Characterization and antimicrobial evaluation of SpPR-AMP1, a proline-rich antimicrobial peptide from the mud crab Scylla paramamosain

Accepted Manuscript Characterization and antimicrobial evaluation of SpPR-AMP1, a proline-rich antimicrobial peptide from the mud crab Scylla paramamosain Chanprapa Imjongjirak, Pawanrat Amphaiphan, Walaiporn Charoensapsri, Piti Amparyup PII:

S0145-305X(17)30198-2

DOI:

10.1016/j.dci.2017.05.003

Reference:

DCI 2887

To appear in:

Developmental and Comparative Immunology

Received Date: 8 April 2017 Revised Date:

3 May 2017

Accepted Date: 3 May 2017

Please cite this article as: Imjongjirak, C., Amphaiphan, P., Charoensapsri, W., Amparyup, P., Characterization and antimicrobial evaluation of SpPR-AMP1, a proline-rich antimicrobial peptide from the mud crab Scylla paramamosain, Developmental and Comparative Immunology (2017), doi: 10.1016/ j.dci.2017.05.003. 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.

ACCEPTED MANUSCRIPT Abstract Antimicrobial peptide (AMP) is an important molecule in the innate immune system. Here, we report the cloning and functional studies of proline-rich AMPs (PR-AMPs) from the three species of mud crab: Scylla paramamosain, S. serrata, and the swimming crab Portunus

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pelagicus. The deduced peptides revealed that they contain the putative signal peptides and encode for mature peptides, which contain sequence architecture similar to a 6.5-kDa prolinerich AMP of the shore crab, Carcinus maenas which showed similarity with the bactenecin7.

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Tissue distribution analysis indicated that the SpPR-AMP1 was expressed in a wide range of adult tissues, with the highest expression levels in the crab hemocyte. Challenge experiments

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showed that the levels of SpPR-AMP1 mRNA expression were up-regulated in the hemocyte after peptidoglycan stimulation. To evaluate the biological properties of mature SpPR-AMP1, peptides were chemically synthesized and recombinantly expressed. SpPR-AMP1 showed strong antibacterial activity against both Gram-positive bacteria Micrococcus luteus and

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in crab immunity.

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Gram-negative bacteria Vibrio harveyi. The results indicate that the SpPR-AMP1 plays a role

ACCEPTED MANUSCRIPT 1

Characterization and antimicrobial evaluation of SpPR-AMP1, a proline-rich

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antimicrobial peptide from the mud crab Scylla paramamosain

3 Chanprapa Imjongjiraka*,1, Pawanrat Amphaiphana,1, Walaiporn Charoensapsrib and

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Piti Amparyupb* a

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Department of Food Technology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand

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National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road,

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Klong 1, Klong Luang, Pathumthani 12120, Thailand

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Keywords: Antimicrobial peptide, Proline-rich, Crab, Scylla paramamosain

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E–mail address: [email protected] (C. Imjongjirak)

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*

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E–mail address: [email protected] (P. Amparyup)

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Corresponding author. Tel.: +66 2 218 5515; Fax: +66 2 254 4314.

Corresponding author. Tel.: +66 2 644 8150 # 81826; Fax: +66 2 644 8190.

These two authors contributed equally to this work and share the first authorship.

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Abstract Antimicrobial peptide (AMP) is an important molecule in the innate immune

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system. Here, we report the cloning and functional studies of proline-rich AMPs

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(PR-AMPs) from the three species of mud crab: Scylla paramamosain, S. serrata,

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and the swimming crab Portunus pelagicus. The deduced peptides revealed that they

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contain the putative signal peptides and encode for mature peptides, which contain

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sequence architecture similar to a 6.5-kDa proline-rich AMP of the shore crab,

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Carcinus maenas which showed similarity with the bactenecin7. Tissue distribution

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analysis indicated that the SpPR-AMP1 was expressed in a wide range of adult

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tissues, with the highest expression levels in the crab hemocyte. Challenge

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experiments showed that the levels of SpPR-AMP1 mRNA expression were up-

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regulated in the hemocyte after peptidoglycan stimulation. To evaluate the biological

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properties of mature SpPR-AMP1, peptides were chemically synthesized and

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recombinantly expressed. SpPR-AMP1 showed strong antibacterial activity against

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both Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Vibrio

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harveyi. The results indicate that the SpPR-AMP1 plays a role in crab immunity.

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1. Introduction The mud crab, Scylla paramamosain, is the commercially important

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crustacean distributed widely throughout the Indo-Pacific region. In recent years,

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mud crab capture and culture have been expanding in Thailand because of the high

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economic value of the species. Mud crabs are traditionally considered to be rather

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hardy organisms against many infectious diseases. However, the occurrence of

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diseases under culture condition is on the rise with the intensification of crab farming

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(Jithendran et al., 2010).

