A collagenous protective coat enables anisopliae to evade immune responses

Chengshu 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 a recognition by camouflaging or modifying their ␤-glucan (9). wide range of . Host hemocytes can recognize and ingest its Indeed, Paracoccidioides brasiliensis displays a transition from conidia, but this capacity is lost on production of hyphal bodies. We ␤-glucan to ␣-glucan in the cell wall upon infection of the lung (10). show that the unusual ability of hyphal bodies to avoid detection Insect pathogens are also reported to engage in several ‘‘hiding’’ depends on a gene (Mcl1) that is expressed within 20 min of the tactics that include changes in cell wall composition that eliminate pathogen contacting hemolymph. A mutant disrupted in Mcl1 is cell surface components associated with non-self recognition, thus rapidly attacked by hemocytes and shows a corresponding reduc- allowing hyphal bodies to circulate freely in the hemolymph (11, tion of virulence to Manduca sexta. Mcl1 encodes a three domain 12). However, the molecular basis of these changes has not been protein comprising a hydrophilic, negatively charged N-terminal determined and it is not clear the extent to which they reflect de region with 14 cysteine residues, a central region comprising novo synthesis of proteins, or morphological and topological rear- tandem repeats (GXY) characteristic of collagenous domains, and rangement of cell surface components. a C-terminal region that includes a glycosylphosphatidylinositol- Recent EST and microarray studies have provided abundant dependent cell wall attachment site. Immunofluorescence assay evidence that sets of functionally related genes are coordinately showed that hyphal bodies are covered by the N-terminal domains induced or repressed by Metarhizium anisopliae in response to host of MCL1. The collagen domain became antibody accessible after related stimuli (13–15). Multiple mechanisms specifically involved treatment with DTT, suggesting that the N termini are linked by in acclimatizing to hemolymph isolated from the lepidopteran interchain disulfide bonds and are presented on the cell surface by model insect Manduca sexta include dramatic changes in lipid extended collagenous fibers. Studies with staining reagents and composition, the accumulation of solutes that increase internal hemocyte monolayers showed that MCL1 functions as an antiad- osmotic pressure, and up-regulation of nonoxidative respiratory hesive protective coat because it masks antigenic structural com- pathways. However, the most highly expressed gene in hemolymph ponents of the cell wall such as ␤-glucans, and because its (5.6% of all ESTs) encoded a collagenous protein of unknown hydrophilic negatively charged nature makes it unattractive to function (13). In this study, we show that transcripts of Mcl1 (for hemocytes. A survey of 54 fungal genomes revealed that seven Metarhizium collagen-like protein) can be detected within 20 min of other species have proteins with collagenous domains suggesting the pathogen contacting hemolymph. Mcl1 encodes a protein with

that MCL1 is a member of a patchily distributed gene family. a hydrophilic N-terminal domain that is presented on the cell IMMUNOLOGY surface within 30–45 min of induction by an extended glycosylated ͉ ͉ ͉ collagen-like protein virulence cell wall proteins fungal pathogen collagenous region. MCL1 functions as an antiadhesive protective coat against phagocytosis and encapsulation because its hydrophilic s the most abundant and diverse land animals, insects have negatively charged nature is unattractive to hemocytes and because Aattracted a variety of pathogens, including viruses and bacteria. it masks the immunogenic ␤-1,3-glucan cell wall structural compo- However, most insect disease is caused by fungi, and their impact nents. Because hyphal bodies (short hyphal lengths and yeast-like on insect populations demonstrates the potential of microbial blastospores) represent the principal stage of replication of the control of insects of medical and agronomic interest (1–3). How- within the host insect hemocoel, the inability to clear these ever, the slow speed of kill and inconsistent results of biologicals in cells allows the fungus to more easily establish itself and kill the host. general compared with chemicals has deterred