Anti- Vibrio Response of Carcinin Pm 1 from Penaeus monodon and Its Heterologous Expression

. Crustins are crucial antimicrobial peptides in shrimp and play very important roles in innate immunity. In this research, a Type I crustin from Penaeus monodon (Carcinin Pm 1) contained 108 residues was studied. The first 16 residues are signal peptide. It contained ten cysteines but did not form an intact whey acidic protein (WAP) domain. Carcinin Pm 1 was observed to widely distribute in all tissues, while highly expressed in intestine. The expression level of Carcinin Pm 1 in hepatopancreas was up-regulated 12-20 times during 4-12h post challenged by Vibrio parahaemolyticus . And the transcription in heart, stomach and gills was also significantly enhanced at 4h post challenge. The mature peptide was expressed successfully in Eschericha coli by fusing to a SUMO protein, with protein production around 8 mg/mL. After cleavage with SUMO protease, carcinin Pm 1 was obtained indicating its potential applications.


Introduction
Antimicrobial peptides (AMPs) are small and active peptides widely distributed in various organisms, as an important part of innate immunity of organisms and exhibited broad-spectrum of antimicrobial activities against variety of bacteria, virus and cancer cells [1].
Penaeus monodon, also called black tiger shrimp, is the second most widely cultured shrimp species in the world. However, the outbreak of diseases seriously reduced the production and economic benefit, especially infections caused by Vibrios [2] or viruses [3]. As invertebrates do not have acquired immune system, they depend on innate immunity to defend invading microbes [4]. Innate immunity includes humoral and cellular immunity. Antimicrobial peptides (AMP) are important humoral immunity molecules, as they can kill invading microbes directly and/or regulate other immune response [5,6]. Several types of AMPs were identified in shrimps, including crustins, penaeidins, antilipopolysaccharide factors (ALFs), stylicins, haemocyanin-derived peptides, lysozymes and histones and derived fragments [6][7][8].
Crustin is one of the largest families of AMPs in invertebrate. There are in total four types of crustins in crustaceans [9]. Type I crustins are constituted by a signal peptide, a cysteine-rich domain with two disulfide bridges and a single WAP (whey acidic protein) domain at the C-terminus. Compared to Type I crustins, the type II crustins contain a long glycine-rich region to the Nterminus of the cysteine-rich domain. Type III crustins, also called single WAP domain (SWD) containing proteins, only contain a short proline and arginine-rich region in front of the WAP domain [10,11]. The type IV crustins (DWD crustins) have two WAP domains [11].
So far, four types of crustins have been found in P. monodon. Only two Type I crustins have been mentioned, while at least ten kinds of Type II crustins were discovered [12]. CrustinPm1 and crustinPm5 exhibit antimicrobial activity only against Gram-positive bacteria, while crustinPm7 is active against both Grampositive and Gram-negative bacteria [13][14][15]. Type III crustin from P. monodon exhibits anti-Gram-positive, but not anti-Gram-negative bacteria activity and it is a competitive inhibitor of subtilisin A [16]. However, the DWD crustins do not show apparent activity [17].
Type I crustins are present mainly in crabs [18,19] but also found in crayfish [20][21][22] and shrimps [23][24][25]. Some shrimps contain more than one isoforms of Type I crustins. The first Type I crustin was isolated in 1999, from the granular haemocytes of the shore crab, Carcinus maenas and named carcinin Cm1. It was heat stable and active only against Gram-positive bacteria [26]. There are in total five type I crustins identified in Marsupenaeus japonicas [24][25].
The type I crustin CarcininPm1, was first reported by Suchao Donpudsa, et.al. [12]. However, it was not fully characterized. In this research, further studies were performed to reveal its sequence similarity with other type I crustin, the tissue distribution and expression trend during Vibrio infection. All the results suggest that it could play a very important role in the innate immunity system of P. monodon.

Bioinformatics analysis of CarcininPm1
The nucleotide sequence of CarcininPm1 was obtained from transcriptome sequencing data of the hepatopancreas of Penaeus monodon. The Open Reading Frame and amino acid sequence of CarcininPm1 were deduced by ORF Finder (http://www.ncbi.nlm.nih.gov/ gorf/gorf.html).The signal peptide was predicted with signalP 4.0 server. DNAman version 6 was used for generating gene structure information. Homologous sequences of CarcininPm1 were obtained by Basic Local Alignment Search Tool (BLASTP)and multisequences alignment was performed with clustalw (https://www.genome.jp/tools-bin/clustalw). The online software ESPript 3.0 was used to generate the alignment result [27]. The physicochemical properties were predicted with the online software Protparam (http://web.expasy.org/protparam/).

