A New Model System for the Study of the Animal Innate Immune Response to Fungal Infections 1

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1 ISSN , Moscow University Biological Sciences Bulletin, 2014, Vol. 69, No. 2, pp Allerton Press, Inc., Original Russian Text J. Plotnikova, O.V. Kamzolkina, F.M. Ausubel, 2014, published in Vestnik Moskovskogo Universiteta. Biologiya, 2014, No. 2, pp PHYSIOLOGY A New Model System for the Study of the Animal Innate Immune Response to Fungal Infections 1 J. Plotnikova a, O. V. Kamzolkina b, and F. M. Ausubel a a Department of Molecular Biology, MGH and Department of Genetics, Harvard Medical School, Boston, MA United States b Department of Mycology and Algology, Biology Faculty, Moscow State University, Moscow, Russia o-kamzolkina@yandex.ru Received January 15, 2014 Abstract The association of the model organism Caenorhabditis elegans and the fungus Pleurotus ostreatus gives the possibility to study the molecular and genetic mechanisms of the early stages of the spatial and temporal interactions of animals with fungal pathogens. We identified the stages of the infection process of P. ostreatus on the nematode C. elegans. We found that prior to penetration inside a worm a fungal toxin paralyzed and immobilized, but did not kill C. elegans. This finding opens the possibility for the further study of the effect of paralyzing toxins on host organisms. The membrane permeability of paralyzed worms increased dramatically and leakage products initiated the growth of directional hyphae towards the nematodes. The hyphae penetrated into live C. elegans animals either through natural openings or directly by piercing the cuticle. Upon contact with the nematode cuticle, P. ostreatus attached to it, formed appressoria-like structures and infection pegs, piercing the cuticle and penetrating inside the nematode body. The small zones around the penetration loci are of special interest for the evaluation of initial contacts between two organisms and for the study of the C. elegans local defense response against fungal infection. Key words: Pleurotus ostreatus, Caenorhabditis elegans, paralyzing toxin, model system for the study of human and animal immunity. DOI: /S INTRODUCTION The nematode C. elegans is one of the most popular model organisms to study the molecular mechanisms of animal pathogenic microbe interactions [1]. The simple anatomy of C. elegans is a great advantage for this research [2]. The nematode length is 1 mm. Its cylindrical body consists of only 959 cells. The nematode head has a mouth and pharynx, its central portion contains intestine and gonads, and the anus is in the tail. The C. elegans intestine consists of only 20 epithelial cells, arranged in 9 rings. The first ring is formed by 4 epithelial cells and the other eight rings are each formed by 2 cells. These epithelial cells are similar to the epithelium of the mammalian digestive tract [3]. Hypodermis (epidermis) and wall muscles surround the intestine, the gonads, and a liquid-filled internal cavity (pseudocelom). A collagen cuticle covers the hypodermis on the outside. C. elegans are primarily hermaphrodites, but produce some males which aids genetic analyses. Specific sinusoidal nematode movement is the result of muscle contractions. Neurons perceive signals and coordinate the nematode s movement and behavior. C. elegans is one of the simplest organisms with a nervous system transmitting 1 The article was translated by the authors. mechanical irritation, chemo and thermotaxis [4]. When reproducing hermaphroditically, each adult produces approximately 300 new genetically identical progeny. This opens the possibility for rapid nematode propagation and maintaining a large population [5]. There is an embryonic stage, four larva stages (L1 L4), and an adult stage in the C. elegans life cycle. At the end of each stage a moulting occurs and a new cuticle is synthesized. After each molt the old cuticle is discarded. The nematode s growth and development depends greatly on temperature. All developmental stages and the rate and kinetics of nematode movement can be analyzed under a light microscope because the nematodes are transparent. C. elegans is widely used to study the development and function of living organisms including carbohydrate metabolism, diabetes, aging, and fat metabolism. The C. elegans genome was the first genome of a multicellular organism that was sequenced [6]. It is easy to activate and inhibit nematode genes and follow the consequence of this procedure. Gene expression can be suppressed by RNAi by simply feeding the nematodes with living E. coli expressing dsrna corresponding to a particular C. elegans gene. Homologs of most human genes are found in C. elegans. Nematode transparency helps to monitor gene expression and infection develop- 45