development. An understanding of fungal-induced immune responses would identify Results the insect defenses and fungal pathogenicity factors that overcome Analysis of MCL1 from M. anisopliae. Structural analysis of the them, and hence identify fungal virulence determinants that could predicted MCL1 protein indicates that it is composed of 605 be manipulated to accelerate host death in a biological control residues (60.4 kDa) that includes an 18-aa secretory sequence and scenario (4). a three domain structure (A, B, and C; Fig. 1A) comprising an Unlike bacteria and viruses that need to be ingested to cause N-terminal domain (domain A) predicted to be globular, acidic (pI disease, fungi penetrate directly through the cuticle. About 1% of 4.9), and highly hydrophilic, a central collagenous domain (domain known fungal species are capable of breaching the cuticle of at least B), and a C-terminal region (domain C) that includes a site for some insect species. These are then fought by the insect innate attachment of a glycosylphosphatidylinositol (GPI) anchor deduced immune responses based on both cellular (5) and humoral (6) with the algorithm of Eisenhaber et al. (16). GPI anchors link to mechanisms. An immune response starts with recognition of patho- ␤-1,6-glucans that protrude from the fungal cell wall, suggesting gen-associated molecular patterns (PAMPs), and many of the that MCL1 is a component of the external protein layer that is molecules and receptors involved are homologous in insects and covalently linked to the underlying skeletal layer of the wall (17). vertebrates (6). For both groups, PAMPs include ␤-1,3-glucans from fungal cell walls (7) as well as nonspecific mechanisms such as surface charge and wettability (8). Various pathways of the immune Conflict of interest statement: No conflicts declared. system then become activated (6), leading to the destruction of the Abbreviations: PAMP, pathogen-associated molecular pattern; GPI, glycosylphosphatidy- pathogen and͞or its removal by cellular reactions such as phago- linositol; SDB, Sabouraud dextrose broth. cytosis or encapsulation in many layers of hemocytes. To cause Data deposition: The sequences reported in this paper have been deposited in the GenBank infection, the fungus has to avoid, subvert, or circumvent this database (accession nos. DQ238488 and DQ238489). system. Given the response of the human immune system to fungal *To whom correspondence should be addressed. E-mail: [email protected]. ␤-glucans, it has been speculated that pathogens may avoid immune © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601951103 PNAS ͉ April 25, 2006 ͉ vol. 103 ͉ no. 17 ͉ 6647–6652 Downloaded by guest on September 27, 2021 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.

The great functional versatility of collagens originates from the domain that is variable in length (absent in the D. hansenii protein). combinational assembly of other domains with the collagen domain Proline is a major component of X and Y of most collagens. It (18). Many collagens, including mammalian collagen type IV that comprised 21.2% of the X and 15.2% of the Y residues in MCL1, comprises basal membranes, have globular noncollagenous do- as compared with mammalian collagens that contain 28.2% Pro at mains (19). However, a search of databases showed no significant X and 38.4% Pro at Y. The average Pro content of the fungal matches to the N-terminal domain of MCL1. It contains 14 cysteine G-X-Y domains at the Y position is 33.8%, as compared with 12.5% residues consistent with multiple intra- and intermolecular bonds. for viruses and 4.2% for bacterial collagens (22). The percentage of The collagenous domain itself comprises 33 Gly-X-Y copies in Pro residues at the X site varied from zero in C. glabrata to 56% in which X and Y are frequently Ser, Asn, or Pro. There were six A. fumigatus (Fig. 6, which is published as supporting information interruptions in the regular Gly-X-Y repeats of the MCL1 protein on the PNAS web site). consisting of two or three residues (Fig. 1B). Such interruptions lead to flexible sites or kinks and are very common in collagens (18). Induction of MCL1 by Hemolymph Constituents. We performed Similar to many bacterial, viral, and invertebrate collagen-like RT-PCR analysis of Mcl1 expression by mycelia suspended in proteins (20), domain B has many (n