Differential Cleaving of Specific Substrates for Cathepsin-Like Activity Shows Cysteine and Serine Protease Activities and a Differential Profile Between Anisakis simplex s.s. and Anisakis pegreffii, Sibling Species Major Etiologic Agents of Anisakiasis.

Humans can contract anisakiasis by eating fish or squid containing live larvae of the third stage (L3) of the parasitic nematodes of the genus Anisakis, majorly from Anisakis simplex s.s. and Anisakis pegreffii, sibling species of the A. simplex s.l. complex. Most cases diagnosed molecularly are due to A. simplex s.s., although A. pegreffii has also been identified in human cases. Cathepsins are mostly lysosomal multifunctional cysteine proteases and can participate in the pathogenicity of parasites. Cathepsin B and L activities were investigated in the two sibling species of Anisakis mentioned. L3 and L4 of both species were collected during their in vitro development, and cathepsin activity was determined in the range of pH 4.0-8.5, using specific fluorogenic substrates. The activity detected with the substrate Z-FR-AMC (N-α-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methyl-coumarin) was identified as cathepsin L (optimum pH = 5.0, range 4.0-6.0, p < 0.001). Activity was highest in L3 freshly collected from fish, especially in A. simplex s.s., and decreased during development, which could be related to virulence, invasion of host tissues, and/or intracellular digestion. Cathepsin B-like activity was not identified with either of the substrates used (Z-RR-AMC [N-α-benzyloxycarbonyl-L-arginyl-L-arginine-7-amido-4-methyl-coumarin] and Z-FR-AMC). With Z-RR-AMC, cleaving activity was detected almost exclusively in L4 of A. simplex s.s. (p < 0.05) with optimum pH = 8.0 (range 7.0-8.5). Assays with class-specific protease inhibitors showed that this activity was mainly due to serine proteases [up to 90% inhibition with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF)], although metalloproteases (up to 40-45% inhibition with 1,10 phenanthroline) and slight cysteine protease activity (<15% inhibition with E64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane]; putative cathepsin B-like) were also detected. These results show differential serine protease activity between sibling Anisakis species, regulated by larval development, at least in A. simplex s.s. The higher cathepsin L and serine protease activities detected in this species could be related to its greater pathogenicity, reported in experimental animals, compared to that of A. pegreffii.

Differential proteolytic activity in Anisakis simplex s.s. and Anisakis pegreffii, two sibling species from the complex Anisakis simplex s.l., major etiological agents of anisakiasis.

Proteolytic activity was studied in two sibling species of Anisakis (Nematoda: Anisakidae), A. simplex s.s. and A. pegreffii, throughout their in vitro development from third larval stage (L3) from the host fish (L3-0h) to fourth larval stage (L4) obtained in culture. Proteases have a significant role in the lifecycle of the parasite and in the pathogen-host relationship. Proteolytic activity peaks were detected at pH 6.0 and 8.5. Protease activity was detected in all the developmental stages of the two species studied at both pH values. These pH values were used for assaying with specific inhibitors which permitted the determination of metalloprotease activity, and, to a lesser extent, that of serine and cysteine protease. Aspartic protease activity was only detected at pH 6.0. At this pH, L4 larvae showed higher proteolytic activity than L3 larvae in both species (p < 0.001), the majority of activity being due to metalloproteases and aspartic proteases, which could be related to nutrition, especially the latter, as occurs in invertebrates. At pH 8.5, proteolytic activity was higher in A. simplex s.s. than in A. pegreffii (p < 0.01). At this pH, the majority of activity was due to metalloproteases in all developmental phases of both species, although, in L3-0h, the activity of these proteases was significantly higher (p < 0.03) in A. simplex s.s. than in A. pegreffii. This could be related to the greater invasive capacity of the former. Serine proteases have frequently been implicated in the invasive capacity and pathogenicity of some parasites. This may be related to the significantly higher activity (p ≤ 0.05) of serine protease in all the larval stages of A. simplex studied at pH 6.0. Thus, there are interspecific differences in proteases that have been related to pathogenesis in nematodes. These differences could thus be contributing to the previously reported differences in pathogenicity between these two Anisakis species.

Fishing area and fish size as risk factors of Anisakis infection in sardines (Sardina pilchardus) from Iberian waters, southwestern Europe.

