BJM 101 1–6 ARTICLE IN PRESS b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y x x x (2 0 1 6) xxx–xxx ht tp://www.bjmicrobiol.com.br/ 1 Microbial Physiology 2 Lectin activity in mycelial extracts of Fusarium 3 species ∗ 4 Q1 Ranjeeta Bhari, Bhawanpreet Kaur, Ram S. Singh 5 Q2 Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala, Punjab, India 6 7 a r t i c l e i n f o a b s t r a c t 8 9 Article history: Lectins are non-immunogenic carbohydrate-recognizing proteins that bind to glycopro- 10 Received 25 February 2015 teins, glycolipids, or polysaccharides with high affinity and exhibit remarkable ability 11 Accepted 12 November 2015 to agglutinate erythrocytes and other cells. In the present study, ten Fusarium species 12 Available online xxx previously not explored for lectins were screened for the presence of lectin activity. Mycelial extracts of F. fujikuroi, F. beomiformii, F. begoniae, F. nisikadoi, F. anthophilum, F. incarnatum, Associate Editor: Rosana Puccia and F. tabacinum manifested agglutination of rabbit erythrocytes. Neuraminidase treatment 13 of rabbit erythrocytes increased lectin titers of F. nisikadoi and F. tabacinum extracts, 14 Keywords: whereas the protease treatment resulted in a significant decline in agglutination by most 15 Fusarium of the lectins. Results of hapten inhibition studies demonstrated unique carbohydrate 16 Lectin specificity of Fusarium lectins toward O-acetyl sialic acids. Activity of the majority of 17 Hemagglutination Fusarium lectins exhibited binding affinity to d-ribose, l-fucose, d-glucose, l-arabinose, d- 18 Carbohydrate specificity mannitol, d-galactosamine hydrochloride, d-galacturonic acid, N-acetyl-d-galactosamine, 19 Culture age N-acetyl-neuraminic acid, 2-deoxy-d-ribose, fetuin, asialofetuin, and bovine submaxillary mucin. Melibiose and N-glycolyl neuraminic acid did not inhibit the activity of any of the Fusarium lectins. Mycelial extracts of F. begoniae, F. nisikadoi, F. anthophilum, and F. incarnatum interacted with most of the carbohydrates tested. F. fujikuroi and F. anthophilum extracts displayed strong interaction with starch. The expression of lectin activity as a function of culture age was investigated. Most species displayed lectin activity on the 7th day of cultivation, and it varied with progressing of culture age. © 2016 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Microbiologia. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1 molecules in cell–cell or cell–matrix interactions. Microbial 22 Introduction lectins include various agglutinins, adhesins, precipitins, tox- 23 2 ins, and enzymes and occur widely in bacteria, protozoa, 24 20 Lectins are carbohydrate-binding proteins or glycoproteins of 3 viruses, and fungi. Lectins have applications in blood typ- 25 21 non-immune origin that play an important role as recognition ing and are known to exert essential functions in the immune ∗ Corresponding author. E-mail: [email protected] (R.S. Singh). http://dx.doi.org/10.1016/j.bjm.2016.04.024 1517-8382/© 2016 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Microbiologia. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Bhari R, et al. Lectin activity in mycelial extracts of Fusarium species. Braz J Microbiol. (2016), http://dx.doi.org/10.1016/j.bjm.2016.04.024 BJM 101 1–6 BJM 101 1–6 ARTICLE IN PRESS 2 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y x x x (2 0 1 6) xxx–xxx 26 recognition process of viral, bacteria, mycoplasmal, and par- Lectin extraction 80 27 asitic infections, as well as in fertilization, cancer metastasis, 4,5 28 and growth and differentiation several cells. After plants, The mycelium was homogenized in PBS (1:1.5, w/v) using an 81 ® 29 mushrooms are the most widely studied group of organisms ultra-high speed homogenizer (Ultra-Turrax T25 basic, IKA- 82 30 for lectins, and these lectins have gained considerable atten- Werke, Staufen, Germany) and then ground in mortar and 83 6,7 19 31 tion due to their promising biological activities. Yeast lectins pestle with acidified river sand for 30 min. The extract was 84 ◦ 32 are known to be involved in cell–cell interactions, pathogene- centrifuged (3000 × g, 20 min, 4 C), and the supernatant was 85 8 33 sis, and cell flocculation. The mitogenic potential of microbial assayed for lectin activity. 