A collagenous protective coat enables Metarhizium anisopliae to evade insect 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 insects. 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- fungus 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