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Antimicrobial peptides (AMPs) are natural compounds that protect the host

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from microbial invasion (Boman, 2003; Hancock and Sahl, 2006; Zasloff, 2002). A

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large number of AMPs have been screened and identified from bacteria

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(bacteriocins), protists, fungi, plants, and animals (Epand and Vogel, 1999; Smith

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and Dyrynda, 2015; Tassanakajon et al., 2010; Tossi and Sandri, 2002; Wang et al.,

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2009; Zasloff, 2002). To date, several AMPs have been reported and characterized in

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various crab species, including a proline-rich peptide (6.5 kDa) (Schnapp et al.,

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1996), callinectin (Khoo et al., 1999; Noga et al., 2011), crustins (Imjongjirak et al.,

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2009; Mu et al., 2010; Relf et al., 1999), scygonadin anionic peptide (Peng et al.,

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2010; Wang et al., 2007), antilipopolysaccharide factors (ALFs) (Hou et al., 2017;

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Imjongjirak et al., 2007, 2011a; Li et al., 2008; Sun et al., 2015; Yedery and Reddy,

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2009; Yue et al., 2010; Zhu et al., 2014), arasin (Imjongjirak et al., 2011b; Paulsen et

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al., 2013; Stensvåg et al., 2008), and hyastatin (Shan et al., 2016a, 2016b; Sperstad et

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al., 2009).

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AMPs are classified into three major groups: (i) linear peptides that form

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amphipathic-helices, (ii) cysteine-rich peptides containing single or multiple

ACCEPTED MANUSCRIPT disulfide bridges and (iii) peptides with an over-representation of some amino acids

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such as proline, arginine, glycine, tryptophan or histidine (Bulet et al., 2004). Among

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these, the proline-rich AMPs are a group of peptides of widespread natural origin

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whose diverse sequences show an unusually high content of proline residues

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(Scocchi et al., 2011).

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A 6.5-kDa proline-rich AMP was initially isolated and partially sequenced

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from hemocytes of shore crab Carcinus maenas, which showed a significant

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sequence similarity with the bovine bactenecin7, a proline-rich AMP (Schnapp et al.,

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1996). The proline-rich AMP, namely, penaeidins, has recently been described from

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several species of shrimp (Cuthbertson et al., 2008; Destoumieux et al., 2000;

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Tassanakajon et al., 2010). However, these peptides display a proline-rich N-terminal

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domain as well as a C-terminal domain characterized by the presence of three

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conserved disulfide bonds. The 14-amino acid long proline-rich AMP without a Cys-

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rich domain, astacidins, has been reported from different crayfish species

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(Jiravanichpaisal et al., 2007). Moreover, the bipartite arrangement of Pro-Arg-rich

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and disulfide-bridged domains is also found in arasins and hyastatin, recently

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identified in spider and mud crab (Imjongjirak et al., 2011b; Shan et al., 2016a;

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Sperstad et al., 2009; Stensvåg et al., 2008) and callinectin from the blue crab (Noga

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et al., 2011). However, genes of proline-rich AMPs have not been reported in

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crustaceans to date.

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novel proline-rich AMP transcript was found to be present in the mud crab S.

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paramamosain EST library. This is a novel AMP gene (hereafter named SpPR-

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AMP1) in the mud crab, which shows 71% amino acid sequence identity to a

In this study, based on the screening of a S. paramamosain EST library, a

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ACCEPTED MANUSCRIPT proline-rich AMP (6.5 kDa) of the shore crab C. maenas (Schnapp et al., 1996).

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Therefore, in the present study, the molecular characterization of a proline-rich

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peptide, SpPR-AMP1 in mud crab S. paramamosain is described. The SpPR-AMP1

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was further characterized for gene expression profiles in various tissues and in

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response to the Gram-positive bacterial cell wall component peptidoglycan (PGN).

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The antimicrobial activity of synthetic peptide and recombinant protein against

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various pathogenic bacteria was also investigated.

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2. Materials and methods

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2.1. Sample preparation

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Adult mud crab, S. paramamosain, (200-220 g body weight), were purchased

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from local fish markets in Bangkok, Thailand, and were cultured in the laboratory for

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a week before processing. The crab hemolymph from three crab pericardial cavities

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was collected with a sterilized syringe. The hemolymph was collected in an

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anticoagulant solution of 10% (w/v) of trisodium citrate dihydrate pH 4.6. The

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hemolymph was immediately centrifuged at 800 × g for 10 min at 4 °C. The

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supernatant was removed and hemocytes were collected for RNA extraction. To

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examine the tissue-specific expression profile of SpPR-AMP1, three healthy

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individuals were selected. Crabs were carefully dissected on ice, and tissues (gills,

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muscle, intestine and hepatopancreas) were collected and pooled from three crabs for

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RNA extraction. All tissue samples were snap-frozen in liquid nitrogen immediately

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after they were obtained from crab and were stored at -80 °C until used for RNA

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isolation.

