Knowledge on how genes are turned on/off during infection and immunity is lacking. Here, we report the coregulation of diverse clusters of functionally related immune response genes in a horseshoe crab, Carcinoscorpius rotundicauda. Expressed sequence tag (EST) clusters for frontline immune defense, cell signalling, apoptosis and stress response genes were expressed or repressed spatio-temporally during the acute phase of Pseudomonas infection. An infection time course monitored by virtual Northern evaluation indicates upregulation of genes in blood cells (amebocytes) at 3-h postinfection, whereas most of the hepatopancreas genes remained downregulated over 72 h of infection. Thus, the two tissues orchestrate a coordinated and timely response to infection. The hepatopancreas probably immunomodulates the expression of other genes and serves as a reservoir for later response, if/when chronic infection ensues. On the other hand, being the first to encounter pathogens, we reasoned that amebocytes would respond acutely to infection. Besides acute transactivation of the immune genes, the amebocytes maintained morphological integrity, indicating their ability to synthesise and store/secrete the immune proteins and effectors to sustain the frontline innate immune defense, while simultaneously elicit complement-mediated phagocytosis of the invading pathogen. Our results show that the immune response against Pseudomonas infection is spatially and temporally coordinated.
During an infection, the innate immune response in multicellular host organisms is initiated by pathogen-recognition receptors (PRRs), which are germline-encoded proteins capable of recognising conserved pathogen-associated molecular patterns (PAMPs) unique to pathogens.1, 2 PRRs are strategically expressed in those cells first exposed to pathogens. Activation of these PRRs leads to the expression of immunomodulated genes that are vital in protecting the host against pathogens. Studies on the genetics of Manduca, Anopheles and Drosophila have generated a great deal of information on innate immune defense, providing some insights on the expression of antimicrobial peptides3, 4 and the functions of endopeptidases, serpins and other immune molecules.5 Furthermore, data mining of the Drosophila and Anopheles genomes and DNA microarray analyses6, 7, 8 have revealed that the Toll and Imd signalling pathways are responsive to microbial infection and induction of the innate immune responses.
The horseshoe crab represents an ancient family of arthropods with >500 million years of evolutionary history. There are four extant species of horseshoe crabs: Limulus polyphemus (in the Eastern seaboard of USA); Tachypleus tridentatus (in China and Japan) and Tachypleus gigas and Carcinoscorpius rotundicauda in South Asia. The C. rotundicauda is the mud-dwelling species, whose habitat contains very high counts of Gram-negative bacteria. Its ability to thrive under such highly infective conditions attests to its possession of a superior frontline innate immune system.9, 10, 11, 12, 13, 14, 15, 16 Like other invertebrates, the horseshoe crab lacks adaptive immunity and relies solely on a very potent innate immune system to combat invading microbes. In the past two decades, the components of the innate immune system of the horseshoe crab have been extensively investigated at the level of individual proteins. This has led to the elucidation of many unique frontline defense molecules such as clotting factors and serine proteases,9, 10, 11, 12 lectins,14, 17 protease inhibitors,18 antimicrobial peptides13, 15 and other humoral factors.19. Most of these molecules are identified from the amebocytes, the major blood cell type. In contrast, only a few immune-related molecules have been identified from the hepatopancreas, which is the immune-responsive functional equivalent to the insect fat bodies and the vertebrate liver. Despite the discovery of these unique molecules in the horseshoe crab, knowledge on their spatial and temporal gene expression profiles and the presence of other innate immune-related molecules responsive to microbial infection remains elusive. Thus, mapping the ensemble of functionally related immune genes, which may be up-/downregulated during bacterial infection represents the first step towards elucidating the pathways contributing to innate immunity.
The horseshoe crab offers significant advantages over its smaller arthropod counterparts since it possesses large amounts of blood and sizeable tissues, which makes the system readily amenable to physiological and molecular manipulations. Furthermore, as a ‘living fossil’, it is expected to harbour an immensely powerful repertoire of innate immune molecules, which act in frontline defense. In view of these advantages, we sought to examine the functional display of expressed sequence tags (ESTs) in the horseshoe crab, in response to infection by Pseudomonas aeruginosa. As a ubiquitous and potent Gram-negative pathogen that has acquired multiple antibiotic resistance, the P. aeruginosa is a major cause of nosocomial infection and is the epitome of opportunistic human pathogens. Therefore, its elimination remains a critical challenge to the medical industry.15 We have recently shown that the horseshoe crab effectively clears a systemic infection by P. aeruginosa (106 cfu/ml) within 6 h, whereas this infection dosage would have been lethal to mice.14
The development of high-throughput methods of gene identification by EST analysis has become a commonly used approach to identify genes involved in specific biological functions. This is especially so in organisms where genome data is unavailable or limited,20 and has accelerated the pace at which new immune functions can be discovered. A growing number of EST databases from Bombyx mori,21 Galleria mellonella22 and other organisms testify to the importance of this technique. Here we report a suppression subtractive cDNA hybridisation approach, to isolate and identify differentially expressed genes from the horseshoe crab, C. rotundicauda, in response to P. aeruginosa infection. In our study, four subtractive cDNA libraries were constructed from the two major immune responsive tissues: hepatopancreas and amebocytes, which are expected to reflect genes that are expressed or repressed during infection. The kinetic profile of transcription of selected genes responsive to infection was studied. By comparing the sequences of cDNA clones from subtractive libraries with known gene sequences deposited in the GenBank, this study has revealed the diversity of genes (frontline immunity, cell signalling, apoptosis and stress responses), which were invoked by infection. We also show that as the major frontline defense tissue which displays acute increases in gene transcription, the blood cells, amebocytes, were intact and functional during Pseudomonas infection, and that phagocytosis, which was recently demonstrated in this species,16 was essentially occurring to clear the microbial pathogens and apoptotic cells.
