Biotechnological Based Subtractive Hybridization and Variations of Gene Content in an Organ of Pathogenic Shrimp
M. Jasmine Priya
The objective of this study was to determine the bacterial pathogen and gene content of the Etroplus suratensis (pathogenic shrimp). PCR-based subtractive hybridization method detected with the help of Escherichia coli on shrimp liver and granulose cells for efficiently detecting the DNAs and applies in to liver pathogen. Seventeen DNAs specific to a mono-key colonizing strain (J140) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (ABO18799). Among the seventeen different clones 14 were unique and other three numbers were each represented at twice. Nine of the clones DNAs were found by sequencing to be absent or very divergent from those in the same direction on disappeared blank region of this liver and granulosa cells. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated E.coli strains by using primer specific for 12 subtracted clones and complementary southern hybridizations indicated that these DNAs are highly polymorphic in the E. coli population with each strain yielding a different pattern of gene-specific PCR amplification. When described about this Polymorphic immune response gene, it helps to identify the previously unknown virulent genes in the pathogens and it provides a new insight of microbial genetic diversity and evolution.
Received: February 07, 2011;
Accepted: May 25, 2011;
Published: September 26, 2011
Fishes are the diverse group of animals, highly specialized for their aquatic
existence and comprising almost half the number of the vertebrate species in
existence today (Hedge et al., 2000). Fishes were
in an intimate contact with their environment which can contain very high concentrations
of bacteria and viruses (Shike et al., 2002).
Very recently, Rane and Barve (2011) described standardization
and optimization of gene content and molecular genetic analysis of D-loop region
in an animal natural fiber. Genes that are present in certain isolates of a
given bacterial species and absent are substantially difficult in others can
be of great interest biologically. Some are strain specific traits such as immune
responsive drug resistance (Bayne et al., 2001).
Later, Pan et al. (2005) studied the immune-relevant
factors responsible for the virus resistance in the WSSV-resistant shrimp, a
suppression subtractive hybridization method was employed to identify differentially
expressed genes and their expression profile in the hepatopancreas of the virus-resistant
penaeid shrimp. Thirty-five genes were identified from more than 400 clones,
of which eight are found for the first time in penaeid shrimp.
Many of the genes or DNA segments specific to individual strains were found
by the special phenotypes they confer (Waldor and Mekalamos,
1996; Gorisman and Ochman, 1996). It was also reported
that the detailed physical mapping 10 or comparisons of sequence data from different
isolates of taxes (Himmelreich et al., 1997;
Bobe and Goetz, 2000a, b; Kanamori,
2000). During the subtractive hybridization 13 allows strain specific DNAs
to be selected directly and is attractive because it eliminates the need to
score any particular phenotype or to do extensive mapping or sequencing at the
outset. However, the subtractive methods developed to date have been unwieldy
technically and tend to be quite narrowly selective (Douglas
et al., 2003; Thangaraj et al., 2011).
Very recently, James et al. (2010) published
a subset of strain specific DNAs are generally obtained and DNA segments with
potentially interesting mixes of pathogenic strain-specific and common sequences
are generally excluded (Tinsley and Nassif, 1996).
The vertebrate immune system is composed of two types of immunity, innate and
adaptive. This kind of immunity emerged early in vertebrate evolution at some
time during the division of the jawless lamprey and the cartilaginous fishes
(Fujiki et al., 2000). An adaptive immune response
is called for when the innate host defenses are defeated or by passed and when
the elimination of a new infection is unsuccessful (Bayne
et al., 2001). There are many commonalities in the immune response
of different vertebrate classes. In Teleost fish, however some unique features,
such as temperature dependent acquired immunity and some limitations on antibody
diversity (Kaattari and Piganelli, 1996). Hence, the
innate immune functions may play a more important role in these animals than
in the more derived homeothermic invertebrates such as fish and amphibians.
The present study is the first approach to the study of immune response of
bacterial infection in a teleost fish of Etroplus. This has been employed
Suppression Subtractive Hybridization (SSH) a new method based upon the Polymerase
Chain Reaction (PCR) and requires far smaller amount of starting materials than
traditional methods. There are number of techniques available to isolate regulated
genes (Van Hal et al., 2000; Aziz
et al., 2010). The choice of technique was somewhat dependent on
the desired result and how much RNA can initially be obtained for subtraction.
