John P. Dalton

Proteinases and associated genes of parasitic helminths

Many parasites have deployed proteinases to accomplish some of the tasks imposed by a parasitic life style, including tissue penetration, digestion of host tissue for nutrition and evasion of host immune responses. Information on proteinases from trematodes, cestodes and nematode parasites is reviewed, concentrating on those worms of major medical and economical importance. Their biochemical characterization is discussed, along with their putative biological roles and, where available, their associated genes. For example, proteinases expressed by the various stages of the schistosome life-cycle, in particular the well-characterized cercarial elastase which is involved in the penetration of the host skin and the variety of proteinases, such as cathepsin B (Sm31), cathepsin L1, cathepsin L2, cathepsin D, cathepsin C and legumain (Sm32), which are believed to be involved in the catabolism of host haemoglobin. The various endo- and exoproteinases of Fasciola hepatica, the causative agent of liver fluke disease, are reviewed, and recent reports of how these enzymes have been successfully employed in cocktail vaccines are discussed. The various proteinases of cestodes and of the diverse superfamilies of parasitic nematodes are detailed, with special attention being given to those parasites for which most is known, including species of Taenia, Echinococcus, Spirometra, Necator, Acylostoma and Haemonchus. By far the largest number of papers in the literature and entries to the sequence data bases dealing with proteinases of parasitic helminths report on enzymes belonging to the papain superfamily of cysteine proteinases. Accordingly, the final section of the review is devoted to a phylogenetic analysis of this superfamily using over 150 published sequences. This analysis shows that the papain superfamily can be divided into two major branches. Branch A contains the cathepin Bs, the cathepsin Cs and a novel family termed cathepsin Xs, while Branch B contains the cruzipains, cathepsin Ls, papain-like and aleurain/cathepsin H-like proteinases. The relationships of the helminth proteinases, and similar proteinases from protozoan parasites and other organisms, within these groups are discussed.

Fasciola hepatica cathepsin L-like proteases: biology, function and potential in the development of first generation liver fluke vaccines.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.

Thioredoxin Peroxidase Secreted by Fasciola hepatica Induces the Alternative Activation of Macrophages

ABSTRACT Alternatively activated macrophages (AAMφ) are primarily associated with the chronic stages of parasitic infections and the development of a polarized Th2 response. We have shown that Fasciola hepatica infection of BALB/c mice induces a polarized Th2 response during both the latent and chronic stage of disease. The activation status of macrophages was analyzed in this model of helminth infection by evaluating the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arg1. AAMφ were recruited to the peritoneum of mice within 24 h of F. hepatica infection and after intraperitoneal injection of parasite excretory-secretory (ES) products. Administration of a recombinant antioxidant thioredoxin peroxidase (TPx), which is contained within the ES products, also induced the recruitment of AAMφ to the peritoneum. In vitro studies showed that this recombinant TPx directly converts RAW 264.7 macrophages to an alternatively activated phenotype characterized by the production of high levels of interleukin-10 (IL-10), prostaglandin E2, corresponding with low levels of IL-12. Our data suggest that the Th2 responses induced by the helminth F. hepatica are mediated through the secretion of molecules, one of which is TPx, that induce the recruitment and alternative activation of macrophages.

An Integrated Transcriptomics and Proteomics Analysis of the Secretome of the Helminth Pathogen Fasciola hepatica PROTEINS ASSOCIATED WITH INVASION AND INFECTION OF THE MAMMALIAN HOST

To infect their mammalian hosts, Fasciola hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum, and penetrate the liver. After migrating through and feeding on the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and produce eggs. Here we integrated a transcriptomics and proteomics approach to profile Fasciola secretory proteins that are involved in host-pathogen interactions and to correlate changes in their expression with the migration of the parasite. Prediction of F. hepatica secretory proteins from 14,031 expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre using the semiautomated EST2Secretome pipeline showed that the major components of adult parasite secretions are proteolytic enzymes including cathepsin L, cathepsin B, and asparaginyl endopeptidase cysteine proteases as well as novel trypsin-like serine proteases and carboxypeptidases. Proteomics analysis of proteins secreted by infective larvae, immature flukes, and adult F. hepatica showed that these proteases are developmentally regulated and correlate with the passage of the parasite through host tissues and its encounters with different host macromolecules. Proteases such as FhCL3 and cathepsin B have specific functions in larvae activation and intestinal wall penetration, whereas FhCL1, FhCL2, and FhCL5 are required for liver penetration and tissue and blood feeding. Besides proteases, the parasites secrete an array of antioxidants that are also highly regulated according to their migration through host tissues. However, whereas the proteases of F. hepatica are secreted into the parasite gut via a classical endoplasmic reticulum/Golgi pathway, we speculate that the antioxidants, which all lack a signal sequence, are released via a non-classical trans-tegumental pathway.

Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles.

Cathepsin L-like activity was demonstrated in the excretory/secretory (E/S) products of Fasciola hepatica newly excysted juveniles (NEJ), 3-week-old, 5-week-old and mature flukes using the fluorogenic substituted 7-amino-4-methylcoumarin substrates Z-phe-arg-AMC, Z-arg-arg-AMC and Z-arg-AMC. Gelatin-substrate polyacrylamide gel analysis revealed that the E/S from each of these stages contained multiple proteolytic enzymes; however, the pattern of proteinases obtained for NEJ E/S differed markedly from that of all other stages examined. The four NEJ proteinases identified were inhibited by leupeptin and Z-phe-ala-diazomethyl ketone indicating that each had cathepsin L-like activity. The E/S products of all four developmental stages contain an enzyme capable of cleaving immunoglobulin at the hinge region, the activity of which is also inhibited by Z-phe-ala-diazomethyl ketone. Using in vitro cell attachment assays we show that the cathepsin L-like proteinase purified from the E/S products of adult F. hepatica can prevent the antibody-mediated attachment of eosinophil to NEJ. These experiments indicate that this proteinase has an important biological function in immune evasion.