Results and discussion
To understand differential immune gene expression in amebocytes and hepatopancreas of the horseshoe crab during P. aeruginosa infection, we constructed suppression subtractive cDNA libraries from the RNAs pooled from 3 and 6 h postinfection (hpi). These two time points were chosen based on studies on Drosophila, which showed the occurrence of acute phase expression of antimicrobial genes at 3–6 h postmicrobial infection.4 In our study, we performed suppression subtractive hybridisation to create EST libraries on amebocytes and hepatopancreas samples of infected and saline-treated horseshoe crabs. These samples were used as both tester and driver. To define, the tester refers to the cDNA population containing sequences of interest whereas the driver refers to the cDNA population used to remove genes present in both conditions. Four cDNA libraries were established: amebocyte forward, amebocyte reverse, hepatopancreas forward and hepatopancreas reverse. The ‘forward’ libraries were constructed using cDNAs from tissues of Pseudomonas-infected horseshoe crabs as the tester and cDNAs from tissues of mock-infected horseshoe crabs as the driver. The ‘reverse’ libraries were constructed vice versa. Additionally, due to the rapid change of immune-related genes over the time course of infection, we carried out virtual Northern analyses of gene expression on 3 + 6 hpi pooled cDNAs as well as cDNAs from seven individual time intervals over 72 h of Pseudomonas-infection. Representative ESTs from each library were used as probes for the Northern analyses.
Characterisation of the ESTs
The EST clones from the four subtractive cDNA libraries, representing gene expression patterns from the amebocytes (Ame) and hepatopancreas (Hp) were sequenced. The ESTs from forward, F (upregulated genes) and reverse, R (repressed genes) libraries of amebocytes and hepatopancreas are referred to as AmeF/AmeR and HpF/HpR, respectively. Detailed information on each library is summarised in Table 1. From the four libraries, a total of 776 randomly selected clones were sequenced, resulting in the characterisation of 447 ESTs. Of these ESTs, 268 (60%) showed significant BLASTx matches (E-value ⩽10− 3) to known identified genes. In the forward libraries, 549 clones were sequenced, yielding 333 ESTs (60.7%) with E-values ⩽10− 3. Of these 333 ESTs, 94 (28.2%) were from the amebocyte and 104 (31.2%) were from the hepatopancreas forward libraries that matched previously known sequences in the database. Of 227 clones sequenced from the reverse libraries, 114 ESTs (50%) with E-values ⩽10− 3 were identified by sequence homology. Of these, 42 ESTs (36.8%) from the amebocyte and 28 ESTs (24.6%) from the hepatopancreas reverse libraries showed significant similarities to known sequences in the database.
Based on the general Expressed Gene Anatomy Database (EGAD),23 the ESTs were classified into nine broad functional categories (Figure 1). The ESTs putatively associated with the immune-related gene clusters (frontline immunity, cell signalling, apoptosis and stress responses), together with their respective NCBI dbEST database accession numbers are shown in Tables 2a, b and 3a, b. The remaining functional clusters of ESTs from the amebocytes and hepatopancreas are catalogued in Supplementary Tables 1a,b and 2a,b. In the scope of this study, only four functional clusters of ESTs (1) frontline immunity, (2) cell signalling, (3) apoptosis and (4) stress response will be discussed. Generally, the forward libraries of both the amebocytes and hepatopancreas contained higher numbers of frontline immunity genes than their corresponding reverse libraries. In contrast, both the amebocyte and hepatopancreas reverse libraries contained more apoptosis-related genes than the forward libraries. The cell signalling and stress response genes were more highly represented in the amebocyte and hepatopancreas reverse libraries, respectively (Figure 1).
