ISJ 5: xxx-yyy, 2008 ISJ 5: 162-179, 2008 ISSN 1824-307X REVIEW The antimicrobial peptides of the immune response of shrimp XF Zhao, JX Wang School of Life Sciences, Shandong University, Jinan, Shandong, China Accepted October 23, 2008 Abstract The cultivation of penaeid shrimp is a worldwide economic activity which has the potential to contribute to increasing shrimp production. However, penaeid shrimps are susceptible to bacterial and viral diseases, and may thus cause significant losses to the aquaculture industry. In view of this, it is imperative to understand the immune response of shrimp against pathogens as this could help in devising efficient strategies to control, and eventually eradicate, shrimp diseases. At present, a considerable number of research studies on the identification and characterization of antimicrobial peptides/proteins (AMPs) in penaeid shrimps. Such research activities will contribute to finding solutions to shrimp diseases. AMPs are widespread in animals and plants, involved in their innate immunity, and considered as the front liners of host defense against pathogens. In penaeid shrimps, eight kinds of AMPs have been found. These are the penaeidins, whey acidic protein (WAP) domain containing proteins [crustins and single WAP domain containing peptides (SWD)], antilipopolysaccharide factors (ALFs), lysozymes, a C-type lectin, histones, anionic hemocyanins, and peritrophins. In this study, the structures, distributions, expression profiles, phylogenetic evolution, and functions of some AMPs are discussed, focusing on the WAP-domain containing peptides and ALF in penaeid shrimp. Key words: antimicrobial peptides; innate defense effectors; innate immunity; penaeid shrimp Introduction The cultivation of penaeid shrimp is an important economic activity in the world. This industry, however, has been suffering serious problems brought by viral and bacterial diseases. One specific disease is the white spot syndrome virus (WSSV) infection which has caused a drastic decline in production and multi-national economic losses. Based on the report of the Fisheries and Aquaculture Department of Food and Agriculture Organization (2007), there is an exceeding 2.4 million tons per annum of shrimp global aquaculture production. However, up to 25 % of this production was estimated to have been lost due to diseases. Given that pathogenic diseases are one significant cause of production and economic loss in the shrimp industry, this phenomenon calls for an urgent understanding of the immune defenses of shrimp. At present, research studies are being conducted to ___________________________________________________________________________ Corresponding author: Jin-Xing Wang School of Life Sciences Shandong University Jinan, Shandong 250100, China E-mail: jxwang@sdu.edu.cn examine the innate immunity of shrimp, and such activities have been continuously contributing to the development of shrimp aquaculture. In fact, it has been found that comparable to insects, the innate defense of shrimp is triggered by the pattern recognition receptors, such as the Gram-negative binding proteins (Vargas-Albores et al., 1997; Yepiz- Plascencia et al., 1998; Jimenez-Vega et al., 2002; Roux et al., 2002; Sritunyalucksana et al., 2002; Romo-Figueroa et al., 2004; Cheng et al., 2005; Du et al., 2007; Lin et al., 2008) and the C-type lectins (Luo et al., 2006; Liu et al., 2007; Sun et al., 2008). Generally, the recognition of non-self activates a proteolytic cascade of serine proteases that amplify the signal and trigger downstream effector responses. This becomes possible through the signal transduction pathways which lead to the elimination of the invader. Moreover, the serine proteinase and its inhibitors was found in shrimp (Okumura, 2007), and the Toll-like receptors and other signal pathway molecules also reported in several shrimp (Arts et al., 2007; Yang et al., 2007; Yang et al., 2008). Anti-microbial peptides (AMPs) are a diverse group of innate immune effector molecules in multi- 162 cellular organisms. They are considered as effector molecules for immune cells that prevent or withstand microbial infection. Similar to those found in other animals, AMPs are also key factors in the innate immunity of shrimp (Bachère et al., 2004). Since AMPs play a significant role in the inherent immunity of shrimp, research studies have been actively focusing on the identification and characterization of AMPs in penaeid shrimp. In fact, eight kinds of AMPs have been found in penaeid shrimps. These are the penaeidins (Destoumieux et al., 1997, 1999, 2000; Cuthbertson et al., 2004; Muñoz et al., 2004; Kang et al., 2004, 2007), whey acidic protein (WAP) domain containing proteins [crustins and single WAP containing peptides (SWD)] (Gross et al., 2001; Amparyup et al., 2008a; Jia et al., in press), antilipopolysaccharide factors (ALFs) (Gross et al., 2001; Liu et al., 2005; Liu 2006; Somboonwiwat et al., 2005, 2008; Tharntada et al., 2008), histones (Patat et al., 2004), hemocyanin (Destoumieux-Garzon et al., 2001; Zhang et al., 2004), lysozymes (Hikima et al., 2003 Sotelo-Mundo et al., 2003; Bu et al., 2008; de la Re Vega et al., 2006; Burge et al., 2007; Xing et al., in press), a C-type lectin (Sun et al., 2008), and peritrophins (Loongyai et al., 2007). This study presents a discussion of the structures, distributions, phylogenetic evolution, expression profiles, and functions of some AMPs, particularly on the WAP-domain containing peptides and ALF from penaeid shrimp. Whey acidic protein (WAP)-domain containing peptides (WDPs) A major milk protein in most mammals, WAP, has eight cysteine residues arranged to form a tightly packed structure called a four-disulphide core (4-DSC) at the carboxyl terminus (Hennighausen and Sippel, 1982). These WAP domain-containing proteins are found to prevail among metazoans (Beg, 1995; Devinoy et al., 1988; Ali et al., 2002; Carro et al., 2004; Furutani et al., 2004). They are further found to have highly diverse biological functions, including proteinase inhibition (Ranganathan et al., 1999; Schalkwijk et al., 1999; Ota et al., 2002), antimicrobial activity (Relf et al., 