Background

Aquaculture in the last fifty years has experienced tremendous growth globally from a production of less than a million tons in the early 1950s to over 50 million tons in the year 2008 (FAO, 2009). Aquaculture has become an economic activity of great importance worldwide due to overfishing of wild populations (Cruz et al., 2012). Asia, since 2008 produced more farmed fish in meeting rising fish food demand (FAO, 2014); this has however been faced with drastic challenges of diseases due to stretches in management practices. Environmental parameters including pH, temperature and salinity affects immune responses in crustaceans and vertebrates, suscepting them to diseases (Cheng and Chen, 2000).

Diseases confronting aquaculture range from bacterial to viral. This study characterizes the molecular and bioinformatics analysis of VirB11 protein in a threatened bacterium (Vibrio harveyi) in maricultured organisms. Vibrio harveyi is a gram-negative bacterium and a causal agent of vibriosis, a major disease in aquaculture farming industries (Sarjito et al., 2009). Studies have revealed V. harveyi as a frequently isolated Vibrio bacteria from cultured fish suffering from vibriosis in Southeast Asia (Tendencia and L.D.de la Peña, 2001; Ruangsri et al., 2004). The bacterium has been identified and reported as an opportunistic pathogen causing serious production losses in many aquaculture species of vertebrates and invertebrates, with varying mortalities from moderate to high in infected adult and juvenile populations respectively (Austin and Zhang, 2006; Nancy and Owens, 2013). This has caused significant loses to the economy of the industry worldwide.

Pathogenicity of V. harveyi relate to a number of factors including; secretion of extracellular products containing substances such as proteases, haemolysins and lipases, phospholipases, outermembrane proteins and adhesive factors to initiate virulence which successfully initiate and maintain disease (Austin and Zhang, 2006; Ningqiu et al., 2008, Cheng et al., 2010). VirB11, a type IV secretion system has been effectively studied in A. tumefaciens for transport of macromolecules such as proteins and DNA across cell envelope (Rego et al., 2010), however, VirB11 is not elucidated in Vibrio harveyi. Previous studies of VirB11 in A. tumefaciens showed that it is a member of a large family of ATPases associated with secretion systems of macromolecules (Planet PJ et al., 2000). VirB11 provides energy for substrate translocation (Atmakuri et al., 2004), and contributes to assembly of T-DNA transfer system (Sagulenko et al., 2001). VirB11-type ATPases function as chaperones to facilitate the movement of unfolded proteins and DNA substrates across the cytoplasmic membrane (Krause et al., 2000; Yeo et al., 2000). This analysis is aimed at a prospective vaccine with VirB11 against vibriosis. Molecular identification and characterization of VirB11 gene was achieved by computational predictions.

1 Materials and Methods

1.1 Strains and growth conditions

V. Harveyi strain HY99 used in this study was isolated from the liver of moribund marine fish E. coioides in Zhanjiang district of the Guangdong province of China and preserved in Guangdong provincial key laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals. Bacteria was selected on LB agar plates supplemented with 2% NaCl, cultured in LB broth supplemented with 2% NaCl, at 37°C for 8 hours, and preserved with 20% glycerol at -80°C. pMD18T (Amp⁺) plasmid was purchased from Takara Biotechnology (Dalian) Co. Ltd. DH5a competent cells were used as host for general cloning purposes.

1.2 PCR amplification of VIrB11 gene

Sense and antisense primers were designed based on VirB11 ORF for PCR amplification. PCR reaction was performed with rTaq DNA polymerase acquired from Takara LTD. PCR reaction consisted 25 ul DNA polymerase, 2 uM each forward and reverse primers, 2 ul DNA template and 19 ul sterile distilled water to make a total of 50 ml PCR mixture. Amplification was carried out at initial denaturation of 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds and extension at 72°C for 10 minutes. Amplified product was separated on 1% agarose by gel electrophoresis for 30 minutes at 110V, and visualized under Tanon 4100 Gel Logic Imaging System. Gels were sliced and purified with DNA Gel Extraction Kit from thermo scientific.

