Bacterial membrane binding and pore formation abilities of carbohydrate recognition domain of fish lectin

Accepted Manuscript Bacterial membrane binding and pore formation abilities of carbohydrate recognition domain of fish lectin Abirami Arasu, Venkatesh Kumaresan, Rajesh Palanisamy, Mariadhas Valan Arasu, Naif Abdullah Al-Dhabi, Munuswamy-Ramanujam Ganesh, Jesu Arockiaraj PII:

S0145-305X(16)30318-4

DOI:

10.1016/j.dci.2016.10.001

Reference:

DCI 2737

To appear in:

Developmental and Comparative Immunology

Received Date: 10 August 2016 Revised Date:

6 October 2016

Accepted Date: 6 October 2016

Please cite this article as: Arasu, A., Kumaresan, V., Palanisamy, R., Arasu, M.V., Al-Dhabi, N.A., Ganesh, M.-R., Arockiaraj, J., Bacterial membrane binding and pore formation abilities of carbohydrate recognition domain of fish lectin, Developmental and Comparative Immunology (2016), doi: 10.1016/ j.dci.2016.10.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Bacterial membrane binding and pore formation abilities of

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carbohydrate recognition domain of fish lectin

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Abirami Arasua,b, Venkatesh Kumaresana, Rajesh Palanisamya, Mariadhas Valan

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Arasuc, Naif Abdullah Al-Dhabic, Munuswamy-Ramanujam Ganeshd, Jesu

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Arockiaraja*

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a

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Faculty of Science and Humanities, SRM University, Kattankulathur 603 203, Chennai, Tamil

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Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology,

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Nadu, India

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India

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of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia

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203, Chennai, Tamil Nadu, India

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Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College

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Interdisciplinary Institute of Indian System of Medicine, SRM University, Kattankulathur 603

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Department of Microbiology, SRM Arts & Science College, Kattankulathur 603 203, Chennai,

* Corresponding author:

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J. Arockiaraj, Division of Fisheries Biotechnology & Molecular Biology

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Department of Biotechnology, Faculty of Science and Humanities

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SRM University, Kattankulathur 603 203

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Chennai, Tamil Nadu, India

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Tel.: +91-44-27452270; Fax: +91-44-27453903

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E-mail: [email protected]

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ABSTRACT Antimicrobial peptides (AMPs) are innate molecules that are found in a wide variety of

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species ranging from bacteria to humans. In recent years, excessive usage of antibiotics resulted

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in development of multi-drug resistant pathogens which made researchers to focus on AMPs as

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potential substitute for antibiotics. Lily type mannose-binding lectin is an extended super-family

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of structurally and evolutionarily related sugar binding proteins. These lectins are well-known

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AMPs which play important roles in fish defense mechanism. Here, we report a full-length lily

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type lectin-2 (LTL-2) identified from the cDNA library of striped murrel, Channa striatus (Cs).

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CsLTL-2 protein contained B-lectin domain along with three carbohydrate binding sites which is

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a prominent characteristic functional feature of LTL. The mRNA transcripts of CsLTL-2 were

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predominantly expressed in gills and considerably up-regulated upon infection with fungus

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(Aphanomyces invadans) and bacteria (Aeromonas hydrophila). To evaluate the antimicrobial

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activity of the carbohydrate binding region of CsLTL-2, the region was synthesized (QP13) and

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its bactericidal activity was analyzed. In addition, QP13 was labeled with fluorescein

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isothiocyanate (FITC) and its binding affinity with the bacterial cell membranes was analyzed.

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Minimum inhibitory concentration assay revealed that QP13 inhibited the growth of Escherichia

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coli at a concentration of 80 µM/mL. Confocal microscopic observation showed that FITC

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tagged QP13 specifically bound to the bacterial membrane. Fluorescence assisted cell sorter

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(FACS) assay showed that QP13 reduced the bacterial cell count drastically. Therefore, the

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mechanism of action of QP13 on E. coli cells was determined by propidium iodide

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internalization assay which confirmed that QP13 induced bacterial membrane disruption.

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Moreover, the peptide did not show any cytotoxicity towards fish peripheral blood leucocytes.

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Taken together, these results support the potentiality of QP13 that can be used as an

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antimicrobial agent against the tested pathogens.

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Lily type lectin

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Antimicrobial peptide

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Fluorescein isothiocyanate

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Membrane disruption

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Flow cytometry

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Confocal microscopy

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1. Introduction In aquatic environment, fishes survive in the midst of a group of pathogenic and non-

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pathogenic microorganism. To counteract the entering pathogens, fishes primarily rely on innate

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immune system which is the fundamental defense mechanism that also plays a key role in

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acquired immunity and homeostasis through a system of receptor proteins (Maciel et al., 2004).

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Notably, they depend on pattern based recognition rather than antibodies for their protection

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against pathogens (Sharon et al., 1998; Kawasaki et al., 1978). Channa striatus, commonly

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known as snakehead murrel is an air breathing freshwater fish which inhabits all types of water

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bodies and has several traditionally identified medicinal benefits. Epizootic ulcerative syndrome

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(EUS) is a fish pathological condition, primarily caused by an oomycete, Aphanomyces invadans

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and later by bacteria such as Aeromonas hydrophila (Arockiaraj et al., 2013 Jayaram, 1981); they

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disrupt the fish epithelial tissues further leading to mass mortality in murrel culture system.

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During the infection state, C. striatus tend to express various immunological components such as

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complement, antimicrobial peptides, C-reactive protein, hemolysin and lectin to eliminate

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pathogens from their system (Arockiaraj et al., 2013).