Immune challenging of shrimps and tissues collection
Individual shrimps (about 16 cm in length) were collected from a prawn breeding base (Dapeng, Shenzhen), and cultured in 50 L glass boxes, each containing 10 L of filtered seawater at 25±1℃ for one day before experiments.
Vibrio parahaemolyticus (SIV) was cultured with Luria-Bertani broth containing 3% NaCl shaken at 30℃. The overnight cultured bacteria were inoculated to a fresh media and cultured until OD540 reached 0.3 (about 5×10 7 CFU/ml). Cells were harvested, washed twice with sterilized PBS and then resuspended with PBS to a final concentration of 1×10 7 CFU/ml. The shrimps were divided into two groups, Group P and Group V. Each group contained at least 20 individuals. For shrimps in group V, 100μl suspended bacteria were injected into the ventral blood sinusand shrimps in group P were injected with 100μl PBS as a control.Hepatopancreas of Group P and Group V shrimps were collected at 0, 2, 4, 8, 12 and 24 h post injection. Heart, gills, stomach, intestine and hemocytes were collected from unchallenged and 4h postchallenged shrimps.
To collect hemocytes, at least 600μl hemolymph was drawn out from pericardial sinus of shrimps with a 1 ml syringe preloaded 100μl anticoagulant (0.1M Sodium Citrate, 0.25M sucrose, 0.01M Tris-HCl, pH 7.6) and centrifuged at 830g, 4°C for 10 min. The collected cells were washed with 1ml anticoagulant and then suspended in 600μl lysis buffer to proceed with the subsequent total RNA extraction.

2.3Total RNAs extraction and cDNAs synthesis
Total RNAs were extracted from all the tissues prepared above using RNeasy Mini Kit (Qiagen, USA), according to the manufacturer's instruction. cDNAs were synthesized with PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan).

2.4Confirmation of the CarcininPm1 gene
Reverse-transcription PCR(RT-PCR) was performed with primers CarcininPm1 conF and CarcininPm1 conR ( Table 1) to amplify the ORF of CarcininPm1. The PCR product was connected to a T-vector (pMD™18-T Vector Cloning Kit, Takara) and sequenced. Table 1.Primers used in the present study.

2.5Tissue distribution of CarcininPm1
Semi-quantitative real-time PCR was used to test the relative expression level of CarcininPm1 in different tissues of P. monodon. One pair of primers (CarcininPm1 F and CarcininPm1 R) was used and EF-1αwas used as a control.

2.7Gene synthesis and vector construction
The mature peptide of CarcininPm1 was fused to the Cterminus of a SUMO protein with His-tag and overexpressed in Escherichia coliBL21 (DE3). The nucleotide sequence of His-SUMO-CarcininPm1 with NdeI and SacI cut sites in both ends was codon optimized for E. coli and chemically synthesized (General Biosystems, Inc., Hefei, China). The synthesized DNA was then linked to pColdIV vector by NdeI and SacI cut sites. The reconstituted plasmid was transformed to E. coli BL21 Rosetta (DE3), named BL21-carpm1.

Protein expression and purification
Adding 20 mL of overnight cultivated BL21-carpm1 intothe fresh 2 L LB broth medium containing ampicillin (50 μg/mL) and then cultivating them at 37 °C with shaking at 200 rpm until the absorbance reached 0.5 at 600 nm. IPTG was added to the culture at a final concentration of 1 mM. The induction was performed at 16°C for 12 hours. Cells were harvested by centrifugation and the cell pelletswere washed and resuspended in PBS. The bacterial suspensions were then disrupted by ultrasonication. The supernatant (soluble fraction) was collected and analyzed by SDS-PAGE. The fusion protein was purified by Ni-NTA Sepharose Fast Flow (Ruidahenghui, Beijing, China), and the his-tagged protein was eluted with buffer contained 50 mM PBS, 300 mM NaCl and 200 mM imidazole. Elutes were further analyzed by SDS-PAGE.
The eluted protein was further dialyzed against 50mM Tris-HCl, 200mM NaCl to remove the imidazole and then quantified by Bradford reagent (Sangon, China).100μg protein was mixed with 1U SUMO protease (General Biosystems, Inc., Hefei, China), and incubated at 4°C overnight to remove the SUMO tag.

Sequence information of CarcininPm1
The nucleotide sequence of CarcininPm1 in this research was obtained from transcriptome data of hepatopancreas of P. monodonand was confirmed by RT-PCR. The deduced amino acid sequence is the same as the CarcininPm1 identified from EST database of P. monodonby Suchao Donpudsa, et.al., and it was classified as Type I crustin [12].The carcininPm1 gene is 327bp in length, encoding a 108-residues peptide and the first 16 residues constituted a signal peptide. The 92 residues mature peptide contained 10 cysteines in total (Figure 1). The first four cysteines were in the cysteine rich region and the rest six cysteines constituted an incomplete WAP domain. The molecular weight is 10.6kDa and the theoretical pI is 5.47.