In total, 60 immune response ESTs from the forward and reverse libraries showed significant matches to known genes in the database (Table 1). A total of 27 ESTs from the amebocyte forward library encode coagulogen (AmeF230). Such abundant representation strongly indicates that the coagulogen gene (a functional homologue of spaetzle in Drosophila) is highly expressed during infection. The role of coagulogen in host defense and hemostasis in the horseshoe crab is well-documented.24 This is strongly linked to our observation of the infection-induced upregulation in both the amebocytes and hepatopancreas of Factor C (AmeF287 and HpF301), a key regulator of lipopolysaccharide (LPS) mediated immune response, which is known to trigger the blood coagulation cascade culminating in the conversion of coagulogen to coagulin. The coagulin gel clot is stabilised by transglutaminase (HpF170 and AmeF98), which is secreted upon LPS-stimulation. Consequently, the insoluble gel clot traps the invading microbes. Transglutaminase catalyses the crosslinking reactions between coagulin and amebocyte cell surface antigens, proxins.25 Interestingly, apoptosis induces the expression of transglutaminase26 that promotes the formation of insoluble clots to minimise cytoplasmic leakages of apoptotic cells.27 Notably, the transglutaminase substrate in the amebocytes (AmeF238) showed early transcriptional activation (Figures 2 and 3) indicating that it probably underwent crosslinkage, being catalysed by transglutaminase. The multimeric forms of transglutaminase substrate have been postulated to form a physical barrier against the spread of the invading pathogen and to possess bactericidal activity.28
Hemocyanin (HMC) is an abundant oligomeric protein constituting ∼90% of the total plasma protein of the horseshoe crab. The HMC EST in the amebocyte (AmeF45) was transcribed within 3 hpi, suppressed between 6 and 12 hpi and then escalated over 24–72 h of infection (Figure 4a). This is consistent with our earlier observation14 that the horseshoe crab effectively clears the Pseudomonas infection after 6 h, hence, the subsequent recovery of HMC expression. As an abundant protein, it is conceivable that the prolonged and relatively high level of transcription of the HMC gene during 24–72 hpi would help to replenish and maintain the high level of the HMC protein. Besides being an important respiratory protein, the HMC plays a crucial role in frontline innate immune response during which it is proteolytically processed by serine proteases to activate its prophenoloxidase (PPO) activity.29 Our recent study has shown that in the presence of Gram-negative bacteria or LPS, the HMC/PPO is activated into phenoloxidase, PO (Jiang et al, unpublished). Indeed, we have also identified a prophenoloxidase activating enzyme, PPOA (HpF149), which was strongly upregulated in the hepatopancreas from 3 hpi to reach a peak at 6 hpi (Figure 4b). The resulting PPO activity of HMC catalyses the oxidation of phenols to quinones, thereby inducing melanisation to oxidatively kill the invading microbe.29 Furthermore, HMC also releases a C-terminal peptide that has antimicrobial activity.30
Proteases are essential for a variety of immune processes, including hemostasis and clot resolution,31 complement activation,32 inflammation33 and tissue remodelling.34 In particular, the role of serine proteases in immunity has been thoroughly studied.11 Interestingly, in this work, we have identified several serine and cysteine proteases, specifically from the forward libraries of both tissues: a 26/29 kDa proteinase (HpF189), Factor C (AmeF287, HpF301), carboxypeptidase B (HpF66), cathepsins B, C and L (AmeF295, HpF193 and HpF276, respectively) and legumain precursor (AmeF202). Factor C is a well-characterised serine protease that plays a pivotal role in LPS-recognition during Gram-negative bacterial infection.9, 10, 11, 13, 35, 36
Ironically, at the other extreme, proteases that act beyond their narrowly constituted physiological function can impose serious damage to host tissues. Thus, hosts have evolved regulatory processes to inactivate proteases associated with the pathological processes and the infective microbes. Most important of these regulatory molecules are protease inhibitors.18, 36, 37 From the forward libraries, we have identified a well-known protease inhibitor, α2-macroglobulin (AmeF244, HpF286). Although earlier studies on the L. polyphemus α2-macroglobulin have shown its expression only in the amebocytes,38 our finding of the α2-macroglobulin ESTs in both the amebocyte and hepatopancreas libraries indicates a more widespread spatial expression of this gene during Pseudomonas infection. To confirm this observation, the tissue distribution of expression of α2-macroglobulin was examined by RT-PCR, which showed that it is indeed expressed in amebocytes, hepatopancreas, heart, intestine and stomach (Supplementary Figure 1).
Our finding of other protease inhibitor ESTs such as Kazal-proteinase inhibitor, HpF219 (Figure 2a), intracellular coagulation inhibitor precursor (AmeF210), intracellular coagulation inhibitor type 2 (AmeF267), Kunitz-like protease inhibitor precursors (AmeF153, HpF232) (Figures 3 and 4), cysteine-rich protease inhibitor (AmeF89) and serine protease inhibitor (HpF282) further reiterates the importance of protease inhibitors in regulating infection-induced proteolytic activation of serine- and cysteine-proteases in innate immune defense. Some of these inhibitors19, 39, 40, 41 have not been previously reported in the horseshoe crab. Another protease inhibitor, a tissue factor pathway inhibitor, TFPI (AmeR235), was isolated from the amebocyte reverse library (Figure 2b). TFPI, which is composed of three Kunitz-type protease inhibitor domains, represents the only physiological inhibitor of hemostasis.42 The TFPI identified in this study differs from the members of the serpin superfamily (limulus intracellular coagulation inhibitor, LICI-1,2,3) of coagulogen inhibitors43 and is also distinctive from the LICI (AmeF210) found in this work (Table 2a). Hence, the C. rotundicauda TFPI-like protease inhibitor represents a novel type of anticoagulant. Therefore, downregulation of TFPI (AmeR235) (Figure 2b) would increase tissue-localised hemostasis, indicating that it plays an important role in the early phase of infection, to entrap and confine the invading pathogen to the infected tissue.