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2.2. Total RNA isolation and first-strand cDNA synthesis The total RNA was extracted from selected tissues of mud crab, S.

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paramamosain, using TRIzol Reagent (Gibco-BRL, USA) and then treated with

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DNase I (Promega, USA) following the manufacturer’s protocol. First-strand cDNAs

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were synthesized from 2 µg of DNA-free total RNA sample and 0.5 µg of oligo

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(dT)18 primers using the ImProm-II™ Reverse Transcriptase System kit (Promega,

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USA) according to the manufacturer’s protocol. The resulting cDNA was stored at -

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20 °C for further processing.

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2.3. cDNA library construction and EST analysis

A cDNA library was constructed from the hemocytes of mud crab, using a

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SuperScript™ Plasmid System for cDNA Synthesis and Plasmid Cloning Kit

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(Gibco-BRL, USA). After sequencing, plasmid DNA was extracted and partially

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sequenced unidirectionally with M13 forward or reverse primers (Macrogen Inc.,

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Korea). The Basic Local Alignment Tool (BLAST) program analysis of the EST

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sequences revealed that an EST of 400 bp was similar to the proline-rich peptide (6.5

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kDa) of the shore crab C. maenas. Therefore, this EST sequence was selected for

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further cloning of the full-length cDNA of SpPR-AMP1.

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2.4. Determination of the full-length cDNA of SpPR-AMP1 To generate the full-length cDNA of SpPR-AMP1, two specific primers,

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BacSpR1 and R2 (Table 1), were designed based on the EST sequence. The full-

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length cDNA of SpPR-AMP1 was isolated through RACE-PCR. The 5′-RACE

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cDNA were performed with SMART™ RACE cDNA Amplification kit (Clontech,

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ACCEPTED MANUSCRIPT USA) according to the manufacturer's protocol using the 5′-RACE-Ready cDNA

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from crab hemocyte as template. The nested PCR strategy was applied to 5′-RACE.

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The amplification reaction and PCR temperature profiles is described in Amparyup

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et al. (2007). Briefly, the 5′-RACE PCR conditions were consisted of 5 cycles of 94

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°C for 45 s, 68 °C for 45 s and 72 °C for 2 min followed by 25 cycles of 94 °C for 45

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s, 55 °C for 45 s and 72 °C for 2 min and the final extension at 72 °C for 10 min. The

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RACE-PCR product was electrophoretically analyzed and 1 µl was used as template

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for the nested PCR amplification which consisted of 25 cycles of 94 °C for 45 s, 55

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°C for 45 s and 72 °C for 2 min followed by the final extension at 72 °C for 10 min.

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The expected DNA fragment was eluted from agarose gel and ligated into pGEM®-T

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Easy vector (Promega, USA). The ligation product was transformed into Escherichia

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coli JM109. The positive recombinant clone was identified by PCR screening with

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M13F and M13R primers. Three of the positive clones were sequenced.

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To isolate the PR-AMP1 mRNA from hemocyte of the three species of mud

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crab, Scylla paramamosain, S. serrata and the swimming crab Portunus pelagicus,

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the amplification of a single fragment of full-length ORF cDNA was carried out by

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PCR using primer BacSpF1 and BacSpR1 (Table 1). The PCR products were cloned

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and sequenced in both directions.

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2.5. Tissue distribution analysis

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RT-PCR was carried out to investigate the transcript expression profile of the

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SpPR-AMP1 transcript in different tissues of S. paramamosain including hemocytes,

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hepatopancreas, gills, intestine and muscle. A pair of SpPR-AMP1 specific primers

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(BacSpF2 and BacSpR2) was used to amplify a PCR product of SpPR-AMP1 (Table

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ACCEPTED MANUSCRIPT 1) using 1 µl of cDNAs synthesized from 2 µg of DNA-free total RNA extracted

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from various crab tissues as a template. A set of elongation factor 1-α gene (EF1-α)

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primers, EF1α-F and R, was used to amplify a product of 149 bp, which served as

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internal control (Table 1). PCR temperature profiles are described in Amparyup et al.

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(2007). In brief, the thermal cycling condition consists of 94 °C for 1min followed by

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25 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s and the final extension

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at 72 °C for 5min. The amplification product was electrophoretically analyzed

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through a 1.8% (w/v) agarose-TBE gel.

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2.6. Sequence analysis

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DNA sequences were further edited with GENETYX (Software Development

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Inc.) and blasted against data in the GenBank (http://www.ncbi.nlm.nih.gov). The

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putative cleavage site of the signal peptide was predicted by the SignalP

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(http://www.cbs.dtu.dk/services/SignalP/). Multiple sequence alignments of amino

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acids were performed using Clustal W (Thompson et al., 1994).

