Abstract
Aims: Anti-lipopolysaccharide factor (ALF) is an antimicrobial peptide (AMP) and a key effector molecule of the innate immune system in crustaceans. However, little is known about the role of its indirect killing against bacteria. The possible regulatory role of this peptide (M-ALF) in kuruma prawns, Marsupenaeus japonicus, was investigated. Materials and Methods: The activities of M-ALF were investigated by antimicrobial activity in vitro and by experimental infection Vibrio penaeicida in vivo with ALF-knock down in kuruma prawn by systemically silencing M-ALF gene through the injection of gene-specific long double-stranded RNA with RNA interference. Results: Synthetic M-ALF had no direct antimicrobial activity against V. penaeicida, whereas ALF-silenced kuruma prawns had significantly higher mortality than untreated prawn after V. penaeicida infection. The data provide compelling evidence that M-ALF plays an indirect protective role against V. penaeicida infection, suggesting the idea that ALF acts as a cytokine-like regulatory molecule, as well as an effector molecule. Conclusion: M-ALF has no direct activity against V. penaeicida, but might be a key molecule in cytokine-like gene regulation in crustaceans.
In multicellular organisms, invading microorganisms are eliminated by the innate immune system, which has both effector and regulatory systems. The effector system involves direct antimicrobial functions, such as phagocytosis and the production of reactive oxygen species and antimicrobial peptides (AMPs). On the other hand, the regulatory system enhances the effector system by mediating antimicrobial functions to attract immune cells to the site of infection (1). The recognition and elimination of foreign substances is an essential function of innate immune host defenses. While invertebrates lack immunoglobulins, which are a feature of the vertebrate adaptive immune system, innate immune system activities such as phagocytosis, encapsulation, nodule formation, blood coagulation and clot formation, melanization and the release of AMPs, are key roles in invertebrate immune defense (2). Thus, we believe that invertebrate species would be most useful in which to analyze the innate immune system in vivo.
Anti-lipopolysaccharide factor (ALF) is an AMP that inhibits the lipopolysaccharide (LPS)-mediated coagulation cascade. ALF was initially isolated and characterized from hemocytes of the horseshoe crab (3). Recently, several ALFs have been isolated and characterized from various prawns, crabs, lobsters and crayfish (4-8). Most research into the role of ALFs in crustaceans has investigated in vitro antimicrobial activity and characterization of ALF gene expression following microbial challenge or infection. Therefore, little is known about functions other than direct killing as an effector molecule. The expression profile of ALFs indicates that mRNA transcript production is induced by bacteria infection or LPS administration (5, 8), suggesting that ALFs play a crucial role in crustacean innate immunity in addition to the effector function. Recently, it has been reported that Penaeus monodon recombinant ALF can reduce white spot virus propagation and prolong the survival of prawns (9). This result supported the idea that ALFs potentially have an indirect protective role in innate immunity against invasive pathogens.
The primary objective of this study was to characterize ALF antimicrobial activity in the kuruma prawn, Marsupenaeus japonicus from the standpoint of a regulatory molecule. The possible role of M-ALF was investigated by examining its antimicrobial activity in vitro and in vivo during experimental infection using RNA interference methods.
Materials and Methods
Experimental prawn. Kuruma prawns, M. japonicus, were obtained from Shimonoseki National Center for Stock Enhancement in Yamaguchi and a shrimp farm in Miyazaki, Japan. All prawn samples used in this study were screened to ensure that they were free of any bacterial or viral pathogens.
For induction examination, LPS derived from Pantoea agglomerans (Macrophi Inc, Takamatsu, Japan) and V. penaeicida (IFO 15640, Institute for Fermentation, Osaka, Japan) were diluted with marine saline. LPS (10 μg/ml) or V. penaeicida (1×104 CFU/ml) were injected into the base of the walking legs of kuruma prawns (10 μl volume). The lymphoid organs were collected at timed intervals after stimulation.
Only prawns weighing 1-2 g were used for experimental infection. The experiments were carried out in aquaria with a 60-liter capacity that contained UV-sterilized marine water at 25°C. The water was changed on alternate days.
Antimicrobial assay. The synthetic M-ALF (synM-ALF) was designed according to a previously reported method (10, 11) and antimicrobial activity was determined by growth inhibition assay. The bacteria used for the antimicrobial assay included Escherichia coli (BL21) and V. penaeicida. The bacteria were centrifuged and adjusted to 1.0×107 colony–forming units (CFU)/ml. Serial ten-fold dilutions of peptides from 10−4 to 10−7 M were made in phosphate-buffered saline (PBS). Each peptide solution was added to the bacterial solution and incubated for 1 hour at 37°C. The E. coli suspensions were then plated on LB agar and cultured for 10 hours at 37°C, and the V. penaeicida suspensions were plated on marine agar (1% polypeptone, 0.2% yeast extract, 0.05 % MgSO4 7H2O, 75% marine water, pH 7.3) and cultured for 20 hours at 25°C. After culturing, the number of CFU was counted.
Preparation of dsRNA. Double-stranded RNAs (dsRNA) were produced using the T7 RiboMAX Express System (Promega, Tokyo, Japan), as previously described (12). Briefly, oligonucleotide primers designed from M-ALF cDNA (GenBank accession number: AB210110), with a T7 promoter sequence added, were used as template for the production of M-ALF-specific dsRNAs. Likewise, a set of primers specific for the intron with the T7 promoter sequence was also made to produce intron dsRNAs (Int dsRNA) as negative control. The primer sequences used in this study were as follows (T7 sequence is in italics): 5’-GGATCCTAATACGACTCACTATAGGAGTCAACAGTGTCCATCTCG-3’ and 5’-GGATCCTAATACGACTCACTATAGGAGAGCATCTGATACCACGAC-3’ for M-ALF dsRNA; 5’-GGATCCTAATACGACTCACTATAGGATCTGTGTCTCCAGCAGTACC-3’ and 5’-GGATCCTAATACGACTCACTATAGGCCTGGAAATGAAGGACAG-3’ for Int dsRNA.