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Lectin is one of the major immune components involved primarily in recognition of

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pathogens (Souza et al., 2011; Melo et al., 2011; Dutta, et al., 2005). During the past decades,

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enormous improvements have been achieved in the study of lectin molecule. The binding of

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lectin is reversible and non-covalent with simple or complex carbohydrate conjugates, whether

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free in solution on cell surface (Tasumi et al., 2002; Tateno et al., 2002). Lectins exist either as

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transmembrane protein located on cell surface or in physiological fluid which act as recognition

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molecules inside the cell. The specificity of lectins is categorized based on the carbohydrate

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recognition domain (CRD). Lectins CRD contained highly conserved amino acid residues at a

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characteristic pattern, which determines the function of the protein (Janeway et al., 2002; Tateno

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et al., 2002). Based on carbohydrate specificity, animal lectins have been classified into several

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types (Kawasaki et al., 1978) including fucose, mannose, sialic acid, N-acetylglucosamine, N-

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acetylgalactosamine and glycan complex. The specificity of binding is achieved all the way

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through hydrogen bridges, van der Waals and hydrophobic interactions between sugar and lectin

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(Tasumi et al., 2002). The animal lectins were divided into four main groups on the basis of the

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structure of the CRD such as C type, galectins, P-type and I-type (Barondes et al., 1994; Kasai et

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al., 1996; Kornfeld et al., 1992; Powel et al., 1995). So far, several lectins have been identified

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from various fish species such as trout, salmon, carp, rohu, channel catfish and blue catfish. Even

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among fish, lectins are highly diversified both structurally and functionally, including specific

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and unique recognition of carbohydrate (Zhang et al., 2012; Ourth et al., 2007). The high affinity

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and specificity of protein-carbohydrate interactions may attribute to multivalent binding that

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produce the cluster effect. Lectins are now intensively investigated to understand their biological

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roles in cell recognition or biodefense and its down-stream mechanism (Ogawa et al., 2011).

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In recent years, gradually the microbes developed resistance against many synthetic drugs

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and antibiotics. We are in need to improve the therapeutic drugs like antimicrobial peptide and

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its derivatives. Antimicrobial peptide showed specific action towards bacterial pathogens and

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disrupts the cell membrane, further targets the intracellular molecules such as DNA. Recent

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research has demonstrated that some of the unique properties of fish peptides, including their

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ability to act even at very high salt concentrations, make them good potential targets for

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development as therapeutic antimicrobials. Further, the stimulation of their gene expression by

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exogenous factors could be useful in preventing pathogenic microbes in aquaculture (Ganz et al.,

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1985; Lehrer, et al., 1983).

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Based on cytotoxicity and pore forming activity, the antimicrobial peptide have been

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discovered from Pardachirus marmoratus, which was active against Gram positive and Gram

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negative bacterial pathogens. Pleurocidins from Pleuronectes americanus have broad-spectrum

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antimicrobial activities (Cole et al., 1997) and inhibit DNA, RNA and protein synthesis. The

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pardaxin toxic peptide was the first identified fish peptide, initially characterized from the Moses

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sole fish Pardachirus marmoratus (Primor et al., 1980; Oren et al., 1996; Cole et al., 1997).

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Thereafter, antimicrobial peptide identification progressed and many more peptides belonging to

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defensin, cathelicidin and hepcidin families were identified from teleost fish. Recently, many

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successful antimicrobial peptide prediction strategies have been proposed which allowed

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researchers to perceive more potential peptides from fish group and other species (Tessera et al.,

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2012; Brahmachar et al., 2004). AMPs are generally short peptides comprising 12-100 amino

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acid residues whose charge plays a key role in their functional property. Some of the potential

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antimicrobial peptides are amphipathic in nature encompass specific antibacterial mechanism

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(Wang, et al., 2004). So far, lectins have been reported to express bactericidal and bacteriostatic

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activity against many pathogens because of its sugar binding specificity. However, the

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antimicrobial role of carbohydrate recognition domains in lectins has not yet been determined.

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Hence, we aimed at identifying the antimicrobial property of the carbohydrate recognition

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domain of lily type lectin-2 and to understand the mechanism of action on the bacterial

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membrane.

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According to our knowledge, the present work on structural and functional

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characterization of striped murrel lily type lectin-2 has not been reported to date. In this study, a

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striped murrel lily type lectin-2 cDNA sequence (designated as CsLTL-2) was identified from

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the constructed cDNA library of C. striatus using Genome Sequence FLXTM (GS-FLXTM) 6

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technology. The protein sequence was subjected to various bioinformatics analysis to

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characterize the CsLTL-2. Followed by; we have determined the gene expression pattern of

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CsLTL-2 in both healthy and infected state. Additionally, we have synthesized a carbohydrate

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binding domain region (CRD) as a peptide from CsLTL-2 which possessed 13 amino acid

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residues and it is designated as QP13. To investigate the peptide antimicrobial activity, we have

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determined minimum inhibitory concentration (MIC) and analyzed its bacterial membrane

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disruption ability. Further, to analyze the localization of the peptide in bacterial cells, QP13 was

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tagged with FITC and visualized with confocal microscope.

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2. Materials and methods

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2.1. Sequencing of CsLTL-2

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The full length CsLTL-2 sequence was identified from the cDNA library of C. striatus

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constructed from the total RNA extracted from the tissue pool including spleen, liver, kidney,

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muscle and gills of C. striatus using genome sequencing FLXTM (GS-FLXTM) technology. The

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detailed description on the construction of normalized C. striatus cDNA library has been

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reported in our earlier studies (Abirami et al., 2013).

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2.2. Bioinformatics analysis of CsLTL-2

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The full-length CsLTL-2 cDNA was analyzed by Expasy translate tool to obtain its

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untranslated region (UTR), coding region and its corresponding polypeptide sequences (Patterton

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et al., 2000). The CsLTL-2 protein sequence was compared with other protein sequences using

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protein BLAST (blastp) (http://blast.ncbi.nlm.nih.gov/Blast) and the regions of similarity

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between the identified sequences were analyzed. The other structural features of the protein

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sequence such as domains and motifs of CsLTL-2 were predicted by comparing with the 7

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PROSITE Database. (http://prosite.expasy.org/scanprosite/). NCBI Domain Database was used

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to predict domain architecture and detect conserved regions of CsLTL-2. Multiple sequence

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alignment of CsLTL-2 was performed to identify regions of similarity with putative structural

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and functional relationships between the related sequences using BioEdit (version7.0.0). The

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evolutionary relationships with other orthologous sequences were predicted by constructing a

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phylogenetic tree of CsLTL-2 was established using Neighbor-Joining Method by MEGA 6.06.