The sardine (Sardina pilchardus) is a fish commonly consumed and appreciated in many countries, although they are more likely to be eaten fresh in western Mediterranean countries such as Spain, Portugal, France or Italy. A molecular epidemiological survey of sardines from 5 fishing areas of the Spanish Mediterranean (Málaga, southern Spain) and Atlantic coasts (southern: Cádiz and Isla Cristina; northern: A Coruña and Ondarroa) was carried out to determine the presence of Anisakis spp. larvae. The highest prevalence of these larvae was observed in fish from A Coruña (28.3%), followed by Ondarroa (5%) and Cádiz (2.5%). No Anisakis larvae were found in fish from Málaga and Isla Cristina. Three Anisakis genotypes were identified: Anisakis simplex sensu stricto, Anisakis pegreffii and a hybrid genotype between these two species. A. pegreffii was the most prevalent species in A Coruña (71% of larvae). Only three Anisakis larvae (9% collected larvae) were located in the musculature of sardines: two were identified as A. pegreffii while the other was a hybrid genotype. Sardine infection was associated with fishing area and fish length/weight (length and weight were strongly correlated; Pearson's correlation 0.82; p<0.001). Risk factor multivariate analysis showed that the risk of infection increases 1.6 times for every additional cm in the length of the sardines from the same fishing area. Comparison of fish of equal length showed that in sardines from A Coruña the risk of parasitization is 11.5 times higher than in those from other fishing areas. Although the risk of infection by Anisakis through consumption of sardines is generally low due to the low epidemiological parameter values (prevalence 10%, mean intensity 1.7 (range 1-5) and mean abundance 0.17), as larger fish are more heavily parasitized, there is an increased risk of infection by Anisakis through consumption of large sardines which are raw or have undergone insufficient treatment (undercooked, smoked, marinated, salted, pickled, freezing,…).

Anthelminthic activity of the cyclotides (kalata B1 and B2) against schistosome parasites.

The risk of reduced sensitivity of the human schistosomes to praziquantel has led to efforts to find new therapies. Here, the cyclotides kalata B1 (kB1), kalata B2 (kB2), MCoCC-1, and MCoTI-II, cyclic peptides extracted from plants and shown to be potent against nematodes and insects, were tested for antischistosome activity. In vitro assays showed that high concentrations (500-1000 µg/mL) of either kB1 or kB2 killed Schistosoma japonicum and Schistosoma mansoni adults within 5 min, whereas MCoTI-II and MCoCC-1 had no effect. Lethal concentrations to kill 50% of the population for kB2 was 15.5 ± 7.4 µg/mL at 1 h for male S. japonicum (Philippine strain). Males were more susceptible than females. kB2 showed higher antischistosome activity than kB1 and killing time was concentration-dependent. Mode of action studies revealed that kB1 and 2 lysed the tegument of adult worms. Lysis of myofibrils was not demonstrated, but longitudinal and radial muscle fibers were distorted, an observation consistent with strong coiling of the parasites after drug exposure. A single dose of kB2 administered either orally or intravenously, reduced worm burdens in S. japonicum-infected mice from 15% to 60%. However, treatment of S. mansoni-infected mice did not result in reduction in worm burdens. Our studies show that kB2 acts as a promising antischistosomal against Philippine S. japonicum, and it or other cyclotides may be developed further as general anthelminthics. With thousands of cyclotides predicted to occur in plants, and the amenability of these peptides to combinatorial variation, there is potential for their exploitation as wide-spectrum anthelminthics.

Proteolytic activity in Hysterothylacium aduncum (Nematoda: Anisakidae), a fish gastrointestinal parasite of worldwide distribution.

Proteases have a significant role in the life cycle of parasites and the pathogen-host relationship, being regarded as important virulence factors. In the parasitic nematode Hysterothylacium aduncum proteolytic activity was measured during in vitro development from third larval stage (L3) to mature adult, using DQ red casein as a fluorogenic substrate. Proteolytic activity was detected in all the developmental stages studied and at all pH values within the range employed (2.0-7.5). The assay with specific inhibitors permitted the determination of metalloprotease activity, and, to a lesser extent, that of aspartate- and cysteine-protease. Serine-protease activity was the lowest of those studied. In L3 recently collected from the host fish (L3-0 h), the greatest activity was found at an optimum pH of 4.0 and was mainly inhibited by 1,10-phenathroline (metalloprotease inhibitor). This metalloprotease activity in L3-0 h (infective stage) may be related to the invasion of the host tissues by this larva. In the other developmental stages, the greatest protease activity was found at pH 5.5, although at pH 4.0 a lower activity peak was detected. On the other hand, our data show that the proteolytic activity of the nematode varies according to the presence of pepsin (an aspartic-protease) in the culture medium. Thus, at pH 4.0, activity was greater in the absence of pepsin, with increasing aspartic-protease activity. Together with the detection of aspartic-, cysteine- and metallo-protease (enzymes involved in digestion in invertebrates) in all the developmental stages of the parasite taking place in the digestive tract of the host fish, this allows us to suggest that the pepsin in the culture medium mimics the predigestion conditions in the habitat of the worm within the host and that the activity detected may have, amongst others, a digestive function.

Collagenolytic activity related to metalloproteases (and serine proteases) in the fish parasite Hysterothylacium aduncum (Nematoda: Anisakidae).

