Cloning and mutagenesis

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Frog antibiotic peptides are seen as a potential weapon to fight various human viral, bacterial and fungal pathogens (Rollins-Smith et al 2005).One of them, Pseudin-2, a subfamily of the Frog Secreted Active Peptides (FSAP) from the skin of the bright green and pink paradoxical frog of the Pseudidae family, Pseudis paradoxa has been recently discovered. Pseudin-2 is a 2685.4 Da peptide made up of 24 residues. The study is particularly relevant since Pseudin-2 is reportedly a potential antibiotic peptide that could eventually be useful to counteract increasingly drug-resistant organisms. Besides antimicrobial features, Pseudin is seen as a promising prospective insulinotropic agent for treating type 2 diabetes. A topical application of a potential antimicrobial peptide like pseudin could be exploited as an effective future therapy in addressing the problem of foot ulcers which affects as many as 1 in every 10 diabetes sufferers according to the Global Diabetes Community (2010).

Pseudin-2, being a relatively newly discovered peptide, has been the subject of relatively few studies, warranting more research to unravel its therapeutic potential. With combinations of 20 existing amino acids, peptide antibiotics are tremendously diverse in sequence and structure, offering opportunities in creating a whole range of novel drugs (Hancock and Scott 2000) but all the features required to make natural peptides suitable therapeutics are not always present, justifying the need to engineer their primary structure to confer those properties to them (Sarah et al 1999) e.g. reduced toxicity (Won and Ianoul 2009); existing desirable properties e.g. potency, selectivity or specificity of antimicrobial activity can also be strengthened. In the case of pseudin, it is desirable to curb toxicity and increase potency. It would be interesting for future synthetic drug design to come up with pseudin analogues with similar or enhanced antimicrobial activity. Multi-l-lysine-substituted analogues have been designed and proved potent against strains of several pathogenic bacteria Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis and Streptococcus (Pal et al 2005).The aim of the study is to thus to generate various pseudin-2 analogues with antimicrobial activity by cloning and mutagenesis. Set objectives are to clone Pseudin-2 and get recombinant colonies, to generate a protein library through NNK or MAX randomisation of the 3rd, 10th and 14th codon of pseudin-2 and to screen the mutant library for Pseudin analogues showing antimicrobial activity.

A gene encoding pseudin-2 will be constructed and ligated downstream from the malE gene into the expression vectors, pMAL-c5X and pMAL-p5X of E. coli which encode a maltose-binding protein (MBP) as a fusion protein. Recombinant colonies will be grown in antibiotic-containing broth and the recombinant plasmid will be purified using a plasmid purification kit. Saturation mutagenesis using NNK or MAX randomisation of three key codons in the pseudin gene will be conducted; a library of mutants with all possible mutations will be generated. The mutants will be ligated to pMAL-c5X or pMAL-p5X and transformed into competent E.coli DH5a cells which will be inoculated onto Ampicillin agar plates. A screen for recombinant colonies will be made using replica plating onto IPTG-containing agar plates. Recombinant colonies will be grown in antibiotic-containing broth and the recombinant plasmid will be purified using a plasmid purification kit. Mutants having antimicrobial activity will be screened and sequenced to get the list of pseudin analogues having antimicrobial activity.

This study will contribute to growing information regarding the role of the various residues in the pseudin-2 structure and antimicrobial activity. It will also give an appreciation of the cloning technique as a method of generating numerous copies of pseudin and inducing the expression of the protein as most of the previous studies (Yasser et al 2008; Pal et al 2005) are conducted using chemically synthesized peptides or by extracting the protein from P.paradoxa skin (Olson et al 2001).

Objectives of proposed work:

  1. To clone Pseudin-2 and get recombinant colonies.
  2. To generate a protein library through NNK or MAX randomisation of the 3rd, 10th and 14th codon of pseudin-2.
  3. To screen the mutant library for Pseudin analogues showing antimicrobial activity.
  4. Will ethical approval be required? NO (Delete as appropriate) :

Strategic relevance of proposed work (not exceeding 750 characters):

The World Health Organization (WHO) has classified the worrying resurgence of communicable diseases observed in numerous countries as the main threat to humans. Microbial induced diseases constitute the most important etiologic factor after cardiovascular disorders to cause death, calling for the need for novel antimicrobial substances (Kamysz 2005).50% of bacteria are resistant to the new antibiotic methicillin and vancomycin (Omilusik 2009).Cationic antimicrobial peptides are hailed as a potential solution to the widespread resistance of bacteria and fungi to current antibiotics for their attractive characteristics. They reportedly eradicate bacteria rapidly, are synergistic with classical antibiotics, neutralize endotoxins and are active in animal models (Hancock and Scott 2000). Furthermore, they exhibit a broad spectrum of activity since they have a relatively non-specific mode of action. In addition, mutations leading to classical antibiotic resistance do not affect them and they do not easily select antibiotic resistant variants (Shai 2002; Huang 2000). They are therefore an attractive model for the design of new antimicrobials some of which are on clinical trials and may substitute drugs against a range of different pathogens in the future.