ϭ 13) consensus N- different media. Mcl1 transcripts were detected during growth in glycosylation sites. Heavy glycosylation would be expected to in- the hemolymph of a diverse array of insects, consistent with the crease rigidity of the domain and produce an elongated structure broad host range of M. anisopliae. However, Mcl1 was not expressed (21). However, the domain lacked the multiple cysteine residues in nutrient-rich artificial media or during starvation conditions, found in many collagens, indicating that it does not form the suggesting that it is only involved in pathogenesis (Fig. 2A). A time intermolecular disulfide bridges required for the high tensile course demonstrated that Mcl1 transcripts began to appear within strength and functioning of structural fibers. 20 min of transfer into M. sexta hemolymph and were still accu- The collagen content of fungi has not been well established. mulating at 4 h (Fig. 2B). Therefore, we conducted unfiltered searches of 54 finished and For MCL1 to function in avoiding the host immune system, it unfinished fungal genomes using the MCL1 collagenous region or must be located on the cell surface. This was verified by using an (GPP)7 (22). Seven species (Clavisopora lusitania, Candida glabrata, indirect immunofluoresence (IIF) assay with rabbit antibodies Debaryomyces hansenii, Aspergillus nidulans, Aspergillus fumigatus, (abA) raised against a peptide sequence from the noncollagenous Coccidioides posadasii, and Coccidioides immitis) have sequences domain A. No fluorescence was detected on conidia or mycelia that include the characteristic Gly-X-Y repeat of collagen domain grown in nutrient broth or minimal medium, but MCL1 was (Fig. 1B). Most of these sequences also had a three-domain detectable on hyphal tips 30–45 min after induction with hemo- structure, including a hydrophilic 5Ј domain containing variable lymph, and levels of staining increased over several hours (Fig. 2 C numbers of cysteine residues, a central domain of variable numbers and D). The accessibility of the N-terminal domain to abA estab- (22–52) of G-X-Y repeats with multiple glycosylation sites, and a 3Ј lishes that it is presented on the surface of the cell. In contrast, the

6648 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601951103 Wang and St. Leger Downloaded by guest on September 27, 2021 Fig. 2. Mcl1 gene induction and protein localization. (A) RT-PCR analysis of Mcl1 expression by wild-type M. anisopliae transferred from Sabouraud dex- trose broth (SDB) cultures to minimal medium (MM), fresh SDB, or hemolymph collected from Manduca sexta (MS), Bombyx mori silkworm (BM), Acheta domesticus (house ) (AD), Leptinotarsa decimlineata (Colorado potato ) (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 dem- onstrating MCL1 production on wild-type mycelia cultured in hemolymph for 40 min (C)or6h(D) and on the surface of a wild-type hyphal body from hemolymph 50 h after inoculation (E). (Scale bar, 5 ␮m.) (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. IMMUNOLOGY

collagenous domain of MCL1 was not available to antibodies (abB) Fig. 3. Differences in the patterns of infection shown by wild type and raised against a constituent peptide sequence, unless cells were ⌬Mcl1. Manduca larvae were injected with conidia and bled at 10-h intervals. pretreated with DTT. This finding suggests that domain B is (A) Wild-type germ tubes emerging from encapsulation 30 h after injection. ⌬ internal to domain A and that disulfide bonds interconnecting the (B) Heavy encapsulation of Mcl1 mutant cells 30 h after injection. The arrows show the emergence of fungal hyphae (note that the center of the capsule is N termini of MCL1 proteins are involved in creating a nonporous melanized). (C) Wild-type hyphal bodies 50 h after injection unhindered by barrier. Without exception, IIF of several hundred hyphal bodies hemocytes. (D) Encapsulation of ⌬Mcl1 hyphal body 50 h after injection. (E) isolated at 10-h intervals from the hemolymph of infected insects Wild-type hyphal body labeled with FITC-conjugated poly(L-lysine) to dem- showed strong, even surface staining with abA (Fig. 2E). However, onstrate negative charge. (F) Calcofluor staining of wild-type hyphal body. (G) like hyphae in hemolymph in vitro, they were not recognized by abB. Calcofluor staining of ⌬Mcl1 hyphal body. (Scale bar, 5 ␮m.)

