A new view on honeybee defense system based on proteinous antibiotics and phytochemicals

Transcription

1 A new view on honeybee defense system based on proteinous antibiotics and phytochemicals Jozef Šim imúth 1, Katarína Bílikov liková 1 and Hans Lehrach 2 1 Dpt. of Molecular Apidolology, Institute of Molecular Biology, Slovak Academy of Sciences, 2 Max-Planck Institute for Molecular Genetics, Berlin, Germany Jozef.Simuth@savba.sk

2 Mechanisms of honeybee defensive system Individual defense Honeybee possess individual defenses, including immune responses toward disease agents Group-level defense Hygienic behaviour of honeybee colony Exogenous defense (EDS) Nutritution Multifunctional potential of proteins and antimicrobial peptides secreted by honeybee in larval diets and honeybee products Plant phytochemicals present in RJ, honey, pollen and propolis

3 Royal jelly proteins and antimicobial peptides as main components of EDS Major proteins accounting for 90% of total RJ proteins and belonged to a protein family consisting of nine members with Mr of kda. The physico-chemical chemical properties of these proteins are similar to typical albunoid proteins (such as ovalbumin, serum albumin) and therefore we proposed to name major royal jelly proteins as apalbumins. Thus, MRJP1 are designated as apalbumin1, MRJP2 as apalbumin2, and so on. The most abundant protein of RJ is apalbumin1 representing 50% of proteinous content of RJ Proteins and peptides from RJ can participate in defense mechanisms of honeybee against microbial pathogens by means of direct inactivation of microorganisms occurring in honeybee products ucts, as well as through induction of cytokines participating in regulation of transcription of defensive proteins and peptides.

4 The quality of larval food determine of exogenous defensive potential of honeybee colony Nutrients are the most influential of environmental stimuli of gene expression of defensive proteins proteins ~ 15% fatty acids ~ 7% honey ~ 10% Royal Jelly

5 Apalbumin 1 determine immunostimulation of honeybee colony 55 kda apalbumin1; 2 - RJ; 3 acacia honey; 4 - apple tree honey; 5 - raspberry honey; 6 - sunflower honey; 7 - pollen pellet; 8 - pollen bread Self-assembly of apalbumin1 and its ability to create of membranes that provides an improved surface for evaporation of water from nectar

6 Apalbumin1 as main physiologically component of beepollen 55 kda apalbumin1; 2 - RJ; 3 - Aacacia; 4 - Apple tree; 5 - Raspberry; 6 - Sunflower 7 - pollen pellet; 8 - pollen bread immobilization of the pollen Formation of filamentous spider web (like networks) in processing of the flower pollen dust to pollen pellet

7 Flow of apalbumin1 in honeybee colony HYPOPHARYNGEAL GLANDS Trophalactic Behaviour Processing Nectar HONEY Processing Flowers Pollen POLLEN KIT Procesing Pollen kit POLLEN BREAD Despite a number of HONEY AND BEE POLLEN physiological data functional aspects on the proteins secreted by honeybee during processing of floral pollen dust to pollen bread and processing of nectar to honey are still unclear.

8 Antimicrobial peptides of Royal Jelly Royalisin Apisimin 51 aa, 5523 Da, pi 7,75 belongs to the family of antibacterial cationic insect peptides, Defensins Fujiwara S. et al. (1990) J.Biol. Chem. 265, antifoulbrood and antifungal factor of larval diet Bíliková, K., et al. (2001) Apidologie 32, aa, 5540 Da, pi 4,68 Serin-valin rich peptide, honeybee specific no homology with other honeybee proteins/peptides, neither with proteins in SwissProt database have been found. Bíliková K. et al (2002), FEBS Letters 528, kda VTCDL... ROYALISIN NILRG... MRJP1 VTCDL... ROYALISIN Tricin-SDS-PAGE. 1. Mw protein marker, 2. royalisin A. Tricin-SDS-PAGE. 1. Mw protein marker, 2. Royal Jelly, 3. Apisimin B. Native PAGE. 1. Royal Jelly, 2. Apisimin