The gene-silencing experiment was performed by injecting the prawns intramuscularly with dsRNA (2.5 μg per prawn). The prawn gill and hepatopancreas were collected two days after dsRNA injection. The total RNAs of these tissues taken from three pooled samples were isolated using Trisol (Invitrogen, Tokyo, Japan) according to the manufacturer's instructions. Real-time PCR with primer combinations were performed using SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen) according to the manufacturer's instructions. The primer sequences used for real time PCR were 5’-GCAACCTATGTGAGCAGAAG-3’ and 5’-AGAGCATCTGATACCACGAC-3’ for M-ALF; 5’-GTCTTCCCCTTCAGGACGTA-3’ and 5’-GAACTTGCAGGC AATGTGAG-3’ for EF-1α used as internal control. PCR conditions were: 1 cycle of 50°C for 30 min and 95°C for 5 min, 40 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 60 s.
Experimental infection. V. penaeicida was plated on TCBS agar (Vibrio spp selective medium, Nissui, Tokyo, Japan) and grown for 48 h at 24°C. A single colony was picked and re-suspended in 5 ml of semi-marine normal medium. The bacterial suspension diluted in sterile 3.2% NaCl, corresponded to 1.25×103 CFU/ml. This dose of bacteria was previously determined to be the dose (administered by the intramuscular route) that killed between 30 and 50% of the prawns. One to 2 g specific pathogen-free prawns were stocked in aquaria with a 60-liter capacity and acclimatized for two days. After acclimatization, the prawns were treated by intramuscular injection between the third and fourth abdominal segment with 2.5 μg (10 μl volume) of dsRNA or marine saline (sterile 3.2% NaCl). After two days, the prawns were injected intramuscularly with 10 μl of marine saline containing V. penaeicida (1.25×103 CFU/ml). The survival rate was recorded daily and the water exchanged on alternative days.
Results and Discussion
We have previously identified and characterized M-ALF and found that its LPS-neutralizing and hemolytic properties are similar to other reported ALFs (5). However, unlike ALFs from other prawns, M-ALF belongs to the cluster II AU-rich element (ARE) category. It has been reported that three or more ARE motif repeats correlate with a higher turnover rate of ARE-mRNAs, and that this motif is found in cellular components largely involved in early responses to pathogen invasion, including certain hematopoietic cell-growth factors, interleukins, interferons, tumor necrosis factor-α, and some proto-oncogenes (13). For this reason, we had concluded that M-ALF functions in a regulatory capacity like a cytokine with respect to responses to infection by foreign substances (5).
In the present study, M-ALF mRNAs were reproducibly induced by LPS or V. penaeicida within 6 hours of stimulation (Figure 1), in agreement with our previous report (5). Unexpectedly, the present study showed that synthetic M-ALF had antimicrobial activity against E. coli, but not against the kuruma prawn pathogen V. penaeicida (Figure 2). Taken together, V. penaeicida induced M-ALF mRNA expression, but the M-ALF did not directly kill the bacteria. A hypothesis that M-ALF acts as a regulatory molecule during V. penaeicida infection in vivo can therefore be raised.
It has been reported that systemic injection of gene-specific dsRNA in prawns causes the silencing of mRNA and its cognate protein (14, 15). The intramuscular injection of the long ALF-specific dsRNA resulted in a very marked reduction of M-ALF mRNA in the gill and hepatopancreas (Figure 3A). There was no reduction in M-ALF mRNA abundance after the injection of marine saline or of nonspecific dsRNA for M-ALF (Int dsRNA).
When the prawns were injected with M-ALF dsRNA or control injected with Int dsRNA or marine saline two days prior to V. penaeicida challenge, the M-ALF dsRNA injection resulted in a statistically significant increase in mortality compared to the control groups (Kaplan-Meier and log-rank test, p<0.0001) (Figure 3B). These results strongly suggested that M-ALF has an important indirect role in defending kuruma prawns against V. penaeicida infection.
Until recently, there has been little knowledge about the cytokine-network system in crustaceans. Based on the assumption that invertebrates, like vertebrates, possess factors regulating responses to infection or wounding, there is presumed to be a cytokine-network system in crustaceans. More recently, it has been reported that the most obvious reason for the presence of cytokines is that these molecules are ancient signals that, regulating reactions fundamental for survival and homeostasis maintenance, have been conserved in structure and function in organisms with quite different evolutionary histories (16). The present study provided clear evidence that M-ALF can function as a cytokine-like regulatory molecule, as well as an effector molecule. The results also indicated that M-ALF might be one of the key molecules involved in cytokine-like regulation of other immune-related genes in crustaceans. These findings might be useful for a better understanding of the function of AMPs and the cytokine-network system involved in regulating homeostasis in other animals.
Acknowledgements
This work was partly supported by a Grant for Open Research and a University-Industry Joint Research Project from the Ministry of Education, Culture, Sports, Science and Technology, Japan. It was also partly supported by grants-in-aid for ‘City area program (development stage, Takamatsu area)'from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Footnotes
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Conflict of Interest Statement
The Authors declare that there are no conflicts of interest.
- Received March 24, 2011.
- Revision received May 11, 2011.
- Accepted May 16, 2011.
- Copyright © 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved