Characterization of cathelicidin gene from buffalo ( Bubalus bubalis )

Wide spread and indiscriminate use of antibiotics is accompanied by the emergence of microorganism that are resistant to these agents. Therefore, new approaches are required to address the problem of antimicrobial resistance. Epithelial linings of living organisms are the source of various antimicrobial peptides. Keeping this in view, the present experiment was designed to characterize one of the important natural antimicrobial peptide gene, that is, cathelicidin gene from the buffalo (Bubalus bubalis) uterine epithelium and explored its potency for use as template for synthesis of novel antimicrobial agents. Total RNA was isolated from epithelial layer of buffalo uterus and reverse transcribed using designed primers. The amplified PCR product was purified and cloned. Positive clone was sequenced and result was analysed using laser gene software (DNA Star, USA). The cDNA of uterus cathelicidin had 516 bases with complete ORF from 6-452 bp. The predicted pre-propeptide comprised of 148 amino acids. Mature active peptide had 18 amino acids and had five each of arginine and tryptophan and four proline residues. From this study, it can be concluded that the buffalo (Bubalus bubalis) uterus expressed a potent antimicrobial peptide and amino acid sequence of mature peptide can be used as template for synthesis of novel antimicrobial agents.


INTRODUCTION
The availability of complete genome sequences and development of information technology have provided a greater opportunity for peptide based drug designing.The field of structure based drug designing is a rapidly growing area and the exposition of genomic, proteomic and structural information has provided new targets and opportunities for drug lead discovery.In the meat industry, the use of antibiotics as growth enhancers is a common practice and extensive use of antibiotic in meat industry causes an alarming increase of antibiotic resistance microbes across the world (Gorbach, 2001).Antibiotic resistance has been posing increasingly serious concern to the public, health specialist and animal food producers.Therefore, there is a need of alternative group of drugs which are active in vivo and are able to act fast and has broad-spectrum activity, do not induce bacterial resistance and have limited or no side effects.Antimicrobial peptides are prevalent throughout the nature as part of the intrinsic defenses of most organisms.These peptides represent ancient host defense E-mail: djkalita@rediffmail.com, kalitadj@vetbifguwahati.ernet.in. Tel: 91-3622337700. Fax: 91-3622337700.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License molecules and act as key elements in non-specific immunity (Ganz and Lehrer, 1989).Their wide spread distribution throughout the animal and plant kingdoms, suggest that antimicrobial peptides have served a fundamental role in the successful evolution of complex multicellular organisms (Bals, 2000).New strategies are required for synthesis of novel antimicrobial agents to deal with the threat of bacterial resistance (Ravi et al., 2011).
Antimicrobial peptides hold promise as broad-spectrum alternatives to conventional antibiotics (Gee et al., 2013).Mamalian defensin and cathelicidin are the two broad classes of natural antimicrobial peptides constitute a large family of endogenous peptide antibiotics with broad spectrum activity against various bacteria, fungi and viruses.Expression of human cathelicidin namely hCAP-18 and LL-37 is reported respectively in the reproductive tract (Malm et al., 2000) and skin epithelial cell (Markus et al., 2012).
Several β-defensin namely, human β-defensin-4 from testis (Garica et al., 2001), cryptidin from mouse sertoli cells (Grandjean et al., 1997); Bin1b from rat epididymis (Li et al., 2001) has been isolated.Antimicrobial peptide gene from buffalo tongue has been sequenced and characterized (Kalita and Kumar, 2009).Synthesis of different length of natural analogue of buffalo lingual antimicrobial peptide and functional study revealed its potency against both gram positive and negative bacteria (Kalita et al., 2009).However till date, antimicrobial peptide gene in the uterine tract has not been characterized.Keeping this in view, the present study was carried out to characterize the cathelicidin from buffalo (Bubalus bubalis) uterine epithelia to elucidate its potency to use as blue print for novel antimicrobial agents.