Characterization of the Posttranslational Modifications of MCL1. ties. Within 10 min of injection, single from the wild-type Western blot analysis of extracts of mycelial cell walls, harvested and ⌬Mcl1 strains had attached to hemocytes or become phago- from insect hemolymph in vitro, confirmed that both abA and abB cytosed, whereas clumps of conidia were encapsulated, showing recognize a polydisperse band with an apparent molecular mass that they are readily recognized as foreign. It was not always ranging as high as 300 kDa (Fig. 2F). Disperse bands are typical of possible to distinguish by microscopic observation between hemo- extensive glycosylation so the protein was treated with N- cyte attachment and subsequent phagocytosis, but propagules of M. glycosidase F to remove all N-chains. Deglycosylation caused the anisopliae are known to survive phagocytosis and grow within host protein to run as a single sharp 75-kDa band consistent with heavy cells (12, 23). Survival of conidia during their initial interactions glycosylation of the collagenous domain, where all but one of the with host cells was not dependent on Mcl1, because Ͼ90% of both consensus N-glycosylation sites are found. The difference with the wild-type and mutant ⌬Mcl1 conidia germinated within 8–10 h. molecular mass predicted from the amino acid sequence (60.4 kDa) However, hyphae and hyphal bodies produced by ⌬Mcl1 conidia is presumably due to the GPI tail (Ϸ5 kDa) and O-mannosylation. continued to recruit hemocytes and were repeatedly encapsulated, whereas wild-type hyphae emerged from capsules and budded off Behavior of Wild-Type M. anisopliae and a ⌬Mcl1 Null Mutant Within hyphal bodies that received little attention from the hemocytes (Fig. Infected Manduca. To study the role of MCL1, we constructed a 3 A–D). Thirty hours after injection of conida, capsule diameters strain of M. anisopliae in which the Mcl1 gene was disrupted. No averaged 75.6 Ϯ 18.1 ␮m(n ϭ 82) and 200.3 Ϯ 29.9 ␮m(n ϭ 73) fluorescence with abA was detected on hyphal bodies of the null in insects infected with the wild type and ⌬Mcl1, respectively. This mutant in hemolymph in vitro or in vivo. Because conidia are of a finding suggests that, during conidial germination, the wild-type can similar size (Ϸ7 ␮m) to blastospores but lack MCL1 protein, we rapidly adapt the composition of the newly formed cell wall in injected conidia directly into the hemocoel of larval M. sexta to response to the hemolymph environment, with resulting changes in study the functional significance of their different surface proper- ligands on cell surfaces from those present on its conidia or on the

Wang and St. Leger PNAS ͉ April 25, 2006 ͉ vol. 103 ͉ no. 17 ͉ 6649 Downloaded by guest on September 27, 2021 Fig. 4. Kinetics of insect survivorship in bioassays. (A) Mortality of Manduca larvae after topical application with 2 ϫ 107 conidia per ml suspensions of wild-type or ⌬Mcl1 mutant strains (control insects were dipped in water). LT50 values were 3.61 Ϯ 0.23 days for wild type and 4.85 Ϯ 0.36 days for the mutant (t ϭ 28.22, P ϭ 0.00062). (B) Mortality of Manduca larvae after injection with 10 ␮lof5ϫ 105 conidia per ml suspensions (control insects were injected with 10 ␮l of water). The LT50 values were 2.12 Ϯ 0.16 days for wild type and 2.83 Ϯ 0.27 days for the mutant (t ϭ 20.49, P ϭ 0.0012).

