A collagenous protective coat enables Metarhiziumanisopliae to evade insect immune responsesChengshu Wang and Raymond J. St. Leger*

Department of Entomology, University of Maryland, College Park, MD 20742

Communicated by John H. Law, University of Georgia, Athens, GA, March 10, 2006 (received for review November 3, 2005)

The ubiquitous fungal pathogen Metarhizium anisopliae kills awide range of insects. Host hemocytes can recognize and ingest itsconidia, but this capacity is lost on production of hyphal bodies. Weshow that the unusual ability of hyphal bodies to avoid detectiondepends on a gene (Mcl1) that is expressed within 20 min of thepathogen contacting hemolymph. A mutant disrupted in Mcl1 israpidly attacked by hemocytes and shows a corresponding reduc-tion of virulence to Manduca sexta. Mcl1 encodes a three domainprotein comprising a hydrophilic, negatively charged N-terminalregion with 14 cysteine residues, a central region comprisingtandem repeats (GXY) characteristic of collagenous domains, anda C-terminal region that includes a glycosylphosphatidylinositol-dependent cell wall attachment site. Immunofluorescence assayshowed that hyphal bodies are covered by the N-terminal domainsof MCL1. The collagen domain became antibody accessible aftertreatment with DTT, suggesting that the N termini are linked byinterchain disulfide bonds and are presented on the cell surface byextended collagenous fibers. Studies with staining reagents andhemocyte monolayers showed that MCL1 functions as an antiad-hesive protective coat because it masks antigenic structural com-ponents of the cell wall such as �-glucans, and because itshydrophilic negatively charged nature makes it unattractive tohemocytes. A survey of 54 fungal genomes revealed that sevenother species have proteins with collagenous domains suggestingthat MCL1 is a member of a patchily distributed gene family.

collagen-like protein � virulence � cell wall proteins � fungal pathogen

As the most abundant and diverse land animals, insects haveattracted a variety of pathogens, including viruses and bacteria.

However, most insect disease is caused by fungi, and their impacton insect populations demonstrates the potential of microbialcontrol of insects of medical and agronomic interest (1–3). How-ever, the slow speed of kill and inconsistent results of biologicals ingeneral compared with chemicals has deterred development. Anunderstanding of fungal-induced immune responses would identifythe insect defenses and fungal pathogenicity factors that overcomethem, and hence identify fungal virulence determinants that couldbe manipulated to accelerate host death in a biological controlscenario (4).

Unlike bacteria and viruses that need to be ingested to causedisease, fungi penetrate directly through the cuticle. About 1% ofknown fungal species are capable of breaching the cuticle of at leastsome insect species. These are then fought by the insect innateimmune responses based on both cellular (5) and humoral (6)mechanisms. An immune response starts with recognition of patho-gen-associated molecular patterns (PAMPs), and many of themolecules and receptors involved are homologous in insects andvertebrates (6). For both groups, PAMPs include �-1,3-glucansfrom fungal cell walls (7) as well as nonspecific mechanisms such assurface charge and wettability (8). Various pathways of the immunesystem then become activated (6), leading to the destruction of thepathogen and�or its removal by cellular reactions such as phago-cytosis or encapsulation in many layers of hemocytes. To causeinfection, the fungus has to avoid, subvert, or circumvent thissystem. Given the response of the human immune system to fungal�-glucans, it has been speculated that pathogens may avoid immune

recognition by camouflaging or modifying their �-glucan (9).Indeed, Paracoccidioides brasiliensis displays a transition from�-glucan to �-glucan in the cell wall upon infection of the lung (10).Insect pathogens are also reported to engage in several ‘‘hiding’’tactics that include changes in cell wall composition that eliminatecell surface components associated with non-self recognition, thusallowing hyphal bodies to circulate freely in the hemolymph (11,12). However, the molecular basis of these changes has not beendetermined and it is not clear the extent to which they reflect denovo synthesis of proteins, or morphological and topological rear-rangement of cell surface components.

