Role of the prophenoloxidase-activating system in invertebrate immunity.

The melanization reaction, which is a common response to parasite entry in invertebrate animals, especially arthropods, is due to the activity of an oxidoreductase, phenoloxidase. This enzyme is part of a complex system of proteinases, pattern recognition proteins and proteinase inhibitors constituting the so-called prophenoloxidase-activating system. It is proposed to be a non-self recognition system because conversion of prophenoloxidase to active enzyme can be brought about by minuscule amounts of molecules such as lipopolysaccharide, peptidoglycan and beta-1, 3-glucans from micro-organisms. Several components of this system recently have been isolated and their structure determined.

Molecular cloning and characterization of prophenoloxidase in the black tiger shrimp, Penaeus monodon.

A cDNA encoding shrimp, Penaeus monodon, prophenoloxidase (proPO) was obtained by screening a hemocyte library by plaque hybridization using a proPO cDNA fragment from freshwater crayfish, Pacifastaceus leniusculus, as a probe. The 3,002 bp cDNA contains an open reading frame of 2,121 bp and a 881 bp 3'-untranslated region. The molecular mass of the deduced amino acid sequence (688 amino acids) is 78,700 Da with an estimated pI of 5.8. Two putative copper binding sites are present and they have a highly conserved sequence around these sites. No signal peptide was detected in the shrimp proPO, as has been previously shown to be the case for all arthropod proPOs cloned so far. The cleavage site of zymogen activation is likely to be between Arg 44 and Val 45. A tentative complement-like motif (GCGWPQHM) is also present. Shrimp proPO mRNA is synthesized in the hemocytes and not in the hepatopancreas. Comparison of amino acid sequences showed that shrimp proPO is more closely related to another crustacean proPO, namely crayfish, than to the insect proPOs.

Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR.

A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of crayfish with the Oomycete Aphanomyces astaci. A set of oligonucleotide primers was designed to specifically amplify A. astaci DNA in the ITS region surrounding the 5.8S rDNA gene. The PCR amplifies a 115bp amplicon. The specificity of the primers was demonstrated by testing on 27 A. astaci strains and against 20 non-A. astaci Oomycetes and 5 fungal species. Most of the non-A. astaci Oomycete or fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. Specificity was also tested against crayfish tissue and some known parasites and bacteria infecting crayfish. A protocol for the extraction of A. astaci DNA from infected crayfish tissue was developed. The optimised method allows the detection of two genome equivalents of purified A. astaci genomic DNA. The method was tested on noble crayfish (Astacus astacus), artificially infected with A. astaci. Detection of A. astaci was possible at the very first time of sampling, which was 2 days after the beginning of spore exposure.

Characterisation of Saprolegnia sp. isolates from channel catfish.

Nineteen channel catfish isolates of Saprolegnia sp. obtained from 5 separate fish farms in Mississippi, which became affected by winter kill syndrome during 1991 and 1996, were investigated with respect to physiological characteristics and genetic variation. Isolates of S. parasitica from crayfish and S. diclina were included for comparison. Most strains of catfish isolates grew well at 20 and 30 degrees C. Repeated zoospore emergence was found in catfish isolates of Saprolegnia sp. and S. parasitica, but not in S. diclina. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was applied for a further characterisation of the isolates. The RAPD analysis among Saprolegnia spp. isolates was constructed from 686 amplified products in 67 separable positions and indicated that the catfish isolates of Saprolegnia sp. are composed of 3 genetically distinct groups.

Sequence analysis of the ribosomal internal transcribed spacer DNA of the crayfish parasite Psorospermium haeckeli.

Two morphotypes of the crayfish parasite Psorospermium haeckeli were isolated from 2 crayfish species of different geographical origin. The oval-shaped sporocysts were obtained from the epidermal and connective tissue beneath the carapace of the noble crayfish Astacus astacus from Sweden and Finland. Elongated spores were isolated from the abdominal muscle tissue of the red swamp crayfish Procambarus clarkii from USA. To compare genetic divergence of 2 morphotypes of the parasite, the ribosomal internal transcribed spacer (ITS) DNA (ITS 1 and ITS 2) and the 5.8S rRNA gene were cloned and sequenced. The analysed region is variable in length, with the ribosomal ITS sequence of the European morphotype longer than the North American one. Sequence diversity is found mainly in ITS 1 and ITS 2 regions, and there is 66% and 58% similarity between the 2 morphotypes, respectively. Thus, analysis of the ribosomal ITS DNA suggests that P. haeckeli forms obtained from Europe and North America are genetically diverse, which supports the previously reported morphological characteristics.

Diptericin expression in bacteria infected Drosophila mbn-2 cells - effect of infection dose and phagocytosis.