2 46 PLOTNIKOVA et al. ment. Tagged with a fluorescent marker, a C. elegans gene product becomes traceable. One can see when and where the gene is switched on. Changes in gene expression can be studied using qrt PCR. C. elegans does not have blood circulation, immune cells or phagocytes. C. elegans, like other invertebrates, has no antibodies and its main defense is innate immunity. C. elegans creates barriers against pathogenic microorganisms by regulating the transcription of defense-related genes [7 8]. C. elegans dwells in soil inhabited by many pathogenic organisms. Moreover, this nematode consumes bacteria that can cause infections. In spite of this, C. elegans is able to survive in soil under harsh conditions and competition for food due to defense barriers created during the evolutionary process. A system of induced defense reactions similar to mammalian immunity was found in C. elegans. The most studied are intestinal infections of C. elegans caused by bacteria including Pseudomonas aeruginosa, Enterococcus fecalis, Salmonella enterica, Staphylococcus aureus, and by fungi including Candida albicans, Cryptococcus neoformans, C. laurentii and Drechmeria coniospora. The defense reactions induced by infections in the digestive tract have been described [9 10]. The nematocidal fungus D. coniospora can attach to the cuticles of some nematode species, primarily around the mouth, form appressorium-like structures and penetrate inside the nematode body. Treatment with proteases decreased the fungal attachment by 40 60% [11]. D. coniospora induces the transcription of defense genes encoding antimicrobial peptides and a kinase cascade regulating the signal transduction in infected nematode cells [12]. The local defense reactions that determine the success or falure of an infection and the ability of a pathogen to overcome the host defense barriers and establish parasitic relationships still need to be studied. It is known that some wood degrading fungi catch and digest freely living nematodes to compensate for the lack of nitrogen. The C : N ratio is close to 100 : 1 in wood, and tree degrading fungi always have a nitrogen deficit. The lack of nitrogen in wood limits fungal growth and development [13]. In the process of evolution only wood decay fungi that created mechanisms of consumption of other organisms (yeasts, bacteria, algae, nematodes) as a nitrogen source have been able to survive [14]. It is known that the fungus Pleurotus ostreatus (Jacq. Fr Kumm.) grows as a saprophyte on wind-fallen twigs and branches of leaf-bearing trees, but can also be a pathogen of weakened live trees. P. ostreatus causes white rot of wood, degrading the wood polymers cellulose, hemicellulose and lignin to monomers. The secretion of various enzymes, degrading both lignin and cellulose open the possibility for P. ostreatus to use a wide range of plant substrates [15]. We showed earlier, that P. ostreatus grown on agar nutrient medium, can use various yeast species including Hanseniaspora uvarum, Rhodotorula minuta, and Saccharomyces cerevisiae, as an additional source of nutrients. It increased the production of P. ostreatus fruiting bodies [16]. Thorn and Barron described the mechanism used by nematocidal fungi to paralyze worms [13]. A nematotoxin was found in secretory outgrowths on hyphae in the mature portions of the fungal colonies which was identified as trans-2- decenedioic acid [17]. Based on these data, we suggested the possibility of infecting the model organism C. elegans with P. ostreatus and tested it in laboratory conditions. The application of this host pathogen combination might give a key advantage in solving some immunity problems, especially at the time of establishing parasitic interrelationships. The great advantage of this system is that both host and pathogen genomes