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The structure of CsLTL-2 protein was predicted based on the resolved structural templates

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available in PDB using I-Tasser program (http://zhanglab.ccmb.med.umich.edu/I-TASSER) and

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the predicted structural models were validated by constructing Ramachandran plot

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(http://mordred.bioc.cam.ac.uk/rapper/rampage.php). The secondary structure was evaluated

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using Polyview-2D (http://polyview.cchmc.org) and its tertiary structure was analyzed using

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PyMol.

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2.3. Experimental fish

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Healthy C. striatus (body weight = 50 ± 5 g) were obtained from a commercial fish farm,

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Tirunelveli (8.73◦ N 77.7◦ E), Tamil Nadu, India. The fishes were carefully transported to the

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laboratory (Division of Fisheries Biotechnology & Molecular Biology, Department of

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Biotechnology, Faculty of Science and Humanities, SRM University) in oxygenated polythene

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bags. The fishes were carefully processed as per the animal handling regulations of the institute.

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The fishes were maintained in 15 rectangular plastic tanks (150 l). The tanks were filled with de-

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chlorinated well water (water quality: dissolved oxygen, 5.8 ± 0.2 mg/l; water temperature, 28 ±

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1 °C and pH, 7.2 ± 0.2) and supplied aeration through an electrical magnetic air pump. The

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fishes were acclimatized for 10 days in the laboratory condition and then they were challenged to

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various pathogenic infections. During the acclimatization period, the fishes were fed up to the

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satiation level twice a day with a handmade semi-moist pellet feed (Arockiaraj et al., 1999). A

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maximum of 15 fish in each tank were maintained during each experiment and three fishes were

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sacrificed for each time point.

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2.4. Pathogen challenge and tissues collection

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In order to investigate the involvement of CsLTL-2 in immune mechanism against EUS

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infection, C. striatus were injected with pathogens such as A. invadans and A. hydrophila. The

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phosphate buffer saline (PBS) was injected (130 µl/fish) as control. For A. invadans injection,

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fish were injected with 130 µl of A. invadans at a concentration of 102 spores. For A. hydrophila

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injection, the fish were injected (130µ l/fish) with A. hydrophila (5 × 106 CFU/ml) suspended in

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1X PBS. Tissues have been collected from C. striatus, such as intestine, heart, liver, spleen,

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kidney, head kidney, brain, gill, skin and muscle were collected before (0 h) and after pathogen

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injection at 3, 6, 12, 24, 48 and 72 h. Blood was collected using sterile syringe from the caudal

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fin and immediately centrifuged at 4°C in 4000 × g for 10 min. The dissected tissues were snap

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frozen immediately in liquid nitrogen and stored at -80°C until total RNA isolated (Dhanaraj et

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al., 2008).

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2.5. RNA extraction, cDNA conversion and gene expression study

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Total RNA from the control and infected fish were isolated using Tri ReagentTM (Life

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Technologies), according to the manufacturer’s protocol with small modifications (Arockiaraj et

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al., 2011). Using 2.5 µg of RNA, first strand cDNA synthesis was carried out using a

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SuperScript® VILO™ cDNA Synthesis Kit (Life technologies) as suggested by the

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manufacturer with slight modifications (Livak et al., 2001; Arockiaraj et al., 2013; Bhatt et al.,

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2013). The resulting cDNA solution was stored at -20 ºC for further analysis. 9

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The relative expression of CsLTL-2 in various collected tissues was quantified by

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quantitative real time polymerase chain reaction (qRT-PCR). qRT-PCR was carried out using a

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Light Cycler 96 Real Time PCR system (Roche, Germany) in 20µL reaction volume containing

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4µL of cDNA synthesized from each tissue, 10µL of Fast SYBR Green Master Mix, 0.5µL each

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of forward and reverse primer (20µM) and 5µL dH2O. The qRT-PCR cycle profile was 1 cycle

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of 95 °C for 10 s, followed by 35 cycles of 95 °C for 5 s, 58 °C for 10 s and 72 °C for 20 s and

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finally 1 cycle of 95 °C for 15 s, 60 °C for 30 s and 95°C for 15 s. The same qRT-PCR cycle

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profile was used for the internal control gene, β-actin. The primers for β-actin of C. striatus were

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designed from the sequence of GenBank Accession No. EU570219 (Forward Primer:

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TCTTCCAGCCTTCCTTCCTTGGTA

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GACGTCGCACTTCATGATGCTGTT. The CsLTL-2 cDNA was amplified using the forward

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primer

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CTGGGTCTGTGTGAGTTTGT (Antisense). After the PCR program, data were analyzed with

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the software provided with qRT-PCR system. To maintain the consistency, the baseline was set

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automatically by the software. The comparative CT method (2-ddCT method) was used to analyze

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the expression level of CsLTL-2 (Benincasa et al., 2004). The results were expressed as relative

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values to that of control.

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2.6. Carbohydrate binding domain synthesis

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The CsLTL-2 carbohydrate binding domain region (QGDGNFVIYPWKP) was

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synthesized and the peptide has been designated as ‘QP13’. To understand the membrane

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binding specificity of QP13, the peptide was tagged with the N-terminal fluorescein

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isothiocyanate (FITC). The peptides were purchased from Synpeptide (China). The purity of the

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peptides was confirmed by HPLC analysis. The peptides were delivered as lyophilized acetate

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salts, diluted in endotoxin-free water and stored at -20 °C until used.

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2.7. Microbial pathogens

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Different bacterial pathogens including both Gram positive and Gram negative organisms

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were used for antimicrobial activity and it was purchased from Microbial Type Culture

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Collection (MTCC), Chandigarh, India. An 18 to 24 h old culture was inoculated into a freshly

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prepared Nutrient agar and allowed to grow at 37 °C for 4 - 6 h to reach their synchronous stage

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after which it was centrifuged at 5000 rpm for 5 min and adjusted to 106 CFU/ml by PBS.

