Native entomopathogenic Metarhizium spp. from Burkina Faso and their virulence against the malaria vector Anopheles coluzzii and non-target insects.

BACKGROUND: Genetically enhanced Metarhizium pingshaense are being developed for malaria vector control in Burkina Faso. However, not much is known about the local prevalence and pathogenicity of this fungus, so we prospected mosquitoes and plant roots (a common habitat for Metarhizium spp.) for entomopathogenic fungi. RESULTS: Our investigations showed that Metarhizium spp. represented between 29-74% of fungi isolated from plant root rhizospheres in diverse collection sites. At low spore dosages (1 × 106 conidia/ml), two mosquito-derived M. pingshaense isolates (Met_S26 and Met_S10) showed greater virulence against Anopheles coluzzii (LT80 of ~7 days) than isolates tested in previous studies (LT80 of ~10 days). In addition, the local isolates did not cause disease in non-target insects (honeybees and cockroaches). CONCLUSIONS: Our work provides promising findings for isolating local Metarhizium strains for application in mosquito biological control and for future transgenic biocontrol strategies in Burkina Faso.

Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana.

The ascomycete fungus Beauveria bassiana is a pathogen of hundreds of insect species and is commercially produced as an environmentally friendly mycoinsecticide. We sequenced the genome of B. bassiana and a phylogenomic analysis confirmed that ascomycete entomopathogenicity is polyphyletic, but also revealed convergent evolution to insect pathogenicity. We also found many species-specific virulence genes and gene family expansions and contractions that correlate with host ranges and pathogenic strategies. These include B. bassiana having many more bacterial-like toxins (suggesting an unsuspected potential for oral toxicity) and effector-type proteins. The genome also revealed that B. bassiana resembles the closely related Cordyceps militaris in being heterothallic, although its sexual stage is rarely observed. A high throughput RNA-seq transcriptomic analysis revealed that B. bassiana could sense and adapt to different environmental niches by activating well-defined gene sets. The information from this study will facilitate further development of B. bassiana as a cost-effective mycoinsecticide.

Insertion of an esterase gene into a specific locust pathogen (Metarhizium acridum) enables it to infect caterpillars.

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.

Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum.

Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ~30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ~16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogeneous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains with customized properties.

MOS1 osmosensor of Metarhizium anisopliae is required for adaptation to insect host hemolymph.

Entomopathogenic fungi such as Metarhizium anisopliae infect insects by direct penetration of the cuticle, after which the fungus adapts to the high osmotic pressure of the hemolymph and multiplies. Here we characterize the M. anisopliae Mos1 gene and demonstrate that it encodes the osmosensor required for this process. MOS1 contains transmembrane regions and a C-terminal Src homology 3 domain similar to those of yeast osmotic adaptor proteins, and homologs of MOS1 are widely distributed in the fungal kingdom. Reverse transcription-PCR demonstrated that Mos1 is up-regulated in insect hemolymph as well as artificial media with high osmotic pressure. Transformants containing an antisense vector directed to the Mos1 mRNA depleted transcript levels by 80%. This produced selective alterations in regulation of genes involved in hyphal body formation, cell membrane stiffness, and generation of intracellular turgor pressure, suggesting that these processes are mediated by MOS1. Consistent with a role in stress responses, transcript depletion of Mos1 increased sensitivity to osmotic and oxidative stresses and to compounds that interfere with cell wall biosynthesis. It also disrupted developmental processes, including formation of appressoria and hyphal bodies. Insect bioassays confirmed that Mos1 knockdown significantly reduces virulence. Overall, our data show that M. anisopliae MOS1 mediates cellular responses to high osmotic pressure and subsequent adaptations to colonize host hemolymph.

A scorpion neurotoxin increases the potency of a fungal insecticide.

The low virulence of the insecticidal fungus Metarhizium anisopliae has stymied its widespread use in controlling insect pests. We show that high-level expression of an insect-specific neurotoxin from the scorpion Androctonus australis in hemolymph by M. anisopliae increases fungal toxicity 22-fold against tobacco hornworm (Manduca sexta) caterpillars and ninefold against adult yellow fever mosquitoes (Aedes aegypti) without compromising host specificity. Prelethal effects include reduced mobility and feeding of the insects targeted.

Fungal peptide Destruxin A plays a specific role in suppressing the innate immune response in Drosophila melanogaster.

Destruxins are a class of insecticidal, anti-viral, and phytotoxic cyclic depsipeptides that are also studied for their toxicity to cancer cells. They are produced by various fungi, and a direct relationship has been established between Destruxin production and the virulence of the entomopathogen Metarhizium anisopliae. Aside from opening calcium channels, their in vivo mode of action during pathogenesis remains largely uncharacterized. To better understand the effects of a Destruxin, we looked at changes in gene expression following injection of Destruxin A into the fruit fly Drosophila melanogaster. Microarray results revealed reduced expression of various antimicrobial peptides that play a major role in the humoral immune response of the fly. Flies co-injected with a non-lethal dose of Destruxin A and the normally innocuous Gram-negative bacteria Escherichia coli, showed increased mortality and an accompanying increase in bacterial titers. Mortality due to sepsis was rescued through ectopic activation of components in the IMD pathway, one of two signal transduction pathways that are responsible for antimicrobial peptide induction. These results demonstrate a novel role for Destruxin A in specific suppression of the humoral immune response in insects.