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2.7. Gene expression analysis in response to PGN challenge The challenge experiment was performed by injection into the last abdominal

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segment of each crab of either saline solution (0.85% NaCl, w/v) as a control, or a

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suspension of the Gram-positive bacterial cell wall component PGN in the same

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volume of saline solution. For each group, hemocytes of S. paramamosain were

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collected from three individual crabs at 0, 6, 12, and 24 h after injection using a 1-ml

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sterile syringe preloaded with 500 µl of an anti-coagulant solution (10% sodium

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citrate, w/v). The collected hemolymph was then immediately centrifuged at 800 × g

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ACCEPTED MANUSCRIPT for 10 min at 4 °C to isolate the hemocytes from the plasma. The hemocyte pellet

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was resuspended in 200 µl of the TRIzol Reagent (Gibco-BRL, USA). RNA isolation

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and first-strand cDNA synthesis were carried out as described above. The

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amplification reaction and thermal profile were performed as described by

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Amparyup et al. (2007) using the specific primers BacSpF2 and BacSPR2, with

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amplification of the EF1-α internal gene fragment as an internal control. Briefly, the

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amplification was performed in a 96-well plate in a 20 µl reaction volume containing

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10 µl of 2x SYBR Green Supermix (Bio-Rad, USA), 2.5 µl of BacSpF2 and

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BacSpR2 primers (12 µM), 5 µl of 1:50 diluted cDNA template. The thermal profile

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for SYBR Green real-time RT-PCR was 95 °C for 5 min followed by 40 cycles of

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denaturation (95 °C for 30 s), annealing (57 °C for 30 s), and extension (72 °C for 30

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s). Amplification of each sample was performed with three replicates. The Ct values

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of the PGN injected samples at each time point were normalized with the saline-

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injected samples and a mathematical model was used to determine the relative

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expression ratio (Pfaffl, 2001). Statistical calculations were carried out using the one-

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way analysis of variance (ANOVA) followed by Duncan’s test and differences were

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considered significant at P<0.05.

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2.8. Recombinant expression of SpPR-AMP1 protein PCR fragment encoding the mature peptide of SpPR-AMP1was amplified

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with specific primers NcoI-BacSp-F and NotI-BacSp-R (Table 1) using Pfu DNA

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polymerase. Recombinant SpPR-AMP1 was expressed in E. coli as N-terminal His6-

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tagged fusion protein using the pET-28b system (Novagen, USA). The recombinant

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plasmid (pET28b-SpPR-AMP1) was confirmed by nucleotide sequencing and then

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ACCEPTED MANUSCRIPT transformed into E. coli Rosetta (DE3) pLysS (Novagen, USA). Protein expression

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was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a

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final concentration of 1 mM and analyzed by 20% SDS-PAGE. The recombinant

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protein was purified by a nickel-nitrilotriacetic acid (Ni-NTA) agarose column

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(Qiagen, USA), as described by the manufacturer. The purified His-tagged protein

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was dialyzed against sodium phosphate buffer (pH 5.8) and then analyzed by 4–20%

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gradient SDS-PAGE. The purified SpPR-AMP1 peptide was subsequently used in

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the antimicrobial activity test.

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The mature peptide of SpPR-AMP1 was synthesized by the peptide

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synthesizer. The N-terminal and C-terminal residues of the peptides was blocked by

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acetylation and amidation, respectively. The synthetic peptide, purified by reverse-

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phase HPLC, was obtained with >95% final purity. Peptides were stored as a

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lyophilized powder at -20 °C and were dissolved in sterile pyrogen-free water

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immediately before use.

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2.10. Antimicrobial assay

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The antimicrobial activities of the synthetic peptide and the recombinant

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peptide of SpPR-AMP1 were determined as minimal inhibitory concentration (MIC).

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Microorganisms used in the antimicrobial activity assay include: (1) Gram-positive

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bacteria: Staphylococcus aureus ATCC25923, Micrococcus luteus ATCC9341, and

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Bacillus cereus ATCC11778 (2) Gram-negative bacteria: Salmonella thyphimurium

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ATCC13311,

Escherichia

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Vibrio

harveyi,

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ACCEPTED MANUSCRIPT parahaemolyticus ATCC17802. The antimicrobial activity method is described in

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Imjongjirak et al. (2007). Briefly, two-fold serial dilutions of synthetic peptide or

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recombinant peptide of SpPR-AMP1 were prepared in 0.2% (w/v) BSA/0.01% (v/v)

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acetic acid. 10 µl of each peptide concentration was added to each corresponding

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well of a 96-well microtiter plate, and each well was inoculated with 90 µl of a

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suspension of mid-log bacteria (105 CFU/ml) in Poor Broth (1% (w/v) tryptone,

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0.5% (w/v) NaCl, pH 7.5). The negative controls were performed as above using

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sodium phosphate buffer (pH 5.8) for the recombinant peptide and sterile pyrogen-

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free water for the synthetic peptide. Cultures were grown for 24 h with shaking at 30

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°C and the growth of bacteria was evaluated by measuring the culture absorbance at

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595 nm using a microplate reader. All tests were carried out in triplicate. The

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minimum inhibitory concentrations (MIC) were taken as the lowest peptide

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concentration at which growth was inhibited after 24 h incubation at 30 °C.