Recent EST and microarray studies have provided abundantevidence that sets of functionally related genes are coordinatelyinduced or repressed by Metarhizium anisopliae in response to hostrelated stimuli (13–15). Multiple mechanisms specifically involvedin acclimatizing to hemolymph isolated from the lepidopteranmodel insect Manduca sexta include dramatic changes in lipidcomposition, the accumulation of solutes that increase internalosmotic pressure, and up-regulation of nonoxidative respiratorypathways. However, the most highly expressed gene in hemolymph(5.6% of all ESTs) encoded a collagenous protein of unknownfunction (13). In this study, we show that transcripts of Mcl1 (forMetarhizium collagen-like protein) can be detected within 20 min ofthe pathogen contacting hemolymph. Mcl1 encodes a protein witha hydrophilic N-terminal domain that is presented on the cellsurface within 30–45 min of induction by an extended glycosylatedcollagenous region. MCL1 functions as an antiadhesive protectivecoat against phagocytosis and encapsulation because its hydrophilicnegatively charged nature is unattractive to hemocytes and becauseit masks the immunogenic �-1,3-glucan cell wall structural compo-nents. Because hyphal bodies (short hyphal lengths and yeast-likeblastospores) represent the principal stage of replication of thefungus within the host insect hemocoel, the inability to clear thesecells allows the fungus to more easily establish itself and kill the host.

ResultsAnalysis of MCL1 from M. anisopliae. Structural analysis of thepredicted MCL1 protein indicates that it is composed of 605residues (60.4 kDa) that includes an 18-aa secretory sequence anda three domain structure (A, B, and C; Fig. 1A) comprising anN-terminal domain (domain A) predicted to be globular, acidic (pI4.9), and highly hydrophilic, a central collagenous domain (domainB), and a C-terminal region (domain C) that includes a site forattachment of a glycosylphosphatidylinositol (GPI) anchor deducedwith the algorithm of Eisenhaber et al. (16). GPI anchors link to�-1,6-glucans that protrude from the fungal cell wall, suggestingthat MCL1 is a component of the external protein layer that iscovalently linked to the underlying skeletal layer of the wall (17).

Conflict of interest statement: No conflicts declared.

Abbreviations: PAMP, pathogen-associated molecular pattern; GPI, glycosylphosphatidy-linositol; SDB, Sabouraud dextrose broth.

Data deposition: The sequences reported in this paper have been deposited in the GenBankdatabase (accession nos. DQ238488 and DQ238489).

*To whom correspondence should be addressed. E-mail: stleger@umd.edu.

© 2006 by The National Academy of Sciences of the USA

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The great functional versatility of collagens originates from thecombinational assembly of other domains with the collagen domain(18). Many collagens, including mammalian collagen type IV thatcomprises basal membranes, have globular noncollagenous do-mains (19). However, a search of databases showed no significantmatches to the N-terminal domain of MCL1. It contains 14 cysteineresidues consistent with multiple intra- and intermolecular bonds.The collagenous domain itself comprises 33 Gly-X-Y copies inwhich X and Y are frequently Ser, Asn, or Pro. There were sixinterruptions in the regular Gly-X-Y repeats of the MCL1 proteinconsisting of two or three residues (Fig. 1B). Such interruptions leadto flexible sites or kinks and are very common in collagens (18).Similar to many bacterial, viral, and invertebrate collagen-likeproteins (20), domain B has many (n � 13) consensus N-glycosylation sites. Heavy glycosylation would be expected to in-crease rigidity of the domain and produce an elongated structure(21). However, the domain lacked the multiple cysteine residuesfound in many collagens, indicating that it does not form theintermolecular disulfide bridges required for the high tensilestrength and functioning of structural fibers.