⌬Mcl1 mutant. Encapsulation of the ⌬Mcl1 mutant continued 50 h bated with unconjugated poly(L-lysine) before treatment with the after injection (Fig. 3D) and only ceased with the manifestation of FITC probe did not fluoresce, indicating that negative surface obvious disease symptoms such as reduced food uptake and soft- charges were neutralized. However, they remained hydrophilic ening of the body. (3.59 Ϯ 0.23 microspheres per ), suggesting that the hydro- The ability of ⌬Mcl1 to survive and cause disease indicates that philicity of blastospores is not due to their electronegativity. To M. anisopliae has additional mechanisms to cope with immune confirm this possibility, cell surface electronegativity was also responses. However, LT50 values from injection assays showed that reduced by treating wild-type blastospores with dicyclohexylcarbo- the ⌬Mcl1 mutant takes a significantly longer time (P ϭ 0.0012) to diimide and ethylenediamine to replace negatively charged carboxyl kill insects than the wild type (Fig. 4). Insects were also dipped in groups with positively charged ammonium groups (24). The deriv- suspensions of conidia to assay infections through the cuticle. At a itized wild-type cells still showed a lower degree of binding to high (2 ϫ 107 conidia per ml) dosage, the ⌬Mcl1 mutant took microspheres (3.87 Ϯ 0.39 beads per spore) than did ⌬Mcl1 significantly longer to kill than the wild type (P ϭ 0.0006) (Fig. 4). blastospores (t ϭ 4.37, P ϭ 0.0243). The difference with untreated At 8 ϫ 106 conidia per ml, ⌬Mcl1 failed to achieve 50% mortality wild-type cells was not significant (t ϭ 1.26, P Ͼ 0.05), and they were before pupation and, at lower (Ͻ5 ϫ 106) dosages, mortality fell to not more readily recognized than untreated cells (Fig. 5). Thus, the Ͻ10%. In contrast, the wild type achieved Ͼ50% larval mortality differential hemocyte response elicited by the ⌬Mcl1 mutant is not at 1 ϫ 106 conidia per ml, suggesting that it would be much more a nonspecific reaction to charge. likely than ⌬Mcl1 to cause high mortality under field conditions, The surface exposure of ␤-glucans was measured by the degree where concentrations seldom exceed 106 conidia per g of soil (2). of binding of Calcoflour white. Strong fluorescence was observed on the ⌬Mcl1 blastospores, but the wild-type blastospores were Hemocyte Monolayer Assays. Given the differences in how hemo- barely visible using the same exposure time (Fig. 3 F and G). This cytes in infected insects behave to wild type and ⌬Mcl1,wealso investigated the effects of deleting Mcl1 on cell surface properties and hemocyte responses in vitro. Cell surface hydrophobicity was measured by using a microsphere adhesion assay. Conidia from the wild type and ⌬Mcl1 were similarly hydrophobic with 15.37 Ϯ 1.21 and 15.14 Ϯ 1.59 beads attached per cell, respectively. Despite conidia and blastospores being very similar in size, this represents Ͼ3-fold more microspheres than adhered to blastospores of the wild type (3.28 Ϯ 0.16 beads per spore) and ⌬Mcl1 (4.94 Ϯ 0.48 beads per spore). The Ϸ30% fewer microspheres adhering to wild-type blastospores demonstrates that MCL1 produces a small but significant (t ϭ 6.81, P ϭ 0.0103) overall increase in cell surface wettability. This finding suggests that MCL1 disruption had un- masked components of blastospores that were also hydrophilic, but less so than MCL1. Hydrophobic Dynabeads exhibit a much greater attraction to insect hemocytes than do hydrophilic beads (t ϭ 21.31, P ϭ 0.0011) (Fig. 5), so loss of hydrophilic MCL1 would be expected to increase attractiveness to hemocytes. Indeed, hemocyte mono- layer assays agreed with the infection studies in showing that ⌬Mcl1 Fig. 5. Recognition of blastospores, conidia, and beads by Manduca hemo- blastospores are recognized at Ͼ3-fold higher efficiency than are cytes in vitro. Monolayers were exposed to wild-type (WT) or ⌬Mcl1 (MU) M. wild-type blastospores (Fig. 5). We investigated whether this could anisopliae cells treated with collagenase (Coll), proteinase K (Pro K), lyticase be due to an ability by hemocytes to distinguish between different (Lyt), DTT, poly(L-lysine) (PL), or dicyclohexylcarbodiimide and ethylenedia- ͞ cell surface charges or degrees of hydrophilicity and͞or to the mine (D E). The Dynabeads tested were M270 (hydrophilic) and M280 (hy- exposure of PAMPs underlying the MCL1 layer. drophobic) beads. Histograms represent the mean % of the test particles that were attached, ingested, or encapsulated by hemocytes (six monolayers and The surface charge of outer cell surfaces was assessed using their associated standard errors) after 1 h. Bars carrying the same letter are not FITC-labeled poly(L-lysine). Only a faint fluorescence was ob- statistically different in terms of mean number of cells or beads recognized by served on conidia, but blastospores of the wild type and the ⌬Mcl1 the hemocytes (Dunnett’s least significant difference multiple comparison mutant were negatively charged (Fig. 3E). Blastospores preincu- method, ␣ ϭ 0.05).

