Download Preparation and characterization of antimicrobial proteins/ peptides

Preparation and characterization of antimicrobial proteins/ peptides from the
heart tissues of consumable vertebrates
Dr.T.S.Saravanan.
The increasing trend of bacterial resistance to classical antibiotics is the major threat to the
human society. This impending crisis has spurred the search for new therapeutic agents with
new modes of actions (Makovitzki et al., 2006) without developing resistance. The antimicrobial
peptides (AMPs) show promise for becoming the next major group of natural antibiotics (Fleury
et al., 1996) to combat this problem. These peptides are either constitutive or inducible, serve as
crucible components in the innate host defenses and represent a potential source of useful natural
antibiotics for pharmaceutical applications (Koczulla and Bals, 2003 & Otvas, 2005) and are
widely distributed from plants and lower invertebrates to higher animals including human beings
and tend to be found in those parts of animals that are most likely to come in to contact with
pathogens from the environment (Chromek et al., 2006). These are mostly small cationic,
membrane active peptides including defensins and cathelicidins. AMPs hold significant
potential activities as therapeutic agents for prophylaxis and treatment of infections, promotion
of wound healing and immune modulation (Mc Dermott, 2009). These endogenous peptides with
broad spectrum of activity against bacteria, fungi and virus may prove more difficult for
microorganisms to develop resistance based on their mode of action. Unlike many natural
antimicrobials synthesized through specialized metabolic pathways, these peptides are expressed
from individual gene and produced by proteolytic processing from precursor molecules.
In developed countries, like USA, Canada, Japan. China etc., recent researches have been
focusing on finding out natural antibiotics from various tissues of invertebrates and vertebrates.
The immune system of multi-cellular organisms includes peptide that protect them from a broad
range of microbes. It is an inevitable fact that upon injury or bacterial infection higher
organisms up-regulate their production level of native antimicrobial peptides (Meister et al.,
1997 & Gallo and Huttner, 1998). Several workers have investigated different types of AMPs.
Xiao et al., (2006) have reported the three cathelicidin antimicrobial peptides named fowlicidin-1
to -3 from chicken. The cathelicidin family of antimicrobial peptides have been identified in
several mammalian species like rabbit (rCAP-18), mouse and rat CRAMP, guinea pig CAP-11,
BMAP-27, 28 and 34, sheep (SMAP-29 and -34), goat MAP-28 and -34, pig (pMAP-23, -36 and
-37) and primates (human LL-37 and rhesus 37) (Zelezetsky et al., 2006). The airway epithelial
cells of human produce β-defensins and cathelicidins (LL-37) (Hiemstra, 2007). Chromek et
al., (2006) described the production and function of the human cathelicidin antimicrobial peptide
LL-37, its precursor hCAP-18 and its ortholog CRAMP in epithelial cells of human and mouse
respectively. Thus a number of antimicrobial peptides have been reported in many tissues and
cell types in a variety of plant and animal species (Brogden, 2005) including cattle and birds.
In India also, some investigators are working in the area of antimicrobial peptides.. The
interaction of indolicidin, a 13 residue peptide rich in tryptophan and proline and its analogues
with model membranes was studied in ‘Centre for Cellular and Molecular Biology, Hyderabad (
Subbalakshmi et al., 1998). In Indian veterinary Research Institute, Izatnagar, Uttar padesh,
Ambika et al., (2008) have cloned and characterized a goat cathelicidin cDNA. The Centre for
Advanced study in Marine Biology, Annamalai University, Parangipettai is also carrying out
researches on antimicrobial proteins from hemolymph pf marine organisms. Recently
Rameshkumar et al., (2009) have also investigated the antimicrobial activity of a crab
(Thalamitta crenata) hemolymph.
The intriguing idea of developing antimicrobial peptides as innovative antibiotics has
been followed up by several biotechnological companies. With the use of protein-biochemical
methods and recombinant DNA technology, the structures of naturally occurring peptides serve
as the starting point for the development of new drugs. The use of antimicrobial peptides from
animal sources as drug is restricted so far by their high costs in the generation of sufficient
quantities. Hence, in this project we have planned to prepare antimicrobial peptides from certain
selective consumable animals in large quantities at cheaper cost against different human
pathogens.
OBJECTIVES OF THE PROJECT
Based on our recent reports (Sundaramoorthy and Saravanan, 2010 & Ph.D thesis of M.
Sundaramoorthy submitted to Bharathisan University, Tiruchirappalli), this project work is
planned to deal with an extensive screening and isolation of antibacterial peptides from the heart
tissues of easily available consumable animals like fish, chicken, goat, cow, pig etc., against
different human pathogens in order to prepare them at large scale. The following are the major
objectives of this project work.
1. Preparation of crude antibacterial proteins from the heart tissues by acid extraction and
screening their antimicrobial activities against human pathogens.
2. Determination of their MICs for the sensitive organisms.
3. Isolation and purification of the antibacterial proteins/peptides by using various methods
like chemical precipitations, SDS-PAGE, ion exchange chromatography and RP-HPLC.
4. Identification of the active peptides from the purified fractions by performing
antibacterial assays against the isolates which were inhibited by the crude extracts and
determination of their MICs.
5. Testing the cytotoxicity of the active peptides for human cells by performing the
hemolytic assays.
6. Determination of the molecular mass of the active peptides using MALDI-TOFF or
ESI/MS/MS.
7. Structural analysis by NMR.
8. Amino acid sequencing of the purified active peptides.
9. Synthesis of the same sequences of the active peptides artificially and to confirm their
antimicrobial properties.
METHODOLOGY
1. Acid extraction
- Matutte et al., (2000).
2. Antimicrobial activities
-Plate assay method (Diamond et al., 1991)
3. MIC
-Microdilution method (Zhu et al., 2007)
4. Cation exchange chromatograpyhy -(Li et al., 2007)
5. RP-HPLC
- Matutte et al., (2000).
YEARWISE WORK PLAN
I
Year
1. Collection and processing of heart tissue samples from animal sources.
2. Collection, conformation and maintenance of clinical isolates.
3. Preparation of crude antibacterial proteins of heart tissues by acid extraction, screening
their antimicrobial activities and determination of MICs for the sensitive organisms
against human pathogens.
4. Isolation and purification of the antibacterial proteins/peptides by different methods,
Identification of the active peptides by antimicrobial assays and MICs.
5. Testing the cytotoxicity of the active peptides for human cells by performing the
hemolytic assays.
II Year
6. Determination of the molecular mass of the active peptides using MALDI-TOFF or
ESI/MS/MS.
7. Performing the structural analysis by NMR.
8. Performing the amino acid sequences of the purified active peptides.
Synthesis the same sequences of the active peptides artificially and confirmation of their
antimicrobial properties.