The collagen content of fungi has not been well established.Therefore, we conducted unfiltered searches of 54 finished andunfinished fungal genomes using the MCL1 collagenous region or(GPP)7 (22). Seven species (Clavisopora lusitania, Candida glabrata,Debaryomyces hansenii, Aspergillus nidulans, Aspergillus fumigatus,Coccidioides posadasii, and Coccidioides immitis) have sequencesthat include the characteristic Gly-X-Y repeat of collagen domain(Fig. 1B). Most of these sequences also had a three-domainstructure, including a hydrophilic 5� domain containing variablenumbers of cysteine residues, a central domain of variable numbers(22–52) of G-X-Y repeats with multiple glycosylation sites, and a 3�

domain that is variable in length (absent in the D. hansenii protein).Proline is a major component of X and Y of most collagens. Itcomprised 21.2% of the X and 15.2% of the Y residues in MCL1,as compared with mammalian collagens that contain 28.2% Pro atX and 38.4% Pro at Y. The average Pro content of the fungalG-X-Y domains at the Y position is 33.8%, as compared with 12.5%for viruses and 4.2% for bacterial collagens (22). The percentage ofPro residues at the X site varied from zero in C. glabrata to 56% inA. fumigatus (Fig. 6, which is published as supporting informationon the PNAS web site).

Induction of MCL1 by Hemolymph Constituents. We performedRT-PCR analysis of Mcl1 expression by mycelia suspended indifferent media. Mcl1 transcripts were detected during growth inthe hemolymph of a diverse array of insects, consistent with thebroad host range of M. anisopliae. However, Mcl1 was not expressedin nutrient-rich artificial media or during starvation conditions,suggesting that it is only involved in pathogenesis (Fig. 2A). A timecourse demonstrated that Mcl1 transcripts began to appear within20 min of transfer into M. sexta hemolymph and were still accu-mulating at 4 h (Fig. 2B).

For MCL1 to function in avoiding the host immune system, itmust be located on the cell surface. This was verified by using anindirect immunofluoresence (IIF) assay with rabbit antibodies(abA) raised against a peptide sequence from the noncollagenousdomain A. No fluorescence was detected on conidia or myceliagrown in nutrient broth or minimal medium, but MCL1 wasdetectable on hyphal tips 30–45 min after induction with hemo-lymph, and levels of staining increased over several hours (Fig. 2 Cand D). The accessibility of the N-terminal domain to abA estab-lishes that it is presented on the surface of the cell. In contrast, the

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- anddown-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.

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collagenous domain of MCL1 was not available to antibodies (abB)raised against a constituent peptide sequence, unless cells werepretreated with DTT. This finding suggests that domain B isinternal to domain A and that disulfide bonds interconnecting theN termini of MCL1 proteins are involved in creating a nonporousbarrier. Without exception, IIF of several hundred hyphal bodiesisolated at 10-h intervals from the hemolymph of infected insectsshowed strong, even surface staining with abA (Fig. 2E). However,like hyphae in hemolymph in vitro, they were not recognized by abB.

Characterization of the Posttranslational Modifications of MCL1.Western blot analysis of extracts of mycelial cell walls, harvestedfrom insect hemolymph in vitro, confirmed that both abA and abBrecognize a polydisperse band with an apparent molecular massranging as high as 300 kDa (Fig. 2F). Disperse bands are typical ofextensive glycosylation so the protein was treated with N-glycosidase F to remove all N-chains. Deglycosylation caused theprotein to run as a single sharp 75-kDa band consistent with heavyglycosylation of the collagenous domain, where all but one of theconsensus N-glycosylation sites are found. The difference with themolecular mass predicted from the amino acid sequence (60.4 kDa)is presumably due to the GPI tail (�5 kDa) and O-mannosylation.

Behavior of Wild-Type M. anisopliae and a �Mcl1 Null Mutant WithinInfected Manduca. To study the role of MCL1, we constructed astrain of M. anisopliae in which the Mcl1 gene was disrupted. Nofluorescence with abA was detected on hyphal bodies of the nullmutant in hemolymph in vitro or in vivo. Because conidia are of asimilar size (�7 �m) to blastospores but lack MCL1 protein, weinjected conidia directly into the hemocoel of larval M. sexta tostudy the functional significance of their different surface proper-