Drosophila haemocytes play a key role in defence against microbial aggression. Their capacity to sense and dispose of bacteria and also to signal to other immune tissues is probably vital to overcome an infection. In this work we used the haemocyte-like mbn-2 cell line to investigate how expression of the antimicrobial peptide diptericin is affected after a high dose bacterial challenge with diaminopimelic acid (DAP)-peptidoglycan Gram-positive and Gram-negative bacteria. We report that diptericin expression is negatively affected by high infection dose and rapid bacterial growth regardless of the type of infection and bacterial virulence and occurs in the absence of mbn-2 cell death. Furthermore we show that the mbn-2 cell population is heterogeneous, containing both phagocytic and nonphagocytic cells and that contact with large numbers of bacteria decreases diptericin expression in the phagocytic cell population.

Molecular cloning and characterization of two serine proteinase genes from the crayfish plague fungus, Aphanomyces astaci.

Two novel genes encoding the serine proteinases, subtilisin (AaSP1) and trypsin (AaSP2), from Aphanomyces astaci were identified. Based on the amino acidconsensus sequences around the catalytic triad of these serine proteinases, degenerated oligonucleotides were designed for isolation of serine proteinase genes from a genomic DNA library. The AaSP1 gene encodes a full-length protein of 515 amino acids as a large precursor of 56 kDa. After cleavage of a predicted leader sequence of 18 residues and a prepeptide of 133 amino acids, the mature enzyme of 364 amino acids is generated with a calculated molecular mass of 39 kDa and a pI of 6.0. The primary sequence of AaSP1 showed similarity to both bacterial subtilisin and fungal subtilisin-like serine proteinases. Southern blot analysis of AaSP1 revealed the presence of at least two subtilisin genes in the A. astaci genome. Northern blot analysis indicated that the size of AaSP1 transcript was 1.6 kb. The AaSP2 gene encodes a prepropeptide of 276 amino acids with a molecular mass of 29 kDa. A mature protein of 237 amino acids is probably generated after cleavage of a 17-residue signal peptide and a 21-amino-acid prepeptide with a predicted molecular mass of 25 kDa and a pI of 6.0. The primary sequence of AaSP2 showed similarity to trypsin enzymes from various organisms. Southern blot analysis revealed the presence of multiple trypsin genes in the A. astaci genome. Northern blot analysis indicated that the size of AaSP2 transcript was 1.0 kb. The regulation of AaSP2 transcription was not controlled by nitrogen catabolic repression. However, the expression of AaSP2 was found to be specifically induced by crayfish plasma, implying a role in pathogenesis toward the crayfish host.

Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus.

The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzing the conversion of prophenoloxidase to an active phenoloxidase, has a molecular mass of about 36 kDa in its active form. This protein was cloned from a blood cell cDNA library and its corresponding cDNA of 1736 base pairs encodes a zymogenic protein (proppA) of 468 amino acids. An antibody raised against a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the beta-1,3-glucan triggered activation of prophenoloxidase in vitro. The C-terminal half of the proppA is composed of a typical serine proteinase domain, with a sequence similar to other invertebrate and vertebrate serine proteinases. The N-terminal half contains a cationic glycine-rich domain, a cationic proline-rich domain and a clip-domain, in which the disulfide-bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian beta-defensins. Antibodies made against both the C- and the N-terminal halves recognize two proppAs under reducing conditions. However, under nonreducing conditions only the anti-C antibody recognized the two proppAs, which suggests that a conformational change takes place upon reduction that allows the anti-N to react with the N-terminal half of proppA. The recombinant clip-domain in crayfish proppA was overexpressed in Escherichia coli and the resulting peptide exhibited antibacterial activity against Gram-positive bacterial strains such as Micrococcus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory concentrations of 1.43 microM and 17.9 microM, respectively. These results suggest that the clip-domains in proppAs may function as antibacterial peptides.

cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.

Prophenoloxidase (proPO), an enzyme that is the terminal component of the so-called proPO activating system, a defense and recognition system in crustaceans and insects, has been purified and cloned from a crayfish blood cell cDNA library. The deduced amino acid sequence codes for a polypeptide with a mass of 80,732 Da, which is close to 76 kDa, the apparent mass of the purified enzyme. proPO contains two copper atoms, and two putative copper-binding sites were found in the deduced amino acid sequence. Sequence comparisons show that these putative copper-binding sites are similar to the corresponding sites in arthropod hemocyanins and also, although the sequence similarities are less extensive, similar to tyrosinases from vertebrates and microorganisms. The purified enzyme is a typical tyrosinase because it hydroxylates monophenols and oxidizes o-diphenols but does not oxidize p-diphenols. If a homogeneous preparation of crayfish proPO were incubated with a homogeneous sample of the proPO activating enzyme, a serine proteinase, the cleavage of proPO by this trypsin-like enzyme was found to occur between Arg-176 and Thr-177.

Trypsin from Pacifastacus leniusculus hepatopancreas: purification and cDNA cloning of the synthesized zymogen.