86 34 lectins has been well established as well as their ability to 9 35 induce mitosis in lymphocytes/splenocytes. Hemagglutination assay 87 36 There are several reports regarding lectins from micro- 10 11,12 ◦ 37 fungi, including Rhizopus stolonifer, Aspergillus fumigatus, Blood samples drawn in Alsever’s solution were stored at 4 C 88 13 14 15 38 A. oryzae, Penicillium marneffei, P. thomii and P. griseofulvum, until further use. Erythrocyte suspension (2%, v/v) was pre- 89 16 17 39 Sclerotium rolfsii and Fusarium solani. Additionally, micro- pared in PBS, and two-fold serially diluted mycelial extract 90 40 fungi exhibiting lectin activity and their possible functions was tested for agglutination of human, goat, pig, sheep, and 91 18 19 41 have been reviewed exhaustively by our group. Singh rabbit erythrocytes. The surfaces of human and rabbit eryth- 92 19–21 42 et al. screened 40 species of Aspergillus for the pres- rocytes were modified with the enzymes, neuraminidase and 93 27 43 ence of lectin activity and discovered wide occurrence protease, as described by Meng et al. and used in the aggluti- 94 44 of lectins in this genera. Few of them have also been nation assay. Hemagglutination was determined visually by an 95 22–24 45 evidenced to possess mitogenic and immunomodulatory appearance of mat formation as an indicative a lectin activity, 96 25 46 properties. whereas button formation was taken as an absence of lectin 97 26 47 Recently, Singh and Thakur reported lectin activity in activity. Lectin titer was defined as inverse of the highest dilu- 98 48 mycelial extracts of eight Fusarium species, namely F. acumi- tion capable of producing visible agglutination of erythrocytes. 99 49 natum, F. chlamydosporium, F. compactum, F. crookwellense, F. 50 culmorum, F. dimerum, F. decemcellulare, and F. coeruleum. The Hapten inhibition assay 100 51 present study attempted to explore lectin activity in ten 52 species of Fusarium, which have been not investigated earlier. Hapten inhibition assay was carried out against a panel of 101 53 The lectins were characterized with respect to their biological carbohydrates according to a method described by Singh 102 19 54 spectrum and carbohydrate inhibition profile. The expres- et al. Lectin was incubated for 1 h with an equal volume of 103 55 sion of lectin activity with respect to culture age was also the test solution in the wells of U-bottom microtiter plates. 104 56 investigated. The present examination could provide useful Erythrocyte suspension was added and hemagglutination was 105 57 information for cataloging lectins and prompt further research established visually following 30 min of incubation. Button for- 106 58 on the possible roles and applications of the lectins isolated mation in the presence of carbohydrates indicated specific 107 59 from Fusarium sp. interaction, while mat formation was taken an absence of 108 interaction between the lectin and the carbohydrate. The min- 109 imum inhibitory concentration (MIC) of each of the specific 110 Materials and methods carbohydrates was determined by serial double dilution of the 111 test solution. MIC was defined as the lowest concentration of 112 60 Maintenance, growth and harvesting of microbial cultures the carbohydrate capable of inducing complete inhibition of 113 lectin-mediated hemagglutination. 114 61 Ten Fusarium species, namely F. fujikuroi (MTCC 9930), F. beomi- The carbohydrates tested as inhibitors were: d-ribose, 115 62 formii (MTCC 9946), F. diaminii (MTCC 9937), F. annulatum (MTCC l-rhamnose, xylose, l-fucose, d-glucose, d-mannose, d- 116 63 9951), F. begoniae (MTCC 9929), F. nisikadoi (MTCC 9948), F. arabinose, l-arabinose, d-galactose, d-fructose, d-mannitol, 117 d 64 staphyleae (MTCC 9911), F. anthophilum (MTCC 10129), F. incar- -sucrose, d-maltose, d-lactose, melibiose, d-trehalose 118 65 natum (MTCC 10292), and F. tabacinum (MTCC 10131) were dihydrate, d-raffinose, maltotriose, inositol, meso-inositol, d- 119 66 procured from Microbial Type Culture Collection (MTCC), Insti- glucosamine hydrochloride, d-galactosamine hydrochloride, 120 67 tute of Microbial Technology, Chandigarh, India. All strains d-glucuronic acid, d-galacturonic acid, N-acetyl-d- 121 68 were maintained on potato dextrose agar slants contain- glucosamine, N-acetyl-d-galactosamine, 2-deoxy-d-glucose, 122 69 ing potato 20.0%, dextrose 2.0%, and agar 2.0%; pH of the 2-deoxy-d-ribose, porcine stomach mucin, bovine submaxil- 123 70 medium was adjusted to 5.6. Agar slants were stored at lary mucin, fetuin, asialofetuin, inulin, pullulan, starch, 124 ◦ ± 71 4 1 C, until further use and were subcultured regularly chondroitin-6-sulphate, N-acetyl neuraminic acid, and N- 125 72 at an interval of two weeks. The cultures were grown in glycolyl neuraminic acid. Simple sugars were tested at a 126 73 Erlenmeyer’s flasks (250 mL) containing 100 mL of mainte- final concentration of 100 mM, whereas glycoproteins and 127 ◦ 74 nance medium without agar and were incubated at 25 C polysaccharides were analyzed at a final concentration of 128 75 under stationary conditions for 7 and 10 days, respectively.