Antibacterial Properties And Efficacy Of Peptide From Honeybees
Honeybees occupy significant place in human food chain as providing about one third of the humans food supply worldwide. Honeybees being efficient pollinators are responsible for production of some vegetables and fruits (cherries, peaches and apricots) at commercial level. Besides of their economic value, honeybees are also seeking great attention as medicinal insect. Honeybees produce some antibiotic substances e. g. , royal jelly, propolis and pollens as defence tool at colony levels. Honey is used in antiseptics and has wound healing feature. Propolis is used in the treatment of diabetes while pollen is used to treat arthritis and incendiary conditions because of its anti-inflammatory and antioxidant characters.
At individual levels, honeybees developed AMPs as a part of more strong and efficient humoral defence against biotic and abiotic stressors (e. g. , parasites, predators, diseases and pesticides). AMPs have massively become a target of research because of their potential usage in clinical applications as a replacement for traditional antibiotics and anti-fungal compounds. From 1950s to 2000s, the number of multidrug resistant (MDR) bacteria with rise of prevalence rate upto 57% have been increased alarmingly. More resistance was observed towards drugs that had been used for humans and animals for the longest time and discovery of alternative drugs to control bacterial infections is need of the hour.
Similar to many conventional antibiotics, AMPs have broad spectrum activity against a wide range of microorganisms, including gram-positive and gram-negative bacteria, fungi, viruses, yeast, and protozoa. AMPs have multiple sites as targets rather than single genes or proteins. The ability of these peptides to target multiple systems makes it difficult for bacteria to gain resistance against them. These peptides disrupt the cell membrane of target organisms, translocate through the cell membrane modifying other primary cellular activities and promote immune response by altering gene expressions, enhancing neutrophil chemotaxis and function, promoting histamine release of mast cells, inhibiting tissue proteases, and stimulating wound healing.
When honeybees are challenged by pathogens, they immediately synthesized antimicrobial peptides in fat bodies and released into hemolymph exhibiting broad spectrum antimicrobial activity against invading pathogen. Four antimicrobial peptides (Abaecin, hymenoptaecin, apidaecin and defensin) have been identified in honeybee hemolymph. The antibiotic action of AMPs depend upon interaction ability between negatively charged bacterial membrane and positively charged AMPs.
The present study centralized around cloning of one of these AMPs genes hymenoptaecin of Apis cerana for assessment of its antimicrobial activity. Hymenoptaecin is placed in gloverin family of glycine rich AMPs. Hymenoptaecin have a molecular weight of 14 kDa and is regulated by immunodeficiency pathway. It shows antimicrobial activity against variety of gram positive as well as gram negative bacteria including some human pathogens.
Hymenoptaecin is expressed as a result of microbial infection and septic wounding. Thus Apis cerana were infected with viable E. coli cells to induce the hymenoptaecin expression. Choi et al. (2008) stimulated bumblebees with PBS or LPS for expression of four AMPs, hymenoptaecin, abaecin, apidaecin and defensin. Expression of the hymenoptaecin gene is generally regulated by the Immunodeficiency pathway. RNA was extracted from infected honeybees and immediately converted into cDNA. Hymenoptaecin gene was amplified using specifically designed forward and reverse primers. Amplified hymenoptaecin gene was comprised of 423 bps containing a coding DNA sequence (cds) of 381 bps (NCBI), a pro-region and his-tag that were added in primers. 6-14 string long histidine tag is added during primer designing for easy purification of recombinant proteins (Singh and Jain, 2013). Amplified hymenoptaecin gene was cloned in BL21 (DE3) bacteria using pET-21a (+) plasmid as a vector. The more efficient vector system for cloning and expression of recombinant protein in E. coli discovered untill now is pET. pET is derivative of pBR322 and uses its origin of replication. pET-21a (+) vector has some special features that help in efficient molecular cloning of target genes; (1) pET plasmids used in cloning transcribed the target gene under control of T7 lac promoters derived by T7 RNA polymerase, (2) have ampicillin resistance gene with copy number of 40-50 copies per cell and (3) pET plasmids have high affinity for 6-his tag residues at its N-terminus promoting the expression of recombinant proteins.
Recombinant plasmid pET-21a (+)-hymenoptaecin gene was produced through the ligation of double digested pET-21a (+) vector and hymenoptaecin gene in the presence of T4 DNA ligase. Restriction enzymes NdeI isolated from Neisseria denitrificans and HindIII isolated from Haemophilus influenza used for restriction of plasmid and gene belong to type II restriction enzymes. Type II restriction enzymes need Mg++ for activity. BL21 (DE3) cells were physiologically altered to enhance their competency to adapt the recombinant plasmid. E. coli hosts are more convenient to use because of their fast replication capacity as their growth cycle takes just 20 min, high expression level of recombinant proteins and easy transformation competency for foreign DNA and free from enzymes involved in recombinant protein degradation.