are known. The P. ostreatus genome was sequenced in 2010 (JGI, Walnut Creek, California, United States) [18]. Our research has focused on the following objectives: to analyze the reaction of the model organism C. elegans after infection by P. ostreatus in situ, to identify the symptoms of the infection, to develop techniques of nematode inoculation with this fungus in the laboratory and to study their interactions at all stages of the infectious process. MATERIALS AND METHODS Objects fungus P. ostreatus (Jacq.: Fr) Kumm. (Basidiomycota, Agaricomycotina, Agaricomycetes, Agaricales, Pleurotaceae, [19], strain NK35 and free living soil round worm Caenorhabditis elegans, strain N2 [5]. Nematode cultivation. A non-pathogenic strain of Escherichia coli was used for C. elegans propagation. We grew synchronized C. elegans until L3/L4 on Nematode Growth Medium, (NGM) on E. coli strain OP50, a uracil auxotroph [1]. Nematodes usually reached L3/L4 stage in 34 hrs at 20 C [5]. P. ostreatus culture and nematode inoculation. P. ostreatus was grown on 2% agar Bacto Malt Extract (Becton, Dickinson and Co., United States) at 22 ± 1 C. The interaction between P. ostreatus hyphae and C. elegans was observed on the thin layer of mycelium grown on 2% water agar. Disks 0.5 cm in diameter cut from P. ostreatus culture were placed in the center of Petri dishes with 2% water agar. The Petri dishes were incubated at 21 C for 7 days. Hyphae grew centrifugally from the disk. The thin hyphal layer covered each Petri dish during this time. Alternatively, P. ostreatus was grown on the surface of thin cellophane at 21 C for 7 days until the cellophane surface was covered evenly with thin layer of mycelium. Hyphae of P. ostreatus NK35 attached tightly to the cellophane surface and gradually started to digest the cellophane. Living C. elegans N2 nematodes were placed on the mycelial surface at the colony periphery. The interac-

3 A NEW MODEL SYSTEM FOR THE STUDY Fig. 1. Pleurotus ostreatus life cycle. The fruiting bodies (1) produce numerous basidia bearing basidiospores (2). Haploid basidiospores give rise to homokaryotic hyphae (3). The fusion of homokaryotic hyphae initiates dikaryotic hyphae with toxocysts (4). Toxocysts contain nematode-paralyzing toxins and induce the growth of dikaryotic hyphae towards nematodes (5). tions of the fungus P. ostreatus and the nematode C. elegans were analyzed 24 hours after application of L3/L4 nematodes. Staining. The contact loci of fungal hyphae and nematode cuticle were visualized by staining with a 1% solution of Trypan blue in lactophenol and subsequent washing in phosphate buffer at ph 7.2. Fluorescent Brightener 28 (Sigma-Aldrich) was applied for the staining of the fungal mycelium. It produced bright fluorescence of hyphal walls and septa at excitation 405 nm. It visualized fungal hyphae inside nematode bodies. Fluorescent and confocal laser scanning microscopy. The interaction between the fungus P. ostreatus NK35 and the nematode C. elegans N2 was studied with fluorescent (Leica D 5000 B, Germany) and confocal (Leica SP1 and SP5, Germany) microscopes. A thin layer of 3% agarose was placed on microscope slides to prevent the disruption of nematodes due to cover slip pressure. The nematodes were immobilized with 25 mm Levamisole hydrochloride (Sigma-Aldrich). We placed 1 µl of this solution in each drop of C. elegans suspension on a microscope slide. Scanning Electron Microscopy. Mycelium of P. ostreatus was grown on circular cover glasses 12 mm diameter (Fisher Scientific CIR-1) at 21 C for 7 days. For this cover glasses were thoroughly cleaned with 96% alcohol and placed in 0.1% water solution of Poly-L-Lysine (Sigma-Aldrich). Then each cover glass was placed in a Petri dish 4 mm in diameter on the surface of 2% water agar and inoculated with P. ostreatus. After the fungal hyphae grew over the surface of the water agar for 7 days at 21 C, 20 L3/L4 nematodes were placed on each cover glass. 