6650 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601951103 Wang and St. Leger Downloaded by guest on September 27, 2021 finding demonstrates that MCL1 renders PAMPs, such as ␤-1,3- suggests that M. anisopliae has evolved multiple strategies that allow glucans, inaccessible to arriving hemocytes. Treatment of wild-type it to grow unhindered by the insect immune system. cells with collagenase or proteinase K prevented labeling with A further unique feature of this research was the identification of antibodies to MCL1 but produced strong fluorescence with Cal- a collagenous Gly-X-Y domain in a fungus. Collagens are the most cofluor and increased their recognition to the levels shown for abundant proteins in the vertebrate body and are essential struc- ⌬Mcl1 (Fig. 5), confirming that a protein component of wild-type tural elements that evolutionary models trace to fibrillar forms in cells blocks hemocyte responses. Additionally, treatment of ⌬Mcl1 early animals (18, 36). The presence of collagen in the cell walls of cells with lyticase (a ␤-1,3-glucanase) greatly reduced labeling with the human pathogen C. albicans had been inferred by immunolog- Calcofluor and phagocytosis compared with untreated cells (t ϭ ical analysis (37), but we found no collagen sequence in the 15.67, P ϭ 0.0051), confirming that hyphal bodies are recognized on published genome of C. albicans. The fungus Ustilago violaceum was contact with ␤-1,3-glucans. also inferred to contain collagenous protein (38), but no gene has been identified. In contrast, the characteristic triple-helical struc- Discussion ture formed by Gly-X-Y motifs has been found in bacterial colla- In the course of infecting a host, pathogens are presented with a gens (39). Demonstrating that MCL1 forms the trimers character- wide array of host environments. Cell surface proteins and secreted istic of collagen fibers is beyond the scope of this paper. However, hydrolases will define the interactions between host and pathogen the MCL1 sequence has all of the features expected of a collagen and together are likely to have a profound impact on the infection and apparently none that would preclude a multimeric helicoidal outcome (25). Because of the functional adaptations of GPI structure. The high proline content of MCL1 domain B that is proteins to localization at the cell surface, they likely comprise the characteristic of most collagens will prevent secondary structure. In majority of Candida spp. surface proteins involved in human addition, the heavy glycosylation also characteristic of collagens is disease (26). The agglutinin-like protein (ALS) cell surface ad- likely to confer an extended conformation on that portion of the hesins of C. albicans are particularly informative in light of their molecule (21). This will project the relatively glycosylation free similarities and differences with MCL1. Although lacking a collag- N-terminal domain into the extracellular milieu. Thus, the exper- enous domain, ALS proteins have a relatively nonglycosylated imental support for the surface location of domain A also makes cysteine rich N-terminal domain that is displayed on the cell surface intuitive sense. An interesting potential model for the structure of by the remainder of the protein that is extended because of its heavy protein motifs in MCL1 is provided by the collectins, a family of glycosylation (27). The central region is so heavily glycosylated that, animal lectins that recognize PAMPs. These also have a cysteine- like MCL1, ALS proteins migrate at three to five times their rich N-terminal domain and a heavily glycosylated collagenous predicted molecular weights (28). However, unlike MCL1, they region with interruptions (kinks). The basic functional unit is a have hydrophobic N-terminal domains that facilitate adhesion to trimer, but the kinks provide flexibility, enabling the trimeric subunits to cover more area. The trimers are stabilized and host tissues (28, 29). There is an abundance of literature identifying assembled into larger oligomers via the cysteine residues (40). The the hydrophobic effect as the driving force for the initial adhesion possibility of interchain disulfide bridges in MCL1 is consistent with of pathogens to host surfaces that establishes infection (reviewed in the ability of DTT to permeabilize the MCL1 sheath around the ref. 30). However, the MCL1 protein has the opposite function of fungus.