1.3 Transformation and selection of positive clones

DNA purification of target amplicon was ligated in to pMD 18T at a ratio of 1:3 vector to insert ratio and transformed in E. coli strain DH5α competent cells. The mixture was incubated on ice for 30 minutes, heat-shocked at 42°C for 45 seconds and returned to ice for 2 minutes. 800 ul LB broth was added and incubated at 37°C in a shaking incubator for 1 hour. Content was centrifuged at 12,000 r/min for 2 minute, 600 ul growth media discarded and bacteria suspended in remaining media for spread. Aliquot of transformed cells (100 ml each) was spread on LB agar plates with antibiotic (Amp⁺). Positive clones were identified by PCR amplification using universal primers M13 forward and reverse, and confirmed by sequencing at Sangon Biotechnology Co. (Shanghai, China).

1.4 Sequence analysis

Bioinformatics analysis of obtained results from sequencing was evaluated on NCBI employing nucleotide BLAST (Basic Local Alignment Tool) program (http://www.ncbi.nih.gov/BLAST) to determine identities and similarities to sequence of interest. EXPASY translate tool server (http://web.expasy.org/cgi-bin/translate/dna_aa) was used to translate nucleotide sequence into protein sequence. Isoelectric point (pI) and molecular weight (Mw) were predicted by EXPASY (http://web.expasy.org/compute_pI/Mw). Putative signal peptides were predicted by Signal Pv4.1 server (http://www.cbs.dtu.dk/services/SignalP) (Petersen et al., 2011) and transmembrane proteins by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/transmembrane proteins). Conserved domain was found by NCBI conserved domain data base (CDD) (Marchler-Bauer et al., 2017). Protein sequences from NCBI Genbank database was used to construct phylogenetic tree and multiple sequence alignment.

2 Results

2.1 DNA cloning

Sense (ATGAACACCATCGATAAAGCTGC) and antisense (GCCCTCGCTTAACAGTCGATTC) primers were designed for PCR amplification of full length VirB11 (Figure 1)

Figure 1 PCR Amplification of VirB11 aided by 2,000 marker. M denotes marker, 1&2 VirB11 genes and-negative control

2.2 Sequence characterization of VirB11 genes

2.2.1 Physiochemical properties

Resulting gene sequence was submitted to GenBank under accession number MH545700. Sequence results showed full length of VirB11 encoded a nucleotide sequence of 1,034 bp coding 338 amino acids.

Physicochemical properties of V. harveyi VirB11 protein (Table 1) was analyzed using ExPASy (http://web. expasy.org/protparam/) software. The molecular structural formula of VirB11 was C₁₆₇₈H₂₆₆₈N₄₆₀O₄₉₄S₁₆ with a total atom number of 5,316. VirB11 was shown to be a stable hydrophilic protein with a theoretical pI value of 6.31.

VirB11 protein subjected to signal peptide prediction using SignalP 4.1 Server showed no significant signal peptide cleavage sites. TMHMM Server 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) also showed VirB11 contained no transmembrane domain. PORST (https://psort.hgc.jp/) subcellular localization prediction showed VirB11 existence and abundance in the cytoplasm. Prediction by SoftBerry-Psite software (http://linux1.softberry.com/berry.phtml?topic=psite&group=programs&subgroup=proloc) revealed the amino acid sequence of VirB11 with four casein kinase II phosphorylation site, six protein kinase C phosphorylation, one N-glycosylation site, five N-myristoylation site, nine microbodies C-terminal targeting signal, one tyrosine kinase phosphorylation site, one cAMP and cGMP-dependent protein kinase phosphorylation site, one cell attachment sequence and one ATP/GTP-binding site motif A (P-loop) as shown in Figure 2.

Casein kinase II phosphorylation site (aa: 202-205, 256-259, 263-266, 308-311), protein kinase C phosphorylation (aa: 110-112, 127-129, 152-154, 181-183, 228-230, 305-307), N-glycosylation site (aa: 54-57 ), N-myristoylation site (aa: 90-95, 177-182, 178-183, 180-185, 267-272), microbodies C-terminal targeting signal (aa: 64-66, 117-119, 128-130, 169-171, 216-218, 282-284, 319-321, 332-334), tyrosine kinase phosphorylation site (aa: 250-257), cAMP and cGMP-dependent protein kinase phosphorylation site (aa: 112-115), cell attachment sequence (aa: 250-252) and ATP/GTP-binding site motif A (P-loop) (aa: 177-184), * as terminator.