ties. Within 10 min of injection, single conidium from the wild-typeand �Mcl1 strains had attached to hemocytes or become phago-cytosed, whereas clumps of conidia were encapsulated, showingthat they are readily recognized as foreign. It was not alwayspossible to distinguish by microscopic observation between hemo-cyte attachment and subsequent phagocytosis, but propagules of M.anisopliae are known to survive phagocytosis and grow within hostcells (12, 23). Survival of conidia during their initial interactionswith host cells was not dependent on Mcl1, because �90% of bothwild-type and mutant �Mcl1 conidia germinated within 8–10 h.However, hyphae and hyphal bodies produced by �Mcl1 conidiacontinued to recruit hemocytes and were repeatedly encapsulated,whereas wild-type hyphae emerged from capsules and budded offhyphal bodies that received little attention from the hemocytes (Fig.3 A–D). Thirty hours after injection of conida, capsule diametersaveraged 75.6 � 18.1 �m (n � 82) and 200.3 � 29.9 �m (n � 73)in insects infected with the wild type and �Mcl1, respectively. Thisfinding suggests that, during conidial germination, the wild-type canrapidly adapt the composition of the newly formed cell wall inresponse to the hemolymph environment, with resulting changes inligands on cell surfaces from those present on its conidia or on the

Fig. 2. Mcl1 gene induction and protein localization. (A) RT-PCR analysis ofMcl1 expression by wild-type M. anisopliae transferred from Sabouraud dex-trose broth (SDB) cultures to minimal medium (MM), fresh SDB, or hemolymphcollected from Manduca sexta (MS), Bombyx mori silkworm (BM), Achetadomesticus (house cricket) (AD), Leptinotarsa decimlineata (Colorado potatobeetle) (LD), Blaberus giganteus (giant cockroach) (BG), Musca domestica(house fly) (MD), and Magicicada septendecim (cicada) (MA). (B) RT-PCR timecourse analysis of Mcl1 expression by wild-type M. anisopliae cultured in M.sexta hemolymph. Indirect immunofluorescence (IIF) with antibody abA dem-onstrating MCL1 production on wild-type mycelia cultured in hemolymph for40 min (C) or 6 h (D) and on the surface of a wild-type hyphal body fromhemolymph 50 h after inoculation (E). (Scale bar, 5 �m.) (F) Western blotanalysis using antibody abB against the collagenous domain of MCL1. Cell wallproteins were extracted from mycelia cultured for 24 h on minimal medium(MM), SDB, or M. sexta hemolymph (HEM). Deglycosylation of proteins fromhemolymph cultures (DG) produced a substantial reduction in molecular mass.The antibody abA gave the same profile (data not shown). Neither antibodycross-reacted with hemolymph components of uninfected insects.

Fig. 3. Differences in the patterns of infection shown by wild type and�Mcl1. 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 �Mcl1 mutant cells 30 h after injection. The arrowsshow the emergence of fungal hyphae (note that the center of the capsule ismelanized). (C) Wild-type hyphal bodies 50 h after injection unhindered byhemocytes. (D) Encapsulation of �Mcl1 hyphal body 50 h after injection. (E)Wild-type hyphal body labeled with FITC-conjugated poly(L-lysine) to dem-onstrate negative charge. (F) Calcofluor staining of wild-type hyphal body. (G)Calcofluor staining of �Mcl1 hyphal body. (Scale bar, 5 �m.)

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�Mcl1 mutant. Encapsulation of the �Mcl1 mutant continued 50 hafter injection (Fig. 3D) and only ceased with the manifestation ofobvious disease symptoms such as reduced food uptake and soft-ening of the body.

The ability of �Mcl1 to survive and cause disease indicates thatM. anisopliae has additional mechanisms to cope with immuneresponses. However, LT50 values from injection assays showed thatthe �Mcl1 mutant takes a significantly longer time (P � 0.0012) tokill insects than the wild type (Fig. 4). Insects were also dipped insuspensions of conidia to assay infections through the cuticle. At ahigh (2 � 107 conidia per ml) dosage, the �Mcl1 mutant tooksignificantly longer to kill than the wild type (P � 0.0006) (Fig. 4).At 8 � 106 conidia per ml, �Mcl1 failed to achieve 50% mortalitybefore pupation and, at lower (�5 � 106) dosages, mortality fell to�10%. In contrast, the wild type achieved �50% larval mortalityat 1 � 106 conidia per ml, suggesting that it would be much morelikely than �Mcl1 to cause high mortality under field conditions,where concentrations seldom exceed 106 conidia per g of soil (2).