1999; Hagiwara et al., 2003), and association to ovulation (Garczynski et al., 1997). In addition, WAP-domain proteins have antiviral functions, specifically against the human immunodeficiency virus (Alvarez et al., 2008). Crustins, which are anti-microbial peptides containing a WAP-domain, were first identified in the shore crab Carcinus maenas, characterized as cysteine-rich 11.5 kDa antimicrobial peptides which function against Gram-positive bacteria (Relf et al., 1999). There have been more than 50 crustins or crustin-like peptides reported to have been found from a variety of decapods, including crabs, lobsters, shrimp, and crayfish (refer to the review of Smith et al., 2008). In this study, they were termed as WAP-domain containing peptides (or WDPs) and were classified into two sub-families, namely, crustins and SWD (the justification for such is discussed below). These WDPs are apparently a large family of antimicrobial peptides ubiquitous among penaeid shrimp. In fact, the cDNAs of crustins and WDPs have been reported to be present in a variety of penaeid shrimp, including Litopenaeus vannamei, Litopenaeus setiferus (Gross et al., 2001; Bartlett et al., 2002; Vargas- Albores et al., 2004), Penaeus monodon (Chen et al., 2004; Supungul et al., 2004, 2008; Amparyup et al., 2008a, b), Marsupenaeus japonicus (Rattanachai et al., 2004), Fenneropenaeus chinensis (Zhang et al., 2007; Jia et al., in press), Farfantepenaeus paulensis, Farfantepenaeus subtilis, Farfantepenaeus brasiliensis, and Litopenaeus schmitti (Rosa et al., 2007). Accordingly, the crustins in shrimp are diverse in amino acid sequences. However, they are conserved with the C-terminus of 12 cysteine residues, thereby leading it to be termed as crustin- domain, in which a single WAP domain is contained. The SWDs have no crustin domain, and only contain a single WAP-domain (8 cysteine residues). Classification of shrimp WDPs Recently, the crustins (WPDs) in crustaceans were comprehensively reviewed by Smith et al. (2008). In the review they discussed three main types of crustins (Crustin type I, II and III) in crustaceans. Here, we focused on the crustins and SWDs in penaeid shrimp, including the new functions of the peptides. There have been several studies that have revealed the presence of crustins and SWDs in different penaeid shrimp species (refer to the above citations), in this study, most of the sequences WDPs in penaeid shrimp and in other crustaceans were collected, including some Expressed Sequence Tags (EST) from the GenBank database. A multiple alignment analysis for the amino acid sequences (Fig. 1) and the phylogenetic analysis of the proteins were performed. The neighbor-joining tree revealed that the WDPs in crustaceans could be divided into four different clusters (Fig. 2), namely, crustins I and II, carcinin and carcinin-like peptides, and the SWD. It is noteworthy to mention that in this study, our classification is considerably different from that of Smith et al. (2008). The crustin type I that they discussed in their study is similar to carcinin and carcinin-like peptide discussed in this study. Similarly, the crustin type II is equivalent to our crustins I and II, and the crustin type III is equivalent to our SWDs. The WDPs in the penaeid shrimp are divided into three classes, namely, crustin I, crustin II, and SWDs. The first class is characterized by the following: (1) a relatively conserved signal peptide, (2) an N-terminal glycine-rich domain, and (3) a C- terminal cysteine-rich domain (12-cysteine crustin domain) with the following signature: C1(X3)C2(XX)C3C4(X16)C5(X6)C6(Xn)C7(X5)C8(X5) C9(X5)C9C10(X3)C11(X5)C12. The second class of crustins have similar sequence domains as class I, but in terms of signal peptide, there are sequence differences between them. Another difference between them lies in the sequence of their crustin domain, as crustin II have the following signature: C1(X2)C2(X7)C3C4(X4)C5(X6)C6(Xn)C7(X5)C8(X5)C9 C10(X3)C11(X5)C12, specifically in the residue numbers between C4 and C5 (Fig.1A). Finally, the 163 164 Fig. 1 Alignment of amino acid sequences of Crustins (A), and SWDs (B), and the domain signature of penaeid shrimp and other Crustacea (C). Fch, Fenneropenaeus chinensis; Lse, Litopenaeus setiferus; Lva, Litopenaeus vannamei; Mja, Marsupenaeus japonicus; Pmo, Penaeus monodon; The sequences of signal peptides are presented in yellow, the identical cysteines that characterize crustin or WAP domain are in purple, and the WAP domains are shown in blue. class III WDPs are single WAP domain-containing peptides (SWD) which are characterized by the following: (1) a highly conserved signal peptide, (2) a proline- and arginine-rich motif between the signal peptide and the WAP domain, and (3) a WAP-domain (8 cysteine residues) in the C-terminus with the signature: C1(X9)C2(X6)C3(X5)C4(X5)C5C6(X3)C(X3)C (Fig.1B). In crustaceans, carcinin and carcinin-like peptides have a signal peptide and a crustin domain, without the N-terminal glycine-rich domain. Moreover, SWDs are significantly different from Crustins I and II, and Carcinins because they have no crustin-domain in their sequences. We therefore consider the idea that the four groups of WDPs in crustaceans should be divided into two sub-families, namely, the crustins (which present crustin-domain in their sequences) and the SWDs (which only have WAP-domain). As such, these two sub-families also have different functions in vitro (as discussed below). Structure comparison of shrimp WDPs with other AMPs To date, a wide variety of AMPs in metazoans have been identified. On the basis of sequence and structural features, these cationic AMPs can be grouped into three classes: (i) the linear peptides which form α-helices and do not contain cysteine residues; (ii) the cyclic peptides which contain cysteine residues; and (iii) the peptides with an over-representation in one or two residues, such as proline, glycine, arginine, and tryptophan (Bulet et al., 2004). Several antibacterial glycine-rich polypeptides have been isolated from various insect species. They are actually considered effective against Gram-negative bacteria and are inactive against