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2.11. Bactericidal assay

The minimal bactericidal concentrations (MBC) of the synthetic peptide and

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recombinant peptide of SpPR-AMP1 against bacteria were obtained by plating out

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the contents of each well showing no visible growth. One hundred microliters of the

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suspension was spread onto LB and incubated at 30 °C for 24 h. All tests were

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carried out in triplicate. The MBC was taken as the lowest concentration at which no

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colonies grew following overnight incubation at 30 °C.

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3. Results and discussion

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3.1. cDNA and protein sequences of SpPR-AMP1 from the crab Scylla

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paramamosain Antimicrobial peptides (AMPs) play a significant role in the defense systems

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of crustaceans (Tassanakajon et al., 2010). Proline-rich AMPs are a group of cationic

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defense peptides of animals characterized by a high content of proline residues, often

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associated with arginine residues in repeated motifs (Scocchi et al., 2011). In our

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effort to discover new antimicrobial peptides from the crab Scylla paramamosain, we

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identified a cDNA clone (SpPR-AMP1) that encodes an ORF with high amino acid

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sequence similarity to the 6.5-kDa antimicrobial peptide of the crab Carcinus

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maenas (Schnapp et al., 1996), from cDNA library of S. paramamosain hemocyte.

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The SpPR-AMP1 cDNA was 466 bp in length and contained an open reading

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frame (ORF) of 180 bp that encoded a predicted product of 59 amino acid residues.

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The 5′-UTR was 45 bp long and the 3′-UTR (241 bp) contained a putative

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polyadenylation site (CATAAA) and one of RNA instability sequences (ATTTA)

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(Fig. 1A). SpPR-AMP1 had a predicted signal peptide (22 amino acids) with a

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cleavage site located between amino acid positions 22 and 23 (A-G). The predicted

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mature peptide (37-amino acids) of SpPR-AMP1 had a calculated molecular weight

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of 4416.17 Da and theoretical isoelectric point (pI) of 12.13. A sequence analysis of

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the mature SpPR-AMP1 revealed the presence of 17 proline residues representing

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45.946% of the whole sequence. Based on antimicrobial peptide database analysis in

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CAMP (Collection of Anti-Microbial Peptides; http://www.camp.bicnirrh.res.in/),

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SpPR-AMP1 belongs to the family of proline-rich AMPs, which is the most similar

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family in sequence architecture to the 6.5-kDa antimicrobial peptide of shore crab,

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Carcinus maenas (71% identity) and Bactenecin7 of Bovine, Bos taurus (66%

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identity). To verify this gene in other crab species for comparative analysis two pairs of

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primers, designed based on the nucleotide sequences of SpPR-AMP1, were

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successfully amplified PR-AMP1 cDNAs from the hemocyte of mud crab S. serrata

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(SsPR-AMP1) and swimming crab Portunus pelagicus (PpPR-AMP1). The SsPR-

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AMP1 and PpPR-AMP1 cDNAs contained a 180 bp and 189 bp ORF encoded a

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peptide of 59 and 62 amino acids, with the first 16 residues predicted to be a signal

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peptide. The putative mature peptides were predicted to have a molecular weight of

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4944.79 and 5300.22 Da and isoelectric point of 12.13 and 12.22, respectively (Fig.

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1B and C). Similar to the SpPR-AMP1 peptide, an analysis of amino acids in SsPR-

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AMP1 and PpPR-AMP1 peptides also revealed 18 and 16 proline residues

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representing 41.86% and 34.783% of the whole sequence. Sequence comparison

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analysis of SpPR-AMP1 peptide revealed that it has a high sequence similarity and is

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homologous, to the SsPR-AMP1 peptide of the mud crab S. serrata (98.3% sequence

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identity) and to the PpPR-AMP1 peptide of the swimming crab P. pelagicus (71%).

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Multiple alignments of PR-AMP1 from crabs show that the signal peptides and

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proline-rich region, contained PRP-motif, in mature peptides of PR-AMP1 from

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three crabs are highly conserved (Fig. 2). Altogether, these findings suggest that the

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PR-AMP1 peptide of three crab species is a member of proline-rich AMPs in crab.