Hemocyte Monolayer Assays. Given the differences in how hemo-cytes in infected insects behave to wild type and �Mcl1, we alsoinvestigated the effects of deleting Mcl1 on cell surface propertiesand hemocyte responses in vitro. Cell surface hydrophobicity wasmeasured by using a microsphere adhesion assay. Conidia from thewild type and �Mcl1 were similarly hydrophobic with 15.37 � 1.21and 15.14 � 1.59 beads attached per cell, respectively. Despiteconidia and blastospores being very similar in size, this represents�3-fold more microspheres than adhered to blastospores of thewild type (3.28 � 0.16 beads per spore) and �Mcl1 (4.94 � 0.48beads per spore). The �30% fewer microspheres adhering towild-type blastospores demonstrates that MCL1 produces a smallbut significant (t � 6.81, P � 0.0103) overall increase in cell surfacewettability. This finding suggests that MCL1 disruption had un-masked components of blastospores that were also hydrophilic, butless so than MCL1. Hydrophobic Dynabeads exhibit a much greaterattraction to insect hemocytes than do hydrophilic beads (t � 21.31,P � 0.0011) (Fig. 5), so loss of hydrophilic MCL1 would be expectedto increase attractiveness to hemocytes. Indeed, hemocyte mono-layer assays agreed with the infection studies in showing that �Mcl1blastospores are recognized at �3-fold higher efficiency than arewild-type blastospores (Fig. 5). We investigated whether this couldbe due to an ability by hemocytes to distinguish between differentcell surface charges or degrees of hydrophilicity and�or to theexposure of PAMPs underlying the MCL1 layer.

The surface charge of outer cell surfaces was assessed usingFITC-labeled poly(L-lysine). Only a faint fluorescence was ob-served on conidia, but blastospores of the wild type and the �Mcl1mutant were negatively charged (Fig. 3E). Blastospores preincu-

bated with unconjugated poly(L-lysine) before treatment with theFITC probe did not fluoresce, indicating that negative surfacecharges were neutralized. However, they remained hydrophilic(3.59 � 0.23 microspheres per spore), suggesting that the hydro-philicity of blastospores is not due to their electronegativity. Toconfirm this possibility, cell surface electronegativity was alsoreduced by treating wild-type blastospores with dicyclohexylcarbo-diimide and ethylenediamine to replace negatively charged carboxylgroups with positively charged ammonium groups (24). The deriv-itized wild-type cells still showed a lower degree of binding tomicrospheres (3.87 � 0.39 beads per spore) than did �Mcl1blastospores (t � 4.37, P � 0.0243). The difference with untreatedwild-type cells was not significant (t � 1.26, P � 0.05), and they werenot more readily recognized than untreated cells (Fig. 5). Thus, thedifferential hemocyte response elicited by the �Mcl1 mutant is nota nonspecific reaction to charge.

The surface exposure of �-glucans was measured by the degreeof binding of Calcoflour white. Strong fluorescence was observedon the �Mcl1 blastospores, but the wild-type blastospores werebarely visible using the same exposure time (Fig. 3 F and G). This

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-typeor �Mcl1 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 �l of 5 � 105 conidia per ml suspensions (control insects were injected with 10 �l 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 hemo-cytes in vitro. Monolayers were exposed to wild-type (WT) or �Mcl1 (MU) M.anisopliae cells treated with collagenase (Coll), proteinase K (Pro K), lyticase(Lyt), DTT, poly(L-lysine) (PL), or dicyclohexylcarbodiimide and ethylenedia-mine (D�E). The Dynabeads tested were M270 (hydrophilic) and M280 (hy-drophobic) beads. Histograms represent the mean % of the test particles thatwere attached, ingested, or encapsulated by hemocytes (six monolayers andtheir associated standard errors) after 1 h. Bars carrying the same letter are notstatistically different in terms of mean number of cells or beads recognized bythe hemocytes (Dunnett’s least significant difference multiple comparisonmethod, � � 0.05).