Gram-positive bacteria, yeasts and mammalian cell lines (Mackintosh et al., 1998). Another example is that of short-chain proline-rich peptides, which are mostly active against Gram-negative bacteria, while the Gram-positive cells remain generally unaffected (Bulet et al., 1999). Furthermore, the cyclic peptides containing cysteine residues, like insect defensins, are active against a wide range of Gram-positive bacteria and only for a few Gram- negative bacteria, fungi and yeasts (Bulet et al., 1999). Crustins I and II are composed of an N-terminal glycine-rich domain, and a C-terminal region which contains 12 cysteine residues (crustin domain) organized in two doublets. These crustins are similar with the two classes of insect AMPs, that is glycine-rich peptides and cyclic peptides containing cysteine residues (Bulet et al., 1999). On the other hand, the SWDs are composed of a short proline- arginine-rich region and a C-terminal region containing 8 cysteine residues (the WAP domain). They are also similar in terms of the two classes of insect AMPs, particularly the cyclic peptides containing cysteine residues and the proline- and arginine-rich peptides. Similar to the penaeidins found in shrimp, the crustins and the SWD are chimera molecules of glycine- or proline-rich AMPs and cysteine-rich AMPs. Chimera-like features often reflect the multi- functional properties of a molecule, such that each domain performs different functions. For example, crustins I and II have glycine-rich and crustin domains, and therefore should have anti-Gram positive and negative bacterial activities. Supungul et al. (2008) reported that crustinPm1 (which belongs to crustin I) exhibited anti-microbial activity against only a Gram-positive bacteria, whereas the rCrus-likePm (crustin II) showed remarkable anti- microbial activity against both Gram-positive and - negative bacteria (Amparyup et al., 2008b). Likewise, the CruFc (crustin II) from F. chinensis exhibited high activity against Gram-positive bacteria but low activity was exhibited against Gram-negative bacteria and fungi (Zhang et al., 2007). The SWDs have Pro- and Arg-rich and WAP domains. They exhibit the following activities: relatively high against Gram-positive and/or negative 165 LseCrus2 AF430078 LvaCrus2 AF430072 LvaCrus AY488497 LvaCrus AY488494 LvaCrus3 AF430073 LvaCrus AY488495 LvaCrus AY488496 LvaCrus AY488493 LvaCrus AY488492 LvaCrus1 AF430071 FpaCrus EF182747 Fsu CrusEF450744 LseCrus EF182748 FbrCrus EF601055 MjaCrus1 AB121740 MjaCrus3 AB121742 MjaCrus5 AB121744 MjaCrus2 AB121741 MjaCrus4 AB121743 LseCrus1 AF430077 LseCrus3 AF430079 FchCrus AY871268 PmoCrus3 BI018073 PmoCrus1 CF415873 PmoCrus4 CF415873 PmoCrus2 BI018072 PmoCrusEE661627 PleCrus2 EF523613 CmaCar AJ237947 CmaCarI AJ821886 CmaCarII AJ821887 CmaCarIII AJ821888 CmaCar AJ427538 CmaCarIV AJ821889 FchCrus DQ097703 PmoCrus EF654658 PmoSWD EU623981 MjaSWD AU176270 LvaSWD AY465833 PmoSWD EU623980 FchSWD EF216349 PmoSWD AY464465 PmoSWD EU62397973 89 56 77 96 99 68 52 100 9099 100 89 98 84 60 71 76 99 95 56 64 5444 12 10 39 0.2 C ru st in I C ar ci ni n- lik e C ru st in II SW D Fig. 2 Phylogenetic analysis of WAP containing proteins/ peptides in penaeid shrimps by MEGA 4. Five thousand bootstraps were performed for the neighbour-joining trees to verify the reliability of the results. Cma, Carcinus maenas; Ham, Homarus americanus; Hga, Homarus gammarus; Par, Panulirus argus; Ple, Pacifastacus leniusculus. Others are the same with Fig. 1. 166 bacteria, moderate against fungi, and strong antiproteinase activity, especially against the bacterial proteinases (Amparyup et al., 2008a; Jia et al., in press). Therefore, they are bi-function peptides. From above results, we can see that the primary structures of AMP are not corresponding to their functions. It need further study for their tertiary structures. Expression profiles and functions of shrimp WDPs The spatio-temporal expression of WDPs in shrimp was not well-understood. Most of them seem to be constitutively expressed in the hemocytes. Apparently, the expression patterns was only reported during the development of the larvae of shrimp, P. monodon. High level expression of a crustin are recorded at all stages of development from the nauplii stage IV to juvenile period(Jiravanichpaisal et al., 2007). Furthermore, the expression patterns of WDPs to bacterial challenge were reported in several shrimps, but the results showed no consistent patterns of change in expression subsequent to bacterial injection. Vargas-Albores et al. (2004) found two isoforms of crustin I in L. vannamei, which showed different expression patterns after bacterial inoculation. First, crustin-P seems to be constitutively expressed, and second, the crustin-I mRNA concentration drops after 6 h. Results also revealed that there was a decrease in the transcribed expression of crustin in P. monodon subjected to bacterial challenge (Supungul et al., 2004). In hemocytes, the M. japonicus crustin-like peptide mRNA was identified, and the expression level of this peptide mRNA increased significantly 1, 3, and 7 days after peptidoglycan feeding (Rattanachai et al., 2004). The Crus Pm1 (crustin I) in P. monodon was also expressed in hemocytes, but the expression profile was not analyzed (Supungul et al., 2008). The mRNA transcript of a Crus-like Pm2 (crustin II) in P. monodon was found to be abundantly expressed in hemocytes and was significantly up-regulated after Vibrio harveyi injection (Amparyup et al., 2008b). Jiménez-Vega et al. (2004) reported that the expression of the SWD gene in L. vannamei hemocytes increased after 3 to 6 h it was inoculated with V. alginolyticus, but slowly returned to non- stimulated levels within 12 to 24 h. The Fc-SWD from Chinese shrimp is constitutively expressed and increased in hemocytes 24 h after bacterial challenge (Staphylococcus aureus and Vibrio anguillarum). The results of the Reverse Transcriptase-Polymerase Chain Reaction (RT- PCR) analysis revealed a weak expression in heart and gill and challenged stomachs in addition to hemocytes. Moreover, results showed that the signal from challenged tissues was stronger than from those