producing a nonadherent cell; to achieve this, it provides a hydro- IMMUNOLOGY The presence of a collagenous domain is not associated with a philic antiencapsulation coat around the fungus that prevents specific lifestyle, i.e., they are present in saprobic fungi as well as recognition by hemocytes. Therefore, it appears analogous in pathogens. The patchy distribution of collagen-like proteins among function to the extracellular polysaccharide capsule produced by bacteria and viruses is supposed to derive from horizontal gene Cryptococcus neoformans to avoid recognition by phagocytes (31). ␤ transfer from multicellular animals (22). The apparently similar A major part of the PAMP, the MCL1 protein blocks is -glucans sparse distribution of the collagenous domain in fungi could also be in the cell wall, but it also contributes to properties of the cell explained by repeated independent instances of gene loss (41), or surface that reduce attraction to insect hemocytes. These include by convergent evolution, particularly as alignment of collagenous physiochemical properties such as wettability, and it is also perti- regions indicates high sequence divergence (Fig. 1B). However, it nent that MCL1 lacks the tripeptide Arg-Gly-Asp (RGD) sequence is interesting to note that three of four C. galabrata collagenous as hemocytes possess an RGD-dependent adhesion mechanism proteins (XP࿝447814, XP࿝447815 and XP࿝447816) have Ͼ50% (32). Rapid encapsulation of hydrophobic conidia despite their overall similarity and locate in tandem within 12 kb on chromosome failure to bind Calcofluor suggests that hemocytes respond to J, indicative of recent duplication events and diverging functions. several criteria including nonspecific mechanisms that can inde- Given that most fungi lack homologs for MCL1 or other proteins pendently induce an immune response. The wettability of blasto- with a collagenous domain, they are evidently expendable in terms spore surfaces has also been noted in other insect pathogens (11) of maintaining the normal organization of fungal hyphae. The roles and could be consequential for infection processes in many ways, they play in the fungi that possess them will probably therefore have besides just avoiding a direct hemocyte response. The negatively to be addressed on a case-by-case basis. charged hydrophilic surface is likely to prevent clumping of cells and attachment to host surfaces thus facilitating dispersal through the Materials and Methods insect. Hydrophilic surfaces are also much more resistant to non For further details, see Figs. 7–9, which are published as supporting specific adsorption of proteins (33), lectins, and other opsonins (30), information on the PNAS web site. minimizing the possibility of opsonization by ␤-1,3-glucan binding proteins. The MCL1 coat probably protects against several ␤-glucan Gene Cloning and Deletion. The cDNA of Mcl1 was fully sequenced, binding proteins in M. sexta. A laminarin-binding M. sexta lectin and the genomic DNA was acquired by using a primer walking kit promotes encapsulation (34), and cell wall ␤-glucans activate the (Seegene) from M. anisopliae strain ARSEF2575. The procedures phenoloxidase cascade (35), so it is significant that encapsulated for construction of gene knockout plasmids and fungal transfor- ⌬Mcl1 propagules were frequently melanized (Fig. 3B). Because mation are provided in Fig. 8. host humoral responses are also triggered by PAMPs (7), MCL1 will likely be involved in avoiding these as well. However, both Cell Wall Protein Isolation and Western Blot Analysis. Thirty-six-hour wild-type and ⌬Mcl1 mutant hyphae survive challenge with the Sabouraud dextrose broth (SDB) cultures were washed three times antimicrobial cercopin A at a level (50 ␮M) sufficient to kill with sterile distilled water and then transferred either to minimum saprobic fungi (C.W. and R.J.S.L., unpublished data). This finding medium (MM) or to isolated Manduca hemolymph as described