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3.2. Tissue distribution and gene expression profile of SpPR-AMP1 mRNA

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The tissue-specific gene expression of the SpPR-AMP1 transcript in normal

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crab was investigated using semi-quantitative RT-PCR. The total RNA from

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ACCEPTED MANUSCRIPT different tissues including hemocyte, gills, intestine, hepatopancreas, and muscle of

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crab were isolated and transcribed into cDNAs and used as the cDNA template for

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PCR analysis. The results showed that the expression level of SpPR-AMP1 mRNA

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was the highest in the hemocyte, followed by a moderate level in the gills, intestine,

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and muscle, and a relatively low level in the hepatopancreas (Fig. 3). In several

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crustaceans, different AMPs have different tissue distribution patterns depending on

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their functional activities. Previously, we found that an arasin-likeSp mRNA was

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mainly expressed in crab hemocytes. However, ALFSp1 and 2 and CrusSp mRNA

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were highly presented in the hemocyte, gills, intestine, and muscle of crabs. The

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different tissue distributions of crab AMPs may be related to their different functions

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against pathogens. However, in several crustaceans, hemocytes are very important

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cells of the innate immune system (Bachère et al., 2004; Gross et al., 2001),

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therefore, SpPR-AMP1 might have functions in crab immunity.

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To further examine the relationship between SpPR-AMP1 and immunity, we

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analyzed the expression patterns of the SpPR-AMP1 transcript in hemocyte after

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PGN challenge at the time course of 0, 6, 12, and 24 h post injection using real time

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PCR. All the results are presented with EF1-α mRNA as an endogenous control. The

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results show that SpPR-AMP1 was significantly up-regulated in hemocyte at 6 h to

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12 h, 5.43- and 5.86-fold higher relative expression level than at 0 h, and decreased

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to the normal level at 24 h after injection with PGN (Fig. 4). In crustaceans, several

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AMPs were examined for the gene expression profiles after microbial cell wall

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component or pathogen stimulation. For proline-rich AMPs, the astacidin 2 mRNA,

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that showed strong antimicrobial activity to both Gram-positive and Gram-negative

326

bacteria, is expressed constitutively and is not up-regulated by LPS injection in

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ACCEPTED MANUSCRIPT crayfish hemocyte (Jiravanichpaisal et al., 2007). However, the primary sequence

328

and proline-rich region of astacidin 2 are not similar to SpPR-AMP1, although they

329

belong to the same group of proline-rich AMPs. Moreover, many AMPs from mud

330

crab have been shown to be induced or up-regulated when the hosts are infected with

331

pathogens (Hou et al., 2017; Imjongjirak et al., 2011b; Shan et al., 2016b; Sun et al.,

332

2015; Yedery and Reddy, 2009; Zhu et al., 2014). For example, transcripts of ALF5

333

of mud crab were found to be significantly up-regulated following LPS, Vibrio

334

parahaemolyticus, PolyI:C, or WSSV challenges (Sun et al., 2015). Thus, our results

335

suggested that SpPR-AMP1 could be involved in the antibacterial response of crab.

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336 337

3.3. Recombinant protein expression of SpPR-AMP1 peptide Recombinant SpPR-AMP1 plasmid was transformed into E. coli Rosetta

339

(DE3) pLysS cells and the recombinant bacterial cell was induced to express by

340

IPTG. The SDS-PAGE analysis indicated that the rSpPR-AMP1 protein was

341

successfully expressed in E. coli cells, and its molecular weight was approximately 6

342

kDa (Fig. 5A). The rSpPR-AMP1 protein was purified by Ni-NTA chromatography.

343

The molecular mass of the purified rSpPR-AMP1 protein was consistent with the

344

theoretically predicted masses (5.73 kDa), as determined by 4–20% gradient SDS-

345

PAGE, indicating that the protein had been purified successfully (Fig. 5B).

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3.4. Antimicrobial activity of SpPR-AMP1 peptide

348

The synthetic SpPR-AMP1 (SpPR-AMP1-S) was first investigated for its

349

potential antimicrobial activities. The MIC and MBC were determined to test its

350

activity of synthetic SpPR-AMP1 (SpPR-AMP1-S) and recombinant SpPR-AMP1

15

ACCEPTED MANUSCRIPT (SpPR-AMP1-R). The MIC and MBC values for these peptides against the Gram-

352

positive and Gram-negative bacterial species are presented in Table 2. The result of

353

antimicrobial activity of SpPR-AMP1-S against bacteria indicated that the SpPR-

354

AMP1-S exhibited strong antibacterial activity against both Gram-positive bacteria

355

Micrococcus luteus (MIC, 1.56 µM) and Gram-negative bacteria Vibrio harveyi

356

(MIC, 0.39 µM). Moderate activity was noted against V. parahaemolyticus (MIC,

357

6.25 µM), Salmonella thyphimurium (MIC, 6.25 µM), and E. coli (MIC, 25 µM).

358

However, no antibacterial activity was observed against Staphylococcus aureus and

359

B. cereus (MIC, >50 µM).

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Based on the antibacterial activity of SpPR-AMP1-S, the most susceptible

361

Gram-positive- and negative- bacteria to this peptide were M. luteus and V. harveyi.