However, the more recent approaches such as SSH and DD-PCR are clearly more
efficient and sensitive than the original techniques involving cDNA subtraction
techniques. The objective of this study was to determine the bacterial pathogen
and its gene content of the Etroplus suratensis (pathogenic shrimp).
MATERIALS AND METHODS
This experiment was carried out during the year of March 2006 to February 2007. The experimental Shrimp juveniles weighing approximately 5 g were collected from the backwaters of Tamilnadu and Kerala. The tank was filled with water and maintained with adequate aeration. The fishes were fed with pelted feed. The water in the holding tank was exchanged daily and the water quality parameters were monitored weekly.
Collection of bacterial pathogens: Fish bacterial pathogens used for the experiments were obtained from the Microbiology Laboratory. Isolates such as Vibrio esturiances and Aeromonas hydrophila characterized earlier were used. In order to prove the pathogenecity profile of bacterial isolates and verify the Kochs postulates, experimental challenge studies were conducted using apparently healthy fishes.
General methods: The Esherichia coli and H. pylori strains
used in this study are listed in Table 1. STD methods (Tiffoche
et al., 1993; Gerwick et al., 2002)
were used for the growth and DNA extraction from E. coli.
Oligonucleotides: E. coli strains-specific oligonucleotide primers used for PCR listed in Table 2. The following gel purified oligonucleotides were used for subtractive hybridizations. Adaptor-1 5GATTATGCACTGTGTCTACCCGAGCTCCGCCCGCGGGCCCGACCA-3 and 3CCGGGCAGGA-5; Adaptor2. 5-GATTATTGCTGAGTGAGTGATATCCCGTCGCACCAGGCCCGGCTCCA-3 and 3-CGGCCGAGGT-5; p1: 5- GATTATGCTGAGTATGATCCCG-3 (This P1 PCR primer matches the long strands of adaptors 1 and 2 at their 5 ends.) NPI 5- AGCTCGCGGCGGGCCCGTCCA-3-3; NP2 5-GGCACCACGCCGGCGTGCA-3. (These NP1 and NP2 nested PCR primer match the internal portions of the long strand of adaptors one and two, respectively).
Overview of subtractive hybridizations: This method involved the digestion
of DNAs from the strain of interest and a reference (tester and driver, respectively)
with restriction endonucleases such as Rsa1 to generate DNA fragment populations
with median sizes of about 0.5 kb. Two different PCR adapters that can join
only to 5 ends of target DNAs because of their own 5 ends lack of
phosphate groups were ligated to different aliquots of tester DNA. These ligated
DNAs were denatured then mixed with an excess of driver DNA (that has no adapters)
and allowed to anneal. The two DNA pools were mixed together also add with additional
denatured driver DNA for further bind tester sequences that are also present
in the driver genome.
|| E. coli strains and used in these studies
|Each strain, except Tx30a WV99, 26695 cag contains the cag
PAI in strain Hp1 contains an internal deletion (D. Kersulyte and D.EB,
unpublished work). *NTCC, National Collection of Type Cultures
|| Name of the experimentally designed primer and primer sequence
Remaining complementary single strands of tester DNA are allowed to anneal
and the adaptor sequences are copied on to their 3 ends. Then PCR was
used to obtain the exponential amplification of tester DNAs with the same adaptor
at each end is suppressed because self-annealing of inverted repeat adaptors
(Fig. 1) which inhibits the binding of PCR primers. Tester
DNAs with an adaptor at only one end undergo linear but not exponential, amplification.
This method offers several substantial advantages over earlier bacterial genome
subtractive methods (i) less DNA was needed; (ii) multiple rounds of hybridization
and physical removal of tester-driver DNA complexes as in ref.13 and are not
needed. Earlier, Tinsley and Nassif (1996) stated that
there was no need for complicated adaptor removal and re-addition with additional
rounds of PCR amplification by difference analysis method.
Driver and tester DNA preparations: Two micrograms of tester and DNAs were each digested to completion with 20 units of AluI (made up where) for 16 h in 200 μL reaction volumes, extracted with phenol and precipitated with ethanol and resuspended in 10 mM Tris HCl, pH 7.5 at a final concentration of 200 ng μL-1. Two aliquots of tester DNA (120 ng each) were ligated separately to 2 μL of the two adapters each in a total volume of 10 μL of (2 μmM final concentration) at 16°C at overnight, using 1 μL of T4 DNA ligase in the buffer supplied by manufacturer. After ligation, 1 μL of 0.2 M EDTA was added and the samples were heated at 70°C for 5 min to inactive the ligase and then stored at -20°C.