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The following bacterial cultures were used for the minimum inhibitory concentration

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(MIC) assay. Vibrio harveyi (7954), Bacillus mycoides (8920), Bacillus subtilis (6051),

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Escherichia coli (9637), Serratia marcescens (3124), Staphylococcus aureus (29213),

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Pseudomonas aeruginosa (15159), Bacillus cereus (2106), Klebsiella pneumonia (27736),

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Streptococcus pyogenes (1927), Listeria monocytogens (1143), Pseudomonas fluorescens

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(2269), Bacillus subtilis (6333), Escherichia coli (10312), Salmonella enterica (1166),

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Micrococcus luteus (6164) and Aeromonas hydrophila (1739). All the bacteria were grown in

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Nutrient agar (HiMedia) and were maintained on 30% glycerol stock.

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2.8. Minimal inhibitory assay of QP13 peptide

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Minimum inhibitory concentration (MIC) values of the peptides were determined using a

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broth micro-dilution method. Briefly, each peptide was serially diluted (600, 300, 150, 75, 37.5

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µM) in 96-well polypropylene micro titer plates with MH broth. Each dilution series included

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control wells without peptide. A total of 50 µl of the adjusted inoculum (approximately 1×106

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cells/ml in MH broth) was added to each well. The MIC was taken as the lowest concentration of

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antimicrobial peptide resulting in the complete inhibition of visible growth after 18 h of

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incubation at 37 °C. Ampicillin was used as positive control for reference. Results are mean

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values of at least three independent determinations.

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2.9. Membrane binding potential of QP13

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2.9.1. Confocal imaging

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The membrane binding ability of FITC tagged QP13 was analyzed against four bacterial

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pathogens V. harveyi, E. coli, B. mycoides and A. hydrophila by confocal microscopy. The

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strains were selected based on the bactericidal activity of QP13 peptide. For imaging,

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exponential growth of bacterial culture was used at a concentration 1x108 CFU in 200 µl of 10

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mM PBS and added 5 or 10 µM peptide then incubated at 37°C for 2 h prior to microscope

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observation. Confocal microscope observation was performed on a Leica TCS SP confocal

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system equipped with a HCX PL APO CS 63× objective. For excitation of FITC, 488 nm laser

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line of an Arlaser was used with emission at 525 nm. The scanning and filter settings were

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carried out as described previously (Eva Harreither et al., 2014).

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2.9.2. Flow cytometry analysis

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Exponentially growing culture of E. coli was taken from overnight grown broth cultures

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and was centrifuged at 7000 rpm for 5 min and the supernatant was discarded. Cell pellets of

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cultures were resuspended in sterile PBS and adjusted to 1x108cells per ml. Two hundred

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microliter of the prepared inoculum was added with 150 µM of QP13 peptide and then incubated

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at 37 ̊C for 180 min. After incubation, the suspension was centrifuged at 1700 Xg for 15 min at

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4 ̊C and incubated at room temperature for 10 min with 5 µM propidium iodide (PI). Then, the

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suspension was analyzed by fluorescent assisted cell sorter (BD FACS). Fifteen thousands events

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were recorded from each solution (three independent assays were performed for each sample).

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The percentage of PI internalized cells in each solution was calculated by CELL-QUEST

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software. Further, the total number of cells in the same sample was counted by flow cytometry.

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The cells were allowed to run for 15 sec and the number of total events and the number of PI

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stained cells were calculated for control as well as peptide treated cells.

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2.10. Cytotoxicity activity of QP13

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The cytotoxicity effect of QP13 was evaluated on fish leucocyte cells. The cells

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(1x107cells per well) were prepared using gradient centrifugation (500 Xg for 30 min) method

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with HiSepLSM (HiMedia). The cytotoxic activity of QP13 was determined by culturing

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peripheral cell in presence of various concentrations of QP13 such as 600, 300, 150, 75 and 37.5

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µM for 4 h in sterile 96 well flat-bottom tissue culture plates. Blank with RPMI AL-120 was

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treated as control. Then, the cells were pelleted (600 X for 30 min) and suspended in fresh

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culture medium. The resuspended cells were incubated with MTT for 2 h and the cells were

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washed with Roswell Park Memorial Institute medium (RPMI). Finally, dimethyl sulfoxide

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(DMSO) was added to solubilize the Formosan and observed for color change. After gentle

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shake, the optical density of the reaction mixture was noted at 490 nm and immediately

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measured in iMarkTM micro plate absorbance reader and the cell viability percentage was

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calculated.

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2.11. Statistical Analysis All the statistical analysis was performed in SPSS (ver. 11.5). The data were subjected to

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one-way ANOVA and the mean comparisons were performed by Tukey’s Multiple Range Test;

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and the significance was determined at P < 0.05 level.

3. Results

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3.1. Sequence analyses

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The full-length CsLTL-2 cDNA was 351 base pairs (bp) with an open reading frame

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(ORF) of 348 bp. The coding sequence of CsLTL-2 encodes a polypeptide of 116 amino acids

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with a theoretically calculated molecular mass of 13 kDa and an isoelectric point of 9.1.

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Nucleotide sequence has been deposited in EMBL GenBank database under the accession

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number HF585139. Scan prosite analysis revealed that the CsLTL-2 polypeptide possessed an

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important homologous bulb type mannose binding domain. Further, within the domain, a

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mannose binding site were observed between residues 30 and 99 along with a specific

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carbohydrate recognition domain (CRD) motif of QxDxNxVxY in three-fold internal repeats (β-

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prism architecture); the repeat 1 at Gln30-Asp32-Asn34-Val36-Tyr38 followed by repeat 2 at Gln59-

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Asp61-Asn63-Val65-Tyr67and repeat 3 with slight changes (LxDxGxLxL) at Leu91-Asp93-Gly95-

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Leu97-Leu99. This β-prism architecture motif specifically involve in D mannose recognition. The

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CsLTL-2 also contains 5 high probability motifs including 3 protein kinase C phosphorylation

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site (39-41, 72-74, 79-81), 1 N-myristoylation site (23-28) and 1 N-glycosylation site (110-113).

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The CsLTL-2 polypeptide sequence neither has a signal peptide region nor a transmembrane

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region. Sequence analysis showed that the CsLTL-2 had a significant sequence identity with

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other known LTL-2 variant groups. It shared highest sequence identity of 77%, 76% and 75%

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with other orthologous sequences from Oplegnathus fasciatus, Larimichthys crocea and

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Epinephelus coioides, respectively.