A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses.

The ubiquitous fungal pathogen Metarhizium anisopliae kills a wide range of insects. Host hemocytes can recognize and ingest its conidia, but this capacity is lost on production of hyphal bodies. We show that the unusual ability of hyphal bodies to avoid detection depends on a gene (Mcl1) that is expressed within 20 min of the pathogen contacting hemolymph. A mutant disrupted in Mcl1 is rapidly attacked by hemocytes and shows a corresponding reduction of virulence to Manduca sexta. Mcl1 encodes a three domain protein comprising a hydrophilic, negatively charged N-terminal region with 14 cysteine residues, a central region comprising tandem repeats (GXY) characteristic of collagenous domains, and a C-terminal region that includes a glycosylphosphatidylinositol-dependent cell wall attachment site. Immunofluorescence assay showed that hyphal bodies are covered by the N-terminal domains of MCL1. The collagen domain became antibody accessible after treatment with DTT, suggesting that the N termini are linked by interchain disulfide bonds and are presented on the cell surface by extended collagenous fibers. Studies with staining reagents and hemocyte monolayers showed that MCL1 functions as an antiadhesive protective coat because it masks antigenic structural components of the cell wall such as beta-glucans, and because its hydrophilic negatively charged nature makes it unattractive to hemocytes. A survey of 54 fungal genomes revealed that seven other species have proteins with collagenous domains suggesting that MCL1 is a member of a patchily distributed gene family.

Differential gene expression by Metarhizium anisopliae growing in root exudate and host (Manduca sexta) cuticle or hemolymph reveals mechanisms of physiological adaptation.

Like many other fungal pathogens Metarhizium anisopliae is a facultative saprophyte with both soil-dwelling and insect pathogenic life-stages. In addition, as M. anisopliae traverses the cuticle and enters the hemolymph it must adapt to several different host environments. In this study, we used expressed sequence tags and cDNA microarray analyses to demonstrate that physiological adaptation by M. anisopliae to insect cuticle, insect hemolymph, bean root exudate (a model for life in the soil), and nutrient rich Sabouraud dextrose broth (SDB) involves different subsets of genes. Overall, expression patterns in cuticle and hemolymph clustered separately from expression patterns in root exudates and SDB, indicative of critical differences in transcriptional control during pathogenic and saprophytic growth. However, there were differences in gene expression between hemolymph and cuticle and these mostly involved perception mechanisms, carbon metabolism, proteolysis, cell surface properties, and synthesis of toxic metabolites. These differences suggest previously unsuspected stratagems of fungal pathogenicity that can be tested experimentally. Examples include the switch-off of cuticle-degrading proteases and a dramatic cell wall reorganization during growth in hemolymph.

Developmental and transcriptional responses to host and nonhost cuticles by the specific locust pathogen Metarhizium anisopliae var. acridum.

Transcript patterns elicited in response to hosts can reveal how fungi recognize suitable hosts and the mechanisms involved in pathogenicity. These patterns could be fashioned by recognition of host-specific topographical features or by chemical components displayed or released by the host. We investigated this in the specific locust pathogen Metarhizium anisopliae var. acridum. Only host (Schistocerca gregaria) cuticle stimulated the full developmental program of germination and differentiation of infection structures (appressoria). Cuticle from beetles (Leptinotarsa decimlineata) repressed germination while cuticle from hemipteran bugs (Magicicada septendecim) allowed germination but only very low levels of differentiation, indicating that the ability to cause disease can be blocked at different stages. Using organic solvents to extract insects we identified a polar fraction from locusts that allowed appressorial formation against a flat plastic (hydrophobic) surface. Microarrays comprising 1,730 expressed sequence tags were used to determine if this extract elicits different transcriptional programs than whole locust cuticle or nonhost extracts. Of 483 differentially regulated genes, 97% were upregulated. These included genes involved in metabolism, utilization of host cuticle components, cell survival and detoxification, and signal transduction. Surprisingly, given the complex nature of insect epicuticle components and the specific response of M. anisopliae var. acridum to locusts, very similar transcript profiles were observed on locust and beetle extracts. However, the beetle extract cluster was enriched in genes for detoxification and redox processes, while the locust extract upregulated more genes for cell division and accumulation of cell mass. In addition, several signal transduction genes previously implicated in pathogenicity in plant pathogens were only upregulated in response to locust extract, implying similarities in the regulatory circuitry of these pathogens with very different hosts.