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The terms such as antimicrobial peptides, toxins etc. used in the field of host pathogen interactions have been in use for over hundred years from now. As the research in this field is progressing the definitions of the terms also went on changing depending on the pathogen and the host and their interplay.
Earlier it was thought that the microbes which infect the host are the primary aggressors which govern the host pathogen interaction. Further studies in the same field prove that the interaction between the host and the pathogen does not always result in a disease. This is due to the fact that the host is embedded with the first line of defence, the innate host immune response, against the microbial infection. The most frequently used terms in this topic are defensins, cathelicidins, histatins, toxins etc.
The topic host pathogen interaction involves the basic concepts of microbial commensalism, formation of colonies, infection and the disease. It is a kind of interplay between the host innate immune system and the virulent nature of the microbe. As a result of the infection, the host innate immunity is stimulated and produces anti microbial peptides which play a major role in defending against the disease caused by the pathogen. Host pathogen interaction results in various levels of responses depending on anti microbial peptides (AMP's) immune factors secreted by the host and also the virulent factors/toxins secreted by the pathogen. Mainly the immune factors i.e., defensins, cathelicidins etc. are produced by the neutrophils, dendritic cells, T-regulatory cells, macrophages etc of the host. While the virulent factors are of different types based on the pathogen and its pathogenicity. For example Bordtella Pertusis releases the virulent factors such as FHA (Filamentous Haemaglutinin) which has Immune Suppression activity and Immuno Modulation activity. ACT (Adenylate Cyclate Toxin) is another virulent factor which targets the host phagocytic cells by binding to complement receptor (CR3) and thus forming pores in the membranes (Carbonetti). The main theme of the project is to isolate and purify the anti microbial peptides released by the host so as to defend the infection caused by the pathogen using various techniques. The purification of the anti microbial peptides helps to know the exact mechanism of the interaction between the host and the pathogen. Thus the innate immune system of the host comes into play when any tissue is infected by the pathogen. The host innate immune system is complemented with some other mechanisms so as to protect the host from infection. Receptors play a vital role in the innate immune response by recognising the invading micro organisms and stimulate the host innate immune response. The components of these several other mechanisms which help in protecting the host are lysozymes, lactoferin, secretary phosphor lipases, complements etc. The anti microbial peptides involved in host immune response are isolated and purified using various techniques such as Dialysis, Column Chromatography, HPLC, SDS PAGE, MALDI etc.
Anti microbial peptides are the cationic, polar molecules and are mainly classified into three families on the basis of their size, structure and amino acid sequence. The most important thing about anti microbial peptides is that they are gene encoded i.e., a single gene codes for single peptide. The three families of anti microbial peptides in humans are
Defencins: Defencins are cationic non glycosylated peptides with 3.5 to 4.5 KDa molecular mass. They are rich in arginine residues. These are again divided into 3 classes. They are
α defensins are concentrated in the Paneth's cells of small intestine and the epithelial cells of various tissues like skin etc. where as β defensins are expressed constitively in the epithelial tract of urinogenital tract and respiratory tract. The genes that are encoding these defensins are located on the 8p23 chromosome as a cluster. Both the α defensins and the β defensins contains 6 cysteine residues with three characteristic disulphide bridges but they differ in their spatial arrangement. The first human β defensin (HPD1) was isolated from hemofilterate which is expressed in the epithelial cells by using affinity chromatography procedure and bio screening. Ó¨ defensins were isolated from rhesus monkey neutrophils because of their circular molecular structure. The first alpha defensin was isolated from neutrophils in the year 1985. While the first beta-defensin was isolated from cow tongue in the year 1991 by Diamond et al. He named it as the tracheal anti microbial peptide.
Cathelicidins: These are also cationic molecules with amphipathic nature. They contain two leucines and 37 residues in length. Until now only one human Cathelicidin i.e. LL-37/hCAP-18 was isolated from the bone marrow of humans. These are constituted in the granules of the myeloid cells and inflamed skin. Apart from possessing antimicrobial activity it also protects against endotoxic shock by binding and neutralising LPS. These are considered as the multi functional effector molecules as they have additional function such as mitogenesis, angiogenesis and chemotactic activity besides their host defending action.
Histatins: This family of anti microbial peptides contain large amounts of histidine residues in large quantities and are much concentrated in the saliva. These are strong anti fungal effectors. These have both candidacidal and cadidastatic activities which are responsible for the interest in the present investigations about the histatins.
The experimental methods used in the present project involve two major steps. They are
Culturing of the epithelial cells
Isolation, purification and characterization of the anti microbial peptides from the cultured epithelial cells.
Culturing of epithelial cells: For the culturing of the epithelial cells first the source is obtained from the fore skin epithelial layer or the air way epithelial cells or the lung epithelial cells and grown on the suitable medium. Then they are infected with suitable pathogen which will stimulate the first line of host defence i.e., the anti microbial peptides, against the toxins released by the pathogens. Then the cells are stimulated for 2 days with heat killed clinical isolate (bacteria) in a serum free medium. Then the supernatants of the stimulated cells are collected for the next processes such as the purification and the characterization of the anti microbial peptides secreted by the cultured cells.
Isolation, Purification and characterization of the anti microbial peptides: The first step involved in the isolation of the anti microbial peptides secreted by the cultured epithelial cells is the cell disruption step. This can be done by many ways such as lysis of the cell or by blending techniques or by sonication process (shearing and cavitation). In the next step the lysed cells are suspended in the buffer and then centrifuged to extract the anti microbial peptides secreted by the cells and the cell debris is separated out.