unchallenged. Consequently, these results suggest that Fc-SWD is an inducible gene and is essential in responding to bacterial infection (Jia et al., in press). The tissue distribution of SWDs in P. monodon was as well analyzed through RT- PCR. Results indicated the presence of all three SWD transcripts in hemocytes. The transcript expression of SWDPm1 was down-regulated upon injection with S. aureus while no change was recorded in temrs of SWDPm2 and SWDPm3 expressions. Contrastingly, the results obtained from the WSSV injection showed that in a biphasic response, there was an up-regulation of the SWDPm1 and SWDPm2 transcripts at 6 h followed by a significant down-regulation by 24 h after infection (Amparyup et al., 2008a). The WDPs are a large family of antimicrobial effectors in shrimp immunity. In fact, more than 30 WDPs, including isoforms and ESTs, have been found in shrimp. Despite this, many of them are poorly characterized for their functions. One of the P. monodon crustins (crustin I), recombinant expressed in E. coli, exhibited an antimicrobial activity against only Gram-positive bacteria, specifically with strong inhibition against S. aureus and Streptococcus iniae (Supungul et al., 2008). Zhang et al. (2007) similarly reported that the recombinant CrusFc (crustin II) had relatively high activities against Gram-positive bacteria and low activities against Gram-negative bacteria and fungi. Moreover, another recombinant crustin II (EF654658) from P. monodon has been recently reported to have a strong activity not only against Gram-positive bacteria, but also against Gram- negative bacteria, such as Escherichia coli 363 and Vibrio harveyi (Amparyup et al., 2008b). In penaeid shrimp, there were less than 10 SWDs found, and two of them were studied for their functions in vitro. The functions of SWD molecule from Chinese shrimp were analyzed (Jia et al., in press). The recombinant Fc-SWD has manifested antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi, as well as a strong inhibitory activity against subtilisin A and protein K with an inhibition constant (Ki) of 2.14 nM and 2.27 nM, respectively; but a much lesser activity against trypsin was recorded. Amparyup et al. (2008a) also analyzed the biological functions of recombinant SWDPm. Based on the results they obtained, recombinant SWDPm exhibits activity against several Gram-positive, but not Gram- negative bacteria and is a competitive inhibitor of subtilisin A with an inhibition constant (Ki) of 1.98 nM. This phenomenon indicates the dual functions of SWDs, that is antimicrobial activity and anti- proteinase activity against pathogenic proteinase. Therefore, these SWDs might have an important role in the immunity of shrimp in vivo. So far, it is generally accepted that both activities could not co-exist in the same (unique) domain. Why does SWD show both anti-microbial and anti-proteinase activities? In fact, similar situations were found in some peptides with a single domain, which is the Avian WAP (AWAP IV) originally found in chicks (Townes et al., 2006). This has a broad-spectrum of antibacterial activities against both Gram-positive and Gram-negative bacteria. In addition to that, the Avian WAP lysate significantly inhibited the activities of the microbial serine proteinases subtilisin and proteinase K. Furthermore, Li et al. (2007) reported the occurrence of a small serine proteinase inhibitor with antimicrobial capability in a diskless-fingered odorous frog, Odorrana grahami. Based on a disulfide-bridged hendecapeptide loop of this serine 167 Crustin II SWD Double WAP domain protein WAP domain Crustin I Fig. 3 The possible divergent evolution of WPDs in crustaceans. proteinase inhibitor, a series of peptides have been synthesized. They found that seven synthetic peptides exhibited trypsin inhibitory activity, while the other five have both the trypsin inhibitory and antimicrobial activities. In terms of SWDs, the findings showed that they have high anti-proteinase activities to bacterial serine proteinases (subtilisin A and proteinase K) and antimicrobial activities. The recombinant WAP domain (Fc-WAPD) shows relatively low activities against the bacterial serine proteinases (Jia et al., in press), and manifests quite a low activity against bacteria. Comparing their sequence, it was found that Fc-SWD contain higher positively charged amino acids than the Fc-WAPD. The net charge of Fc-SWD is +4, while that of Fc-WAPD is − 2. In addition, it is generally known that most antimicrobial cationic peptides have the same unique features, that is, they are both polycationic (having a net positive charge of more than +2) and fold into amphipathic structures (having both a hydrophobic and a hydrophilic domain). As such, these characteristics enable them to interact with the negatively charged surface molecule of bacteria and to interact with and penetrate into the negatively charged cytoplasmic membranes of most bacteria (Hancock, 1997). This is therefore the reason for the high anti-microbial activity of Fc-SWD compared to that of Fc-WAPD’s low activity against bacteria (Jia et al., in press). The possible divergent evolution of the WAP domain in crustaceans The WAP domain was initially identified in the primary milk protein of rats and mice (Hennighausen and Sippel, 1982). WAP proteins have one or more WAP domains containing about 50 amino acids with eight highly conserved cysteine residues that form a four-disulphide core (4-DSC). Amino acids in the WAP domain, except for the conserved cysteine residues, are significantly diverse, and proteins with a WAP motif perform variety of functions. In fact, a large biological diversity exists between the proteins that contain one or two WAP domains, with many being identified as proteinase inhibitors or AMPs. The most studied WAP proteins, for example, are the elafin and antileukoproteinase, which are two serine-proteinase inhibitors with anti-microbial and inflammatory activity (Bouchard et al., 2006). In crustaceans, several WAP domain-containing proteins were found. In addition to the aforementioned four WAP-containing proteins, double WAP domain containing proteins were also reported