9 The oligomerics form of apalbumin1 contained heterodimers with peptide apisimin MALDI-TOF MS of oligomeric apalbumin1 Apalbumin1 formed 420 kda subunit assembled in noncovalent structure. Šimúth J., (2001) Apidology 32, 69-80; Bíliková K., et al., (2002) FEBS Letters 528,

10 Slovak Academy of Sciences Antifungal properties of apisimin and royalisin the peptides of royal jelly Test fungus: Botrytis cinerea A apisimin B C royalisin D - apisimin (2.10-5M) - apisimin (1.10-4M) - royalisin (5.10-6M) - royalisin (2,5.10-5M) - negative control without addition of antifungal agent F - positive control hydrogen peroxide (5.10-4M) A B C D E Bíliková K., Wu G., Šimúth J. (2001) Apidologie 32, Bíliková K., Šimúth J. (unpublished) E control H2O2 F

11 Plants as a source of antimicrobials. Alternatives to chemotorapy plants phytochemicals The health condition of honey depends substantially on physiologically active substances present in propolis, pollen and nectar, which are not determined by the honeybee genome. Plants make over 100,000 small-molecule compounds, many if not most of which have antimicrobial activity. The honeybee and the plant represent a complex symbiotic system, which evolved in complete mutual dependence.

12 Bioprevention based on propolis components in treatment of honeybee colony aginst pathogens Propolis contains water soluble substances with antiviral activity and ability to inhibit a key enzyme of gene expression, the DNA-dependent RNA polymerase, through blocking its binding to the promotor of a given gene. This observation contribute to explanation to antiviral and antimicrobial properties of propolis. Šimúth J., et al. (1986) Inhibition of bacterial DNA - dependent RNA polymerases and restriction endonuclease by UV-absorbing components of propolis. Die Pharmazie 4, The propolis extracts from the various states of Brazil showed significant inhibition of Paenibacillus larvae. Brazilian propolis samples were more effective than the few collected in MN, USA. Bastos E.M. Simone M. Jorge D.M. Soares A.E. and Spivak M. (2008) J Invertebr Pathol., 97, (2008)

13 F A C T O R S REDUCING EFFECTS of EXOGENOUS DEFENSE SYSTEM E D S Loss of biodiversity of plants One-direction breeding for honey production Antibiotic era

14 Allelic variations of genes originally found in wild species, but gradually lost through domestication, breading and application of antibiotics. 16 Genes 10 genes 8 genes Wild species Early domesticates Modern varieties Such lost alleles can not be recovered by going back to the wild ancestors in honeybee.

15 Drastic changes of honeybeeh oneybee-plant symbiotic system 1. In the course of breeding directed to higher yields, most plants have lost many genes representing natural antibiotics, which are now no more avalilable to honeybess. 2. The honeybee breeding and continous domestication of the bees with higher honey yields have resulted in the loss of genetically determined resistence to pathogens due to allelic variation.

16 Antibiotics Era 1. Antibiotics have indirectly suppressed the function of genes of natural immunity in the honeybee, because they have taken over their role. 2. In the present era of extensive use of antibiotics, the honeybee breeding to resistence or tolerance of diseases cannot be considered a process in which the resistance or tolerance is the main criterion. 3. Antibiotics have destroyed symbiotic microorganisms such as lactobacillic type, which, under normal conditions, act in the honeybee digestion tract as probiotics and have natural mmunostimulatory and antibiotic activities. 3. The primary effects of prohibition of antibiotics in beekeeping is the restoration of natural immunity of honeybees.

17 Conclusions 1. Exogenous defensive system of honeybee colony based on proteins and petides of royal jelly and phytochemicals is the key factor on prevention against micobial, fungal and viral pathogenes. 2. The level of honeybee defensin(s) in larval diets is an important antifaulbrood factor and potential marker of antimicrobial capacity honeybee colony. 3. The research could be oriented on finding new immunostimulators and antimicrobial substances originated from plants.