МОЛЕКУЛЯРНАЯ БИОЛОГИЯ, 2011, том 45, № 4, с. 601-609
TEHOMHKA. ^^^^^^^^^^^^^^ TPAHCKPHnTOMHKA
UDC 579.25
MOLECULAR DETECTION AND CLONING OF THERMOSTABLE HEMOLYSIN GENE FROM Aeromonas hydrophila © 2011 Vijai Singha, b*, Indra Manib, c, Dharmendra Kumar Chaudharyb, Pallavi Somvanshid
aProgramme d'Epigenomique, Institute of Systems and Synthetic Biology Universite d'Evry Val d'Essonne-Genopole-CNRS
UPS3201, 5 rue Henri Desbrueres, 91030 Evry, France bNational Bureau of Fish Genetic Resources, Canal ring road, PO- Dilkusha Lucknow-226002, India cDepartment of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, India dBioinformatics Centre, Biotech Park, Sector-G, Jankipuram, Lucknow-226021, India
Received June 02, 2010 Accepted for publication September 29, 2010
Aeromonas hydrophila is a major bacterial pathogen associated with hemorrhagic septicemia in aquatic and terrestrial animals including humans. There is an urgent need to develop molecular and immunological assays for rapid, specific and sensitive diagnosis. A new set of primers has been designed for detection of thermostable hemolysin (TH) gene (645 bp) from A. hydrophila, and sensitivity limit for detection of TH gene was 5 pg. The TH gene was cloned, sequenced and analyzed. The G+C content was 68.06%; and phylogeny was constructed using TH protein sequences which had significant homology with those for thermostable and other hemolysins present in several bacterial pathogens. In addition, we have predicted the four and eight T-cell epitopes for MHC class I and II alleles, respectively. These results provide new insight for TH protein containing antigenic epitopes that can be used in immunoassays and also designing of thermostable vaccines.
Keywords: Aeromonas hydrophila, thermostable hemolysin, phylogeny, epitopes, vaccine.
INTRODUCTION
Fish and fishery products are an important food component in major parts of the world population, particularly in developing countries where fish is a major source of protein. There is an increasing awareness about the nutritional and health benefits of fish consumption from the last two decades. The low fat content of some fish and the presence of polyunsaturated fatty acids in a red meat fish increase the dietary and health significance of seafood consumption and are known to reduce the risks of coronary heart diseases [1]. However, there is a major issue to control of an infection and diseases in fishes.
Aeromonas hydrophila (A. hydrophila) causes soft tissue infections; meningitis and diarrhea in human hemorrhagic septicemia, fin and tail rot in fish, thus, resulting in high mortality rate in commercial aquacultures [2]. There are many virulence factors which are basically proteins contribute significantly to patho-genesis and play a major role in developing fish diseases. The virulence factors are directly associated with reducing strengthen of the immune system of fishes. A. hydrophila secretes number of proteins and enzymes such as proteases, DNase, RNase, elastase, lec-ithinase, amylase, lipase, gelatinase, chitinase [3, 4], cytotoxic enterotoxins [5], and 3 hemolysins: a-hemol-ysin, P-hemolysin and an unknown type. The aerol-
* E-mail: vijaisingh15@gmail.com; vijai.singh@issb.genopole.fr
ysin gene has been reported as P-hemolysin [6—8]. Lipase is an important extra-cellular enzyme having lipolytic activity that was considered to be a virulence factor affecting host immune system functions by generating of free fatty acids. For a molecular detection of A. hydrophila PCR amplification of the lipase gene has been used [9, 10].
The detection of A. hydrophila was based on the virulence gene amplification using PCR techniques. Two unrelated extracellular toxins, such as aerolysin and hemolysin of A. hydrophila, entail in the development of hemorrhagic septicemia and also play a crucial role in the pathogenesis in fish and other animals. Aerol-ysin of A. hydrophila binds to specific glycosylphos-phatidyl inositol-anchored proteins (GPI) of host red blood cells (RBCs) causing their P-hemolysis [11, 12]; while a-hemolysin plays a vital role in the partial destruction of RBCs. Genes coding both virulent proteins are located in the genome of A. hydrophila in diverse position and resembled only 18% in the sequence homology [13, 14].
