TUMSAT-OACIS Repository - Tokyo
University of Marine Science and Technology(東京海洋大学)
Identification and characterization of theantibacterial substances from pearl oyster
Pinctada fucata by bacterial inoculation andnuclei implantation
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言語: English
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公開日: 2017-06-26
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キーワード (En):作成者: 林, 海生
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https://oacis.repo.nii.ac.jp/records/1438

博士学位論文の要約
Summary of doctoral dissertation
専 攻Major

応用生命科学氏 名Name

論文題目
Title

Identification and characterization of the antibacterial substances from pearl oyster
Pinctada fucata by bacterial inoculation and nuclei implantation（アコヤガイ由来抗菌物質の性状に関する研究）林

海生

Pearl culturing industry started in Japan, contributing enormous value to the marine
economic development. Pearl oyster Pinctada fucata is one of the most important bivalves for theproduction of seawater pearl known as Akoya pearl. They are routinely subjected to wounding
and pathogen invasion as a result of nuclei implantation and pearl harvesting operations. Diseases
caused by infectious organisms, including viral, bacterial, fungal, protozoan, and metazoanorganisms, during the cultivation practices occur frequently, resulting in significant economic
losses. In order to control disease and enhance the yields and quality of pearl, it is necessary toinvestigate the defense factors and to clarify their function and mechanisms. Many defense
factors have been found in bivalves to prevent the colonization by microbes, including lectin(agglutinin), antimicrobial peptides (AMPs), peptidoglycan-recognition proteins, lysozymes,
antibacterial proteins, and other substances (pro-phenoloxidase, protease inhibitors, lysosomal
enzymes). They possess a broad-spectrum microbicidal activity and serve as a primary function in
innate immunity defense. However, less information of defense factors or antibacterial substancesin pearl oyster was reported. Are these traditional defense factors present in pearl oyster? Are
there any novel ones instead of them playing the roles in bacteria defense?
The research objectives of this study are (1) to identify the presence of antibacterial activity
in organs of pearl oyster and whether there are any antibacterial substances induced by bacterial
inoculation or nuclei implantation. (2) To clarify the structural properties and antibacterial
properties of these antibacterial substances. The long-term objectives of this thesis project are toclarify the roles of these antibacterial proteins in the immune defense system and the woundhealing process, and to develop novel antibiotics for disease control in aquaculture and for the
potential clinical application.
In the first part of this study, in order to investigate the antimicrobial activity and whether
there are antibacterial substances induced by immune stimulations, pearl oysters were inoculated
by injection with Vibrio parahaemolyticus into the adductor muscle and inserted a nucleus into
the gonad by a nuclei implantation surgery, respectively. Then, acid extracts (AEs) were preparedby 0.1% trifluoroacetic acid (TFA) from different tissues, and antibacterial activity was assayed.
Protein components of AEs were analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and the antibacterial properties were investigated. In the second part,to clarify the antibacterial substances from pearl oyster after inoculation, antibacterial proteinswere separated from the gill by gel filtration chromatography, following by high-performanceliquid chromatography (HPLC) using a Superdex 200 column and a TSkgel G3000 column. The
protein components were analyzed by both SDS- and native-PAGEs staining with Coomassie
Brilliant Blue. The antibacterial proteins were in-gel digestion and then characterized by
Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).
In addition, antimicrobial peptides (AMPds) were extracted by 1% TFA mixed acid medium fromdigestive gland with heating processing, and purified by solid phase extraction (SPE) and HPLC
with a TSKgel amide-80 column and a reserved-phase (RP) C18 column. The purified peptides
were characterized by MALDI-TOF MS. Finally, to clarify the antibacterial substances from pearl
oyster after nuclei implantation, antimicrobial proteins were separated from digestive gland andmantle respectively. The antibacterial protein was partially characterized by MALDI-TOF MS
and partial amino acid sequences were predicted by De novo sequencing.
As results, (1) the acid extracts from the gill by bacterial inoculation (AEg-H), mantle(AE2-m-6) and digestive gland (AE2-d-6) of pearl oyster by nuclei implantation showed stronger
antibacterial as well as the bactericidal activity than those from the control. These experimental
results support the hypothesis that the bacterial inoculation and implantation could induce
antibacterial substances in pearl oyster. It provides us a new insight to study the antibacterialsubstances related to the bacterial defense in marine bivalves by immune stimulation. (2) It isfound that various antibacterial substances, including antibacterial proteins (APg-1 of 210 kDa,
APg-2 of 30 kDa, APd250 of about 250 kDa, and APm of about 114 kDa) and antibacterial
peptides (AMPd-1 with m/z of 656.01, AMPd-2 with m/z of 658.32, and AMPd-3 with m/z of1167.42) are present in pearl oyster accumulating in the gill, mantle, and digestive gland. These
antibacterial substances are potentially induced in these tissues or released therefrom serving as adefending function against the bacterial invasion. These new finding antibacterial proteins APg-1,APg-2, and APd250 were not identified as the traditional defense factors by MALDI-TOF MSand partial amino acid sequences, suggesting that they might be novel antibacterial factors. (3) It
is first found that the new antibacterial protein APg-1 possesses LAO activity, which could
specifically catalyze the oxidation of L-Lys and L-Phe. APg-1 is specifically effective against V.
parahaemolyticus and its antibacterial and bactericidal actions were mediated by the H 2O2
producing from the oxidation of L-amino acid substrates. These results suggest that thisantibacterial factor in pearl oyster is possibly different from those in other bivalves.
In this study, various antibacterial substances including antibacterial proteins and peptides ,which are accumulated in the gill, mantle, and digestive gland, were identified and characterizedfrom the pearl oysters by the bacterial inoculation and nuclei implantation.