Subtractive hybridization: Three micro liters of driver DNA (600 ng) was added to 1 μL (12 ng) of each of the adaptor-ligated tester DNAs (50:1 ratio). One microliter of 5 x hybridization buffer (2.5 M NaCl/250 mM Hepes, pH 8.3/1 mM EDTA) was added to each tube the solutions were overlaid with mineral oil and the DNAs were denatured (1.5 min, 90°C) and allowed to anneal for 1.5 h at 60°C. After this first hybridization, the two samples (first with adaptor 1, the second with adaptor 2) were combined, 300 ng more of heat-denatured driver DNA was added in 3 μL of 1 x hybridization buffer and the sample was allowed to hybridized for an additional 14 h at 65°C (without intermediate denaturation). This final 13 μL reaction was diluted 200 μL with dilution buffer (50 mM NaCl/5 mM EDTA), heated at 65°C for 10 min and stored at -20°C until use in PCR.
Two sequential PCRs were carried out. The first PCR contained 1 μL of genomic DNA prepared as described, 2 μL of PCR primer P1 (10 μM) and 47 μL of a PCR master mix prepared using the advantage cDNA PCR core kit (total volume 50 μL). This first PCR was incubated at 72°C, 2 min and then subjected in to 25 cycles of 95°C, 30 sec; 72°C for 1.5 min. The amplified products were then diluted 20 fold in 10 mM Tris HCl pH 7.5 and 1 μL of each diluted samples was used in the second PCR with 2 μL of PCR primers with NPI and NP2 in 10 μM each and advantage cDNA PCR core kit (total volume, 50 μL) for 10 cycles of 90°C, 30 sec with 68°C, 30 sec and 72°C 1.5 min.
Construction of SSH libraries: Total tissue extracted RNA was used to
synthesized cDNA for SSH according to the manufacturer instructions. Briefly
cDNA has been synthesized from challenged fish (tester-cDNA) and unchallenged
fish (driver-cDNA) then RNA treated with an oligo (dT) containing primer. Tester
and driver-cDNA were separately digested with Real shorter blunt ended molecules.
Two ester populations were ligated to two different adapters. The tester cDNA
was hybridized with an excess of driver-cDNA to remove common cDNA transcripts,
generating the (Single Subtractive Hybridization) SSH+library. The tester cDNA
were hybridized with water instead of driver cDNA to provide unsubstracted cDNA
it act as a control.
Preparation of driver cDNA probes: DIG labeled probes were made by PCR labeling according to the manufacturers instructions (Roche Diagnostics) using primers listed in Table 1 also with tester-cDNA as a template. Driver-cDNA probes were made by PCR labeling according to the manufacturers protocol using 5 PCR primer (5-AAGCAGTGGTATCAACGCAGAGT-3) SmartTM cDNA library construction Kit.
The present study is ascertaining to know the Etroplus host and its bacterial pathogenesis through amenable genetic analysis. An infection disease is the manifestation through amenable genetic analysis. An infection disease is the manifestation of a dynamic series of event that occur between the host and pathogen that are defined by the interaction of pathogen-expressed virulent factors and the surveillance and defense systems of the host. Expression of both host and pathogen components is highly co-ordinated and the stimulus for any given response by the pathogen.
Subtractive hybridization: Tested of this bacterial genome by subtractive
hybridization method (Fig. 1) using isogenic E. coli
strains that differed only by the presence or absence of the 37 kh cag PAI.
