. In E. coli, transcription of the preproenzyme coding sequence from a bacterial promoter results primarily in the accumulation of unsecreted, enzymatically inactive polypeptides, immunologically related to the authentic protease. In Aspergillus nidulans expression of the cloned gene, probably from its own promoter, results in the secretion into the culture medium of polypeptides which, compared to the authentic protease, are similar in specific activity, but differ in the character of their asparagine-linked oligosaccharides.
True fungi (Eumycota) are heterotrophic eukaryotic microorganisms encompassing ascomycetes, basidiomycetes, chytridiomycetes and zygomycetes. The natural systematics of the latter group, Zygomycota, are very poorly understood due to the lack of distinguishing morphological characters. We have determined sequences for the nuclear-encoded genes actin (act) from 82 zygomycetes representing all 54 currently recognized genera from the two zygomycetous orders Mucorales and Mortierellales. We also determined sequences for translation elongation factor EF-1alpha (tef) from 16 zygomycetes (total of 96,837 bp). Phylogenetic analysis in the context of available sequence data (total 2,062 nucleotide positions per species) revealed that current classification schemes for the mucoralean fungi are highly unnatural at the family and, to a large extent, at the genus level. The data clearly indicate a deep, ancient and distinct dichotomy of the orders Mucorales and Mortierellales, which are recognized only in some zygomycete systems. Yet at the same time the data show that two genera - Umbelopsis and Micromucor - previously placed within the Mortierellales on the basis of their weakly developed columella (a morphological structure of the sporangiophore well-developed within all Mucorales) are in fact members of the Mucorales. Phylogenetic analyses of the encoded amino acid sequences in the context of homologues from eukaryotes and archaebacterial outgroups indicate that the Eumycota studied here are a natural group but provide little or no support for the monophyly of either zygomycetes, ascomycetes or basidiomycetes. The data clearly indicate that a complete revision of zygomycete natural systematics is necessary.
Several pathogenic isolates of Metarhizium anisopliae, Beauveria bassiana, and Verticillium lecanii when grown in buffered liquid cultures containing comminuted locust cuticle as composite carbon source (good growth occurred on most monomeric and polymeric cuticular constituents), produced a variety of extracellular enzymes corresponding to the major components of insect cuticle, e.g., endoproteases, aminopeptidase, carboxypeptidase A, lipase, esterase, chitinase, and N-acetylglucosaminidase. Considerable variations occurred in levels of production between species and even within a species, but endoproteases were exceptional as production of them was high with all the isolates. Cuticle-degrading enzymes were produced rapidly and sequentially in culture. The first activities to appear (<24 hr) were those of the proteolytic complex; chitinases were always produced substantially later. Chitin was probably masked by protein as a fluorescent chitin-specific lectin and calcofluor (binds to β-glucans) stained mainly cuticles from which protein had been removed (by protease or KOH).
Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.
An extracellular elastinolytic metalloproteinase, purified from Aspergillus fumigatus isolated from an aspergillosis and patient/and an internal peptide derived from it were subjected to N-terminal sequencing. Oligonucleotide primers based on these sequences were used to PCR amplify a segment of the metalloproteinase cDNA, which was used as a probe to isolate the cDNA and gene for this enzyme. The gene sequence matched exactly with the cDNA sequence except for the four introns that interrupted the open reading frame. According to the deduced amino acid sequence, the metalloproteinase has a signal sequence and 227 additional amino acids preceding the sequence for the mature protein of 389 amino acids with a calculated molecular mass of 42 kDa, which is close to the size of the purified mature fungal proteinase. This sequence contains segments that matched both the N terminus of the mature protein and the internal peptide. A. fumigatus metalloproteinase contains some of the conserved zinc-binding and active-site motifs characteristic of metalloproteinases but shows no overall homology with known metalloproteinases. The cDNA of the mature protein when introduced into Escherichia coli directed the expression of a protein with a size, N-terminal sequence, and immunological cross-reactivity identical to those of the native fungal enzyme. Although the enzyme in the inclusion bodies could not be renatured, expression at 30 degrees C yielded soluble enzyme that showed chromatographic behavior identical to that of the native fungal enzyme and catalyzed hydrolysis of elastin. The metalloproteinase gene described here was not found in Aspergillus flavus.