Besides the above-mentioned factors such as coagulogen, proteases and protease inhibitors that regulate infection-induced hemostasis, the histones H2A (AmeF288), H2B (AmeF248) and H3 (AmeF82) were present in the amebocyte forward library. This concurs with recent evidence suggesting that histones possess antimicrobial activities44 and endotoxin binding capabilities.45
PAFAH (AmeR218), a platelet-activating factor acetylhydrolase, plays a role in hydrolysing a platelet-activating factor (PAF),46 which mediates inflammatory activity.47 Thus, the Pseudomonas infection-mediated suppression of PAFAH was probably required for PAF to accumulate to an effective level for the production of antimicrobial proinflammatory cytokines. Therefore, the downregulation of PAFAH in the acute phase of infection (Figure 2b) suggests a mechanism that the host might employ to enable PAF-mediated control of pathologic inflammation.
Cyclophilin (CYP) comprises a ubiquitous family of proteins,48 which plays a role in immunity by binding to the immunosuppressant drug cyclosporin A, resulting in the blockage of cytokine gene transcription in activated T cells.49 Two CYP ESTs (HpR14, HpR151) were isolated from the hepatopancreas reverse library. Downregulation of the CYP, for example, HpR14 (Figures 2b and 3b) during the acute phase of Pseudomonas infection indicates that CYP participates in the modulation of other genes involved in the host innate immune response.
Previously, the complement system was thought to exist only in deuterostomes. Here, we have identified ESTs encoding complement components C3 (HpF160), C2/Factor B (HpF192) and C4 (HpF226) from the hepatopancreas forward library. Invertebrate C3 isolated from the sea urchin is upregulated during LPS injection.50 Some of the known roles for C3 are opsonisation of the invading microbes,51 formation of membrane attack complex32 and clearance of apoptotic cells.52 Our EST data suggest the involvement of complement system in frontline immune defense against Pseudomonas infection. Recently, we have isolated and extensively characterised the cDNAs of complement factors and their corresponding proteins, C3 and C2/Bf, from the horseshoe crab, C. rotundicauda16 (an ancient protostome). It is therefore conceivable that a formidable and complex host–pathogen interaction mechanism already exists >500 million years and that the vertebrate complement system evolved from that of the early invertebrates.
During microbial infection, the recognition of PAMPs by host cell Toll/Toll-like receptors and IMD triggers complex signalling cascades to the nucleus, leading to the expression of effector molecules. NFκB and MAPK pathways are major cytoplasmic regulators of innate immunity.53, 54 Recent evidence indicates that G-protein signalling through the inositol-1,4,5-trisphosphate pathway potentially plays a role in the amebocyte degranulation upon LPS stimulation.55 In addition, we observed that Factor C (AmeF287 and HpF301) was upregulated in amebocytes and hepatopancreas. Factor C released from degranulated amebocytes9, 24 as well as extracellular Factor C secreted by the hepatopancreas36 probably activates the G protein pathway.56 On the other hand, downregulation of AmeR13, a regulator of G-protein signalling 7 (RGS7), which promotes GTPase activity, indicates prolonged activity of G proteins for the signalling action of Factor C.36 However, in view of the inositol phosphate pathway, the appearance of a variant surface glycoprotein phospholipase C, VSG PlpC (HpF125) (Figure 2a) is intriguing as this enzyme cleaves a glycosylphosphatidylinositol anchor of the VSG, a Trypanosome coat protein.57 Therefore, the involvement of VSG PlpC as a potential cell signalling molecule in the horseshoe crab during Pseudomonas infection remains uncertain.
The calcineurin (CaN) homologue in the vertebrates is a calcium- and calmodulin-dependent protein serine/threonine phosphatase, which plays an essential role in T-cell receptor-mediated signal transduction leading to the transcriptional activation of cytokines.58 Thus, upregulation of both of the calcineurin-like phosphoesterase/metallo-phosphoesterase (HpF134) and CaN substrate (HpF6) (Figures 3 and 4 and Table 3b) in response to Pseudomonas infection is indicative of a parallel existence of T-cell receptor-like mediation of signal transduction in this ancient species and that these genes possibly interact during Pseudomonas infection.
The horseshoe crab coagulogen and Drosophila spaetzle are proposed to be structurally and functionally equivalent,59 with the latter functioning in a signal transduction cascade upstream of Drosophila Toll.36, 60 Therefore, coagulogen, which appeared in 27 ESTs, was most highly expressed and probably serves multiple roles as a direct pathogen recognition protein in frontline immune defense, acting as a coagulant to immobilise invading pathogens, as well as priming cell signalling events during infection.