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The deduced amino acid sequence of CsLTL-2 was aligned with the other LTL-2 family

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members among fish. Even though the length of the amino acids varied between fish and other

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species, many residues remained conserved among them. The multiple sequence alignment of

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CsLTL-2 showed that the amino acid residues between 3 and 116 are much conserved in all the

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fish species taken for analysis especially the three CRDs (Fig. 1).

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3.2. Phylogenetic and structural analysis

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The phylogenetic tree of CsLTL-2 with other species was conducted using Neighbor-

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Joining (NJ) Method (Fig. 2). The phylogenetic tree showed that CsLTL-2 was closely related to

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other fish LTL-2 groups from Platycephalus indicus belonging to Perciformes. CsLTL-2 has

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95% bootstraps identity with fish group. LTL-2 forms three different clades of fish, microbes and

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plants.

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The secondary structural elements analysis of CsLTL-2 showed that the protein possesses

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43.10% extended strand region (50 residues), 35.35% coils (41 residues) and 21.55% (25

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residues) occupied by beta turn region. No helix region was observed in the protein structure of

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CsLTL-2. Five different 3D models of CsLTL-2 protein were predicted using I-Tasser server

332

based on the sequence; and the quality of the predicted models were evaluated using

333

Ramachandran Plot analysis. The analysis indicated that among the 116 amino acids of CsLTL-

334

2, model-1 shared 90 amino acid residues in favored region (88.40%), 10 residues in allowed

335

region (6.01%) and 6 residues in outlier region (5.50%). Hence, model-1 was selected as the best

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model for further analysis and is given in Fig. 3. The model was observed in PyMol surface

337

view. The observation indicated that the carbohydrate recognition domain 1 region was present

338

at random coil which was derived as peptide and its antimicrobial activity was analyzed.

339

3.3. Gene expression studies

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Real Time PCR was used to investigate the expression of CsLTL-2 gene product in

341

various tissues of C. striatus. The analyses were validated using housekeeping gene, β-actin. The

342

results showed that the gills expressed the highest level of CsLTL-2 followed by skin, intestine,

343

blood, heart, spleen, liver, head kidney, kidney, brain and muscle (Fig. 4). The statistical analysis

344

indicated that all the distribution levels are significantly (P < 0.05) different compared to the

345

distribution level in muscle. Since the expression of CsLTL-2 is higher in gills, it was used to

346

evaluate the regulation of CsLTL-2 during the biological stress induced by fungus A. invadans,

347

and the bacteria A. hydrophila. The fungus, A. invadans infected fish expressed the maximum

348

expression (P < 0.05) at 48 h post-infection (p.i) when compared to other time points (Fig. 5A).

349

In A. hydrophila infected murrels, significantly highest expression (P < 0.05) was observed at 24

350

h p.i and finally reached to the basal level (Fig. 5B) at 72 h p.i.

351

3.4. Structural properties and bactericidal activity of QP13 peptide

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QP13 peptide was synthesized chemically and the purity of the peptide was confirmed by

353

HPLC (92%). The molecular weight of the peptide was calculated as 1523.90. The sequence

354

contained two proline residues among which the one at the C terminal tend to involve in

355

membrane disruption activity. Since the peptide sequence was more probable to act as an

356

antimicrobial peptide, we analyzed the bactericidal activity of QP13 peptide by determining the

357

minimum inhibitory concentration values of the peptide against various bacterial pathogens. 16

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Totally, seventeen bacterial pathogens were subjected for this analysis. The peptide inhibited the

359

growth of three bacterial cultures (V. harveyi, E. coli and B. mycoides) at low concentration of 75

360

µM concentration. It also inhibited the growth of M. luteus, S. enterica and S. aureus up to a

361

concentration of 150 µM, however, the peptide did not inhibit the growth of other tested bacteria

362

even at the maximum concentration (600 µM) (E-Supplementary Table 1). Also, the number of

363

cells present in the peptide-treated culture and control were calculated by FACS. A significant

364

decrease in the total number of cells was observed in E. coli, V. harveyi and B. mycoides when

365

compared with their corresponding untreated cells. In case of peptide-treated V. harveyi, only

366

25% cells were live, in E. coli, it is 38% and in B. mycoides, 47% cells were live when compared

367

with the bacterial cells treated with scrambled peptide. Notably, there is no significant change in

368

the total number of cells in case of A. hydrophila which has been used as a negative control for

369

this study (Table 1). A dose response analysis was performed by counting cells using FACS to

370

evaluate the effect of QP13 on the four bacterial cells and the results were interpreted as

371

percentage of live cells and the results were in accordance with the MIC results (E-

372

Supplementary Figure 1). This clearly demonstrated that QP13 exhibited bactericidal activity

373

against the above mentioned bacterial cells.

374

3.5. Confocal imaging

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FITC tagged QP13 were used to understand the binding specificity of the peptide with the

376

bacterial membrane. In vitro cell-binding experiments were thus performed with four different

377

bacteria treated with QP13 peptide. Results showed that the peptide bind to the membrane of

378

three bacterial strains, such as V. harveyi, E. coli and B. mycoides, whereas the peptide did not

379

show any binding affinity with the bacterial membrane of A. hydrophila (Fig. 6). This was in

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accordance with the result of the bactericidal assay, where the peptide did not show any

381

bactericidal activity against A. hydrophila.

382

3.6. Bacterial membrane disruption activity of QP13

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Flow cytometric analysis was performed to understand the bacterial membrane disruption

384

activity of the peptide QP13. The bacterial samples treated with peptide QP13 and PI

385

fluorochromes were made to run through the cell sorter and the cells were categorized into two

386

groups based on PI fluorescence. In the cytogram (Fig. 7), R2 represent the PI negative viable

387

cells and R3 shows the PI positive viable cells. Propidium iodide staining of nucleic acids in

388

cells indicated that the bacterial cell membrane got compromised, thus it allowed the PI to enter

389

into the cell. Results showed that peptide treated E. coli cells showed significantly higher amount

390

of PI stained cells than the control cells.