(1) The antibacterial substances derived by bacterial inoculation
In this study, it is found that gill has a significant function in response to bacterial
inoculation basing on the increasing antibacterial activity in this tissue after bacterial injection.
Recent findings also showed that the gill of bivalve is one of the major portals of entries formicrobes. In the normal condition, the gill is highly exposed to the microorganism in the marine
environment due to its large surface and the filter-feeder character of bivalves. Bivalves have an
open circulatory system that bathes the organs in hemolymph. Histological studies revealed that a
lot of hemocytes and effective glycoproteins are secreted from gill filaments for immune defense.
In addition, immunological studies indicated that most of the immune-related genes have a hightissue-specific expression in the gill. In this research, bacteria was inoculated by injection into theadductor muscle. It is assumed that bacteria was spread from the site of inoculation into the waterflow paths and distributed to other tissues. When V. parahaemolyticus suspension firstly flows
through the gills, which serve as the filter for expelling most of the pathogen impinging on it, asignificant response to the bacterial invasion might result in the accumulation of antibacterial
substances in this tissue. Therefore, gill has a significant function in immune response to thebacterial inoculation.
The antibacterial protein APg-1 was found in the gills, which are potentially released in thistissue serving as the defending function against the bacterial invasion. APg-1 possesses LAO
activity and bacterial cell binding ability, and its antibacterial and bactericidal activities aremediated by the H2O2 producing during the oxidation of L-amino acids. It is recognized that
antibacterial mechanism of LAO proteins relays on the H2O2 from the oxidation of L-amino acidand the depletion of amino acid required for the bacterial growth, as well as the bacterial cellbinding ability. Therefore, it is a possibility that the bactericidal action of APg-1 is involved in
the bacterial combination processing and the biochemical reaction, which produce H2O2 as amediator for defense against the bacteria invasion. To date, numbers of LAO proteins have beenreported from various vertebrate animals. In contrast, only a few examples of LAO were found ininvertebrates, such as marine mollusk. To my best knowledge, the novel antibacterial protein
APg-1 is the first example of marine bivalve-derived LAO protein, which presents in pearl oyster
and may play the important roles in the bacterial defense. These findings also suggest that theantibacterial factor and its defense mechanism in pearl oyster are possibly different from those inother bivalves.
(2) The antibacterial substances derived by implantation
The pearl oyster is prone to operational injury followed by the pathogen invasion because of
the nuclei implantation. Although the pearl oyster has been proved to possess a considerable
capacity to recover from the wound and a rapid response to the acute bacterial infection, thedefense-related factors response to the nuclei implantation remained unknown. The physiologicalresponse of the nuclei implantation was considered to be composed of a comprehensive system
including inflammatory response, reactive oxygen species (ROS) response, and the innate
immune response. Antimicrobial proteins and peptides play a major role in innate immunity byinteracting directly with bacteria and killing them. These multifunctional proteins, such asdefensins, cathelicidins, and peptide LL-37, also have wound healing activity andreceptor-mediated effects on eukaryotic cells. In this study, antibacterial proteins (APd250 andAPm) were found in the mantle and digestive gland by nuclei implantation, whether they play theroles related to the bacteria defense and the inflammatory response still remains unclear.
Based on the above results that various antibacterial substances was present in pearl oyster
and the new finding antibacterial proteins APg-1, APg-2, and APd250 were not identified as thetraditional defense factors, it is a possibility that these antibacterial factors are involved in thebacteria defense and their mechanisms are possibly different from those in other bivalves.
To my best knowledge, it is first finding that various antibacterial factors are present in pearl
oysters. Further studies should be carried out based on the current findings. The structures and theroles of these antibacterial proteins in the immune defense system and the wound healing process
should be first clarified to contribute to the development of novel management strategies fordiseases control and the long-term sustainability of pearl industry. It is anticipated that furtherstudies could provide more information in the discovery and development of new antibiotic
agents for potential use in aquaculture and in human medicine.