Although cell signalling is expected to regulate many crucial defense mechanisms, a sizeable proportion (23%) of the cell signalling ESTs was present in the amebocyte reverse library at 3–6 hpi (Figure 1c). It is conceivable that the cell signalling genes were repressed during this acute phase, and may only be de-repressed at a later phase of infection. Supporting this hypothesis was the downregulation of several cell signalling ESTs as they appeared in the reverse libraries of both the hepatopancreas and amebocytes (Tables 2 and 3). Firstly, MAPK (AmeR65), a major arm in intracellular signalling, was suppressed. Secondly, PAFAH (AmeR218), which hydrolyses and attenuates the bioactivity of the PAF, a mediator of inflammation,46 was repressed (Figure 2b), indicating the suppression of cell signalling47 and the accumulation of PAF, which mediates inflammatory response. Thirdly, SARM (AmeR209), which codes for sterile α and Toll interleukin-1 receptor (TIR) motif-containing protein 1, and TRAF4 (HpR202), a signal-transducing adaptor protein, were also downregulated (Figures 2b and 4d). Our discovery of TRAF4 and SARM provides a strong indication on the existence of a complex network of Toll-like receptor signalling pathway in this species. Nevertheless, the presence of TRAF4 (Figures 2b, 3b and 4d) and SARM (Table 3a) in the reverse libraries consistently suggests substantial suppression of cell signalling genes at this acute phase of infection.
It is plausible that, with some exceptions, most of the cell signalling genes may not need to be upregulated until after the pre-existing high levels of extracellular frontline innate immune proteins are exhausted from the initial antimicrobial combat during the early phase of infection. Equally likely are the post-translational modifications like phosphorylation/dephosphorylation that could change the signalling protein profile rather than the expression/repression of signalling genes.
Pathogen-mediated apoptosis may be viewed as a means to disable the host from properly mounting an immune defense. On the other hand, the host can also bring about apoptosis to minimise the spread of infection from infected cells to healthy cells, since apoptosis of the infected host cell permits other cells to ingest the apoptotic bodies containing internalised pathogens.61 Therefore, whether the occurrence of apoptosis during Pseudomonas infection of the horseshoe crab is pathogen- or host-mediated is of particular interest. Recent studies have reported that apoptosis induced by many stimuli such as TNF-α depends on the integrity of the mitochondrial respiratory chain,62 and that its role in the generation of reactive oxygen species (ROS) can lead to apoptosis.63
In this study, 13 ESTs related to apoptosis genes were identified, mostly from the reverse libraries. Of these, cytochrome c oxidases (COX) were found in both tissues. COX is a terminal enzyme of the mitochondrial respiratory chain,64 which plays dual functions in energy generation and apoptosis. COX I, II and III proteins have been previously isolated from the Atlantic horseshoe crab, L. polyphemus.65 As abrogation of host cell apoptosis is often beneficial for the pathogen,66 it can be postulated that P. aeruginosa has mediated the repression of the COX I, II and III genes (Figures 2b, 3 and 4c and d), a mechanism which the pathogen has employed for its survival in the host. At the same time, the invading Pseudomonas has established various effects like latent infection61 and inhibition of the immune cell oxidative burst, although the latter is important in the host innate immune defense against pathogens.67 It appears that the host has counterbalanced the pathogen-mediated repression of COX genes by upregulation of another mitochondrial EST, the amine oxidase (HpF292), which generates H2O2, causing oxidative damage68 and apoptosis.69
Another gene, sensitive to apoptosis gene, SAG, which is a member of the zinc RING finger family of proteins, is evolutionarily conserved in diverse organisms and has been shown to function as an antioxidant to protect cells from metal ion- or ROS-induced apoptosis.70 Hence, SAG plays an antiapoptotic function. In the 3 + 6 hpi pooled cDNA library, SAG (AmeR204) appeared downregulated (Figure 2b and 3a). Virtual Northern analysis of separate time points, however, showed the two major isoforms of SAG to be strongly represented at 3 hpi and downregulated at 6 hpi (Figures 3a and 4c). Taken together, this observation indicates that, as an antiapoptotic factor, the SAG isoforms probably played opposing roles at different time points of Pseudomonas infection, preventing host cell apoptosis at 3 hpi and causing apoptosis of the acutely infected cells at 6 hpi.
Thus, in view of the expression of amine oxidase and SAG 1 and 2 on the one hand and the repression of COX enzymes on the other, there appears to be a fine balance of forces between the up- and/or downregulation of apoptosis and immune cell oxidative damage during host–pathogen interaction, which is beneficial for the survival of either the host or the pathogen as the modulation of immune response progresses.
As many as 33 ESTs (Table 1) were found to show high homology to known stress-related genes. During an infection, the host generates ROS as a cytotoxic process.71 However, the host employs detoxification mechanisms to protect itself against excessive ROS. Four upregulated ESTs show high homology to genes involved in detoxification (Tables 2 and 3): (i) copper chaperone for superoxide dismutase, Cu-SOD (HpF51, see Figure 2a); (ii) catalase (AmeF105); (iii) amine sulfotransferase (HpF41) and (iv) glutathione S-transferases (GSTs): HpF11 (Figures 2a, 3b and 4b), HpF20, AmeF81, AmeF182. SOD converts free radicals into H2O2, which is further detoxified into nontoxic components by catalase. Amine sulfotransferase and the family of GST enzymes facilitate the coupling of glutathione to endo- and xeno-biotics, which bear electrophilic functional groups, hence detoxifying them.72 Thus, in response to P. aeruginosa invasion, GSTs were expressed to cope with oxidative stress due to pathogen-induced release of free radicals. Other than GST, another glutathione-dependent ROS-scavenger, selenoprotein W (AmeF49), was isolated from the amebocyte forward library. Selenoproteins are known to protect cells from oxidative stress.73 Contrary to expectation, thioredoxin, TRX (HpR168) was found in the hepatopancreas reverse library (Figure 2). TRX is known to play a role in homeostasis, detoxification and regulation of cytokine expression through modulation of NFκB upon induction by LPS.74 Its promoter contains regulatory elements that are responsible for oxidative stress.75 It is conceivable that the downregulation of TRX implies a tight control of this gene during Pseudomonas infection. It is also plausible that pre-existing TRX protein was sufficient to maintain the redox potential of the host cell. Perhaps, other stress-response genes like Cu-SOD, GSTs and amine sulfotransferase played a major role in protecting cells from oxidative stress during this acute phase of infection.
