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

Abstract

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.

Fig. 1.

A schematic structure of MCL1 (A) and the alignment (clustalw) of MCL1 domain B with collagenous regions from other fungal sequences (B). Up- and down-pointing arrows indicate N-glycosylation sites and cysteine residues, respectively. SP, signal peptide; GPI, glycosylphosphatidylinositol-anchor site. Asterisks show consensus sites. Proteins XP_444847, XP_447814, XP_447815, and XP_447816 are from Candida glabrata; XP_407169 is from Aspergillus nidulans; EAL85438 is from Aspergillus fumigatus; and XP_460045 is from Debaryomyces hansenii.

Fig. 2.

Mcl1 gene induction and protein localization. (A) RT-PCR analysis of Mcl1 expression by wild-type M. anisopliae transferred from Sabouraud dextrose broth (SDB) cultures to minimal medium (MM), fresh SDB, or hemolymph collected from Manduca sexta (MS), Bombyx mori silkworm (BM), Acheta domesticus (house cricket) (AD), Leptinotarsa decimlineata (Colorado potato beetle) (LD), Blaberus giganteus (giant cockroach) (BG), Musca domestica (house fly) (MD), and Magicicada septendecim (cicada) (MA). (B) RT-PCR time course analysis of Mcl1 expression by wild-type M. anisopliae cultured in M. sexta hemolymph. Indirect immunofluorescence (IIF) with antibody abA demonstrating MCL1 production on wild-type mycelia cultured in hemolymph for 40 min (C) or 6 h (D) and on the surface of a wild-type hyphal body from hemolymph 50 h after inoculation (E). (Scale bar, 5 μm.) (F) Western blot analysis using antibody abB against the collagenous domain of MCL1. Cell wall proteins were extracted from mycelia cultured for 24 h on minimal medium (MM), SDB, or M. sexta hemolymph (HEM). Deglycosylation of proteins from hemolymph cultures (DG) produced a substantial reduction in molecular mass. The antibody abA gave the same profile (data not shown). Neither antibody cross-reacted with hemolymph components of uninfected insects.

Fig. 3.

Differences in the patterns of infection shown by wild type and ΔMcl1. Manduca larvae were injected with conidia and bled at 10-h intervals. (A) Wild-type germ tubes emerging from encapsulation 30 h after injection. (B) Heavy encapsulation of ΔMcl1 mutant cells 30 h after injection. The arrows show the emergence of fungal hyphae (note that the center of the capsule is melanized). (C) Wild-type hyphal bodies 50 h after injection unhindered by hemocytes. (D) Encapsulation of ΔMcl1 hyphal body 50 h after injection. (E) Wild-type hyphal body labeled with FITC-conjugated poly(l-lysine) to demonstrate negative charge. (F) Calcofluor staining of wild-type hyphal body. (G) Calcofluor staining of ΔMcl1 hyphal body. (Scale bar, 5 μm.)

Fig. 4.

Kinetics of insect survivorship in bioassays. (A) Mortality of Manduca larvae after topical application with 2 × 10⁷ conidia per ml suspensions of wild-type or ΔMcl1 mutant strains (control insects were dipped in water). LT₅₀ values were 3.61 ± 0.23 days for wild type and 4.85 ± 0.36 days for the mutant (t = 28.22, P = 0.00062). (B) Mortality of Manduca larvae after injection with 10 μl of 5 × 10⁵ conidia per ml suspensions (control insects were injected with 10 μl of water). The LT₅₀ values were 2.12 ± 0.16 days for wild type and 2.83 ± 0.27 days for the mutant (t = 20.49, P = 0.0012).

Fig. 5.

Recognition of blastospores, conidia, and beads by Manduca hemocytes in vitro. Monolayers were exposed to wild-type (WT) or ΔMcl1 (MU) M. anisopliae cells treated with collagenase (Coll), proteinase K (Pro K), lyticase (Lyt), DTT, poly(l-lysine) (PL), or dicyclohexylcarbodiimide and ethylenediamine (D/E). The Dynabeads tested were M270 (hydrophilic) and M280 (hydrophobic) beads. Histograms represent the mean % of the test particles that were attached, ingested, or encapsulated by hemocytes (six monolayers and their associated standard errors) after 1 h. Bars carrying the same letter are not statistically different in terms of mean number of cells or beads recognized by the hemocytes (Dunnett's least significant difference multiple comparison method, α = 0.05).