The entomopathogenic fungus, Metarhizium anisopliae, produces three distinct types of proteinases during growth on cockroach cuticle. These were separated by analytical isoelectric focusing and characterized according to their substrate specificity and inhibition patterns as Pr1 subtilisin-like proteinases (four isoforms pI range approximately 9.3-10.2), a thermolysin-like metalloproteinase (pI approximately 7.3), and trypsin-like serine Pr2 proteinases (two major isoforms, pI approximately 4.4 and 4.9 and two minor isoforms, pI approximately 5.2). Preparative isoelectric focusing was used to separate the four Pr1(2) components produced during growth on cockroach cuticle with isoelectric points of 10.2 (m = 30.2 kDa), 9.8 (m = 28.5 kDa), 9.3 (m = 29.5 kDa), and 9.0 (m = 31.5 kDa). Two of the isoforms were also produced, at diminished levels, during growth on elastin or cellulose presumably as a result of carbon and nitrogen derepression. The pI 10.2 Pr1 differed from the other isoforms in preferring alanine over bulky hydrophobic groups at P2 and P3, in discriminating against proline at P2 and in its lack of sensitivity to tetra-butyl-oxycarbonyl-Gly-Leu-Phe-chloromethyl ketone. Differences in the N-terminal amino acid sequences confirmed that the four isoforms are related products of at least two distinct genes. The isoforms showed similar primary specificities, with the aromatic P1 phenylalanine being 10- to 16-fold more reactive than a P1 leucine residue reflected principally in Kcat. However, methionine (containing a long unsubstituted side chain) was also a good substrate for each isoform confirming the low selectivity of their S1 subsites. The isoforms all degraded a variety of solubilized cuticle proteins, with high-molecular-weight acidic proteins being preferentially hydrolyzed. The metalloproteinase is active against the Pr1 substrate succinyl-(Ala)2-Pro-Phe-7-amino-4-coumarin trifluoromethyl, but differs from the Pr1 isoforms in being inhibited by 1,10-phenanthroline and phosphoramidon. The potential role of the metalloproteinase in pathogenicity is discussed.
Osmotically sensitive mutants of Neurospora crassa are unable to grow on medium supplemented with 4% NaCl, have altered morphologies and cell-wall compositions, and are resistant to dicarboximide fungicides. Osmotic-1 (os-1) mutants have a unique characteristic of forming protoplasts that grow and divide in specialized liquid medium, suggesting that the os-1+ gene product is important for cell-wall assembly. A cosmid containing the os-1+ locus of N. crassa, isolated from a genomic cosmid library by chromosomal walk from a closely linked gene, was used to subclone the os-1+ gene by functional complementation of an os-1 mutant. Analysis of the sequence of complementing DNA predicts that os-1+ encodes a predicted protein similar to sensor-histidine kinases of bacteria and a yeast osmosensor-histidine kinase. Importantly, the predicted os-1+ protein is identical to the N. crassa nik-1 predicted protein that was identified by using polymerase chain reaction primers directed against histidine kinase consensus DNA sequences. Our results indicate that nik-1 and os-1 encode the same osmosensing histidine kinase that plays an important role in the regulation of cell-wall assembly and, probably, other cell responses to changes in external osmolarity.
The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press
Protein kinase C (PKC) plays an important role in the control of proliferation and differentiation of a wide range of cell types, and fungi are no exception. Previous results reported by us on the effects of the phorbol ester, 12-myristate-13-acetate phorbol (PMA) and other PKC effector molecules, on dimorphism in Sporothrix schenckii suggested the presence of this enzyme in the fungus and its involvement in the control of morphogenetic transitions. The work summarized here confirms the presence of PKC in yeast and mycelium extracts of S. schenckii. Different isoforms of this enzyme were found to be present in the yeast and mycelium forms of the fungus and were identified by Western blot analysis using affinity purified anti-PKC isoforms specific antibodies: the gamma and zeta isoforms were detected in both the yeast and mycelium forms of the fungus, while the beta isoform was only detected in the yeast form. The presence of PKC was confirmed biochemically by measuring total enzyme activity in both forms of the fungus. No significant differences were observed for the PKC activity level recorded for both the mycelium and yeast forms of the fungus (p < or = 0.05). These data confirm the presence of PKC activity in Sporothrix schenckii and constitutes the first evidence concerning the differential expression of PKC isoforms in the mycelium and yeast forms of a dimorphic fungus, supporting the possible involvement of this important signal transduction enzyme in the control of morphogenesis in this fungus.