Specific primers have been used to detect a fragment of gene which encodes an aerolysin from a hemolytic strain of A. hydrophila [15]. Aerolysin gene was detected in A. hydrophila isolated from drinking water, fish and various food samples [16]. The PCR assay has been used to amplify the hemolysin and aerol-ysin genes in A. hydrophila and A. veronii isolates of clinical samples [17]. Aerolysin and hemolysin are the
common genes of almost all pathogenic A. hydrophila which were isolated frequently from diarrhea patients but these were noticeably absent in the A. caviae and A. veronii strains [18]. The detection of three major genes such as hemolysin, aerolysin and lipase ofA. hydrophila isolated from fish samples was carried out by PCR [10, 19, 20]. Hemolysin and aerolysin were detected by PCR amplification in all virulent strains of A. hydrophila isolated from clinical and environmental samples [21]. Hexaplex PCR were used by targeting the aerolysin (aer), GCAT (gcat), serine protease (ser), nuclease (nuc), lipase (lip) and lateral flagella (laf) genes to detect the virulence factors in different Aeromonas spp. isolated from water samples of trout and infected fish farms. In these, the major strain was A. sobria and dominant virulence genes were aer and nuc [22]. Three virulent genes, aerolysin, hemolysin and DNase, were frequently observed in clinical as compared to environmental samples; while aerolysin, hemolysin and serine protease genes were present in all P-hemolytic strains of Aeromonas species [23]. The strains of A. hydrophila and A. veronii biovar sobria were tested for presence of potential virulence hemolytic genes of aerolysin and hemolysin. A. hydrophila isolates were positive for both the genes, while A. veronii biovar sobria ones were negative [24].
There is also constant need to develop immunoas-says for easy and user friendly techniques for diagnosing and controlling pathogens. The use of immunoassays for identification and characterization of the pathogens require presence of an antigen or antibody. The whole bacterial cell can be used for the immu-noassay, but generally the common antigenic epitopes give cross reactivity to them. Therefore, purified specific proteins are required for the diagnostics of A. hydrophila. Earlier, a set of primers were designed for amplification of complete open reading frame (ORF) of aerolysin gene from A. hydrophila was cloned, sequenced and expressed [25, 26]. The gene for another toxin protein, hemolysin secreted by A. hydrophila, was amplified, cloned and expressed for the control of A. hydrophila infections [14]. The presence of thermostable related hemolysin (trh) gene coding a new type of hemolysin known as thermostable hemolysin in three clinical isolates of Aeromonas veronii biovar vero-nii strains was confirmed by PCR amplification followed by cloning and sequencing [27]. However, the development of an immunoassay early diagnostics for identification of A. hydrophila is particularly important for the infection control and prevention of spread. The cloning, expression and purification steps for protein production are quite time consuming and laborious process, while recently in silico tools are easily accessible for prediction and mapping of antigenic epitopes from protein sequences. A use of in silico technique assists in designing the subunit vaccines against pathogens independent of their availability and without a need to grow these microbes in vitro [28, 29].
An epitope, also known as antigenic determinant is a region of the antigen which is specifically recognized by the immune system, i.e. by antibodies, B-cells, or T-cells. The region of the antibody that recognizes the epitope is called the paratope. Antibodies recognize and interact directly with the antigen. T-cell receptors recognize only the antigen that is combined with either class I or class II major histocompatibility complex (MHC) molecules. The two major subpopulations of T-lymphocytes are the CD4+ T helper (TH) cells and the CD8+ T cytotoxic (TC) cells. TH cells secrete cytokines that regulate the immune response upon recognizing the antigen combined with class II MHC.T C cells recognize the antigen combined with class I MHC and give rise to cytotoxic T-cells (CTLs) which display the cytotoxic ability. T-cell epitopes are presented on the surface of antigen-presenting cells (APC) where they are bound to MHC molecules. T-cell epitopes presented by MHC class I molecules are typically between 8 and 11 amino acids in length, whereas MHC class II molecules may be longer. An epitope mapping of proteins is used for development of new vaccines and diagnostic approaches. Prediction of epitopes in the putative protein of hepatitis B virus with Propred and Propred1 is a new strategy to produce antigens for diagnosis. Synthetic peptides can be used as vaccines to induce either humoral or cell-mediated immunity that requires the understanding of the nature of T-cell and B-cell epitopes [30, 31]. Identification of significant T-cell epitopes from secretory and cell surface virulent proteins of the M. tuberculosis H37Rv strain was done. Promiscuous epitopes from HTL and CTL were recognized [32]. The prediction of T-cell epitopes of surface proteins hemagglutinin and neuraminidase of influenza A virus H5N1 was reported. Highly conserved and specific epitopes were predicted for the influenza A virus hosts and were shown to be strain specific [33]. The complete genome of the hepatitis B virus was used for identification of the T-cell epitope in silico [34].
The aim of the study was to develop a TH based molecular assay for sensitive and speci
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