3.2Alignment of CarcininPm1 with other Type I crustins
CarcininPm1 showed 70 % identity with crustinI-5 from Penaeus japonicas (ANA91277.1), and 33%-40% sequence alignment indicated that the ten cysteines and their flanking residues are conserved. Notably, dentity with the other four crustins in Figure 2. The CarcininPm1 and CrustinI-5 contained less cysteine residues in the mature peptide region compared to the other four crustins.

3.3Tissue distribution of CarcininPm1 in P. monodon
The tissue distribution results (Figure 3) showed that CarcininPm1 could be detected in all tissues of P. monodon tested. It was highly expressed in intestine and the expression level in gills was relatively higher compared to that in the other four tissues.

3.4Response of CarcininPm1 to Vibrio challenge
The expression of CarcininPm1 in hepatopancreas of P.monodon was up-regulated significantly when the shrimp was challenged by SIV( Figure 4A). About 12-20 times upregulation was detected at 4 and 12h post challenge. And then the expression returned to the normal level post 24 hours. This is in accordance with the transcriptome sequencing result, which showed that the expression level of CarcininPm1 increased 16 times at 3h post challenge. The expression level of CarcininPm1 in other tissues also increased significantly at 4h post infection ( Figure 4B). The most significant upregulation was in heart, stomach and gills (12-15 fold). The transcription was up-regulated about 8 times in hemocytes and about 3 times in intestine.

3.5Vector construction, recombination expression of CarcininPm1
The mature peptide of CarcininPm1 contained 92 residues was linked with SUMO protein with His-tag ( Figure 5). The His-SUMO-CarcininPm1 was linked to pCold IV vector. The molecular weight of CarcininPm1 was 10.64 kDa as shown on SDS-PAGE ( Figure 6C).

Purification of CarcininPm1
The His-SUMO-CarcininPm1 fusion protein was highly expressed in E.coli induced with IPTG at low temperature and half protein was in the supernatant ( Figure 6A). The fusion protein was purified with a Ni-NTA column and eluted by 500mM imidozol as a pure protein ( Figure 6B). The yield of purified SUMO-CarcininPm1 was around 8 mg/mL, measured by bradford reagent.And then the SUMO tag was successfully removed by cutting with a SUMO protease, shown that a ~10.64 kDa band was detected on SDS-PAGE ( Figure 6C).

Discussion
Type I crustins normally contain a cysteine-rich domain and a WAP domain. The cysteine-rich domain forms two disulfide bridges and the WAP domain contains four disulfide bridges [20]. However, the WAP domain of CarcininPm1was not intact, as it lacked two cysteins, the second and seventh cysteines in the WAP domain. And these two cysteines normally form a disulfide bridge. This indicates that there were in total three disulfide bridges in the WAP domain of CarcininPm1. The MjCru I-4 and 5 are also ten-cysteine crustins [24]. And there are some Type Icrustins contain sevencysteines in the WAP domain [21]. However, most of Type I crustins have 12 cysteines with an intact WAP domain [23].
Although there are many types of crustins discovered in P. monodon, the trancriptome sequencing data showed that most of them were not up-regulated during Vibrio challenge, but the expression level of CarcininPm1 increased about 15 times post infection, which means that it might play an important role in the innate immunity of P. monodon. The most significant upregulation was in hepatopancreas, heart, stomach and gills. As the normal expression level in gills was high, CarcininPm1 was abundantly transcribed in gills post challenge. The up-regulationcould be observed at 2h post challenge, which means that CarcininPm1 responded to Vibrio challenge in a very short time. And the upregulation lasted at least 12 hours in hepatopancreas. Besides Vibrio, Type I crustinscould also be upregulated by other pathogens. A type-I crustin from red swamp crayfish Procambarus clarkii was significantly induced by Staphylococcusaureus,Vibrio anguillarum and Aeromonas hydrophila stimulations, with the relative expression level only increased about 5 times. It could also be induced by WSSV in hemocyte from 48 h post-infection [20]. MjCru I-1 was also observed to upregulate about 15 and 10 times at 12h post challenged by S. aureusand V. anguillarum, respectively [25].
In this research, we used a novel method to express CarcininPm1, and the expression and purification were successful, which indicates that this method could be applied for overexpression of other crustins.
In summary, CarcininPm1 is a very important molecule in P. monodon during fighting against Vibrios. However, it is still unknown about how it functionedin vivo and the in vitro activities need to be further clarified.