Forager and Hall (2007) directly cloned a gene 60-EGP in pET-21 a(+) vector leading to the transformation into BL21 (DE3) by heat shock for expression. Transformed colonies were confirmed through colony PCR and restriction analysis of recombinant DNA. Cloned hymenoptaecin gene was expressed through IPTG induction. Overnight grown culture (O. D: 0. 4-0. 6) was induced by addition of 50mM IPTG. Supernatant containing hymenoptaecin peptide was harvested by centrifugation of culture because of its extracellular expression and used for assessment of antimicrobial peptide. IPTG is the structural mimic of lactose that attaches to the lacI thus initiating the transcription of recombinant protein hymenoptaecin under the control of T7 promoter. The hymenoptaecin peptide was induced extracellularly through IPTG as it is released into hemolymph upon induction in vivo.
Liu et al. (2012) cloned and characterized the heat shock protein AccHsp27. 6 that is involved in cellular defence. AccHsp27. 6 gene was isolated and amplified using specific primers. AccHsp27. 6 was ligated into pET-30a(+) and transformed into E. coli BL21 for cloning. Expression of cloned AccHsp27. 6 was induced chemically by Isopropy l-β-D-thiogalactopyranoside (IPTG). Induced culture was centrifuged to harvest the bacterial cells. Shen et al. (2012) studied the antibacterial activity of royalisin, an antimicrobial peptide that is produced in the royal jelly of honeybees. To study the mechanism of royailsin present in the royal jelly of Apis cerana cerana, a recombinant RAcc-royalisin was cloned and expressed in E. coli BL21. The agar dilution assays with inhibition zone showed that RAcc-royalisin inhibits the growth of Gram-positive bacteria. RAcc-royalisin displayed the minimum inhibitory concentrations (MIC) of 62. 5 µg/ml, 125 µg/ml, and 250 µg/ml against gram-positive bacterial strains, Bacillus subtilis and Micrococcus flavus and Staphyloccocus aureus in the microplate assay, respectively. Antibacterial mechanism of royalisin against gram-positive bacteria suggested the application of recombinant royalisin in food and pharmaceutical industries as an antimicrobial agent.
Ayaad et al. (2012) isolated hymenoptaecin antimicrobial peptide from honeybees induced through E. coli and its antimicrobial activity was assessed against bacterial strains E. coli, staphylococcus aureus, Klebsiella pneumoniae and Bacillus subtilis through agar well diffusion method. Elhag et al. (2017) analyzed the antimicrobial peptide stomoxynZH1 having wide spectrum potential obtained from Hermetia illucens (L). StomoxynZH1, 189-basepair gene was cloned into the pET-32a expression vector and expressed in the Escherichia coli as a recombinant protein with thioredoxin. Expression was induced through IPTG and supernantant harvested as a result of centrifugation was used for antibiotic assay. Antimicrobial activity of StomoxynZHI was assessed againstm Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice)-10, and fungus Sclerotinia sclerotiorum indicating a potential benefit in controlling antibiotic-resistant pathogens.
Casteels et al. (1993) assessed the antimicrobial activity of cationic glycine rich polypeptide hymenoptaecin against different gram positive and gram negative bacterial strains including E. coli, Haemophilus influenzae, Streptococcus pneumoniae, Klebsiella aerogenes, Staphylococcus aureus and Bacillus subtilis etc. In present study, antimicrobial activity of hymenoptaecin was assessed against different gram negative and gram positive pathogenic bacterial strains, e. g. , E. coli gram negative bacteria, Klebsiella pneumoniae gram negative bacteria, pseudomonas aeruginosa gram negative bacteria, Haemophilus influenzae gram negative strain, Streptococcus pneumoniae gram positive bacterial strain and Staphylococcus aureus gram positive bacteria by agar well diffusion method. . Hymenoptaecin has shown antibacterial activity against Haemophilus influenzae but no significant activity has shown against E. coli, B. subtilis, S. pneumoniae and K. pneumoniae same as analyzed by Casteels et al. (1993).
While hymenoptaecin has shown antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa in contrast to Casteels et al. (1993) line of work inferring the antibacterial act. Hymenoptaecin as antimicrobial peptide not only can be used against pathogenic bacterial strains as a potent alternative of conventional antibiotic but also act as immunoregulator by some other mechanisms e. g. , hymenoptaecin can enhance the immune responses of host by activation of other immune cells and chemicals other than just killing the pathogens.