in shrimp [the L. vannamei secretory leukocyte proteinase inhibitor, EF467169; the M. japonicus double WAP domain-containing protein, EU095018; the F. chinensis double WAP domain- containing protein (our unpublished data)]. Many identified genes that code for WAP proteins in human are clustered on chromosome 20q12–13.1 (Bouchard et al., 2006). The results of Southern analysis show that a large family of sequences related to the crustins is present in L. vannamei genome (Bartlett et al., 2002). This may indicate some similarities in gene locations. Based on the domain structure and functions of the WAP- containing proteins in crustaceans, we therefore propose the possible divergent evolution of WAP domain in crustaceans (Fig. 3). In this proposal, the different groups may have different functions, including antimicrobial and anti-proteinase activities among others. ALF factors ALF, a basic peptide, was initially found as a potent anticoagulant from horseshoe crabs, Limulus polyphemus and Tachypleus tridentatus, which inhibited the endotoxin mediated activation of the coagulation cascade (Tanaka et al., 1982). Thereafter, several studies demonstrated that the ALF from hemocytes of the horseshoe crab L. polyphemus have similar characteristics with that of the binding and neutralizing lipopolysaccharide (LPS). Additionally, ALF was indicated to have a strong antibacterial activity, particularly on the growth of Gram-negative bacteria (Morita et al., 1985; Aketagawa et al., 1986; Muta et al., 1987). In shrimp, the cDNA clones homologous to the horseshoe crab ALFs were initially identified in hemocytes of P. monodon and L. setiferus by means of EST analysis (Gross et al., 2001; Supungul et al., 2004). In recent years, there have been a growing number of studies on shrimp ALF available to provide pertinent information. To note, several ALFs have been isolated and characterized from hemocytes in penaeid shrimp, F. chinensis (Liu 168 http://www.ncbi.nlm.nih.gov/pubmed/9033483?ordinalpos=14&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Fig. 4 Alignment of ALFs-based amino acid sequence. All sequences of ALFs are from GenBank. Fch, Fenneropenaeus chinensis; Fpa, Farfantepenaeus paulensis; Ham, Homarus americanus; Lse, Litopenaeus setiferus; Lsc, Lst; Litopenaeus stylirostris:Litopenaeus schmitti; Lva, Litopenaeus vannamei; Mja, Marsupenaeus japonicus; Mol, Macrobrachium olfersii; Pmo, Penaeus monodon; and Ple, Pacifastacus leniusculus, Lpo, Limulus polyphemus. 169 et al., 2005; Zhou et al., 2008) M. japonicus (Nagoshi et al., 2006), P. monodon (Supungul et al., 2004; Somboonwiwat et al., 2005; Tharntada et al., 2008), L. vannamei (de la Vega et al., 2008), L. setiferus (Gross et al., 2001), F. paulensis (EF601051, EF601054), L. schmitti (DQ991357), and L. stylirostris (DQ010421). Classification of shrimp ALF Twenty-nine crustacean ALF sequences collected from GenBank were aligned using the Alignment Editor of the MEGA 4 software (Tamura et al., 2007). The results obtained by the multiple alignment (Fig. 4) revealed that all the molecules of ALFs contain two preserved cysteine residues which form a disulfide bridge. It was also found out that ALF contained a relatively conserved sequence of a positively-charged amino acid residue cluster within the disulfide loop. This structure of the β- hairpin loop in shrimp ALF suggests a conservation of the LPS binding activity. With the use of neighbor joining method of MEGA 4, a phylogenetic tree was constructed (Tamura et al., 2007). To attain and assess the reliability of the tree, bootstrapping using 5,000 replications was as well performed. The phylogenetic tree analysis categorized the various ALF proteins into three main groups. Accordingly, most ALFs from shrimp belong to cluster I, including the ALFs from L. setiferus (white gulf shrimp); then cluster II contains the L. stylirostris (blue shrimp) and Eriocheir sinensis (Chinese mitten crab). Finally, cluster III contains the ALFs from L. vannamei (Pacific white shrimp), P. monodon (black tiger shrimp) and other crustacean Pacifastacus leniusculus (signal crayfish), L. polyphemus (Atlantic horseshoe crab), and Tachypleus tridentatus (horseshoe crab) (Fig. 5). It can be observed that in L. vanamei and P. monodon, two kinds of ALFs. Expression profiles and functions in shrimp innate immunity The transcription of ALFs in some species was specified in tissues. In Chinese shrimp (F. chinensis), for example, the ALF (Cluster I) has high expression in hemocytes, gills, and intestine but exhibited low expression in ovary, hepatopancreas, and muscle (Liu et al., 2005). Similar observations had been reported in P. monodon, in which ALF (Cluster I) was constitutively expressed in hemocytes, hearts, gills, intestines, and lymphoid organs, while there was no observed transcription in the hepatopancreas (Supungul et al., 2004). Another example is that in the kuruma shrimp (M. japonicus), in which the ALF (Cluster I) was reported to be expressed at higher levels in hemocytes, lymphoid organs, hearts, intestines, and gills. The expression of ALFs, on the other hand, was found to be at lower levels in stomachs, hepatopancreas, and muscles (Nagoshi et al., 2006). In L. vannamei, ALF1 (which belongs to Cluster III ) was found to have high mRNA levels in the lymphoid organ and heart, intermediate levels in the gills, eyestalk, and hemocytes, and very low levels in the muscle and hepatopancreas (de la Vega et al., 2008). The aforementioned reports are considerably in contrast with the patterns of many other AMPs in shrimp which are carried out primarily in hemocytes (Munoz et al., 2002; Bachère et al., 2004; Kang et al., 2004). It is important to note that in shrimp, however, it is clear that ALF transcription, although it is tissue-specific, occurs in multiple organs and could thereby provide systemic protection against pathogens. The transcription of the ALF is induced upon bacterial challenge in several shrimp (Supungul et al., 2004; Liu et al., 2005; Nagoshi et al., 2006). In a study, ALF was induced upon WSSV infection or exposed to UV-inactivated WSSV (Liu et al., 2006). On the other hand, in L. vanname, the