24 h later the hyphal mats with captured nematodes were fixed in 2.5% glutaraldehyde in phosphate buffer at ph 7.2 for 12 h at 4 C, washed with the phosphate buffer ph 7.2, and transferred through an alcohol series: 30, 50, 70, 96, 100%, 1 hour at each concentration. The absolute (100%) alcohol was changed 3 times. After this the samples were dried at critical point in a semiavtomatic apparatus Critical Point Dryer (Samdri-795, United States), mounted on metal stubs 12 mm in diameter, and covered with a Cromium layer 600 Å in vacuum in a Gatan High Resolution Ion Beam Coater 681, United States. Samples were analyzed using a Scanning Electron Microscope (SEM JEOL-7401F, Japan). RESULTS AND DISCUSSION The fungus P. ostreatus growing on decaying wood, primarily angiosperm trees, produces fruiting bodies known as oyster mushroom (Fig. 1, 1). There are numerous basidia with four haploid basidiospores in gills on the underside of the fruiting bodies (Fig. 1, 2). Dikaryotic basidia line the surface area of the gill. The

4 48 PLOTNIKOVA et al. (a) (b) (c) Fig. 2. Directional growth of P. ostreatus hyphae towards a nematode (a); hyphal growth inside the nematode (b); fungal penetration through natural openings (c). nuclei in the basidia undergo karyogamy, fusion, and form diploid nuclei that quickly undergo meiosis. Each of four haploid nuclei of different mating types migrate into one of 4 basidiospores formed on a basidium. The basidiospores are haploid, having half the complement of genes. If conditions are favorable, the basidiospores germinate. Starting from basidiospore germination until the newly formed spore production, P. ostreatus passes mono and dicaryotic stages of its development. Basidiospores produce monokaryotic hyphae (Fig. 1, 3). The haploid hyphae conjugate with another haploid hyphae of a compatible mating type. They undergo plasmogamy, to create a dikaryotic cell, with two genetically different haploid nuclei (Fig. 1, 4). P. ostreatus spends most of its life cycle at this stage. Diploid hyphae penetrate into wood or living trees, grow fast and accumulate a large mycelial mass inside the tree trunk. The typical feature of the Basidiomycetes is a clamp connection [20]. The clamp connections are formed by the terminal hyphae during elongation. This terminal segment contains 2 nuclei. Once the terminal segment is long enough it begins to form clamp connections. At the same time, each nucleus undergoes mitotic division to produce two daughter nuclei. As the clamp continues to develop, it uptakes one of the daughter nuclei and separates it from its sister nucleus. While this is occurring the remaining nuclei begin to migrate from one another to opposite ends of the cell. Once all steps have completed a septum forms, separating each set of nuclei. P. ostreatus evolved to produce nematode paralyzing toxin(s) and to consume nematodes as a source of nitrogen. The fungus produces numerous secretory vesicles (toxocysts) on the surface of the mycelium (Fig 1, 4). Toxocysts are a specific feature of P. ostreatus. Each toxocyst consists of a thin leg and a round head 1 µm diameter. They are the outgrowths of fungal hyphae (Figs. 2a 2c and 4a). Our observation showed that nematodes crawled over the surface of young growing hyphae not bearing the toxocysts. If a nematode touches a toxocyst usually localized on the central portion of a fungal colony, the toxocyst envelope sticks to the nematode s surface and bursts. The toxin penetrated through worm cuticles and the nematodes were paralyzed in 30 seconds [14]. They coiled up fast. The toxin caused a serious disruption of the feeding apparatus of nematodes immediately. The tissues surrounding the pharynx were disrupted and the pharynx was displaced and the elongated shape of worm s head changed to roundish (Fig. 3d). The toxin might change the permeability of the nematode cuticle, and leached signal molecules might induce the observed growth of lateral absorbing hyphae of P. ostreatus towards the paralyzed nematodes (Figs. 2a 2c). The presence of nematodes appeared to stimulate toxocyst formation. Hyphae penetrated primarily through the worm s natural orifices: mouth, anus, vulva (Figs. 2a 2c, 3b, and 4c) and eggshells (Figs. 4b). Some fungal hyphae attached to the worm cuticle. In this case an appressorium-like outgrowth of a hypha appeared at the infection locus (Fig. 3b). A piercing cuticle penetration peg was formed at the bottom of the appressorium-like outgrowths (Fig. 3b). The fungal hyphae became visible in paralyzed nematode bodies 12 hrs later (Figs. 2b, 2c, 3a, and 3c). Figure 3c shows hyphae that penetrated through the mouth, grew towards the tail, and formed