Sample collection, RNA extraction and RT-PCR
A total of five numbers of uteri was collected from apparently healthy buffalo based on the observation and pre rectal clinical examination before slaughter from local abattoir in ice after washing by chilled phosphate buffer saline (pH 7.4).RNA was isolated using TRI Reagent TM (Sigma-Aldrich, USA) following manufacturer protocol.The purity and integrity of RNA was checked spectrophotometrecally (A260/A280) and 1% agarose gel electrophoresis, respectively.Reverse transcription of uterus RNA was done with specific primer designed from published conserve sequences (Accession No.NM_174001, Accession No. NM_174150, Accession No. NM_174831, Accession No.174827) and oligonucleotide sequences for forward 5 / GGACCATGCAGACCCAGA 3 / and reverse primers were 5 / TGTCCAGAAGCCCGAATC3 / ).One-Step RT-PCR kit (Qiagen Inc., Chatsworth, CA) was used to carry out the reverse transcription as well as amplification in a single step.The reaction mixture was prepared by adding 5x RT-PCR buffer 10 µl, 10 mM dNTP mixture 2 µl, primer both forward and reverse (20 pmoles/µl) 2 µl each, RT-PCR master enzyme mix 2 µl, template RNA (50 ng/µl) 5 µl and nuclease free water 27 µl.The different cycle condition in PCR was: reverse transcription for 30 min at Kalita 759 50°C, initial PCR activation for 15 min at 95°C, denaturation for 1 min at 94°C, annealing for 1 min at 52°C and extension for 45 s at 72°C (35 cycles) and final extension was done at 72°C for 5 min.1.0% agarose gel electrophoresis was performed for confirmation of amplified PCR product and specific product was purified by 'DNA Elution Kit' (Sigma -Aldrich, USA.

Cloning, sequencing and in silico analysis
The purified PCR product was ligated to pGEM-T Easy (Promega, Madison, USA) cloning vector and transformed into freshly prepared E. coli DH5 competent cells (Chung et al., 1989).
Plating was done on LB agar containing ampicillin (50mg/ml), IPTG and X-gal (25 mg/ml).The plates were incubated for overnight at 37°C and white recombinant colonies were picked up after completion of incubation period.Plasmids were isolated (Sambrook and Russel, 2001) from the overnight grown culture and insert release was confirmed by EcoR1 digestion.The positive recombinant plasmids of five clones were sequenced and analyzed using Lasergene Software (DNA Star, USA) with other published sequences.The amino acid sequence was predicted and the amino acid distribution in different domain was analyzed using clustal W analysis programme (DNA Star, USA).
The predicted pre-propeptide of buffalo uterus cathelicidin precursor peptide contain 20 strongly basic, 19 strongly acidic, 48 hydrophobic and 42 polar amino acids.The amino acid alignment with different buffalo cathelicidin congeners revealed 120 conserved amino acids (Figure 4).Four additional amino acids (132 to 135) were observed in both uterine cathelicidin as compared to buffalo indolicidin.The deduced amino acid sequences atg cag acc cag agg gcc agc ctc tcg ttg ggg cgg tgg tca ccg tgg ctt ctg ctg ctg ggg ctt gtg 69 gtg tcc tcg acc agt gcc cag gac ctc agc tac agg gag gcc gtg ctt cgt gct gtg gat cag ctc aat 138 gag cgg tcc tca gaa gct aat ctc tac cgc ctc ctg gag cta gac ccg cct ccc aag gat gat gca gat 207 ctg ggc act cga aag cct gtg agc ttc acg gtg aag gag act gtg tgc ccc agg acg act cag cag ccc 276 acg gag cgg tgt gac ttc aag gag gaa ggg cgg gtg aag cag tgt gtg ggg aca gtc acc ctg gac ccg 345 tcc aat gac cag ttt gac cta aac tgt aat gag ctc cag agt gtc agg ata cgc ttt cca tgg cca tgg 414   of cathelicidin showed 31 conserved amino acids with different species of animals.The percentage similarity and divergence of uterine cathelicidin with publish sequences at amino acid level is depicted in the Figure 5.