Then the anti microbial proteins are concentrated and partially fractionated by adding salt to the antimicrobial peptide solution this salt is solvated by the water and thus decreases the solvation of the proteins present in the solution. Thus all the hydrophobic tails of the proteins are exposed out and interact with each other resulting in the aggregation of the anti microbial peptides as the precipitate.
The next step involves the removal of the salt from the protein sample using gel filtration chromatography and dialysis. Dialysis is a process which is used to separate smaller molecules from larger molecules. This method involves a semi permeable membrane. During dialysis process, at the equilibrium point the concentration of the salt added is the same on either sides of the dialysis bag. Due to the variation in the volumes of the dialysis bag inside and outside, the salt concentration is decreased in the anti microbial peptide solution.
Then the anti microbial peptides solution is used for the HPLC (High Performance Liquid Chromatography) process. There are various types of liquid chromatography for the separation depending on the type of the samples i.e., the anti microbial peptides solution. There are mainly four types of liquid chromatography. They are as follows
Size exclusion Chromatography: This is a simple chromatography technique for the separation of peptides. The peptides are separated on the basis of their sizes
Normal phase Chromatography: This is the oldest form of liquid chromatography technique used for the separation of peptides. This technique of separating peptides is governed by the adsorbent-solute interaction. The intensity of interaction depends on the polarity i.e., if the polarity is high then the time of retention is also high.
Ion exchange Chromatography: This form of chromatography is mainly based on the ionic forces between the polar mobile phase ( usually water containing minute amounts of alcohol or salts ) and the stationary phase (containing fixed sites of acids or bases.) If the concentration of the salt added is increased then the movement of the components of the sample down the column also increases. This technique is run at a specific pH which creates charge differences in the sample components.
Reversed phase Chromatography: in this technique the polar solvent while the stationary phase is non polar. Most of the analytical liquid chromatography separations on done on bonded phases in the reverse phase mode.
In general, this liquid chromatography process is mainly used for the purification and characterization of the anti microbial peptides from the contaminating materials. It is a purely separating technique. In this process the obtained anti microbial peptides supernatants are diafiltered against a suitable buffer solution and then introduced on to a affinity column which is equilibrated with suitable buffer. Then the peptides secreted by the cells are bound to the column. Then slowly the effluent is applied to the column for eluting the bounded peptides. This process is done several times so as to increase the efficacy of the column to with hold the anti microbial peptides secreted by the cells. At the end of the column with the help of a light beam, the detector present detects the components of the eluted materials and is recorded on the record chart. Thus both the qualitative and quantitative information about the eluted anti microbial peptides can be known.
Analysis of gene expression:
Anti microbial peptides are not only the antibiotics but also acts as the mediators. Thus their regulation of gene expression is strict. The different defensins shows different expression depending on the signalling path ways and different stimuli. Thus they act as the effector molecules. For example the expression of human β defensin-1 is found to be constitutive where as the expression of the human β defensin-2 is unregulated by the inflammatory and infectious stimuli. Some times the expression of these defensins and cathelicidins is unregulated by the various factors such as interleukins, tumour necrosis factor-α etc. epithelial cells expresses receptors which are help full in detection of the infection and the inflammation and secreting the anti microbial peptides as effector molecules. As the anti microbial peptides are antibiotic in nature, they show synergistic activity along with other anti microbial molecules such as lysozyme, lactoferrin etc. The anti microbial peptides are strongly believed to act as mediators due to their patterns of expression and also gene regulation.
By the discovery of these anti microbial peptide molecules such as hBD1, hBD2etc. confirmed that not only epithelia of primates, insects, plants and some mammals are shielded with a chemical defence system but also human epithelial system is protected.
The anti microbial peptides discovered confirms the hypothesis of stating that these anti microbial peptides are a part of the innate host immunity by responding vigorously against the infection. This may pave the path for the development of new era in antimicrobial therapy as it proves to be a future promising advancement in the treatment of infectious diseases related to the epithelial or gastric or mucosal cell types.
The only disadvantage of using these anti microbial peptides as drugs is that their biological activity is still not known clearly and the other reason is that they are highly expensive in producing in sufficient amounts. Until now the facts regarding the antimicrobial peptides make clear that they can also be useful as the mediators of the inflammation in addition to antibiotics
Anti microbial peptides acts as an integral part of the host's innate immunity i.e., the first line of defence. Thus finally this discovery of antimicrobial peptides in the biotechnology and pharmaceutical field proves to be an advanced step in treating the skin disease and respiratory diseases thus paving novel path for the development of antimicrobial therapy
The idea of developing anti microbial peptides as innovative drugs by several biotechnological companies is worth while. With the scope of biotechnology and related areas such as biochemical assays, r-DNA technology etc the using of anti microbial peptides as a source for the development of drugs is possible. Even the derivatives of the anti microbial peptides are under the process in the pharma field which gives much scope for them in the near future.
The antimicrobial peptides are useful in treating the diseases.
This can be used in the field of gene therapy in the near by future.
As the antimicrobial peptides are protein molecules they are used in the production of vaccines.
Used in the production of drugs in the future but right now its use as drugs is restricted due to its unknown biological importance and function.
As it is very much expensive various strategies are put-forth for its reduction of the cost in designing the drugs using antimicrobial peptides.