362

Therefore, the antibacterial activity of the recombinantly expressed SpPR-AMP1 (

363

SpPR-AMP1-R) against M. luteus and V. harveyi was determined. The SpPR-AMP1-

364

R displayed the antibacterial activity against M. luteus (MIC, 6.25 µM) and V.

365

harveyi (MIC, 3.13 µM). However, the MIC and MBC of SpPR-AMP1-S were lower

366

against M. luteus (MIC, 1.56 µM) and V. harveyi (MIC, 0.39 µM) than those of

367

SpPR-AMP1-R. The possible reason for the slightly different activity of SpPR-

368

AMP1-S and SpPR-AMP1-R against bacteria may be due to several factors, such as

369

the composition of amino acid in the peptide, total net charge of the protein, or three-

370

dimensional structure.

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have been previously characterized in the shore crab, C. maenas, also named a 6.5

373

kDa proline-rich AMP (Schnapp et al., 1996), that showed high similarity to SpPR-

374

AMP1 from mud crab. This peptide was isolated from crab hemocyte and was

The first proline-rich AMPs in crustaceans that exhibit antibacterial activities

16

ACCEPTED MANUSCRIPT effective against both Gram-positive M. luteus and Gram-negative Psychrobacter

376

immobilis (Schnapp et al., 1996). In agreement, SpPR-AMP1 peptide of mud crab

377

has similar antimicrobial activity against M. luteus compared with 6.5 kDa proline-

378

rich AMP (Schnapp et al., 1996). Moreover, SpPR-AMP1 peptide is mainly active

379

against Gram-negative bacteria. These results show clearly that the SpPR-AMP1

380

peptide is a crustacean proline-rich AMP exhibiting antimicrobial activity. Although

381

AMP containing the proline-rich regions have been reported in several crustacean

382

species including crab arasin, crab callinectin, crabs and shrimp penaeidins, and

383

shrimp SWD, these peptides often show characteristics of the bipartite arrangement

384

of Pro-rich and Cys-rich domains, indicating that these peptides are functionally a

385

different class of AMPs in crustaceans.

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Our results clearly demonstrate that SpPR-AMP1 is a proline-rich AMP,

387

which exhibits antimicrobial activity against Gram-positive and Gram-negative

388

bacteria. In conclusion, proline-rich AMPs similar to a 6.5 kDa proline-rich AMP of

389

the shore crab, C. maenas, were identified from hemocyte of three crab species S.

390

paramamosain, S. serrata and Portunus pelagicus. The SpPR-AMP1 transcript was

391

expressed in hemocyte and up-regulated after PGN challenge. SpPR-AMP1 possess

392

antibacterial activity against Gram-positive and Gram- negative bacteria. All of these

393

results suggest that SpPR-AMP1 is a potent immune effector in the immune defense

394

of crab.

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Acknowledgments

397

This work was supported by grants from the Ratchadaphiseksomphot

398

Endowment Fund awarded to Chanprapa Imjongjirak and Piti Amparyup. Pawanrat

17

ACCEPTED MANUSCRIPT 399

Amphaiphan is the recipient of the 90th Anniversary of Chulalongkorn University

400

Fund (Ratchadaphiseksomphot Endowment Fund).

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ACCEPTED MANUSCRIPT Figure Legends

537

Fig. 1. The full-length nucleotide (above) and predicted amino acid (below)

538

sequences of SpPR-AMP1 (A), SsPR-AMP1 (B) and PpPR-AMP1 (C) cDNAs

539

from the mud crab Scylla paramamosain, S. serrata and the swimming crab

540

Portunus pelagicus. The numbers on the right of the sequence give the positions of

541

the last nucleotide on each line. The proposed start and stop codons are in bold. The

542

predicted signal peptides are in bold and shaded in gray. The putative RNA

543

instability sequences (ATTTA) and polyadenylation signal (CATAAA) are in bold

544

and underlined. Circle indicates the proline residues.

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Fig. 2. Multiple alignment of deduced amino acid sequence of crab PR-AMP1

547

with the Bovine Bactenecin and crabs Bac-likes. The amino acid sequence of the

548

mud crab Scylla paramamosain (SpPR-AMP1), S. serrata (SsPR-AMP1), the

549

swimming crab Portunus pelagicus (PpPR-AMP1), the shore crab Carcinus maenas

550

(CmAMP6.5; P82964) and the Bovine Bos taurus (Bac7; NP_776426), were

551

collectively compared. The Pro-Arg-Pro (PRP) motifs are underlined. Grey indicates

552

conservation in five species. Dark grey indicates conservation in three or four

553

species. The predicted signal peptides are in bold and underlined.

