Lage Cerenius

The prophenoloxidase-activating system in invertebrates.

Summary:  A major innate defense system in invertebrates is the melanization of pathogens and damaged tissues. This important process is controlled by the enzyme phenoloxidase (PO) that in turn is regulated in a highly elaborate manner for avoiding unnecessary production of highly toxic and reactive compounds. Recent progress, especially in arthropods, in the elucidation of mechanisms controlling the activation of zymogenic proPO into active PO by a cascade of serine proteinases and other factors is reviewed. The proPO‐activating system (proPO system) is triggered by the presence of minute amounts of compounds of microbial origins, such as β‐1,3‐glucans, lipopolysaccharides, and peptidoglycans, which ensures that the system will become active in the presence of potential pathogens. The presence of specific proteinase inhibitors prevents superfluous activation. Concomitant with proPO activation, many other immune reactions will be produced, such as the generation of factors with anti‐microbial, cytotoxic, opsonic, or encapsulation‐promoting activities.

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.

The proPO-system: pros and cons for its role in invertebrate immunity

Melanisation is an important immune response in many invertebrates. Recent evidence also strongly implies that the melanisation (prophenoloxidase activating) cascade is intimately associated with the appearance of factors stimulating cellular defence by aiding phagocytosis and encapsulation reactions. However, some controversy exists in the field, and at least in flies and mosquitoes, the successful combat of some pathogens does not seem to be dependent on phenoloxidase activity. This may be because of redundancy among separate immune mechanisms, inappropriate testing, species differences or a combination thereof. Recently, by using RNA interference against phenoloxidase or in specific host-pathogen interactions where the pathogen prevents melanin production by the host, convincing data have confirmed the importance of this cascade in invertebrate innate immunity.

Proteolytic cascades and their involvement in invertebrate immunity

Bacteria and other potential pathogens are cleared rapidly from the body fluids of invertebrates by the immediate response of the innate immune system. Proteolytic cascades, following their initiation by pattern recognition proteins, control several such reactions, notably coagulation, melanisation, activation of the Toll receptor and complement-like reactions. However, there is considerable variation among invertebrates and these cascades, although widespread, are not present in all phyla. In recent years, significant progress has been made in identifying and characterizing these cascades in insects. Notably, recent work has identified several connections and shared principles among the different pathways, suggesting that cross-talk between them may be common.

Antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish Pacifastacus leniusculus.

ABSTRACT In a study of genes expressed differentially in the freshwater crayfish Pacifastacus leniusculus infected experimentally with the white spot syndrome virus (WSSV), one protein, known as antilipopolysaccharide factor (ALF), was chosen, among those whose transcript levels increased upon viral infection, for further studies. ALF RNA interference (RNAi) experiments in whole animals and in cell cultures indicated that ALF can protect against WSSV infection, since knockdown of ALF by RNAi specifically resulted in higher rates of viral propagation. In a cell culture of hematopoietic tissue (Hpt) from P. leniusculus, quantitative PCR showed that knockdown of ALF by RNAi resulted into WSSV levels that were about 10-fold higher than those treated with control double-stranded RNA (dsRNA). In addition, RNAi experiments with other crayfish genes that had been found to be up-regulated by a WSSV infection did not result in any changes of viral loads. Thus, the cell culture does not respond to dsRNA in a similar manner, as shown earlier for dsRNA injected into shrimp, which gave a higher degree of resistance to WSSV infection. If ALF transcription in whole animals was stimulated by the administration of UV-treated WSSV, a partial protection against a subsequent challenge with the active virus was conferred to the host. This is the first crustacean gene product identified with the capacity to interfere with replication of this important pathogen.

Phenoloxidase is an important component of the Defense against Aeromonas hydrophila infection in a crustacean, Pacifastacus leniusculus

The melanization cascade, in which phenoloxidase is the terminal enzyme, appears to play a key role in recognition of and defense against microbial infections in invertebrates. Here, we show that phenoloxidase activity and melanization are important for the immune defense toward a highly pathogenic bacterium, Aeromonas hydrophila, in the freshwater crayfish, Pacifastacus leniusculus. RNA interference-mediated depletion of crayfish prophenoloxidase leads to increased bacterial growth, lower phagocytosis, lower phenoloxidase activity, lower nodule formation, and higher mortality when infected with this bacterium. In contrast, if RNA interference of pacifastin, an inhibitor of the crayfish prophenoloxidase activation cascade, is performed, it results in lower bacterial growth, increased phagocytosis, increased nodule formation, higher phenoloxidase activity, and delayed mortality. Our data therefore suggest that phenoloxidase is required in crayfish defense against an infection by A. hydrophila, a highly virulent and pathogenic bacterium to crayfish.