391

3.7. Cytotoxicity of QP13

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In order to evaluate the cytotoxic property of QP13 against host cells, we examined the

393

cytotoxic activity of QP13 against peripheral blood leucocytes of C. striatus (CsPBL). The

394

results showed that QP13 exhibited negligible toxicity against CsPBL even at higher

395

concentrations (1200 µM) which was similar to the PBS treated cells, whereas the positive

396

control showed 100% activity against the leucocytes (Supplementary Figure 2).

397

4. Discussion

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The constant risk of microbial infection in aquaculture persuades significant economic

399

losses which demands better strategies to prevent or treat infections caused by those pathogens.

400

Since abundant usage of antibiotics resulted in drug resistance among bacterial pathogens, it 18

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made those antibiotics less efficient and there is a search for new strategy which is more specific

402

against those pathogens (Heuer et al., 2006; Holo et al., 1991). Antimicrobial peptides have been

403

considered as evolutionary ancient weapons because of their broad spectra activity and are

404

involved in direct destruction of various microorganisms. Lectins specifically act against

405

bacterial membranes based on the specificity of sugar present in the membrane and are reported

406

to exhibit antibacterial activity against a broad spectrum of bacterial pathogens. However, the

407

antimicrobial activity of carbohydrate recognition domain of LTL-2 has not been identified yet.

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Therefore, in the present study, we identified a cDNA that encode a LTL-2 protein from

409

constructed cDNA library of C.striatus. Protein BLAST analysis showed that CsLTL-2 amino

410

acid sequence showed much high homology to the existing LTL-2 sequences of fish, monocot

411

plants and microbes of mannose-binding lectins. This LTL-2 also shows more than 50%

412

similarity to the mannose binding lectin of mono cotyledonous plants including Allium

413

ampeloprasum, A. sativum and A. squamosa as reported by Tsutsui et al. (2003). Conserved

414

domain search results suggested that LTL-2 belonged to bulb-type D-mannose specific lily type

415

lectin containing the beta-prism architecture domain (QXDXNXXXY) with three-fold internal

416

repeats. The third repeat of mannose binding sites slightly varied from plants group but

417

conserved among fish group. This domain architecture specifically recognizes mannose

418

carbohydrates and mediates a wide variety of biological processes, such as cell-cell and host-

419

pathogen interactions and other innate immune responses (Tsutsui et al., 2003). The domain

420

pattern was similar to Galanthus nivalis, Clivia miniata, C. asiaticum and Narcissus

421

pseudonarcissus which had three specific conservative mannose-binding sites (Smeets et al.,

422

1997) as observed in skin mucus lectin (Suzuki et al., 2003; Tsutsui et al., 2006) and intestine

423

mucus lectin (Tsutsui et al., 200, and 2006). Prosite analysis suggested that the deduced amino

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424

acid sequence of LTL-2 had many process sites, such as glycosylation and phosphorylation sites,

425

which are all implying the existence of possible post-transcriptional processes. Phylogenetic analysis revealed that CsLTL-2 is closely related to other fish LTL-2 and

427

the group remained evolutionarily conserved, indicating that CsLTL-2 might be one type of lily

428

type lectin. The deduced amino acid sequences of CsLTL-2 were aligned with the other fish

429

LTL-2 sequences. Even though the length of the amino acids varied among fish species, many

430

conserved residues were observed especially in CRD. CRD is necessary for the sugar binding

431

activity of lectins and its carbohydrate binding specificity mainly determined by the position of

432

hydrogen bond donors and acceptors in the conserved motifs of Ca2+ -binding site 2 in CRDs

433

(Zelensky et al., 2005). Structural analysis showed that CsLTL-2 consists of larger number of

434

coils and β sheet which is similar to the XRD structure of carp lectin (Capaldi 2015). Moreover,

435

Pusztai (2008) stated that the tertiary structure depends on the relative orientation of the β-sheets,

436

which forming the β-prism lectins that may be further classified as β-prism I and β-prism-2 fold

437

or β-trefoil lectins.

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Tissue distribution analysis clearly indicated that CsLTL-2 is expressed in all the tissues,

439

however, the level of CsLTL-2 distribution varied in each tissue. The maximum expression was

440

observed in gills, suggesting that LTL-2 may play a crucial role in first line defense mechanism

441

against several microbial pathogens; also gills are highly exposed to the environment. A similar

442

pattern of tissue distribution was observed in another lectin CsLTL-1 reported from C. striatus

443

(Arasu et al., 2014). Previously, Nakamura et al. (2001) reported that LTL-2 from conger eel

444

(Congerins) were expressed in skin and gills, whereas the lactose binding lectin from Japanese

445

eel (AJL-2) was expressed only in skin (Tasumi et al., 2002). Tsutsui et al. (2003) reported

446

pufflectin from Puffer fish which was expressed in exterior tissues like gills, skin, oral cavity

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wall and esophagus. Pathogen-induced gene expression analysis showed that expression of

448

CsLTL-2 was significantly increased by both bacterial and fungal pathogens. This confirmed that

449

CsLTL-2 is highly expressed at the time of pathogenic infections. In our previous studies, we

450

have identified that expression of CsLTL-1 was up-regulated at the time of bacterial and fungal

451

infections. Thus, we could conclude that lectins play a vital role in defense mechanism against

452

EUS causing pathogens.

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Antimicrobial peptides (AMPs) are effective molecule of innate immune mechanism that

454

has evolved different mechanisms to inactivate bacterial pathogen. The majority of them exhibit

455

amphipathic nature that allow interaction with, and damage of membranes. The mechanisms of

456

actions of AMPs have been studied extensively and were proven to be different from those of

457

antibiotics (Brogden. 2005). The naturally occurring antimicrobial peptide from frog, the

458

odorranain-HP exhibited antimicrobial activity against Helicobacter pylori with a MIC of

459

20µg/ml and in addition Staphylococcus aureus was more sensitive to this peptide. (Lihua Chen

460

et al., 2007). It is a well-known fact that CRD is responsible for the specific binding ability of

461

lectins; however, the antimicrobial role of CRD has never been studied. In order to visualize the

462

bactericidal activity of CRD, we have synthesized the CRD and observed their antimicrobial

463

activity. Exposure of QP13 to bacterial cells induced bactericidal activities against bacterial

464

pathogens such as V. harveyi, E. coli and B. mycoides. PI uptake assay revealed the membrane

465

permeabilizing capacity of the peptide. Also, a significant loss in cell count was also observed by

466

FACS analysis. All these results together established the bactericidal activity of QP13 peptide.