Another interesting cluster of stress-response ESTs was heat-shock proteins. Hsp 90α (AmeF123), Hsp 70s (AmeF112, AmeF253, HpF102) and Hsp 40 (HpF24) were identified in the forward libraries. The Hsps 90, 70 and 40 are molecular chaperones, which assist protein folding, solubilise aggregated proteins and degrade damaged proteins.76 Stress-related oxidative damage increases chaperone levels. Thus, a high representation of Hsp ESTs (Tables 2 and 3) in the immune-responsive tissues of the horseshoe crab clearly indicates infection-induced Hsp response to stress.
Kinetic profile of transcriptional response to Pseudomonas infection
Although virtual Northern analysis of 3 + 6 hpi pooled cDNAs yielded some general clues to the expression of genes over the acute phase of infection (Figure 2), it was insufficient to reveal the kinetic profile of gene expression, since the true levels of transcripts at 3 and 6 hpi may be compromised through pooling of reciprocal representations of the mRNAs at the two time points of infection. Thus, based on the premise that the immune-related genes undergo rapid change over the time course of infection, we carried out virtual Northern analyses of gene expression in amebocyte and hepatopancreas from naïve and Pseudomonas-challenged animals over 72 h of infection, using representative ESTs from each forward and reverse library (Figures 3 and 4). In the forward libraries, changes in gene expression upon bacterial challenge were apparent, while in the reverse libraries, there was repression of transcription of various genes. A consistent increase in the amebocyte gene expression was detected at 3 hpi in the forward libraries (Figure 4a and b). It is also notable that while some genes from the same functional group in the same tissue were induced, others were repressed. Recently, Ng et al14 demonstrated that the horseshoe crab rapidly clears a systemic infection by 106 cfu/ml P. aeruginosa within 6 hpi. This suggests that the pre-existing innate immune defense proteins in the plasma acted immediately in frontline antimicrobial combat, while transcriptional upregulation of the corresponding genes was imminently mounted within 3 h of infection, such that nascent immune response proteins may be translated to replenish the diminishing store of pre-existing proteins and effectors in order to sustain the immune defense.
Interestingly, the hepatopancreas did not display a distinctive profile of transcriptional activation. Of the ESTs examined, only the genes encoding GST and PPOA were upregulated (Figure 4b), while the remaining genes were either only slightly activated later or were unresponsive and showed general suppression of transcription (Figure 4d) to this regime of Pseudomonas challenge over the 72 h period.
Ameboyctes are intact and functional during Pseudomonas infection
Phagocytosis is essential in host defense against microbial pathogens and in the clearance of apoptotic cells. Both microbial and apoptotic cells are delivered on a common route for degradation. We have recently demonstrated the role of amebocytes in the phagocytosis of pathogens.16 The importance of the amebocytes to such frontline innate immune defense was exemplified by its ability to remain largely intact and granulated (Figure 5) during the course of infection. Thus, we propose that in response to infection, the spatial and temporal difference in expression and interactions of gene clusters, namely, frontline immune defense, cell signalling, apoptosis and stress response allow for the compensation of/gain-of-functions to overcome the infection. Since the amebocytes of the bacteria-challenged horseshoe crab showed increased gene transcription and continued to exhibit morphological integrity indicating their physiological and biochemical competency, the amebocytes would be expected to synthesise the cognate proteins and retain their ability to store and/or secrete those newly synthesised proteins. Hence, exemplary to expectation, being the first to encounter the invading pathogens, the amebocytes would phagocytose the foreign bodies, rapidly transcribe their immune response genes and remain mostly intact to continue synthesising a formidable array of innate immune molecules to sustain the antimicrobial action.
Consistent with our recent discovery of a primitive yet complex opsonic complement defense system in the horseshoe crab,16 we propose that during Pseudomonas infection, the amebocytes actively and acutely express immune-related genes and simultaneously invoke a complement-mediated phagocytosis of the invading pathogen.
The primary goal of establishing the horseshoe crab EST database was to identify potential genes involved in the immune response against Gram-negative bacterial infection. As a ‘living fossil’, which lacks adaptive immunity, the horseshoe crab is an ideal experimental model as it has sustained an exceptionally powerful innate immune system. It offers a unique isolated system to study the genetics of innate immunity during host–pathogen interaction. Thus, mapping and clarifying the functional immunogenomics, particularly those that show homology in structure and function to human counterparts would be requisite to offering solutions to clinical problems associated with Pseudomonas-induced inflammation and sepsis.