DISCUSSION
The nucleotide sequence of uterine cathelicidin showed a highly conserve 5' nucleotides region and a limited similarity in 3' region with other published sequences, which is the common structural features of the cathelicidin gene (Ganz et al., 1989;Storici and Zanetti, 1993).The deduced pre-propeptides of uterus cathelicidin cDNA had conserved N-terminal and diverse heterogeneous C-terminal was in agreement with other cathelicidin congeners (Popsueva et al., 1996;Wu et al., 1999).Alanine at 29 was conserved in most of the congeners and it might be the probable site of elastase mediated proteolytic cleavage to separate the signal sequence from the prosequence.The signal sequence of uterine cathelicidin comprised of 1-29 hydrophobic stretch of amino acid residues and corroborated with other congeners (Storici et al., 1992;Das et al., 2006).
Valine at 130 was common to all most all pre-propeptide including uterine cathelicidin indicating the common processing sites to yield the mature carboxyl terminal peptides (Storici and Zanetti, 1993;Skerlavaj et al., 1996).The prosequence of uterine cathelicidin comprised of 101 amino acid residues from 30 to 130 residues, which are highly identical to the cathelin motif, an inhibitor of thiol proteases (Ritonja et al., 1989).The cathelin like prosequence is the hallmark for all cathelicidin as it prevents the tissue injury and inflammation by neutralizing the high cationic charge of mature peptides by the presence of clusters of negatively charged amino acids (Storici et al., 1992;Selested et al., 1992;Ganz, 1989).The cathelin domains also have anti-protease activity and thus confer stability during storage (Ganz, 1989).Uterine cathelicidin had 18 amino acid residues in the mature peptide from 131 to 148.The glycine was the last residues in most of the congeners along with the uterine cathelicidin and it might probably act as amide donor in post translation amidation of the C-terminus (Skerlavaj et al., 1996;Bradbury, 1991).The C-terminal amidation is essential for its optimum antimicrobial activity which increases the lipolysaccharide binding ability and enhances the outer membrane permeabilization (Sitaram and Nagaraj, 1999;Timothy andRoberte, 1997, Peters et al., 2010).The C-terminal amidation of peptides provides an additional hydrogen bond for α-helix stabilization (Dennison et al., 2005).The amino acid sequence of uterine cathelicidin had 4 extra stretch of amino acids namely arginine, phenylalanine, proline and tryptophan from 133 to 136, respectively.Thus, the mature peptide of uterine cathelicidin had 5 arginine, 5 tryptophan and 4 proline.The positively charged arginine is essential to initiate interaction with negatively charged outer bacterial surface (Timothy and Roberte, 1997;Boman, 1991).The tryptophan influences the localization of these peptides in to membrane interfaces (Schiffer et al., 1992).
Proline is an important amino acid that enhances the microbicidal activity by forming flexible helical kink and more ordered structure, which increases membrane permeability (Park et al., 2002).Besides, prolines also provide some protection against nonspecific proteolytic degradation of proteases secreted by microorganisms (Vanhoof et al., 1995).Peptide rich in arginine, trypto-phan and proline are shown to have strong microbicidal activity against gram positive and gram negative bacteria (Selested et al., 1992), fungi (Subbalakshmi and Sitaram, 1998) and virus (Robinson(jr) et al., 1998).Expression of antimicrobial peptide with high number of arginine along with tryptophan and proline by buffalo uterine epithelium help this species to be free from several infectious reproductive disorders as compared to cattle and other species.The mature peptide domain had more variation compared to other region and it is attributed as, genes involved in immunity and host defense is rapidly diverged and had undergo positive selection to fight competently in antigerm war (Emes et al., 2003;Crovella et al., 2005).

Conclusion
From the present study it can be concluded that, mature amino acids sequence of uterine cathelicidin is comprise of 5 arginine, 5 tryptophan and 4 proline residues and the domain can serve as blue print for synthesis of a novel antimicrobial agent.

Figure 2 .
Figure 2. Nucleotide and amino acid sequence of buffalo uterus cathelicidin (Accession No. EF 050453).Single under line indicates the nucleotide sequence of cathelicidin and double underline of single letter indicates the corresponding amino acid sequence.Numbering in the left shows the numbers of nucleotide (bold) and amino acids (italic) respectively.The putatitive cleavage site of the signal sequence (↑) and prosequence (↓) is indicated by an arrow, the stop codon by an asterisk.The mature peptide is shown on boldface.

Figure 3 .
Figure 3. Percent divergence and similarity of buffalo uterine cathelicidin at nucleotide level with different published cathelicidin sequences.

Figure 4 .
Figure 4. Alignment of amino acid sequence of buffalo uterus cathelicidin with buffalo testis and indolicidin.The consensus residues were included within the same box.

Figure 5 .
Figure 5. Percent divergence and similarity of buffalo uterine cathelicidin at amino acid level with different published cathelicidin sequences.