Which were equivalents about at 2% of the E. coli genome: strain 1480
wild type (cag+) was used as tester and Cag (C) (Calcium Channel
Gene) (Huntington Diseased Gene) was used as a driver. Subtracted DNAs were
cloned, the inserts were PCR amplified and products were spotted on hybridization
filters. Clones specific to the cag PAI were then identified by probing these
filters with labeled genomic DNAs from the cag tester strain and the cage driver
and also with the set of DNA fragments that span the entire cag PAI (data not
||Representative dot-blot hybridization of products of subtractive
hybridization by using genomic DNAs of E. coli strains J170 as tester
and ABO18799 as driver. Cloned subtractive hybridization products were PCR-amplified
using primers NPI and NP2 and spotted on Hybond N+ filters (Materials
and Methods) and probe with labeled genomic DNAs, as indicated. The clones
shown by DNA sequencing to have J170 DNAs either not present in the ABO18799
genome or substantially different from sequence in ABO18799 (as summarized
in Table 3) are circled
The hybridization results indicated that more than 90% of clones had derived
from the cage PAI and 14 of them were one pass sequenced. This result indicates
that many different DNAs specific to a given strain can be obtained by the subtractive
Two unrelated E. coli strains were tested next for differences in gene content, by using a strain well suited for rhesus monkey colonization (J 170) as tester and the strain whose genome was sequenced (26695) as driver. Each strain is plasmid free and it contains the cag PAI and the ice A1 gene. DNAs that recovered after subtraction were cloned. PCR amplified and arrayed for hybridization with labeled tester and driver DNAs as above. About 27 of 52 clones tested by hybridization were judged to contain strain J170 specific sequences, although the differences among clones were apparently less than in the first trial with isogenic cage of E. coli strains as will be explained. In contrast none of the 8 random clones (generated without subtractive hybridization) appeared to be specific (J170, 1480, 266695). Fourteen were unique three others were each represented twice which indicated that 17 different clones had been obtained. Nine of the cloned DNAs were found by sequencing to be absent or very divergent from those in the same direction on disappeared blank region of this liver and granulose cell.
Protein homologies: Eleven of the seventeen clones exhibited significant protein homologies with other databases entries, as summarized in Table 3. Five clones had homologies to part of putative restriction-modification (R-M) proteins. Two matched overlapping portions of type I specificity subunits (HsdS) shown in Table 3 but differed from one another size and sequence and thus must derive from different hsd5 genes three putative hsd 5 genes were found in the 16953 genome sequence ref. 29. Two clones had homology to Bcg-1 (Table 3) a two- subunit enzyme that is unusual in cleaving DNA at fixed distance on each side of the recognition site. Although quite difference BcgI homologs are encoded in strains 166943 (HP 1471; ref) PCR tests mapped these two types of homologs to the same locus. However the DNA sequence of the HP 1208 gene for J166 is known and does not contain the sequence found in clone F3. Thus the clones represent the different locus. The fifth clone exhibited protein homology to a type of II DNA methyltransferase. Two other clones exchibited homology to proteins that might be metabolic as well as immunoresponsive function. Five clones exhibited protein level homology to particular gene for hypothetical proteins of unknown homologs in other microbes. The remaining seven clones did not exchibit significant functions, two of which had equivalent function unknown protein homology to entries in current databases.
|| Features of E. coli specific clones with significant
The present study is ascertaining to known the Etroplus host and its bacterial
pathogenesis through amenable genetic analysis. Though, Fujiki
et al. (2000) suggested a manifestation of the infectious diseases
were a dynamic series of events mainly that occurred between the host and pathogen
it might be defined as an interaction of pathogens expressed through virulence
factors also depends upon the surveillance and resistant power of the host.
Currently, Sriputhorn and Sanoamuang (2011) stated that
the expression of both host and pathogen components is highly co-ordinated and
the stimulus for any given response by the pathogen is often a prior change
in defense gene expression by the host. This dynamic interplay determines the
character, course and outcome of the pathogenic process. Much remains to be
elucidated about host-pathogen interactions at the molecular level and the most
informative model systems are likely to be those in which both the pathogen
and host. Mainly, they were amenable to genetic systems for a wide variety of
pathogenic microorganisms; rather less progress has been made in the host organisms
that have traditionally been used to model infectious disease (Alonson
and Leong, 2002). The present findings of this result also agreed by Bayne
and Gerwick (2001) on immunological efficiency reflected gene content in
another variety of the fish.
Recently an exciting approach has been to make use of the abilities of certain
bacterial pathogens to infect nonmammalian model organisms with well-defined
genetic systems, including the invertebrates Drosophilla melanogaster
and Caenorhabdities elagans and the plant Arabidopsis thaliana. The ability
of these models to mainly provide the infection of mammals derived from the
fact that many fundamental host defense strategies. Velculescu
et al. (1995) and Jorgensen et al. (2000)
explained the evolutionarily ancient and are conserved diverse taxa are the
microbial virulent factors, since they were used for evade from those defensive
process. However, these model organisms lack defense systems that play important
roles in mammalian host-pathogen interactions, including leukocytes, innate
cellular immunity and adaptive immune systems.