Wang and St. Leger PNAS ͉ April 25, 2006 ͉ vol. 103 ͉ no. 17 ͉ 6651 Downloaded by guest on September 27, 2021 (13) for up to 24 h. Fungal cell wall proteins were extracted as before Hydrophobic (M280) and hydrophilic (M270) Dynabeads (2.8 ␮m (17). In-solution deglycosylation was conducted by using a Glyco- in diameter, Dynal) were used as references to test the effects of Profile II kit (Sigma). Two predicted antigenic regions, A1 (PGP- nonspecific surface properties on hemocyte responses. After incu- NASPDQIKKHRD; residues 59–73 of the N-terminal domain) bation of the monolayer coverslips for1hat28°C, the percentage and B1 (NGKPGSGNNGANGSN; residues 421–435 of the col- of test particles recognized by the hemocytes (the number of lagenous domain) were synthesized and conjugated with keyhole particles bound or ingested by hemocytes relative to the number limpet hemocyanin. The antibodies were raised in New Zealand added) was determined in five different fields of vision using the 40ϫ objective. Data shown were calculated from 600 or more cells White rabbits (Sigma) and designated as abA and abB. Western blot ͞ ͞ analyses were conducted as described (42). or beads monolayer insect and six insects per treatment. Characterization of Fungal Cell Surface Properties. Indirect Immunofluorescence (IIF). Fungal cells grown in hemolymph A microsphere adhesion assay of cell surface hydrophobicity was conducted by in vitro or in vivo were prepared for IIF as described (37). Antibodies using 0.6-␮m latex polystyrene beads (Sigma) (44). The were were diluted 500-fold and FITC-conjugated-goat anti-rabbit Ig G 7 suspended in 0.1 M KNO3 solution (2 ϫ 10 cells per ml, pH 6.5), (Sigma) was used for secondary labeling. Control samples of cells and the suspensions were mixed with microspheres suspended in were treated as above but minus either the primary or secondary the same buffer in a ratio of 20 beads͞one spore. Three replicates antibodies. for each treatment were performed, and a total of 50 spores were counted for each replicate. To modify cell surface charge, blasto- ⌬ Insect Bioassay. Virulence of wild-type and Mcl1 mutant conidia spores were treated with dicyclohexylcarbodiimide and ethylenedi- was assayed against newly molted fifth-instar larvae of M. sexta as amine. By this method, carbodiimide-activated carboxylate groups described (4). Thus, conidia were applied topically by immersion of are substituted with positively charged ammonium groups from the larvae or by injecting the rearmost proleg with 10 ␮l of an aqueous ethylenediamine (24). FITC-labeled poly(L-lysine) was used to suspension containing 5 ϫ 105 spores per ml. Each treatment was assay surface charge (8). Staining with Calcofluor White was used replicated three times with 10 insects per replicate, and the exper- to measure the exposure of ␤-glucans on the cell surfaces of iments were repeated twice. Mortality was recorded every 12 h, and wild-type and mutant hyphal bodies (45). estimated lethal time values for 50% mortality (LT50) were used to compare speed of kill between strains with the t test as before RT-PCR. Mycelia from 36-h SDB cultures (0.1 g wet weight) was (4). Additional infected insects were bled at 10-h intervals for washed twice with sterile water before transfer into minimal microscopic observation of fungal development within the insect medium, fresh SDB, or hemolymph collected from seven insect haemocoel. species. At different time points, RNA (0.5 ␮g) was extracted and converted into single-strand cDNA using an anchored oligo(dT) Hemocyte Monolayer Assay. Hemolymph was collected in prechilled primer (ABgene, Surrey, U.K.). Complementary DNA samples saline buffer (43) from day 2 fifth-instar larvae and applied as a diluted 500-fold were used as template for PCR. Primers designed suspension of 2 ϫ 106 cells per ml onto glass coverslips (10-mm for the small subunit ribosomal RNA were used as the reference. diameter). The coverslips were incubated in Grace’s medium at 28°C for 2 h and then washed twice with 0.5 ml Grace’s medium Statistical Analysis. Student’s t test was used for the pairwise comparisons of means given in the text. Dunnett’s least significance (43). Wild-type and ⌬Mcl1 blastospores (harvested from fungal difference multiple comparison method in the program SPSS (ver- cultures grown in hemolymph in vitro for72h)orconidiawere ϫ 3 sion 11.0.0) was used to compare the different treatments shown in washed twice with PBS, and 2 10 cells were applied to the Fig. 5. hemocyte monolayers to assay recognition. In some experiments, fungal cells were fixed in 4% formaldehyde or pretreated for1hin This work was supported by National Research Initiative of the U.S. PBS containing either DTT, poly(L-lysine), lyticase, proteinase K, Department of Agriculture Cooperative State Research, Education, and or collagenase (Sigma) (the enzymes at 200 ␮g͞ml) before assaying. Extension Service Grant 2003-353-02-13588.

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