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finding demonstrates that MCL1 renders PAMPs, such as �-1,3-glucans, inaccessible to arriving hemocytes. Treatment of wild-typecells with collagenase or proteinase K prevented labeling withantibodies to MCL1 but produced strong fluorescence with Cal-cofluor and increased their recognition to the levels shown for�Mcl1 (Fig. 5), confirming that a protein component of wild-typecells blocks hemocyte responses. Additionally, treatment of �Mcl1cells with lyticase (a �-1,3-glucanase) greatly reduced labeling withCalcofluor and phagocytosis compared with untreated cells (t �15.67, P � 0.0051), confirming that hyphal bodies are recognized oncontact with �-1,3-glucans.

DiscussionIn the course of infecting a host, pathogens are presented with awide array of host environments. Cell surface proteins and secretedhydrolases will define the interactions between host and pathogenand together are likely to have a profound impact on the infectionoutcome (25). Because of the functional adaptations of GPIproteins to localization at the cell surface, they likely comprise themajority of Candida spp. surface proteins involved in humandisease (26). The agglutinin-like protein (ALS) cell surface ad-hesins of C. albicans are particularly informative in light of theirsimilarities and differences with MCL1. Although lacking a collag-enous domain, ALS proteins have a relatively nonglycosylatedcysteine rich N-terminal domain that is displayed on the cell surfaceby the remainder of the protein that is extended because of its heavyglycosylation (27). The central region is so heavily glycosylated that,like MCL1, ALS proteins migrate at three to five times theirpredicted molecular weights (28). However, unlike MCL1, theyhave hydrophobic N-terminal domains that facilitate adhesion tohost tissues (28, 29). There is an abundance of literature identifyingthe hydrophobic effect as the driving force for the initial adhesionof pathogens to host surfaces that establishes infection (reviewed inref. 30). However, the MCL1 protein has the opposite function ofproducing a nonadherent cell; to achieve this, it provides a hydro-philic antiencapsulation coat around the fungus that preventsrecognition by hemocytes. Therefore, it appears analogous infunction to the extracellular polysaccharide capsule produced byCryptococcus neoformans to avoid recognition by phagocytes (31).

A major part of the PAMP, the MCL1 protein blocks is �-glucansin the cell wall, but it also contributes to properties of the cellsurface that reduce attraction to insect hemocytes. These includephysiochemical properties such as wettability, and it is also perti-nent that MCL1 lacks the tripeptide Arg-Gly-Asp (RGD) sequenceas hemocytes possess an RGD-dependent adhesion mechanism(32). Rapid encapsulation of hydrophobic conidia despite theirfailure to bind Calcofluor suggests that hemocytes respond toseveral criteria including nonspecific mechanisms that can inde-pendently induce an immune response. The wettability of blasto-spore surfaces has also been noted in other insect pathogens (11)and could be consequential for infection processes in many ways,besides just avoiding a direct hemocyte response. The negativelycharged hydrophilic surface is likely to prevent clumping of cells andattachment to host surfaces thus facilitating dispersal through theinsect. Hydrophilic surfaces are also much more resistant to nonspecific adsorption of proteins (33), lectins, and other opsonins (30),minimizing the possibility of opsonization by �-1,3-glucan bindingproteins. The MCL1 coat probably protects against several �-glucanbinding proteins in M. sexta. A laminarin-binding M. sexta lectinpromotes encapsulation (34), and cell wall �-glucans activate thephenoloxidase cascade (35), so it is significant that encapsulated�Mcl1 propagules were frequently melanized (Fig. 3B). Becausehost humoral responses are also triggered by PAMPs (7), MCL1will likely be involved in avoiding these as well. However, bothwild-type and �Mcl1 mutant hyphae survive challenge with theantimicrobial cercopin A at a level (50 �M) sufficient to killsaprobic fungi (C.W. and R.J.S.L., unpublished data). This finding

suggests that M. anisopliae has evolved multiple strategies that allowit to grow unhindered by the insect immune system.