Proteases play a vital role in both the life cycle of parasites and the parasite-host relationship and are considered important virulence factors. In the present study, the presence of proteases with collagenolytic activity was investigated in the fish nematode Hysterothylacium aduncum during in vitro development. Collagenolytic activity was found in all studied developmental stages of the nematode (third [L3] and fourth [L4] larval stages and adults). In L3, the activity was maximum at pH 6.5 and, in the other stages, at 7.0. Pepsin is known to favour in vitro development of the worm, but, in this study, collagenolytic activity was shown to be significantly greater when no pepsin was added to the culture medium (at pH 6.5, p = 0.011). At pH 7.0, most activity was observed in the immature adult, after the final moult, suggesting that the collagenolytic activity may be involved in remodelling of the cuticle and in sexual maturity. On the other hand, at pH 6.5, activity may be related to tissue migration by L3 within the host. Using specific inhibitors, it was demonstrated that most of the collagenolytic activity detected in all the developmental stages was due to metalloproteases (40 to 100%), although serine proteases were also detected in L4 and adults (10 to 30%).

Cathepsin B- and L-like cysteine protease activities during the in vitro development of Hysterothylacium aduncum (Nematoda: Anisakidae), a worldwide fish parasite.

Proteinases play an important role as virulence factors both in the life-cycle of parasites and in the pathogen-host relationship. Hysterothylacium aduncum is a worldwide fish parasite nematode which has been associated with non-invasive anisakidosis and allergic responses to fish consumption in humans. Cysteine proteinases have been associated with allergy to plant pollens, detergents and dust mites. In this study the presence of two types of cysteine proteinases (cathepsin B and cathepsin L) during in vitro development of H. aduncum is investigated. Specific fluorescent substrates were used to determine cathepsin activities. The activity detected with substrate Z-FR-AMC was identified as cathepsin L (optimum pH=5.5; range 3.5-6.5). Cathepsin B activity was only identified with Z-RR-AMC (optimum pH=7.0-7.5; range 5.0-8.0). The start of cultivation led to increased activity of both cathepsins (1.8-fold for cathepsin B and 6.3-fold for cathepsin L). These activities varied according to the developmental stage. Cathepsin B activity decreased after M4, returning to its initial level. Cathepsin L activity also decreased after M4, but still maintained a high level (4-6 times the initial level) in adult stages. Having considered these activity variations and the optimum pH values, we suggest that cathepsin L has a role in digestive processes while cathepsin B could be involved in cuticle renewal, among other possible functions.

CO2-fixing enzymes and phosphoenolpyruvate metabolism in the fish parasite Hysterothylacium aduncum (Ascaridoidea, Anisakidae).

CO2 stimulates the development of many of the intestinal helminths that are able to fix CO2 by means of phosphoenolpyruvate carboxykinase (PEPCK), such as Hysterothylacium aduncum. We determined the activity of CO2-fixing enzymes such as PEPCK and phosphoenolpyruvate carboxylase (PEPC), although no significant activity was detected for pyruvate carboxylase or carboxylating-malic enzyme. The former act on phosphoenolpyruvate (PEP) to yield oxalacetate. In the helminths studied, PEP has a vital role in glucidic metabolism. Consequently, we determined the activity of other enzymes involved in the crossroad of PEP, such as pyruvate kinase (PK), lactate dehydrogenase and malate dehydrogenase. All enzymes detected showed significant variations in activity during the in vitro development of the parasite from the third larval stage to mature adult. Fixing of CO2 by PEPCK decreased during development (from 228 to 115 nmol min(-1) mg(-1) protein), while that by PEPC increased (from 19 to 46 nmol min(-1) mg(-1) protein). This enzyme, which is rare in animals, could play a part in detecting levels of free phosphate, releasing it from PEP when required for processes such as glycogenolysis, glycolysis and adenosine 5'-triphosphate (ATP) synthesis. PK, which showed increasing activity during development up to immature adult (from 56 to 82 nmol min(-1) mg(-1) protein), could act in combination with PEPC to obtain energy in the cytosol (in the form of ATP) and in the mitochondria (possible destination of the pyruvate formed), compensating for the decrease in activity of PEPCK.

Anisakis simplex: CO(2)-fixing enzymes and development throughout the in vitro cultivation from third larval stage to adult.

We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).

CO(2)-fixing enzymes during moulting from third larval to fourth larval stage of Anisakis simplex and Hysterothylacium aduncum (Nematoda: Anisakidae).

The fixing of CO(2) is an important metabolic process for many organisms. In the anisakid nematodes, CO(2) has been shown to be necessary for their development, at least in vitro. The presence of CO(2) stimulates the moulting (M3) of the larvae from the third (L3) to the fourth (L4) stage and prolongs the survival, at least, in vitro. We determined the activity of CO(2)-fixing enzymes, common to many organisms, in two anisakids: Anisakis simplex, a parasite of cetaceans, and Hysterothylacium aduncum, a parasite of fish. Although no activity was detected for pyruvate carboxylase or carboxylating-malic enzyme, we detected phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) activity. In A. simplex, PEPCK was clearly higher than that of PEPC throughout the moulting process studied. In H. aduncum, although the activity of both enzymes was of similar magnitude, they showed different behaviour; PEPCK activity decreased after the moulting to L4, PEPC activity increased so that the ratio PEPCK/PEPC activity decreased from 1.90 before moulting to 0.59 after.