467

According to the statement of Ashida et al. (1994), in the host organism, the pathogenic microbes

468

are recognized as unknown particles when the pattern recognition receptors bind to the sugar

469

compound of the microbial cell wall, thus commencing the activation of peptide for killing the

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pathogens. Therefore, QP13 might involve in defense mechanisms because the peptide is one of

471

pattern recognize molecule which is capable to bind with sugar sites especially mannose sugar. Confocal microscopy results showed that FITC-tagged QP13 specifically binds to the

473

membrane of bacteria such as V. harveyi, E. coli and B. subtilis. But the peptide did not bind to

474

the membrane of A. hydrophila. Thus, we can conclude that the peptide has specificity towards

475

bacterial membrane sugars which can be further used to specifically target those bacterial

476

membranes. Anderson et al. (2003) identified cyclic peptides with similar membrane targeting

477

activity, where its membrane binding activity was determined by confocal microscopy. In

478

confocal microscopy images, it is clear that the membrane remained intact in case of E. coli and

479

B. subtilis, whereas the cells of V. harveyi was disintegrated even at shorter duration of peptide

480

exposure. This is in accordance with MIC results, where V. harveyi cells exhibited the activity at

481

lower concentration. MIC assay showed that QP13 exhibited bactericidal activity against

482

bacteria. The mechanism of action of QP13 on bacterial membrane was determined by

483

flowcytometry assay by analyzing the PI internalization by bacterial cells. Results showed that

484

QP13 significantly induced membrane disruption of bacterial cells. Furthermore, QP13 reduced

485

the cell counts of bacterial cells when compared with controls. Hence, we may possibly suggest

486

the QP13 encompass bacterial membrane binding ability along with membrane disruption

487

activity. Blondelle et al. (1990) and Dempsey et al. (1991) reported Melittin derived from the

488

venom of the European honeybee, Apis mellifera a cell-lysing and membrane-active peptide

489

against both bacterial and eukaryotic cells. Frecer et al. (2004) suggests that increased outer

490

membrane disruption is due to the high binding affinity of peptide with LPS in E. coli.

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491

We assessed the cytotoxicity of the QP13 peptide and no cytotoxicity influence was

492

observed against fish PBLs. No effect on cell viability and proliferation was detected with MTT 22

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for the tested peptide even at the highest concentration applied fish leucocytes. Kumaresan et al.

494

(2015) reported that the fish lysozyme derived antimicrobial peptide (IK12) was active against

495

Salmonella enterica and remained non-toxic to the fish leucocytes even at higher concentration.

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In conclusion, this study depicts the function of CsLTL-2 in innate immunity of C.

497

striatus during pathogenic condition. Bioinformatics analysis showed that basic characteristic

498

features of CsLTL-2 were in accordance with other LTL. Basal and temporal gene expression

499

pattern demonstrated the immune role of CsLTL-2 against EUS causing pathogens. Further, we

500

have demonstrated the membrane binding potential of the carbohydrate recognition domain

501

towards selected bacterial pathogens and also observed its bactericidal activity. Mechanism of

502

action of the peptide, QP13 was found to disrupt the membrane of the bacterial cells which is the

503

key mode of its action. Also, cytotoxicity assays confirmed that QP13 was non-toxic to host

504

cells, thus comprising a high potential to be developed as a therapeutic or bacterial membrane

505

targeting agent in aquaculture.

506

Acknowledgement

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This research is supported by DBT’s Prestigious Ramalingaswami Re-entry Fellowship

509

(D.O.NO.BT/HRD/35/02/2006 and BT/RLF/Re-entry/27/2011) funded by Department of

510

Biotechnology, Ministry of Science and Technology, Government of India, New Delhi. Also, the

511

authors extend their sincere appreciation to the Deanship of Scientific Research at King Saud

512

University for its funding through the Prolific Research Group (PRG-1437-28).

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leaves and roots of garlic (Allium sativum L.). Plant Molecular Biology 867, 223-234.

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mucus lectins in fish. Comparative Biochemistry and Physiology Part B 136, 723-730.

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S.No

Experiment

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Table 1 A number of total events and the number of PI stained cells of various bacterial cells with scrambled and QP13 peptides were determined by FACS. Results showed that three of the QP13-treated bacterial pathogens bacterial pathogens, E. coli, B. mycoides and V. harveyi showed maximum PI internalisation, whereas no significant PI staining of A. hydrophila was observed. Also, QP13 significantly reduced the bacterial cell counts of E. coli, B. mycoides and V. harveyi Events/15 sec 51,208

Number of PI stained cells 1,495

% of PI stained cells 2.91

Scrambled peptide + V. harveyi

2

QP13 + V. harveyi

12,802

8,984

70.18

3

Scrambled peptide + E. coli

74,572

942

1.26

4

QP13 + E. coli

28,337

5

Scrambled peptide + B. mycoides

50,003

6

QP13 + B. mycoides

23,501

7

Scrambled peptide + A. hydrophila

8

QP13 + A. hydrophila

20,859

73.61

1,095

2.19

14,262

60.69

80,710

2,985

3.69

82,995

4,012

4.83

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Figure legends Fig. 1. The alignment of CsLTL-2 with other orthologous sequences from Oplegnathus

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fasciatus; lily type lectin from Fundulus heteroclitus, Orochromis niloticus, Epinephelus coioides and Larimichthys crocea; skin mucus lectin from Takifugu rubripes was performed using BioEdit software. Aligned sequences showed that the three carbohydrate recognition domain (CRD) remained conserved among all the LTL sequences and are highlighted in red

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boxes.

Fig. 2. Phylogenetic analysis of C. striatus lily type lectin-2 with 14 other species was

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constructed by the Neighbor-Joining method. The tree is based on an alignment corresponding to full length amino acid sequence using clustal W and MEGA 6. The numbers at the branches denote bootstrap majority consensus values on 1000 replicates. The scale bar represents a genetic distance of 0.5. The tree comprised three groups such as fish, plants and microbes which are clearly indicated by brace brackets.