infection with pathogenic bacterium, Vibrio penaeicida or fungus, Fusarium oxysporum, did not cause any significant change in the LvALF1 mRNA levels compared to saline-injected controls. An considerable increase was recorded in the LvALF1 mRNA expression in WSSV-infected shrimp at 54-h time point (de la Vega et al., 2008). Similarly, in L. vannamei, ALF was significantly up-regulated in the infected viral hepatopancreas (Robalino et al., 2007). This up- regulation of antimicrobial proteins as a response to viral infection has also been reported in Drosophila (Zambon et al., 2005). Despite these multitude studies, the mechanism in anti-viral responses of ALF still needs to be clarified. The results reported by different authors indicate the inconsistent expression patterns of the ALFs after the shrimps have been injected with bacteria or viruses. As such, this further indicates the different functions in vivo of the ALFs. To date, only a few ALFs and their characteristics have been analyzed. In P. monodon, ALFPm3, a predominant antimicrobial peptide, was identified in both the unchallenged and V. harveyi- challenged shrimp (Supungul et al., 2004). In their study, a strong activity against multiple Gram- positive and Gram-negative bacteria and filamentous fungi (Somboonwiwat et al., 2005) has been recorded. Another study found out that a synthetic peptide, corresponding to the LPS-binding domain of Mj-ALF from M. japonicus, exhibited a LPS-neutralizing and hemolytic activities on LPS- sensitized human red blood cells (Nagoshi et al., 2006). Moreover, ALF was found to have a potential in interfering with the replication of WSSV in crayfish P. leniusculus (Liu et al., 2006). The in vivo function of ALF in protecting shrimp from bacterial, fungal, and viral infections was also studied in L vanamei through the RNA interference (RNAi) method (de la Vega et al., 2008). The injection of double-stranded RNA (dsRNA) corresponding to the LvALF1 message resulted in a significant reduction of the abundant LvALF1 mRNA levels. Following knockdown of the LvALF1, the shrimps were challenged with low pathogenic doses of pathogenic V. penaeicida, or F. oxysporum. The results showed a significant increase in the mortality among the LvALF1 low-level shrimps, specifically those with V. penaeicida and F. oxysporum infections, compared to the control shrimps. The result showed that this gene functions in protecting shrimp from both bacterial and fungal infections. In the viral challenge using WSSV, the ALF dsRNA injection caused no significant increase in mortality 170 Fig. 5 Phylogenetic analysis of ALFs from shrimp using the sequence information from Fig. 4. compared to the non-specific dsRNA controls in the L. vannamei (de la Vega et al., 2008). Other experiments that focused on ALF RNAi in freshwater P. leniusculus indicated that ALF can protect against WSSV infection, as observed in the ALF low-level through RNAi, which specifically resulted in higher rates of viral propagation (Liu et al., 2006). The differences in the two above-mentioned reports emphasize the diverse taxonomic groups of crustacea and their varying responses to infection and immunity mechanisms (de la Vega et al., 2008). Possible mechanisms of LPS binding and antimicrobial activity ALFs contain two conserved cysteine residues which form a disulfide bridge. They also contain a relatively conserved sequence of a positively charged amino acid residue cluster within the disulfide loop, which is regarded as ALF’s functional domain. Hoess et al. (1993) reported the high resolution structure of a recombinant Limulus-ALF (L-ALF). In their report, they stressed that it contains an N-terminal α-helix (that opens into a α-helix in its final turn), a simple four-stranded anti-parallel β- sheet, and two C-terminal α-helices. The three helices form a bundle that packs against the β-sheet and encloses a hydrophobic and highly-aromatic core. Their study performed a structural analysis of L-ALF through the X-ray crystallography, and the results demonstrated the β-hairpin loop with an alternating series of hydrophilic (mainly basic amino acids) in the central disulfide-bonded loop region thus obtaining an amphipathic protein molecule (Hoess et al., 1993). This amphipathic loop structure is believed to be a LPS-binding motif which can bind a single fatty acid with the phosphoglucosamine portion of lipid A (the membrane anchor of LPS). This amphipathic loop of L-ALF were compared to the ALF sequences (Cys1st to Cys2nd ) of shrimp, and the results revealed similarity between the alternating residues pattern (Fig. 4). As such, the structure of the β-hairpin loop in shrimp ALF suggests that there is a conservation of LPS binding activity. Somboonwiwat et al. (2008) also studied the binding activity of rALFPm3 and found that it could strongly bind to both Gram negative and Gram positive bacterial cells. Further analysis demonstrated that ALFPm3 could bind to both the immobilized LPS and lipoteichoic acid (LTA), with a high dissociation constant (Kd) of 1.26×10 −8 and 1.34×10−8 M, respectively. This suggested that they Lva DQ208701 Lva DQ208706 Lva DQ208702 Lva DQ208705 Lva DQ208703 Lva DQ208704 Lsc DQ991357 Fpa EF601054 Fpa EF601051 Fpa EF601052 Fpa EF601053 Fch AY859500 Fch-Alf-2 Pmo EU617325 Pmo EF523562 Pmo EF523559 Pmo EF523563 Ham EU625516 Mol EU289220 Ham EU625517 Lva ALF2 Mja AB210110 Esi DQ793214 Lse BE846661 Lst DQ010421 Ple EF523760 Lpo 1307201A Ttr P07087 Lva ALF1 Pmo EF523561 100 100 100 99 96 55 76 58 78 39 76 91 44 25 80 93 93 96 58 51 29 60 71 39 80 89 0.1 171 I II III Lva AF387661 Lva AX006138 Lva AF387662 Lva AF390145 Lva AF390139 Lva Y14926 Lva AF387663 Lva DQ206403 Lva Y14928 Lva Y14927 Fpe EU333491 Lva AF387660 Fpe EU333492 Lva AF390143 Lva DQ211700 Lva AF390142 Lva AF390140 Lva AF390144 Lva AF390141 Lst DQ010422 Lva AY351656 Lse AY039204 Lse AY039206 Lse AY039202 Lse AY039203 Fch AY260151 Fch DQ308408 Pmo AF475082 Pmo AY326471 Lsc AY956420 Lse AY039207 Lva DQ211701 Lva AF390149 Lva AF390147 Lva DQ206402 Fbr EF450745 Fsu EF450742 Fpa AY956416 FPa AY956417 Lsc AY956418 Lse AY039205 Lsc AY956419 Lst AY351655 Lva DQ211699 Lva AF390146 Lva DQ206401 