A further unique feature of this research was the identification ofa collagenous Gly-X-Y domain in a fungus. Collagens are the mostabundant proteins in the vertebrate body and are essential struc-tural elements that evolutionary models trace to fibrillar forms inearly animals (18, 36). The presence of collagen in the cell walls ofthe human pathogen C. albicans had been inferred by immunolog-ical analysis (37), but we found no collagen sequence in thepublished genome of C. albicans. The fungus Ustilago violaceum wasalso inferred to contain collagenous protein (38), but no gene hasbeen identified. In contrast, the characteristic triple-helical struc-ture formed by Gly-X-Y motifs has been found in bacterial colla-gens (39). Demonstrating that MCL1 forms the trimers character-istic of collagen fibers is beyond the scope of this paper. However,the MCL1 sequence has all of the features expected of a collagenand apparently none that would preclude a multimeric helicoidalstructure. The high proline content of MCL1 domain B that ischaracteristic of most collagens will prevent secondary structure. Inaddition, the heavy glycosylation also characteristic of collagens islikely to confer an extended conformation on that portion of themolecule (21). This will project the relatively glycosylation freeN-terminal domain into the extracellular milieu. Thus, the exper-imental support for the surface location of domain A also makesintuitive sense. An interesting potential model for the structure ofprotein motifs in MCL1 is provided by the collectins, a family ofanimal lectins that recognize PAMPs. These also have a cysteine-rich N-terminal domain and a heavily glycosylated collagenousregion with interruptions (kinks). The basic functional unit is atrimer, but the kinks provide flexibility, enabling the trimericsubunits to cover more area. The trimers are stabilized andassembled into larger oligomers via the cysteine residues (40). Thepossibility of interchain disulfide bridges in MCL1 is consistent withthe ability of DTT to permeabilize the MCL1 sheath around thefungus.

The presence of a collagenous domain is not associated with aspecific lifestyle, i.e., they are present in saprobic fungi as well aspathogens. The patchy distribution of collagen-like proteins amongbacteria and viruses is supposed to derive from horizontal genetransfer from multicellular animals (22). The apparently similarsparse distribution of the collagenous domain in fungi could also beexplained by repeated independent instances of gene loss (41), orby convergent evolution, particularly as alignment of collagenousregions indicates high sequence divergence (Fig. 1B). However, itis interesting to note that three of four C. galabrata collagenousproteins (XP�447814, XP�447815 and XP�447816) have �50%overall similarity and locate in tandem within 12 kb on chromosomeJ, indicative of recent duplication events and diverging functions.Given that most fungi lack homologs for MCL1 or other proteinswith a collagenous domain, they are evidently expendable in termsof maintaining the normal organization of fungal hyphae. The rolesthey play in the fungi that possess them will probably therefore haveto be addressed on a case-by-case basis.

Materials and MethodsFor further details, see Figs. 7–9, which are published as supportinginformation on the PNAS web site.

Gene Cloning and Deletion. The cDNA of Mcl1 was fully sequenced,and the genomic DNA was acquired by using a primer walking kit(Seegene) from M. anisopliae strain ARSEF2575. The proceduresfor construction of gene knockout plasmids and fungal transfor-mation are provided in Fig. 8.

Cell Wall Protein Isolation and Western Blot Analysis. Thirty-six-hourSabouraud dextrose broth (SDB) cultures were washed three timeswith sterile distilled water and then transferred either to minimummedium (MM) or to isolated Manduca hemolymph as described

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(13) for up to 24 h. Fungal cell wall proteins were extracted as before(17). In-solution deglycosylation was conducted by using a Glyco-Profile II kit (Sigma). Two predicted antigenic regions, A1 (PGP-NASPDQIKKHRD; residues 59–73 of the N-terminal domain)and B1 (NGKPGSGNNGANGSN; residues 421–435 of the col-lagenous domain) were synthesized and conjugated with keyholelimpet hemocyanin. The antibodies were raised in New ZealandWhite rabbits (Sigma) and designated as abA and abB. Western blotanalyses were conducted as described (42).