Trypsin was purified from crayfish, Pacifastacus leniusculus, hepatopancreas, and the gene that encoded this enzyme was cloned from a hepatopancreas cDNA library. Crayfish trypsin is synthesized as a zymogen according to the sequence of the putative precursor peptide. The authenticity of the trypsinogen is supported by the deduced amino acid sequence and confirmed by the N-terminal amino acid sequence of the mature protein. The enzyme has features characteristic of a trypsin, such as a specific binding pocket. Sequence comparison shows that crayfish trypsin is similar to those of other species, with the exception that six cysteine residues present in vertebrates are missing. Some structural characteristics, such as the length of the signal peptide and a calcium binding site, are similar to bacterial trypsin.

Molecular characterization of the fish-pathogenic fungus Aphanomyces invadans.

Aphanomyces invadans (Saprolegniaceae) is a peronosporomycete fungus associated with the serious fish disease, epizootic ulcerative syndrome (EUS), also known as mycotic granulomatosis. In this study, interspecific relationships were examined between A. invadans isolates and other aquatic animal pathogenic Saprolegniaceae, and saprophytic Saprolegniaceae from EUS-affected areas. Restriction fragment length polymorphisms and sequences of ribosomal DNA confirmed that A. invadans is distinct from all other species studied. A sequence from the internal transcribed spacer region ITS1, unique to A. invadans, was used to design primers for a PCR-based diagnostic test. Intraspecific relationships were also examined by random amplification of polymorphic DNA using 20 isolates of A. invadans from six countries. The isolates showed a high degree of genetic homogeneity using 14 random ten-mer primers. This provides evidence that the fungus has spread across Asia in one relatively rapid episode, which is consistent with reports of outbreaks of EUS. Physiological distinctions between A. invadans and other Aphanomyces species based on a data set of 16 growth parameters showed remarkable taxonomic congruence with the molecular phylogeny.

A cell adhesion protein from the crayfish Pacifastacus leniusculus, a serine proteinase homologue similar to Drosophila masquerade.

A cDNA encoding a protein resembling masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster, was identified in hemocytes of the adult freshwater crayfish, Pacifastacus leniusculus. The crayfish protein is similar to Drosophila masquerade in the following aspects: (a) overall sequence of the serine proteinase domain, such as the position of three putative disulfide bridges, glycine in the place of the catalytic serine residue, and the presence of a substrate-lining pocket typical for trypsins; (b) the presence of several copies of a disulfide-knotted motif in the putative propeptide. This masquerade-like protein is cleaved into a 27-kDa fragment, which could be detected by immunoblot analysis using an affinity-purified antibody against a synthetic peptide in the C-terminal domain of the protein. The 27-kDa protein could be immunoaffinity-purified from hemocyte lysate supernatant and exhibited cell adhesion activity in vitro, indicating that the C-terminal domain of the crayfish masquerade-like protein mediates cell adhesion.

Structure and biological activity of a 1,3-beta-D-glucan-binding protein in crustacean blood.

The prophenoloxidase activating system, an enzyme cascade present in arthropod blood, has been shown to be involved in defense and recognition reactions. This system is converted to its active form by fungal 1,3-beta-D-glucans through binding to a plasma protein, a 1,3-beta-D-glucan-binding protein. Here the molecular cloning and carbohydrate composition of the 1,3-beta-D-glucan-binding protein from the freshwater crayfish Pacifastacus leniusculus are reported. It is also demonstrated that this protein can act as an opsonin, stimulating phagocytic uptake of yeast particles by isolated blood cells. The deduced amino acid sequence of 1,339 residues shows no significant similarity to proteins with similar functions in other animals such as the mannan-binding and lipopolysaccharide-binding proteins present in mammals. However, a short sequence motif with similarity to the active site of microbial 1,3-1,4-beta-D-glucan 4-glucanohydrolases was found to occur twice in the 1,3-beta-D-glucan-binding protein.

A novel clade of protistan parasites near the animal-fungal divergence.

Sequences of nuclear-encoded small-subunit rRNA genes have been determined for representatives of the enigmatic genera Dermocystidium, Ichthyophonus, and Psorospermium, protistan parasites of fish and crustaceans. The small-subunit rRNA genes from these parasites and from the "rosette agent" (also a parasite of fish) together form a novel, statistically supported clade. Phylogenetic analyses demonstrate this clade to diverge near the animal-fungal dichotomy, although more precise resolution is problematic. In the most parsimonious and maximally likely phylogenetic frameworks inferred from the most stably aligned sequence regions, the clade constitutes the most basal branch of the metazoa; but within a limited range of model parameters, and in some analyses that incorporate less well-aligned sequence regions, an alternative topology in which it diverges immediately before the animal-fungal dichotomy was recovered. Mitochondrial cristae of Dermocystidium spp. are flat, whereas those of Ichthyophonus hoferi appear tubulovesiculate. These results extend our understanding of the types of organisms from which metazoa and fungi may have evolved.