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Fig. 3. Tissue-specific expression of SpPR-AMP1 in hemocytes, gills, intestine,

556

hepatopancreas and muscle of Scylla paramamosain by RT-PCR analysis. EF1-α

557

was used as a control housekeeping gene to indicate and standardize the amount of

558

cDNA template in each of the various tissues. Gel images shown are representative

559

of those seen from three independent trials.

ACCEPTED MANUSCRIPT 560 Fig. 4. Relative gene expression profile of SpPR-AMP1 mRNAs in hemocyte of

562

Scylla paramamosain after peptidoglycan (PGN) injection. The cDNAs from three

563

individual crabs at each time point (0, 6, 12 and 24 h) were pooled and used as

564

templates for real-time RT-PCR analysis of the SpPR-AMP1 transcripts. Relative

565

expression levels of mRNA were calculated according to Pfaffl (2001) using the

566

EF1-α as an internal reference gene. The average relative expressions are

567

representative of three independent repeats±1 S.D. (error bars). Asterisks indicate

568

statistical significant difference between means compared to 0 h post PGN injection

569

(**P < 0.01).

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Fig. 5. SDS-PAGE analysis of the recombinant SpPR-AMP1 (SpPR-AMP1-R)

572

protein. (A) Expression analysis of SpPR-AMP1-R protein after induction with 1

573

mM IPTG for 0 to 5 h in the crude protein extract (CPE) of E. coli Rosetta (DE3)

574

pLysS. (B) Expression analysis of SpPR-AMP1-R in protein fractions of the bacterial

575

lysate. (C) Purification of SpPR-AMP1-R protein by Ni-NTA chromatography. Tn-

576

CPE : the IPTG-induced protein fraction (n = 0, 1, 2, 3, 4 and 5 h); SPF : the soluble

577

protein fraction; IPF : the insoluble protein fraction; PP : the purified protein fraction

578

(20 µg). CPE, SPF and IPF samples were resolved on 20% reducing SDS-PAGE gel.

579

PP was analyzed by SDS-PAGE on a 4–20% gradient gel under a reducing

580

condition. Gels were visualized with Coomassie Brilliant Blue staining. Lane M is

581

the protein ladder. Arrows indicate the SpPR-AMP1-R protein.

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ACCEPTED MANUSCRIPT Table 1. Primer sequences used for amplification of PR-AMP1 genes from crab.

BacSpF1 BacSpR1 BacSpF2 BacSpR2 EF1α-F EF1α-R NcoI-BacSp-F

Purpose

TCCCACGTCAACACACAGCCCCCAG GTTTATGCAGGGGTACATTCAGGGT AATGCGTCTGCTGTGGCTCCTGGTG TAACTCCAACACGAAGACAAAAGCAGCG GGTGCTGGACAAGCTGAAGGC CGTTCCGGTGATCATGTTCTTGATG CATGCCATGGGCCATCATCATCATCATCAT ATGGCTTCTGCTGGGTACTTTCCCG ATAAGAATGCGGCCGCTCAGCGCCAAGGAT AAGGCCGT

RT-PCR, Full-length cDNA RT-PCR, RACE-PCR , Full-length cDNA RT-PCR, Real-time PCR, RT-PCR, RACE-PCR , Real-time PCR, RT-PCR RT-PCR Recombinant protein expression Recombinant protein expression

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NotI-BacSp-R

Sequence (5’-3’)

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Primer name

ACCEPTED MANUSCRIPT Table 2. Antimicrobial activity of the synthetic and the recombinant SpPR-AMP1 peptides.

SpPR-AMP1-S MICa

MBCb

MICa

MBCb

(µM)

(µM)

(µM)

(µM)

>50

>50

0.78–1.56

1.56

>50

>50

Gram positive bacteria: Micrococcus luteus ATCC9341

nd

nd

3.13–6.25

6.25

nd

nd

50

nd

nd

50

nd

nd

0.195–0.39

3.13

1.56–3.13

6.25

3.13–6.25

6.25

nd

nd

Bacillus cereus ATCC11778 Gram negative bacteria: Escherichia coli ATCC35218 Vibrio harveyi V. parahaemolyticus ATCC17802 a

3.13–6.25 12.5–25

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Salmonella thyphimurium ATCC13311

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Staphylococcus aureus ATCC25923

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Microorganisms

SpPR-AMP1-R

MIC values are expressed as the interval of concentration [a]–[b], where [a] is the highest

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concentration tested at which microbial growth can be observed and [b] is the lowest concentration that causes 100% growth inhibition. b

MBC values are expressed as the lowest concentration at which no colonies grew following

overnight incubation at 30 °C.

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nd = not determined.

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ACCEPTED MANUSCRIPT Highlights •

A proline-rich AMP, SpRR-AMP was identified from crab Scylla paramamosain.

•

SpPR-AMP was highly expressed in hemocyte and up-regulated after PGN stimulation. SpPR-AMP exhibited antimicrobial activity against bacteria.

•

SpPR-AMP is a potent immune effector in crab immunity.

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•