Lva AX006136 Lva Y14925 Fch DQ153253 Fch DQ308407 Fch AY669323 Fch DQ154152 64 75 58 99 43 88 99 12 67 29 10 99 97 97 46 99 60 11 2 99 39 19 99 53 31 47 99 99 80 76 92 85 71 67 59 86 50 25 0.000.050.100.150.200.25 IV II V III Fig. 6 Phylogenetic analysis of penaeidins from shrimp using the sequence information from GenBank. The UPGMA tree was obtained using MEGA with complete deletions of gaps. Bootstraps (5000) were performed for the UPGMA trees to verify repeatability and reliability of results. Fch, Fenneropenaeus chinensis; Fpa, Farfantepenaeus paulensis; Fpe, Fenneropenaeus penicillatus; Fsu, Farfantepenaeus subtilis; Fbr, Farfantepenaeus brasiliensis; Lva, Litopenaeus vannamei; Lst, Litopenaeus stylirostris; Lsc, Litopenaeus schmitti; Lse, Litopenaeus setiferus, and Pmo, Penaeus monodon 172 are at least one of the target molecules for the ALFPm3 on Gram-negative and Gram-positive bacteria, respectively. Assuming that ALF binds to bacterial cells before they exterminate the cells, it is still unknown as to how such extermination is performed. ALF has both the ability to inhibit the endotoxin or LPS mediated coagulation system and to exhibit strong anti-microbial activity against the Gram- negative and Gram-positive bacteria and fungi. Thus, ALF is also one of the pivotal effectors in shrimp immunity. nes (Bu t al., 2008). -type lectin molecule in the nate immunity of Chinese shrimp. ther antimicrobial peptides/proteins Penaeidins Penaeidins, initially isolated from the Pacific white shrimp L. vannamei (Destoumieux et al., 1997), are also a large family of AMPs that have been detected in several penaeid shrimp, including L. setiferus, M. japonicus, P. monodon, F. chinensis (Bachère et al., 2004; Kang et al., 2004, 2007; Cuthbertson et al., 2008), L. stylirostris (AAY33770), L. schmitti (AAX58698; AAX58697), Fenneropenaeus penicillatus (ABY56821), F. paulensis (AAX58696), F. subtilis (ABO93321), and F. brasiliensis (ABO93324). In fact, among the AMP family, the penaeidins are considered the most well-characterized in terms of the level of gene expression and biological activities. There are four classes of penaeidins (penaeidins 2, 3, 4, and 5) that have been characterized so far (Bachère et al., 2004; Kang et al., 2007) (Fig. 6). This classification and characterization of penaeidin isoforms have been summarized in the database, PenBase (Gueguen et al., 2006) and were reviewed by several authors (Bachère et al., 2004; Cuthbertson et al., 2008). The evolution pattern of these peptides was likewise analyzed (Padhi et al., 2007). Table 1 presents the primary characteristics and functions of penaeidins. Lysozymes Lysozyme (muramidase, EC.3.2.1.17), an important antibacterial protein, catalyzes the hydrolysis of bacterial cell walls and acts as a non- specific innate immunity molecule against the invasion of bacterial pathogens (Jollés and Jollés, 1984). Initially found among eukaryotes and prokaryotes, the lysozymes are classified into six types. These are the chicken-type lysozyme (c- type), goose-type lysozyme (g-type), plant lysozyme, bacteria lysozyme, T4 phage lysozyme (phage-type), and invertebrate lysozyme (i-type) (Hikima et al., 2003). Similarly, these lysozymes had been reported in several shrimps, such as L. vannamei (Sotelo-Mundo et al., 2003; de-la-Re- Vega et al., 2006; Burge et al., 2007; Xing et al., in press), M. japonicus (Hikima et al., 2003), P. monodon, (Xing et al., in press), P. semisulcatus (Xing et al., in press) and F. chinensis (Bu et al., 2008). Furthermore, most lysozymes identified in shrimps belong to a c-type lysozyme, like those from L. vannamei (AF425673), M. japonicus (BAC57467), P. monodon (B1784440), and F. chinensis (AAV83994). In addition, there were i-type lysozymes identified in shrimp, such as lysozymes from L. vannamei (BF023863, BF024192) and L. setiferus (BF024309) (Hikima et al., 2003). In some penaeid shrimp, the lysozymes are well-characterized, and they possess lytic activity against a range of Gram-positive and Gram- negative bacterial species, including pathogenic Vibrio spp. (Hikima et al., 2003; de-la-Re-Vega et al., 2006). Hikima et al. (2003) used the RT-PCR analysis and reported that the lysozyme from M. japonicus was strongly expressed in samples from hemocytes, moderately expressed in the epidermis, and weakly expressed in the gills, midgut, and muscle. Furthermore, the post-infection expression profile of a lysozyme EST was analyzed using the macro-array, Northern blot, and real-time PCR. The results revealed that the hemocyte lysozyme expression during the V. penaeicida challenge was significantly lower at 12 h after the infection, but had returned to control levels within 24-96 h after the challenge as observed in the surviving shrimps (de Lorgeril et al., 2005). Similarly, the lysozyme mRNA from Chinese shrimp (FcLyz) was analyzed by semi- quantitative RT-PCR. The lysozyme was actually expressed in various tissues of the unchallenged shrimp and the expression of FcLyz was increased in the bacterial-challenged tissues of the hemocytes, heart, hepatopancreas, and gills as compared to the mock (saline)-challenged o e C C-type lectins have diverse functions. Aside from their agglutinating activity and opsonic effects, some C-type lectins perform antimicrobial activities. A hepatopancreas specific C-type lectin, designated Fc-hsL, has been found from the hepatopancreas of the Chinese shrimp, F. chinensis (Sun et al., 2008). This type of lectin was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated after the bacterial and viral challenge. In addition to Fc-hsL’s calcium- dependent agglutinating and binding activity to some Gram-positive and Gram-negative bacteria, it also has high anti-microbial activity against some Gram-positive and Gram-negative bacteria and fungi. Therefore, Fc-hsL may act as a pattern recognition receptor and an effector in O Hemocyanin, as the main protein component of hemolymph, is prevalent in several invertebrate animals. It typically represents up to 95 % of the total amount of protein (Sellos et al., 1997). Meanwhile, the hexamer is the predominant form in the most primitive crustacean Decapoda, such