As testified by Roberts (2007), more than 2.5 million people in England are diabetic, 90 % of whom suffer from type 2 diabetes; 5% of the NHS budget is exhausted on treatment of diabetes and related concomitant infections. In a time when the incidence of diabetes is escalating, unhampered by anti-diabetic drugs, finding novel compounds with a potential for long-term glycaemia control is particularly relevant. There is the additional challenge of containing diabetes-related foot infections notably foot ulcers against which scarcely any effective potent topical antimicrobial is available (Guzman 2010). Pseudin-2 analogues, [Lys18]-pseudin-2 and [Phe15]-pseudin-2 have reportedly been found to favour insulin release at low concentrations (10-9 and10-6 M) (Yasser et al 2008). Controlling diabetes minimises the risk of complications like blindness, heart disease, kidney problems and amputation therefore potential treatments are essential and any innovative research conducted on them is relevant and should be encouraged since that would be of benefit to the type 2 diabetic patients. Pseudin-2 would be seen as an incretin mimetic (Abdel-Wahab et al 2008) serving as a route for treatment after implementing lifestyle and dietary changes and help regulate blood glucose. The work proposed is in line with the research priorities of Diabetes UK (2008) which encourages research to cure and prevent Diabetes with its accompanying complications including infections.

Numerous previous studies have highlighted the potential of the antimicrobial peptide Pseudin from Pseudis paradoxa; Pal et al (2005) found that Pseudin-2 had moderately high bacteriocidal action with an MIC of 20 µM against highly antibiotic-resistant E.coli strains and was bacteriostatic against Staphylococcus aureus. Olson et al (2001) reported activity against the fungus Candida albicans as well with an MIC of 130 µM. Furthermore, in contrast to other antimicrobial peptides whose toxicity against mammalian cells is a major concern, low haemolytic activity was observed against human red blood cells (Pal et al 2005). A topical application of a potential antimicrobial peptide like pseudin could be exploited as an effective future therapy in addressing the problem of foot ulcers which affects as many as 1 in every 10 diabetes sufferers according to the Global Diabetes Community (2010).

The proposed study will serve as a stepping stone for other pseudin-related studies to build on it. It can be viewed as a contribution to gathering more information since research on pseudin is still in its infancy and deeper studies are required before it can make its way to the clinical trial as a drug.

Details of proposed investigation (Maximum of 5 sides of A4, Arial font 11):

Background to the project

Cationic antimicrobial peptides (AMPs) are gene-encoded peptides of the host defence system made up of 12-50 amino acids, with at least 2 positive charges conferred by lysine and arginine residues and about 50% hydrophobic amino acids (Hancock et al 2000).They have been discovered from the skin of frogs from families ranging from Pipidae, Discoglossidae, Hyperoliidae, Ranidae, Hylidae, Iomedusa, Agalychnis and Litoria and the best-known peptides are caeruleins, tachykinins, bradykinins, thyrotropin- releasing hormone, (Barra and Simmaco 1995), brevinins, esculentins, magainins, ranatuerins and temporins (Conlon et al 2004). Unlike numerous antibiotics or secondary metabolites that halt microorganisms over a number of days by hindering the action of key enzymes, most of the vertebrate antimicrobial peptides neutralise microbes quickly by disrupting the membrane or permeating it and targeting anabolic reactions (Barra and Simmaco 1995).