Vaccines against the zoonotic trematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica.

Schistosoma japonicum, Fasciola hepatica and F. gigantica are digenetic trematodes and, therefore, possess similar life cycles. While schistosomiasis japonica has for a long time been recognised as a major disease of both humans and animals, infection with fasciolids has only been considered of relevance to animals. However, a number of recent reports indicate that fasciolosis is becoming a serious public health problem, especially in South America, Egypt and Iran (sporadic cases are also on the increase throughout Europe). Vaccines targeted at animals could play an important role in controlling these three diseases in animals and, by blocking transmission of infection, have a concurrent beneficial effect on disease in humans. Approaches towards identifying and producing vaccines against these parasites are similar and are discussed in this review.

Fasciola hepatica infection downregulates Th1 responses in mice

Immune responses induced with helminth parasites have been extensively studied, but there is limited information on those to Fasciola hepatica, especially on the subtype of T cell induced with this parasite. We investigated the local and systemic T cell responses of different strains of mice following oral infection with doses of metacercariae from F. hepatica. Spleen cells from BALB/c and 129Sv/Ev mice given a low‐dose (5 metacercariae) infection exhibited a Th2 response, producing high levels of the cytokines IL‐4 and IL‐5, and low levels of IFN‐γ and IL‐2. In contrast, C57BL/6 mice showed a mixed Th1/Th2 response. A more marked polarization to a Th2 response was observed in BALB/c, 129Sv/Ev exposed to a high‐dose (15 metacercariae) infection and the C57BL/6 mice also exhibited a clear Th2 response. IL‐4 defective (IL‐4−/−) C57BL/6 mice infected with 5 metacercariae produced less IFN‐γ and more IL‐5 compared to their wild‐type C57BL/6 counterparts, suggesting that IL‐4 is important in establishing the Th2 type response in murine fasciolosis. However, the secretion of IFN‐γ and IL‐2 was completely suppressed in the high‐dose infection and this was also observed in IL‐4−/− mice. Thus, liver flukes may secrete molecules that downregulate Th1 responses. T cell responses in the mesenteric (MLN) and hepatic lymph nodes (HLN) were also examined since newly excysted juveniles infect through the intestinal wall of their host before migrating to the hepatic tissue. Cells from both MLN and HLN secreted higher levels of IL‐4 and IL‐5 compared to spleen cells. We also observed a difference in cytokine profiles secreted by the MLN and HLN, which may reflect responses to antigens liberated by newly excysted juveniles and hepatic stage parasites, respectively.

Progress in Development of Liver Fluke Vaccines

Infection of ruminants by Fasciola spp continues to cause large economic losses worldwide. Recent results from several laboratories have demonstrated that animals can be significantly protected against infection by vaccination with defined Fasciola antigens. Apart from reducing fluke burdens, some vaccines can elicit a concurrent reduction in parasite egg production. The expectation of a commercially feasible vaccine that might also reduce parasite transmission in the field is now realistic, although major hurdles still exist. Here, Terry Spithill and John Dalton review the results of several recent vaccine trials and discuss the future prospects for vaccine development.

Thiol proteases released in vitro by Fasciola hepatica

Immature Fasciola hepatica release 11 distinct proteases when cultured in vitro for 16 h as revealed by gelatin-substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Six of these proteases are active in the pH range 4.5 to 8.0. Five are acid proteases, being most active in the pH range 3.0 to 4.5. The majority of proteases released in vitro by immature flukes are also released by mature flukes; however, a 40-kDa protease released by immature flukes is a very minor protease released by mature flukes. The activity of all proteases is inhibited by leupeptin, L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane, phenylmethylsulfonyl fluoride and iodoacetamide and enhanced or stabilized by the reducing agents cysteine and dithiothreitol. Therefore, all F. hepatica in vitro-released proteases identified by gelatin-substrate SDS-PAGE are thiol proteases.

Proteolytic degradation of host hemoglobin by schistosomes.

Schistosomes acquire amino acids for growth, development, and reproduction by catabolizing hemoglobin obtained from ingested host erythrocytes. While the biochemical pathway(s) involved has not been determined definitively, a number of proteases including schistosome legumain and cathepsin L-, D-, B- and C-like enzymes have been ascribed roles in the degradation of hemoglobin to diffusible peptides. Transcripts encoding these schistosome proteases, which appear to be expressed in the gastrodermis and cecum of the schistosome, have been reported. Because these enzymes are candidate targets at which to direct novel anti-schistosomal therapies, the comparative biochemistry of these and their counterpart mammalian proteases is now the focus of research in a number of laboratories. This paper reviews reports dating from 40 years ago to the present on how schistosomes digest host-derived hemoglobin, and interprets apparent anomalies in some earlier compared to later reports, the latter having benefited from the availability of PCR and gene cloning technologies. More specifically, the review concentrates on five proteolytic enzymes, and their associated genes, which have been ascribed key roles in the pathway of hemoglobin degradation.