A total of 60 frontline immune response genes that represent previously characterised ESTs have been identified from both the amebocyte and hepatopancreas cDNA libraries. Among these genes, coagulogen was most highly expressed, suggesting its important role in innate immunity. Various complement components, members of the Toll-like receptor signalling pathway, apoptosis and stress response were elucidated. The amebocytes appeared to be acutely responsive to Pseudomonas infection while the hepatopancreas probably acted as a long term ‘immune-reserve’. The high levels of pre-existing frontline innate immune proteins in the plasma were probably summoned into immediate antimicrobial combat. While the frontline immunity genes and a limited number of cell signalling genes in the amebocytes underwent an upsurge in transcriptional activation, the transcription of the corresponding genes in the hepatopancreas seemed to remain somewhat repressed or delayed. It is not surprising that the majority of amebocyte cell signalling genes were downregulated (Figure 1c) at the early phase of infection since desensitisation might have occurred. Such spatial and temporal expression of the host genes that were differentially up-/downregulated during Gram-negative bacterial infection highlights the intricate complexity of the host–pathogen interaction and antimicrobial response. We propose that the participation and interplay of functionally related clusters of genes in different tissues encoding innate immunity, cell signalling, apoptosis and stress response occur during a systemic infection by a dose (106 cfu/ml) of P. aeruginosa, to successfully clear the pathogen within 6 hpi.14 This dose of P. aeruginosa would have been lethal to mice. Many of these pathogen-responsive genes could be developed as strategic antimicrobial candidates. Our findings contribute towards the understanding and resolution of the pathogen-recognition gene clusters; how they are exploited and/or modulated during the acute phase of infection; and how different immune-responsive tissues coordinate their actions in a concerted and timely manner. These findings will ultimately offer the much needed insights into such systems in the vertebrates and humans.
Materials and methods
P. aeruginosa was used as an inoculum as it is a known opportunistic and clinically significant pathogen.15 The horseshoe crab, C. rotundicauda, was collected from the Kranji estuary in Singapore. Infection of the horseshoe crab was performed as described by Ng et al.14 Briefly, P. aeruginosa ATCC 27853 was cultured overnight in tryptic soy broth (Difco) at 37°C. Bacteria were pelleted at 5000 g for 5 min at 4°C, washed in saline and resuspended in the original culture volume in saline. Serial dilution and colony count were performed to determine colony-forming units (cfu). Following bacterial titration, the horseshoe crabs were injected with a sublethal dose of 1 × 106 cfu/ml of P. aeruginosa. Crude plasma was collected by cardiac puncture into 100 mM PMSF and centrifuged at 150 g for 15 min to separate the cell-free plasma from amebocytes. The hepatopancreas was removed via ventral excision at various hours postinfection (3, 6, 9, 12, 24, 48, 72 hpi). Each time point constitutes three experimental animals. The amebocytes and hepatopancreas were stored at − 80°C for extraction of total RNA. Naïve tissue samples were obtained under the same conditions after mock infection with pyrogen-free saline as an inoculum.
Total RNA was isolated using TRIzol™ reagent (Invitrogen) and mRNA was purified using the Oligotex-direct mRNA mini kit (Qiagen). The quality of the total RNA from the different tissue samples was assessed by electrophoresis in 1.2% formaldehyde agarose gel. mRNA samples were stored at − 80°C until use.
Construction of cDNA libraries by suppression subtractive cDNA hybridisation
Double-stranded cDNA was synthesised with SMART™ cDNA synthesis kit (Clontech). Briefly, the first strand cDNA synthesis reaction consist of 100 ng mRNA, 1 μM 3′ SMART CDS Primer II A, 1 mM SMART II A Oligo, 1 × first-strand buffer, 2 mM dithiothreitol, 50 U Powerscript reverse transcriptase and 1 mM dNTP. The reaction was performed at 42°C for 1 h followed by a 10-fold dilution of the resulting cDNA in TE buffer. Second strand cDNA utilised 4 μl of the first strand diluted cDNA as template, 1 × Advantage 2 PCR buffer, 0.2 mM dNTP, 0.2 μM 5′ PCR Primer II A, and 1 × Advantage 2 polymerase mix. PCR was performed as follows: 95°C (1 min), proceeding to 15–19 cycles of 95°C (15 s), 65°C (30 s), and 68°C (6 min) in a DNA gradient cycler AC 1234 Thermocycler (MJ Research, Inc.). CHROMA-SPIN columns were used for nucleotide removal and size-fractionation of the cDNA. The double-stranded cDNA was blunt-ended by digestion with RsaI. Suppression subtractive cDNA hybridisation was performed with PCR-Select cDNA subtraction kit (Clontech).