Several technical aspects of this bacterial genome subtraction protocol merit
attention. First the recovery of strain specific DNAs with unrelated strains
(J140VS1780) was lower than that with isogenic control strains (cag+ and cag).
This may reflect the base substitution and restriction fragment length differences
between tester and driver DNAs which lower the efficiency with which driver
DNAs titrate homologous tester sequences. Second half of J140-specific clone
contained patches of sequence that were matched to those strain 1780. Such DNAs
tend to be lost in other subtractive hybridization protocols (Brown
and Curtiss, 1996; Tinsley and Nassif, 1996), although
many of them could be important phenotypically (Coffey et
al., 1995; Seifert, 1996). Their recovery here
may reflect incomplete pairing with driver DNA which allows annealing with complementary
tester DNA strands and thereby amplification (Fig. 1). In
most of the cases, the nature and sizes of the DNA segments remain to be defined.
Some will certainly represent members of divergent gene families that are mostly
carried on E. coli strains (Savan and Sakai, 2002).
Others may represent only a single gene that are strain specific in their distributions.
A third important class may derive from much larger strain specific in their
distribution of DNA segments such as PAIs and may contain new genes affecting
colonization or diseases.
The present study also has demonstrated up-regulation of complement C7 and
DRTP transcription by both RT-PCR and virtual Northern analysis. Many clones
related to C7 complement and DRTP transcripts were sequenced in this SL5 library.
A similar result was reported in rainbow trout liver library in response to
Vibrio injestion with highest number of clones being for DRTP (n = 12) (Bayne
and Gerwick, 2001; Ellis, 2001). The derived C7
and DRTP aminoacid sequences are 100% (AF281336). The derived C7 and DRTP aminoacid
sequences are 100% (AF281336) and (AF81355) identical to rainbow trout sequences,
respectively. Complement C7 is the part of the assembly of the late complement
components to form a membrane attack complex (Janeway et
al., 2001). The DRTP shares 34% sequences homology with CD59 which is
complement inhibitor whose function is to protect against the lytic effect of
the complement membrane attack complex (Lee and Goetz, 1998).
The increase transcription of complement C7 and DRTP may balance one another
and restore homeostasis during the APR to bacterial infection. Interestingly
number of researchers discovered another detoxification related EST derived
aminoacid sequence showed 26% identity to a novel soluble saxitoxin and tetrodotoxin
binding plasma protein of puffer fish (Yotsu-Yamashita
et al., 2001; Bayne and Gerwick, 2001) and common
carp by Agouz and Anwer (2011).
Since the exact functions of both O-methyl transferases of SBP in fish are
unknown, it will be very important to elucidate their role of infection distinguished
by Hedge et al. (2000). Pathogens are often first
recognized by virtue of their carbohydrate surfaces and a number of animal lectins
are up regulated in infected salmon. Tachylectin was up regulated during infection
in all three tissues tested (Aranishi and Mano, 2000).
To date this fibrinogen like lectin has only been discovered in the Japanese
horseshoe crab (Tachypleus tridentatus).
These results been showed that when the infection raised by any one of the
pathogenic organisms the immune modulation of gene detected through the subtractive
hybridization. Lectins from the C-type lectin super family were also represented
in the subtracted libraries and three distinct C-type lectins were found to
be up regulated in response to infection (Lund and Olafsen,
1998). While, C-type lectin 2-1 was originally isolated from carp and found
to be constitutively expressed in liver and up-regulated in liver during the
APR (Bayne et al., 2001; Alonson
and Leong, 2002) and it specify an interesting difference in tissue specific
modulation of expression between these two species. However, such mixtures of
matching and divergent sequences are also potentially important evolutionarily
in facilitating the formation of new genotypes by recombination during mixed
infection and in thereby sometimes fascilitating quite dramatic changes in bacterial
phenotype. Beyond these organisms that colonize inconstant and potentially hostile
niches, such gene exchange can often be more potent than de novo mutation as
The authors gratefully acknowledged to our Malankara Catholic College Correspondent
Fr. Prem Kumar (M.S.W) given encouragement and support for preparation of this
research manuscript. We wish to extend the thanks to colleagues of S. Muthukumar,
Dr. Francis Borgio, P.S. Sumi, Mary Sujin and Mary Jeeva for sample collection
and all supports of this research study. The second author thankful to the Women
Scientist Scheme (WOS-A), DST for financial assistance.
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