Indirect Immunofluorescence (IIF). Fungal cells grown in hemolymphin vitro or in vivo were prepared for IIF as described (37). Antibodieswere diluted 500-fold and FITC-conjugated-goat anti-rabbit Ig G(Sigma) was used for secondary labeling. Control samples of cellswere treated as above but minus either the primary or secondaryantibodies.

Insect Bioassay. Virulence of wild-type and �Mcl1 mutant conidiawas assayed against newly molted fifth-instar larvae of M. sexta asdescribed (4). Thus, conidia were applied topically by immersion oflarvae or by injecting the rearmost proleg with 10 �l of an aqueoussuspension containing 5 � 105 spores per ml. Each treatment wasreplicated three times with 10 insects per replicate, and the exper-iments were repeated twice. Mortality was recorded every 12 h, andestimated lethal time values for 50% mortality (LT50) were usedto compare speed of kill between strains with the t test as before(4). Additional infected insects were bled at 10-h intervals formicroscopic observation of fungal development within the insecthaemocoel.

Hemocyte Monolayer Assay. Hemolymph was collected in prechilledsaline buffer (43) from day 2 fifth-instar larvae and applied as asuspension of 2 � 106 cells per ml onto glass coverslips (10-mmdiameter). The coverslips were incubated in Grace’s medium at28°C for 2 h and then washed twice with 0.5 ml Grace’s medium(43). Wild-type and �Mcl1 blastospores (harvested from fungalcultures grown in hemolymph in vitro for 72 h) or conidia werewashed twice with PBS, and 2 � 103 cells were applied to thehemocyte monolayers to assay recognition. In some experiments,fungal cells were fixed in 4% formaldehyde or pretreated for 1 h inPBS containing either DTT, poly(L-lysine), lyticase, proteinase K,or collagenase (Sigma) (the enzymes at 200 �g�ml) before assaying.

Hydrophobic (M280) and hydrophilic (M270) Dynabeads (2.8 �min diameter, Dynal) were used as references to test the effects ofnonspecific surface properties on hemocyte responses. After incu-bation of the monolayer coverslips for 1 h at 28°C, the percentageof test particles recognized by the hemocytes (the number ofparticles bound or ingested by hemocytes relative to the numberadded) was determined in five different fields of vision using the40� objective. Data shown were calculated from 600 or more cellsor beads�monolayer�insect and six insects per treatment.

Characterization of Fungal Cell Surface Properties. A microsphereadhesion assay of cell surface hydrophobicity was conducted byusing 0.6-�m latex polystyrene beads (Sigma) (44). The spores weresuspended in 0.1 M KNO3 solution (2 � 107 cells per ml, pH 6.5),and the suspensions were mixed with microspheres suspended inthe same buffer in a ratio of 20 beads�one spore. Three replicatesfor each treatment were performed, and a total of 50 spores werecounted for each replicate. To modify cell surface charge, blasto-spores were treated with dicyclohexylcarbodiimide and ethylenedi-amine. By this method, carbodiimide-activated carboxylate groupsare substituted with positively charged ammonium groups from theethylenediamine (24). FITC-labeled poly(L-lysine) was used toassay surface charge (8). Staining with Calcofluor White was usedto measure the exposure of �-glucans on the cell surfaces ofwild-type and mutant hyphal bodies (45).

RT-PCR. Mycelia from 36-h SDB cultures (0.1 g wet weight) waswashed twice with sterile water before transfer into minimalmedium, fresh SDB, or hemolymph collected from seven insectspecies. At different time points, RNA (0.5 �g) was extracted andconverted into single-strand cDNA using an anchored oligo(dT)primer (ABgene, Surrey, U.K.). Complementary DNA samplesdiluted 500-fold were used as template for PCR. Primers designedfor the small subunit ribosomal RNA were used as the reference.

Statistical Analysis. Student’s t test was used for the pairwisecomparisons of means given in the text. Dunnett’s least significancedifference multiple comparison method in the program SPSS (ver-sion 11.0.0) was used to compare the different treatments shown inFig. 5.

This work was supported by National Research Initiative of the U.S.Department of Agriculture Cooperative State Research, Education, andExtension Service Grant 2003-353-02-13588.

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