Briefly, the tester cDNA was divided into two aliquots. Each aliquot was ligated to either adaptor 1 or 2R. The adaptor 1-ligated and adaptor 2R-ligated tester cDNAs were separately denatured at 98°C for 90 s and then hybridised at 68°C for 8 h with excess driver cDNA. The two primary hybridisation samples were then combined without denaturing. Simultaneously, freshly denatured driver cDNA was added to further enrich differentially repressed genes and the mixture was hybridised at 68°C for 16 h. Subtraction was performed at a 1 : 40 ratio of tester to driver cDNA. Following this, the secondary hybridisation mixture was subjected to two rounds of suppression PCR to selectively enrich differential transcripts. The first round of PCR with primer 1 (IndexTerm5′-CTA ATA CGA CTC ACT ATA GGG C-3′) was carried out at 75°C for 5 min and 94°C for 25 s, followed by 27 cycles of 94°C for 30 s, 66°C for 30 s and 72°C for 90 s. A second round of PCR using nested primers 1 (IndexTerm5′-TCG AGC GGC CGC CCG GGC AGG T-3′) and 2R (IndexTerm5′-AGC GTG GTC GCG GCC GAG GT-3′) were employed to further enrich differential cDNAs. To perform this, primary PCR products were diluted 10-fold and used as a template for 12 cycles of PCR at 94°C for 30 s, 68°C for 30 s and 72°C for 90 s each. Subtracted cDNAs were ligated into pGEM-T Easy vector (Promega) and transformed into Escherichia coli TOP10 competent cells. The blue-white colony selection method was applied to select positive clones.
DNA sequencing and analysis of ESTs
Randomly selected clones were sequenced unidirectionally using T7 primer (IndexTerm5′-TAA TAC GAC TCA CTA TAG GG-3′) in Big Dye Terminator (Ver 3.1) reactions with an ABI Prism model 3100A sequencer. Vector sequences were manually removed with the DNAMAN ver. 4.15 software (Lynnon BioSoft), thereby leaving the insert for BLASTx nonredundant searches for matches to known sequences in GenBank.77 A sequence is considered to be significantly matched when the E-value is ⩽10− 3 and the alignment length is more than 10 amino acids.23, 78
Virtual Northern analysis
Virtual Northern analysis enables the study of gene transcription using minimal amounts of mRNAs, which are reverse-transcribed and amplified by RT-PCR into cDNAs.79 The analysis of the transcription of genes before and after infection using mRNAs pooled from 3 + 6 hpi was first performed to offer a general profile on genes which may be up- or downregulated by Pseudomonas infection. Furthermore, to define the kinetics of expression of various functional groups of ESTs over the 72 h of infection, the transcription of selected members of each functional group of immune responsive genes was monitored over individual time points (0, 3, 6, 12, 24, 48 and 72 hpi). Each time point used mRNAs pooled from three infected animals. To ensure unbiased amplification of the cDNAs for all tissues taken at each time point, the mRNAs were subjected to the same number of cycles of RT-PCR. To this end, the number of PCR cycles was predetermined by several rounds of optimisation of the PCR conditions.
To analyse the expression of selected clones, cDNAs were synthesised as described above. The double-stranded cDNA (400 ng) was resolved in a 1% agarose gel and transferred onto a nylon membrane (Hybond N +, Amersham) by capillary action. DNA probe fragment was obtained by PCR amplification of the insert using Clontech nested PCR primers 1 and 2R, followed by RsaI digestion to remove the cDNA subtraction adapters. The probe fragment was [32P]-labelled with Rediprime™ II labelling kit (Amersham). For loading controls, the Northern membrane was simultaneously hybridised with endogenous gene coding for ribosomal protein L3 (RpL3). Hybridisation was carried out at 68°C overnight in 1% SDS, 6 × SSC (0.9 M sodium chloride and 0.1 M trisodium citrate), 100 μg/ml calf thymus DNA (Sigma), 0.1% Ficoll 400 (Sigma), 0.1% polyvinylpyrrolidone and 0.1% bovine serum albumin. The membrane was subjected to stringency washes in 2 × SSC 0.1% SDS at room temperature, 1 × SSC 0.1% SDS at 37°C and 0.2 × SSC 0.1% SDS at 42°C, respectively. The nylon membrane was then exposed to X-ray film (Kodak) and developed using a Kodak M35 X-OMAT Processor.
Analysis of the amebocytes
The horseshoe crab was infected with P. aeuruginosa as described above. At 0 h (naïve), 3 and 6 hpi, blood was collected by cardiac puncture, directly into tubes containing a fixative (10% formalin in 0.5 M NaCl) at the final ratio of 1 : 5 (v/v). After 30 min of fixation in an ice bath, the samples were centrifuged at 150 g for 10 min. The supernatant was removed, and the amebocytes were resuspended in fresh fixative and viewed under bright field microscopy.
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This work was supported by a grant (03/1/21/17/227) from the Agency of Science, Technology and Research (A*STAR), Singapore. We thank Ms Kaitian Peng (an A*STAR-funded undergraduate scholar of the Imperial College, London, UK) for help with sequencing some ESTs.
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Ding, J., Tan, K., Thangamani, S. et al. Spatial and temporal coordination of expression of immune response genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda. Genes Immun 6, 557–574 (2005). https://doi.org/10.1038/sj.gene.6364240
- immune-response gene clusters
- Pseudomonas infection
- expressed sequence tags (ESTs)
- transcript profiling
- spatial and temporal immune gene expression
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