Differential Expression and Characterization of Antimicrobial Peptide genes in

Megalobrama amblycephala against bacterial pathogens

Objectives:
1. Tissue distribution analysis of Histone and Chemokine antimicrobial peptide
transcripts in various tissues of Megalobrama amblycephala.
2. Characterization of Histone in fish and its oocyte-specific expression pattern
during oogenesis and embryogenesis.
3. Expression analysis of identified antimicrobial peptide transcripts in normal
and bacterial infected (4 Gram Positive and 4 Gram Negative) Megalobrama
amblycephala.
4. in-situ hybridization analyses of identified antimicrobial peptides.
5. Synthesis of antimicrobial peptides and their activity analyses against bacterial
pathogens.
Introduction:
Antimicrobial peptides play key roles in innate immunity. Antimicrobial peptides
are endogenous antibiotics that have been isolated from a multitude of animal and
plant species and are now recognised as crucial effectors of the innate immune
system. These peptides and proteins interact directly with bacteria and kill them. First
identied in frogs and insects, antimicrobial peptides are now known to be
widespread throughout the animal kingdom. Although many are cationic amphiphilic
alpha helices, such as the frog magainins, they show remarkable diversity in structure
and size. A small number of these agents have been identied in teleost sh. Parasin, a
peptide derived from the N-terminus of histone H2A, was found in Parasilurus
asotus and a much larger protein related or identical to histone H2B was found in the
skin mucus of another catsh Ictalus punctatus.
Histones are central components of nucleosomes and have vital roles in the
transmission of heritable gene expression patterns and in the epigenetic regulation of
nucleosome mobility. In eukaryotes, numerous histone variants have been identified
from almost all histones, and the nucleosome incorporation of histone variants can
alter the local chromatin structure to facilitate cellular processes such as transcription
or development regulation.
There is a growing body of evidence that histones, in addition to their roles in
chromatin formation and regulation of transcription, may constitute a component of
the system of innate immunity in both invertebrates and vertebrates, including

humans. Histones H2A and H2B are secreted from epithelial cells on the surface of
the human placenta and contribute to the antimicrobial activity of amniotic fluid (Kim
et al., 2002). In addition to the intact histones, fragments of histone H2A with potent
antimicrobial activity have been isolated from a range of tissues. The best known is
buforin I, comprising residues (139) of histone H2A, that was identified in an extract
of stomach tissue of the toad Bufo bufo gargarizans (Kim et al.,1996).
Materials and Methods:
Bacterial challenge
The fish will be injected intraperitonealy with the isolated Gram Positive
(Bacillus subtilis, Streptococcus, Staphylococcus, and Lactococcus lacti ) and Gram
negative bacteria (A. hydrophila, E. coli, Vibrio sp. and Pseudomonas sp.) suspended
in 1X phosphate buffer saline (100 ml/fish). Samples will be collected before (0 h),
and after injection (3, 6, 12, 24 and 48 h) and would be immediately snap-frozen in
liquid nitrogen and stored at -80C until the isolation of total RNA. Using a sterilized
syringe, the blood (0.5-1.0 ml per fish) will be collected from the fish caudal fin and
immediately centrifuged at 4000g for 10 min at 4C to allow blood cell collection for
total RNA extraction. PBS (1X) were prepared and served as control (100 ml/fish).
All samples will be analyzed in three duplications and the best representative data
would be chosen as described by Livak et al 2001.
Total RNA extraction and cDNA conversion:
Different tissues (Liver, Kidney, Spleen, Intestine, Heart, Gills, Brain, Skin,
Muscle and Blood) will be collected from both normal and parasite infected
Megalobrama amblycephala. Total RNA will be extracted from each tissue using
Trizol reagent method.
Gene expression analysis by qRT-PCR:
The relative expression of Pleurocidin in the Liver, Kidney, Spleen, Intestine,
Heart, Gills, Brain, Skin, Muscle and Blood will be measured by quantitative real
time polymerase chain reaction (qRT-PCR). In 20 ml reaction volume containing 4 ml
of cDNA from each tissue, 10 ml of Fast SYBR Green Master Mix, 0.5 ml of each
primer (20 pmol/ml) and 5 ml dH2O. After the PCR program, data will be analyzed
with ABI 7500 SDS software (Applied Biosystems). To maintain consistency, the
baseline was set automatically by the software. The comparative CT method will be
used to analyze the expression level of Pleurocidin. All samples were analyzed in
three duplications.

In situ hybridization:
Tissues collected for in situ hybridization will be manually dechorionated and
then fixed overnight at 4 C in 4% phosphate-buffered paraformaldehyde. Fixed
embryos will be washed briefly in 1X PBST to remove the fixative, transferred to
100% methanol and stored at 20 C for a minimum of 24 hr. The cDNAs amplified
by the primer pairs cloned into the pGM-T vectors (Tiangen, Beijing). Whole-mount
in situ hybridization using digoxigenin (DIG)-labeled RNA riboprobe will be carried
out as reported previously (Thisse and Thisse, 2008). Each template plasmid
linearized by restriction enzyme digestion, followed by in vitro transcription reaction
with T7 or SP6 RNA polymerase to generate the antisense or sense RNA riboprobes.
Tissues hybridized with appropriate riboprobes at 60C, incubated with anti-DIG
antibodies conjugated with alkaline phosphatase (AP) and stained with Roche BM
Purple AP substrates to produce purple, insoluble precipitates. Photographs will be
taken with a fluorescence microscope (Shen et al. 2010; Thummel et al. 2004).
Peptide Synthesis:
The amino acid sequences of histones peptides were predicted from nucleic acid
sequences as previously described Peptides will be synthesized by N-9fluorenylmethoxycarbonyl (Fmoc) chemistry at Sangon Biotech.
Antimicrobial properties Assays:
a. Bacteriostatic Assay
b. Bactericidal assay
c. Hemolytic assay
d. Cell proliferation,