2.2.2 Homology analysis with similar protein sequences of Vibrio strains

VirB11 protein sequence of V. harveyi HY99 was subjected to homology comparison with Vibrio strains including V. campbellii and V. owensii through NCBI Blast (http://blast.ncbi.nlm.Gov/Blast.cgi). The results showed VirB11 protein was relatively stable in Vibrio.

Multiple alignment of deduced amino acid sequence of VirB11 from V. harveyi with close relatives retrieved from Genbank. Amino acid sequence abbreviations are as follows: The dark and grey shaded regions represent identical and similar region respectively and the unshaded portions indicated differences (Figure 3).

2.2.3 Phylogenetic analysis

Phylogenic tree of deduced amino acid sequence showed closeness of V. harveyi with Vibrio jasicida, V. owensii and V.campbellii (Figure 4).

2.2.4 Analysis of VirB11 structure domain

The putative conserved domains detected by NCBI conserved domain search program (http://ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi) showed VirB11 conserved with a P-loop NTPase domain (residues 60 to 942) shown in Figure 5. The results of VirB11 showed P-loopNTPase superfamily, AAA superfamily and RecA-like_NTPases superfamily. SOPMA secondary structure prediction (https://npsa-prabi. ibcp.fr/cgi-bin/secpred_sopma.pl) for VirB11 is shown in Figure 6. Tertiary structure prediction by SWISS-MODEL online software (http://swissmodel.expasy.org/) for a 3-D structure model showed VirB11 gene to be a Type IV secretion system protein in Figure 7 below.

3 Discussion

Secretion systems are used by many pathogens to deliver protein effectors to eukaryotic cells during the cause of infection (Green and Mecsas, 2016). In this bioinformatic study, VirB11 full length 1,034 bp coded 388 amino acids (aa). The predicted protein was weakly acidic with an isoelectric point of 6.31 and an estimated molecular weight of 37,703.39 Da. Signal P v4.1 server predicted no remarkable signal peptides showing VirB11 was not a secreted protein, no remarkable transmembranes were predicted. Casein kinase II phosphorylation site, protein kinase C phosphorylation, N-glycosylation site, N-myristoylation site, microbodies C-terminal targeting signal, tyrosine kinase phosphorylation site, cAMP and cGMP-dependent protein kinase phosphorylation site, cell attachment sequence and ATP/GTP-binding site motif A (P-loop) are indicated in Figure 2 above. Multiple alignment and phylogenic tree analysis of deduced amino acid sequence showed closeness of V. harveyi with Vibrio jasicida, V. owensii and V.campbellii.

Conserved domain database predicted a conserved P-loop NTPase domain between residues 60 to 942. P-loop NTPases are involved in cellular functions; members of the P-loop NTPase domain are characterized by a conserved nucleotide phosphate-binding motif thus Walker A motif involved in nucleotide binding and hydrolysis (Atmakuri et al., 2004). VirB11 uses NTP hydrolysis to provide energy for secretion or movement of macromolecules across membranes (Christie and Vogel, 2000; Christie, 2001). Walker A residues are required for binding phosphate groups (Yeo et al., 2000), and disrupt the capacity of C-terminal domain of VirB11 to self-assemble (Rashkova et al., 1997). AAA ATPases are energy-dependent unfoldases whose activities are associated with substrate remodeling (Christie, 2004). Secondary structure analysis of VirB11 presented α-helix, β-turn, stretch fragment and random coil. 3-D structure model showed VirB11 gene to be a Type IV secretion system protein.

4 Conclusion

VirB11 was cloned in V. harveyi and subjected to bioinformatics analysis to understand its function and candidacy for drugs against Vibrio bacteria pathogens. Studies reveal the potential of VirB11 for vaccine development to elicit protection against Vibrosis in maricultured organisms.

Authors’ contributions

DA carried out the cloning, molecular studies, analysis and drafted the manuscript. MES participated in the cloning of the gene. UFM participated in the molecular studies and performed the bioinformatics analysis. SC and HP conceived the study, and participated in its design, EDA and WL participated in its coordination. All authors read and approved the final manuscript.

Acknowledgments

Supported by Natural Science Foundation of Guangdong Province (2017A030307033;2017A030313174); Natural Science Foundation of Guangdong Ocean University (C17379); Undergraduate Innovative and Entrepreneurial Team Project (CCTD201802); Shenzhen modern agriculture and marine biological industry support plan project (2017042631005389); Science and Technology Program of Guangdong Province (2015A020209163).

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