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Fig. 3. Tertiary structure of CsLTL-2 was predicted by I-TASSER program. The C terminus and N terminus ends are marked in black bold letters. Three mannose binding sides are highlighted in different color balls.

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Fig. 4. Tissue distribution analysis of CsLTL-2 gene from various tissue samples of C. striatus quantified using real time PCR. Data are given as a ratio to CsLTL-2 expression in muscle. The

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error bar represents the SD. The expression is significantly different at P < 0.05 level by one-way ANOVA and Tukey’s Multiple Range Test between the bar carries different alphabet.

Fig. 5. Time course of CsLTL-2 expression in gills at 0, 3, 6, 12, 24, 48 and 72 h post-injection with (5A) Fungus, A. invadans and (5B) Bacteria, A. hydrophila. The error bar represents the SD. The significant difference of CsLTL-2 expression between the challenged and the control group were indicated with asterisks.

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Fig. 6. Confocal microscopic images showing interaction of FITC-tagged QP13 peptides with the membrane of bacterial pathogens. For better understanding of binding affinity of peptide with bacterial membrane, each image show three images: Left top – only fluorescence, Right top – Only light microscopic images, Left bottom – shows both fluorescent as well as light

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microscopic images. The assay was performed against four bacterial pathogens: (A) No or weak binding of FITC-tagged QP13 on A. hydrophila, (B) Strong binding of FITC-tagged QP13 on B. mycoides, (C) Damaged cells of V. harveyi along with few peptide bound cells and (D) Strong

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binding of FITC-tagged QP13 on E. coli.

Fig.7. Dot Plot graph showing the internalization of PI in E. coli cells by FACS. (A) E. coli cells

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treated with PBS showing 99% unstained cells (R2) and 1% PI stained cells (R3). (B) E. coli

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cells treated with QP13 showing 18% unstained cells (R2) and 82% PI stained cells (R3).

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CRD1 MSRNCLFRFQEFRKGDYLVSNNGNFKAIFQGDGNFVIYTW MSRNYLSKNDELRKGDYLMSNNREFKAVFQDDGNFVVYGW MSRNYLARFDELRKGDYLMSNNGEWKAVFQGDGNFVIYGW MSRNFLSKNDEFRRGDYLVSNNKQFKAIFQDDGNFVIYGW MSINVLEKGSELKRGDSVLSKNSQWIALFQQDGNFVVYRT MSRNYLSKNAELRRGDYLLSNNGQWKAIFQDDGNFVIYGW MSRNYVSKNDELRRGDYLMSNNGQWKAVFEDDGNLVIYGW

C.striatus O.fasciatus F.heteroclitus O.niloticus T.rubripes E.coioides L.corcea

KPIWASDTANSDVVRLCMQDDCNLVMYNKDGTPRWQTNSH KPVWASDTAGSDAVRLCMQADCNLVMYNKDSEPRWHTNSA KPVWSSDTSGTDVVRLVMQEDCNLVMYNKEGVGRWVSNTH KPLWASDTYGSDAVRLCMQADCNLVMYNNCDTPRWSTNSY EPVWASDTFGMDPTRLCMQGDCNLVMYNDEDKPRWHTNTS KPVWASDTYGSDAQRLCMQADCNLVMYNKCDEPRWHTNSA KPVWTSETNGSDAIRLCMQADCNLVMYSQCDSPRWQTNSA

C.striatus O.fasciatus F.heteroclitus O.niloticus T.rubripes E.coioides L.corcea

RPSCTSCSLYLTDEGVLVLTKDREEIWNSNQSRGMKKSNCNMCRLHLTDDGELVVNRECDGIWSSADSRGMKRSGDHLSRLHLSDEGKLHIYNGPDELWNSTQSSGVKG GPAANLCRLQLTDDGKLVVNRECKEIWSSEKSKGMKKGSCKTCVLSLTDEGQLVLKKDGQEIWNSDHDHGMKKSECNMCRLQLTDDGKLVVNRECDEIWSSAESKGMKKPKCNMCRLQLTDDGKLVLYRECDEIWSSASSKGM--

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CRD3

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80 80 80 80 80 80 80

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Fig. 1.

40 40 40 40 40 40 40

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C.striatus O.fasciatus F.heteroclitus O.niloticus T.rubripes E.coioides L.corcea

116 116 117 116 116 116

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Channa striatus (HF585139) 98 Platycephalus indicus (BAE79274) Leiognathus nuchalis (BAE79275) 55

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42 Epinephelus coioides (AEG78370) 29

Oplegnathus fasciatus (AAV35592)

60

75

FISH

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Oncorhynchus mykiss (AAM21196) 89

Gadus morhua (AEK21705)

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Aspergillus oryzae (XP009817559) Mycobacterium xenopi (ZP009980532) 70

MICROBES

Mycobacterium fortuitum (ZP11005898)

100 95

Mycobacterium smegmatis (YP887965)

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Allium sativum (AAB64237)

Allium ampeloprasum (AAC37361)

39

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Fig. 2.

PLANTS Polygonatum multiflorum (AAC49413) Galanthus nivalis (AEK21705)

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Distribution of CsLTL-2/ β-actin, folds

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c

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12 PBS Control

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Fungus Challenged 10

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8 6 4

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48

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mRNA expression of CsLTL-2/ β-actin, folds

Fig. 5A.

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Fungus post-challenged time (hour)

Bacteria Challenged 20

PBS Control

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mRNA expression of CsLTL-2/ β-actin, folds

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Fig. 5B.

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Fig. 5.

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Bacteria post-challenged time (hour)

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72

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Fig. 6.

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Fig.7.

(B) QP13 treated E. coli cells.

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(A) PBS treated E. coli cells

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Research Highlights An AMP QP13 was synthesized from lily type lectin 2 of murrel.

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Binding affinity of QP13 labeled with FITC was analyzed.

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Confocal observation showed FITC tagged QP13 bound to bacterial membrane.

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FACS showed QP13 reduced the bacterial cell count drastically.

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PI internalization confirmed QP13 induced membrane disruption.

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