as Penaeus setiferus or P. monodon (Sellos et al., 1997). The hemocyanin, in relation to crustaceans, primarily functions as anthropods’ oxygen carrier. It is also the multi-functional proteins involved in physiological processes, such as osmoregulation, protein storage or enzymatic activities. In some reports, the fragments generated from the C- terminus of hemocyanin of L. vanamei and L. stylirostris have exhibited a high anti-fungal activity (Destoumieux-Garzon et al., 2001). Contrary to most 173 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=63109228 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=62002137 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=62002135 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=164451887 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=62002133 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=143598606 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=143598723 Table 1 Antimicrobial peptides/proteins in penaeid shrimp AMPs Expression tissues Functions References 1. Penaidins Penaeidin II Penaeidin III Penaeidin IV Penaeidin V Primarily in hemocytes and in highly vascular tissues Broad spectrum of anti-Gram- positive and anti-fungal activities and weak activity against Gram-negative strains Destoumieux et al., 1997, 1999, 2000; Bachère et al., 2004; Cuthbertson et al., 2004, 2008; Kang et al., 2004, 2007 2. WDPs Crustin I Hemocytes Anti-Gram-positive activity Supungul et al., 2008 Crustin II Hemocytes Anti Gram-positive and anti- Gram-negative activities Zhang et al., 2007; Amparyuo et al., 2008 SWD Hemocytes Anti-Gram-positive, anti- Gram-negative, anti-fungal and anti-proteinase activities Amparyup et al., 2008; Jia et al., in press 3. ALFs ALF I High expression in hemocytes, gills and intestine and low expression in ovary, hepatopancreas and muscle Anti-Gram-positive, anti- Gram-negative and anti- fungal activities; LPS- neutralizing and hemolytic activities; interference with the replication of WSSV Gross et al., 2001; Supungul et al., 2004; Liu et al., 2005; Nagoshi et al., 2006 ALF II ? ? (DQ010421, BE846661) ALF III High level in lymphoid organ and heart, intermediate levels in the gills, eyestalk and hemocytes and very low levels in the muscle and hepatopancreas Anti-Gram-positive, anti- Gram-negative and anti- fungal activities de la Vega et al., 2008; Somboonwiwat et al., 2005; Zhou et al., 2008 4. Lysozymes c-type lysozyme Hemocytes, lymphoid organ, hepatopancreas, gill, heart, midgut, muscle, epidermis, and eyestalk Anti Gram-positive and anti- Gram-negative activities Sotelo-Mundo et al., 2003; Hikima et al.,2003; de-la- Re-Vega et al., 2006; Burge et al., 2007; Xing et al., in press; Bu et al., 2008 i-type lysozyme ? ? (BF023863, BF024192) (BF024309) 5. C-type lectin HsL Hepatopancreas Anti-Gram-positive, anti- Gram-negative and anti- fungal activities Sun et al., 2008 6. Hemocyanin-derived peptides C-terminal fragment 7.9 kDa, 8.3 kDa Hepatopancreas Anti-fungal activity Destoumieux-Garzón et al., 2001 Hemocyanin subunits Hepatopancreas Anti-viral activity Zhang et al., 2004 7. Histones H2A, H2B, and H4 Hemocytes Anti-Gram-positive activity Patat et al., 2004 8. Peritrophin ovary Anti Gram-positive and anti- Gram-negative activities Loongyai et al., 2007 174 Hemocytes: Penaeidins Crustins SWD, ALF Lysozyme Histones Hepatopancreas: Hemocyanin C-type Lectin ALF Lysozyme Lymphoid organ: ALF Lysozyme Intestine: Lysozyme Penaeidins ALF Gill: Lysozyme ALF Penaeidin Epidermis: Lysozyme Penaeidin Fig. 7 Distribution of anti-microbial peptides/proteins in shrimp. AMPs with highly-cationic charges, the antifungal substances present a negative net charge at physiological pH with a pI ranging from 5.65 to 6.54. Zhang et al. (2004) also reported that shrimp hemocyanin had antiviral property as it inhibited the virus replication. Histone proteins are primarily involved in DNA packaging and regulation of DNA replication and transcription. A number of reports have shown that histone proteins or histone-derived peptides from various vertebrates possess antimicrobial activity (Hirsch, 1958; Robinette et al., 1998; Fernandes et al., 2002). Patat et al. (2004) as well reported that the hemocyte histone H2A, a mixture of histones H2B and H4 and an H1-derived fragment in L. vannamei, have anti-microbial activity against the tested bacterium. They believed that histone proteins or histone-derived peptides can be secreted to the cytoplasm from the nucleus and be localized in it along with other antimicrobial peptides. Shrimp peritrophin, a major protein in jelly layer (JL) and cortical rods (CRs) (Du et al., 2006; Loongyai et al., 2007), was inducing expression in hemocytes, heart, stomach, intestine and gill, and was constitutively expressed in ovary (Du et al., 2006). The recombinant peritropnin exhibited a chitinase activity and efficiently inhibited the growth of Vibrio harveyi and S. aureus, with minimum inhibitory concentrations of 2.4 and 15.7 μM, respectively (Loongyai et al., 2007). Conclusions To summarize, shrimps have efficiently developed and used their innate immune system in defense against pathogenic microorganisms. To date, eight kinds of AMPs have been identified in penaeid shrimp, namely, penaeidins, WDPs (crustins and SWDs), ALFs, lysozymes, anionic hemocyanin, histones, and a C-type lectin. These kinds of AMPs have different distribution and expression profiles and functions in shrimp (Table 1 and Fig. 7). Furthermore, there are several subclasses or isoformes for each kind of AMPs in one species. An example is the three subgroups of WDPs (Crustin I, II, and SWD) in Chinese shrimp. This only suggests that they had different functions in vivo in the shrimp. Even though large groups of AMPs were found in penaeid shrimp, there is still a limited number of studies conducted about the AMPs’ antibacterial properties and their other functions. In view of this, there is a need to have more studies that will delve on the functions and the anti-bacterial properties of AMPs, especially with respect to the diverse bioactivities of the natural proteins in vivo. 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