Peptides can be used for battling antibiotic-resistant bacterial infections or septic shock (Finlay and Hancock 2004). Other potential applications include topical applications for preventing sexually transmitted diseases (Rana et al 2006) including HIV/HSV (Reddy et al 2004), Meningococcal meningitis, diabetic wounds e.g. foot ulcers, gastric helicobacter infections, impetigo (Gunaratna et al 2002; Reddy et al 2004), treating eye infections (Migenix, 1998). Creams with snail's mucin containing antimicrobial peptides are currently marketed for topical applications treating skin infections and acne inflammation (Cottage 2007). Efforts have also been directed at developing magainin analogs into anticancer drugs (Boman 1995). Furthermore, as attempts persevere to alter the immune system of the vectors or their symbionts to confer to the vectors the ability to eradicate the parasites, peptide antibiotics are seen as a potential weapon in fighting insect-borne diseases like malaria, trypanosomiasis, and filariasis (Ham et al 1994; Gwadz et al 1989). Besides using AMP as proteins, genes encoding AMP can be delivered as gene therapy. Genetically altered bacteria making the antimicrobial in situ can be used for targeting pathogens, which is particularly relevant to the treatment of dental caries, Crohn disease, and other disorders in which disturbances in natural microflora play a role and host-microbe balance must be preserved (Palffy 2009).Peptides are also used for food preservation as exemplified by Nisin, produced by certain strains of Lactococcus lactis subsp. Lactis (Joerger 2003). Antimicrobial peptides are appealing for therapeutics since they are rapidly produced at low metabolic expenses, stored easily in abundance and readily available shortly following an infection, to rapidly counteract a wide range of microbes (Zhoa 2003).The 20 existing amino acids confer tremendous diversity in sequence and structure of peptides, presenting opportunities in creating a whole range of novel drugs (Hancock and Scott 2000)

Frog peptides are seen as interesting and potentially useful molecules that could be effective against a range of human pathogens (VanCompernolle et al 2005), viral, bacterial or fungal. In the 1960s, a 24 amino acids long antibiotic peptide bombinin secreted from the skin of the frog Bombina variegata was isolated but discouraging high haemolytic activity restricted applicability (Csordas and Michl 1970).Since then, large numbers of various peptides have been discovered with antimicrobial potential. Gaegurin, for instance, from a Korean frog is described as having large spectrum of activity with mild haemolytic activity, rendering it a potential antibiotic (Suh et al 1996). Recently, high amounts of peptides were discovered in Litoria chloris, the Australian red-eyed tree frog which blocked HIV without harming T cells; the peptides appeared to target the HIV virus probably by insertion into its outer membrane envelope and punching holes in it (VanCompernolle et al 2005).

Recently, Pseudin-1, Pseudin-2, Pseudin-3 and Pseudin-4 antimicrobial peptides with structural similarity have been discovered which are secreted from the skin of the bright green and pink paradoxical frog from the Pseudidae family, Pseudis paradoxa, inhabiting Trinidad and the Amazon basin (Olson et al 2001). Pseudins, a subfamily of the Frog Secreted Active Peptides (FSAP) are cationic, amphipathic and helical (Olson et al 2001).Pseudin-2, the most abundant and powerful 2685.4 Da peptide comprises 24 residues (GLNALKKVFQGIHEAIKLINNHVQ).In aqueous solutions pseudin-2 coils randomly while in those emulating the hydrophobicity of the cell membrane e,g 50% trifluoroethanol/water, it assumes an a-helical conformation (Yasser et al 2008). Kim et al (2007), in a study attempting to link structure to mechanical action of pseudin-2 in microorganisms and liposomes, suggested that the antimicrobial activity of Pseudin-2 is ascribed to the punching of holes in the target cell membrane through its oligomerisation according to the toroidal pore model in zwitterionic liposomes and the barrel-stave model in anionic liposomes.

A study by Pal et al (2005) showed that strains of several pathogenic bacteria Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis and Streptococcus were effectively acted upon by multi-l-lysine-substituted analogues. Besides antimicrobial features, Pseudin is seen as a promising, prospective insulinotropic agent for treating type 2 diabetes as an incretin mimetic (University of Ulster 2008).Yasser at al (2008) demonstrated that Pseudin-2 and derivatives, particularly a [Lys18]-pseudin-2 derivative, enhanced release of insulin from a clonal B-cell line, BRIN-BD11 via Ca2+ independent pathways. The insulin- releasing features of [Lys18]-pseudin-2 are largely similar to the gut hormones GLP-1(7-36) amide and GIP (McClenaghan and Flatt 1999).

Natural peptides do not always possess all the features required to make them suitable therapeutics, validating the need to engineer their primary structure to confer those properties to them (Sarah et al 1999) e.g. stability, reduced toxicity (Won and Ianoul 2009); existing desirable properties e.g. potency, selectivity or specificity of antimicrobial activity can also be strengthened. For instance, Pal et al (2005) demonstrated that gradually raising the cationicity of pseudin-2 by replacement of several residues with l-lysine in the hydrophilic part of the peptide enhanced the antimicrobial property. An analogue [D-Lys3,D-Lys10,D-Lys14]pseudin-2 in particular, had a significantly increased antimicrobial effect against E.coli and S.aureus and, low haemolytic and cytolytic activity against human erythrocytes. Replacing Asn with Lys at codon 3 was found to double the antimicrobial activity against E.coli and S.aureus probably from a reduction in destabilisation of the a-helix besides greater positive charge (Pal et al 2005).