This short review describes the molecular evolution and phylogeny of various defense molecules participating in the host defense of horseshoe crab. It is well known that invertebrate animals, which lack adaptive immune systems, have developed various defense systems, so called innate immunity, that respond to common antigens on the surface of potential pathogens. The systems include hemolymph coagulation, melanization, cell agglutination, antimicrobial action, active oxygen formation, and phagocytic action. Among them, hemolymph coagulation and phenoloxidase-mediated melanization, in addition to cell agglutination, are directly induced by foreign substances, that result in the engulfment of invading microbes. The immobilized invaders are finally killed by antimicrobial substances released mainly from many kinds of hemocytes. In the past two decades, we have investigated biochemically various defense molecules, using horseshoe crab as a model animal, and established extensively their molecular structures. These results now make it possible to discuss evolution and phylogeny of the defense molecules at a molecular level, in comparison with those derived from vertebrate animals. Here, the authors will describe the present state of our knowledge concerning molecules mainly associated with innate immunity.
We sequenced the nuclear small subunit of ribosomal DNA (SSU rDNA) from seven species within the insect-pathogenic order Entomophthorales. These sequences were aligned with other published SSU rDNA sequences and phylogenies were inferred using phenetic and cladistic methods. Based on three different phylogenetic methods the Entomophthorales (excluding Basidiobolus ranarum) is monophyletic; B. ranarum was more closely related to chytrids from Chytridiales and Neocallimasticales than to Entomophthorales, as was proposed by Nagahama et al. (Mycologia 87: 203-209, 1995). Nuclear characters (large nuclei containing conspicuous condensed chromatin and lack of a prominent nucleolus) were of predictive value for the monophyly of the family Entomophthoraceae. Conidial characters separate the Entomophthoraceae, which only includes obligate pathogens, into at least two lineages: one lineage with uninucleate conidia and another with multinucleate conidia. The two species of Conidiobolus studied were paraphyletic in our analyses and only distantly related to each other. This information may prove to be important in the use of these fungi as biocontrol agents.
To survive and replicate in vertebrate hosts, protozoan and fungal invaders must be capable of securing host iron. Successful pathogens obtain the metal from either extraction of heme, binding of siderophilins, binding of siderophores, and/or iron pools within host cells. The actual strategy can vary with the availability of iron in the particular host milieu. As a corollary, hosts have developed an elaborate iron withholding defense system. Conditions that can compromise the system as well as procedures that can strengthen it are reviewed.
We describe the third generation of the CAP sequence assembly program. The CAP3 program includes a number of improvements and new features. The program has a capability to clip 5' and 3' low-quality regions of reads. It uses base quality values in computation of overlaps between reads, construction of multiple sequence alignments of reads, and generation of consensus sequences. The program also uses forward-reverse constraints to correct assembly errors and link contigs. Results of CAP3 on four BAC data sets are presented. The performance of CAP3 was compared with that of PHRAP on a number of BAC data sets. PHRAP often produces longer contigs than CAP3 whereas CAP3 often produces fewer errors in consensus sequences than PHRAP. It is easier to construct scaffolds with CAP3 than with PHRAP on low-pass data with forward-reverse constraints.
Ambient pH regulates the expression of virulence genes of Metarhizium anisopliae, but it was unknown if M. anisopliae can regulate ambient pH. Mutants of M. anisopliae altered in production of oxalic acid were evaluated for the interrelationship of ambient pH, buffering capacity added to media, growth, and generation of extracellular proteases and ammonia. Wild-type and acid-overproducing mutants [Acid(+)] grew almost as well at pH 8 as at pH 6, but acid-non-producing [Acid(-)] mutants showed limited growth at pH 8, indicating that acid production is linked to the ability to grow at higher pH. Production of ammonia by M. anisopliae was strongly stimulated by low levels of amino acids in the medium when cells were derepressed for nitrogen and carbon. Likewise, although Aspergillus fumigatus and Neurospora crassa produced some ammonia in minimal media, addition of low levels of amino acids enhanced production. Ammonia production by A. fumigatus, N. crassa and M. anisopliae increased the pH of the medium and allowed production of subtilisin proteases, whose activities are observed only at basic pH. In contrast, protease production by the Acid(+) mutants of M. anisopliae was greatly reduced because of the acidification of the medium. This suggests that alkalinization by ammonia production is adaptive by facilitating the utilization of proteinaceous nutrients. Collectively, the data imply that ammonia may have functions related to regulation of the microenvironment and that it represents a previously unconsidered virulence factor in diverse fungi with the potential to harm tissues and disturb the host's immune system.