5 A NEW MODEL SYSTEM FOR THE STUDY (a) 49 (b) (c) (d) Fig. 3. P. ostreatus penetration through mouth and hyphal growth inside worm body. Directional and trophic hyphae in the nem atode body: trophic hyphae are perpendicular to the directional hyphae (a); P. ostreatus hyphae inside the nematode: apices of the fluorescent hyphae stained with Calcofluor 9 directed towards worm tail (b); fungal appressoria on C. elegans cuticle (c); worm head deformation after contact with toxocyst (d). (a) (b) (c) (d) Fig. 4. Scanning Electron Microscopy (SEM) of P. ostreatus and infected nematodes. Toxocyst on a mycelial clamp (a); penetra tion of fungal hyphae inside a C. elegans egg (b); hypha entering through the nematode mouth (c); Cross section of a nematode body: all internal structures degraded and utilized by the fungus and only the cuticle remained (d). Bar 1 µm. thick runners with short thin lateral hyphae. Hyphae grew in the pseudocoelom in a longitudinal direction along the nematode body from head to tail. The fungal hyphae did not break cell membranes, and paralyzed nematodes pierced with fungal hyphae remained alive and slightly moved their heads upon touching with a glass stick. Later, hyphae filled the nematode bodies, digested all organelles with their hydrolases, and uti lized the worm content excluding the cuticle (Fig. 4d). The tips of fungal hyphae excrete digestive enzymes. The enzymes break down their food leaving a space filled with nutrients that the hyphae continue growing into, absorbing, and digesting again as the nutrients are consumed. Young adults at the L3 larval stage MOSCOW UNIVERSITY BIOLOGICAL SCIENCES BULLETIN Vol. 69 No

6 50 PLOTNIKOVA et al. responded faster: their energetic swimming movement converted to slow motion of the paralyzed bodies. Large adults were less sensitive to the toxin and looked normal for 48 hours. We found that the growth of P. ostreatus hyphae was stimulated in the presence of nematodes due to the consumption of their nutrients. In 72 hours the radial hyphae grown on 2% water agar in 9 cm Petri dishes with nematodes were 20% longer than without nematodes under the same conditions. The tight contact of the fungus with nematode cuticle and its deformation were visible on scanning electron microscope pictures after fungal hyphae were misplaced. The nematode cuticle invaginates under the attached hyphae. CONCLUSION Our results showed that the association P. ostreatus and C. elegans can potentially be used to study the local defense reactions of nematodes. In comparison with some bacteria and fungi penetrating through mouth and inducing defensive genes in epithelial cells of the digestive system, P. ostreatus can penetrate through cuticle and induce defense genes in hypodermis. P. ostreatus is a facultative pathogen of immunocompromised trees. Its fruiting bodies are used widely as food and they are known as oyster mushrooms. P. ostreatus grows well on artificial medium. It has a remarkable ability to digest both plant and animal tissues changing its balance towards consumption of carbohydrates when plant food is available and towards proteins when animal food sources become available. It highlighted the great adaptative ability of the fungus. We identified the initial contacts of P. ostreatus and the nematode C. elegans. We found that the fungus paralyzed C. elegans, penetrated into their living bodies either through natural openings or directly by piercing the cuticle and colonized them. Our results showed the possibility to study further the local defense reactions of C. elegans against fungal infection. P. ostreatus has a long biotrophic stage while infecting nematodes. Fungal hyphae grew in the pseudocoelom and caused minimal damage of worm internal organs at the initial stages of infection. Nematodes pierced by several hyphae grown from mouth towards tail compartment were alive and were able to move. The association of the model organism C. elegans and fungus P. ostreatus opens the possibility to study the molecular and genetic mechanisms of the initial stages of the spatial and temporal interactions of humans and animals with fungal pathogens. REFERENCES 1. Brenner, S., The genetics of Caenorhabditis elegans, Genetics, 1974, vol. 77, no. 1, pp Hall, D. and Altun, Z.F., C. elegans. 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