Purpose and outcomes of the proposed research

The aim of the study is to generate various pseudin-2 analogues with antimicrobial activity by cloning and mutagenesis.Pseudin-2 is a relatively newly discovered peptide that has been the subject of relatively few studies, warranting more research to unravel its therapeutic potential. It is interesting for future synthetic drug design to come up with pseudin analogues with similar or enhanced antimicrobial activity. This study will add to accumulating information regarding the role of the various residues in the pseudin-2 structure and antimicrobial activity. It will also give an appreciation of the cloning technique as a method of generating numerous copies of pseudin and inducing expression of the protein as most of the previous studies (Yasser et al 2008; Pal et al 2005) are conducted using chemically synthesized peptides or by extracting the protein from P.paradoxa skin (Olson et al 2001).

Research plan

Proposed Methodology

Cloning of pseudin

The pseudin-2 oligonucleotides will be ordered from the New England Biolab, phosphorylated and annealed.

The gene will be ligated into the appropriately-cut expression vector, pMAL-c5X which provides a means of expressing and purifying proteins produced from a cloned gene or open reading frame. It will be inserted downstream from the malE gene of E. coli, which encodes maltose-binding protein (MBP) modified for binding to amylase more strongly, giving a MBP fusion protein (Guan et al 1988;Maina et al 1988).The fusion protein is cytoplasmically-expressed when using pMAL-c5X because of the absence of the signal sequence in the malE gene on these vectors which also generally produce more fusion protein than the pMAL-p5X series where the signal sequence is present, allowing protein export to the periplasm.pMAl-p5X will also be used and protein expression form the two vectors will be compared. The interesting features of these chosen plasmids are the presence of the powerful "tac" promoter and the malE translation initiation signals to give high-level expression of the cloned DNA sequence (Amann et al 1985; Duplay et al 1984). Not only can a fast, one-step purification of the fusion protein be achieved using MBP's affinity for maltose (Kellerman et al 1982). Furthermore, the vectors carry the lacIq gene coding for the Lac repressor which will keep pseudin-2 expression low in the absence of IPTG induction which is particularly important given that the pseudin protein is toxic to the cell and results in death (Hebaishi,H 2010 pers. comm.,12 March).The ligation mixture will be incubated overnight and a culture of E.coli DH5a will be set up and made competent using ice-cold CaCl2.

5 batches of competent cells will be transformed (i) the pMAL/pseudin ligation, (ii) pMAL self ligation, (iii) Hindlll and Xmnl digested pMAlc5X (iv)uncut pMAlc5X,(v) E.coli with no plasmid. Cultures will be set up on nutrient agar plates with Ampicillin.

The presence of the Pseudin gene in the plasmid of picked recombinant colonies will be confirmed from running a gel electrophoresis after conducting a PCR followed by Bam Hl digestion of the PCR product.

Recombinant colonies will be grown in antibiotic-containing broth and the recombinant plasmid will be purified using a plasmid purification kit.0

Further confirmation of recombinant clones will be done by sequencing the recombinant plasmid of each colony and aligning the sequences to that of the pseudin-2 using the multiple sequence alignment tool on NCBI.

Mutagenesis

Saturation mutagenesis using NNK or MAX randomisation of three key codons in the pseudin gene will be conducted; a library of mutants with many potential mutations will be constructed. In a study, Pal et al (2005) found that an analogue [D-Lys3,D-Lys10,D-Lys14]pseudin-2 in particular, in contrast to others had a significant antimicrobial effect against E.coli and, low haemolytic and cytolytic activity against human erythrocytes. Changing other codons to lysine-encoding ones had no additional effect on antimicrobial activity but instead increased haemolysis. In the light of the finding, the third, tenth and fourteenth codon will be randomised. NNN is not used owing to its unnecessary degeneracy and also since a sizeable portion of the library will contain premature stop codons especially because of the multiple codon-randomisations.NNK and NNS codons are preferred since they reduce the over-representation of the commonly-occurring variants (Patrick and Firth 2003). MAX randomisation will be used for its numerous advantages. While randomising NNN and NNG/T codons generates exponentially-rising redundant gene libraries with increasing randomised codons, MAX randomisation generates non-redundant libraries (Hine et al 2005).

The mutants will be ligated to pMAL-c5X or pMAL-p5X and transformed into competent E.coli DH5a cells which will be inoculated onto Ampicillin agar plates.

A screen for recombinant colonies will be made using replica plating onto IPTG-containing agar plates. Recombinant colonies will be grown in antibiotic-containing broth and the recombinant plasmid will be purified using a plasmid purification kit.

Mutants having antimicrobial activity will be screened and sequenced.

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