The prophenoloxidase activating system and its role in invertebrate defence.

Invertebrates have an open circulatory system and must therefore have rapid and immediate noninducible defence and coagulation mechanisms to entrap parasites and prevent blood loss after wounding. As in most animals, these processes are mainly carried out by the blood cells or, as they are called in arthropods, hemocytes. The hemocytes of arthropods and other invertebrates have been shown to be important in defence, since they are responsible for phagocytosis of small foreign particles such as bacteria or fungal spores and form capsules around parasites that are too large to be internalized by individual hemocytes. Because the hemocytes obviously can react to and remove a foreign particle that has succeeded in gaining entry into the body cavity of an arthropod, it appears as if these animals can differentiate non-self from self, and thus a system that can carry out this process ought to be present. Agglutinins or lectins may be one candidate for such a system.l-s Another likely candidate is the so-called prophenoloxidase (proPO) activating system: and recently evidence has accumulated mainly from work done on crustaceans that this may be the case. The prime reason why this system early was proposed to function in recognizing foreign particles was the finding that the enzyme phenoloxidase in crayfish blood was turned into its active form by fungal cell wall p-1,3-gl~cans.~~* This was later also confirmed to be the case in several insect species”” and other in~ertebrates.l~-’~ Other microbial products such as lipopolysaccharides and peptidoglycans from bacterial cell walls are also active as elicitors of the proPo system.*s.16 Thus, regardless of which events follow after the proPo system is activated, it is clear that it can react to foreign microbial polysaccharides and as such can be defined as a recognition system. Recent research has also provided clear evidence that, upon activation of the proPo system, factors are produced that will aid in the elimination of foreign particles such as parasites within the body cavity. This brief overview will report some of these studies, which have mainly been carried out on arthropods, and where

Host prophenoloxidase expression in freshwater crayfish is linked to increased resistance to the crayfish plague fungus, Aphanomyces astaci.

The crayfish plague (Aphanomyces astaci) susceptible freshwater crayfish Astacus astacus and the resistant species Pacifastacus leniusculus were compared with respect to differential haemocyte count and expression of prophenoloxidase and peroxinectin. A major difference found was that resistant crayfish continuously produced high levels of prophenoloxidase (proPO) transcripts and that these levels could not be further increased, whereas in susceptible crayfish proPO transcript levels and resistance were augmented by immunostimulants. In As. astacus this could be registered as higher proPO transcript levels in the semigranular population of haemocytes and to an increased survival time after experimental infections with A. astaci.

Analysis of genetic diversity in the crayfish plague fungus, Aphanomyces astaci, by random amplification of polymorphic DNA

Abstract Arbitrary primers and the DNA polymerase chain reaction (PCR) technique were applied to study genetic variation between different strains of the crayfish plague fungus, Aphanomyces astaci. Eight different primers among 15 tested were chosen for a comparative analysis of the different A. astaci strains. On average, each primer gave rise to 5–8 bands and a majority of the bands was polymorphic for at least some strains. Two main groups among the different Swedish isolates were clearly discernible. One group included isolates from the indigenous crayfish species Astacus astacus and one isolate from As. leptodactylus originating from Turkey. The other main group included fungal isolates from both As. astacus and the introduced North American crayfish Pacifastacus leniusculus. In this latter case it seems likely that the introduced American signal crayfish has served as a vector and transmitted the pathogen to the indigenous crayfish.

Physiological adaptation of an Aphanomyces astaci strain isolated from the freshwater crayfish Procambarus clarkii

Physiological, epidemiological and genetical properties of an Aphanomyces astaci strain (Pc) isolated from the warm water crayfish, Procambarus clarkii, were compared to other A. astaci strains iso ...