BackgroundThe liver fluke Fasciola hepatica is a major pathogen of livestock worldwide, causing huge economic losses to agriculture, as well as 2.4 million human infections annually.ResultsHere we provide a draft genome for F. hepatica, which we find to be among the largest known pathogen genomes at 1.3 Gb. This size cannot be explained by genome duplication or expansion of a single repeat element, and remains a paradox given the burden it may impose on egg production necessary to transmit infection. Despite the potential for inbreeding by facultative self-fertilisation, substantial levels of polymorphism were found, which highlights the evolutionary potential for rapid adaptation to changes in host availability, climate change or to drug or vaccine interventions. Non-synonymous polymorphisms were elevated in genes shared with parasitic taxa, which may be particularly relevant for the ability of the parasite to adapt to a broad range of definitive mammalian and intermediate molluscan hosts. Large-scale transcriptional changes, particularly within expanded protease and tubulin families, were found as the parasite migrated from the gut, across the peritoneum and through the liver to mature in the bile ducts. We identify novel members of anti-oxidant and detoxification pathways and defined their differential expression through infection, which may explain the stage-specific efficacy of different anthelmintic drugs.ConclusionsThe genome analysis described here provides new insights into the evolution of this important pathogen, its adaptation to the host environment and external selection pressures. This analysis also provides a platform for research into novel drugs and vaccines.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0632-2) contains supplementary material, which is available to authorized users.

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH 2]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite. drug design ͉ malaria ͉ structural biology ͉ protease T here are 300-500 million cases of clinical malaria annually, and 1.4-2.6 million deaths. Malaria is caused by apicomplexan parasites of the genus Plasmodium, with Plasmodium falciparum the most lethal of the 4 species that infect humans. Clinical manifestations begin when parasites enter erythrocytes, and most antimalaria drugs, such as chloroquine, exert their action by preventing the parasite development within these cells (1). As a result of the rapid spread of drug-resistant parasites, there is a constant need to identify and validate new antimalarial targets.Intraerythrocytic parasites have limited capacity for de novo amino acid synthesis and rely on degradation of host hemoglobin (Hb) to maintain protein metabolism and synthesis, and an osmotically stable environment within the erythrocyte (1-4). Within the erythrocytes, malaria parasites consume as much as 75% of the cellular Hb (1). Hb is initially degraded by the concerted action of cysteine-, aspartyl-, and metalloendoproteases, and a dipeptidase (cathepsin C) within a digestive vacuole (DV) to di-and tripeptide fragments (5, 6). These fragments are suggested to be exported to the parasite cytoplasm, where further hydrolysis to release free amino acids takes place [supporting information (SI) Fig. S1; see refs. 7 and 8].The release of amino acids involves 2 metallo-exopeptidases: an alanyl aminopeptidase, PfA-M1 (9, 10), and a leucine aminopeptidase, PfA-M17 (7,11,12). We have demonstrated that the aminopeptidase inhibitor bestatin, an antibiotic and natural analogue of the dipeptide Phe-Leu derived from the fungus Streptomyces olivoretticul, prevents P. falciparum malaria growth in culture (13,14). More recently, it was shown not only that synthetic phosphinate dipeptide analogues that inhibit metallo-aminopeptidases prevented the growth of wildty...

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 trypsinlike 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. Fasciola hepatica is a helminth (worm) parasite with a worldwide distribution. Although traditionally regarded as a parasite of livestock, particularly sheep and cattle, that results in a large economic loss to the agricultural community it has recently emerged as an important human infection in many regions of the world, including South America, Iran, Egypt, and mainland South-East Asia (1). Dormant larvae contained within cysts adhere to vegetation and emerge as infective juveniles (newly excysted juveniles (NEJs)) 1 in the duodenum following ingestion by animals or humans. They infect their hosts by rapidly penetrating the intestinal wall and entering the peritoneal cavity where they break through the liver capsule. After 8 -12 weeks of consistent burrowing, feeding, and growth within the liver parenchyma they move to their final destination within the bile ducts where they mature and produce enormous numbers of eggs (2). The two distinct clinical phases of fasciolosis are directly related to the migration of the parasites: acute fasciolosis, which manifests as fever, abdominal pain, weight loss, and hepatomegaly, is associated with...

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular “machinery” required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

Alternatively activated macrophages (AAM) 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. AAM were recruited to the peritoneum of mice within 24 h of F. hepatica infection and after intraperitoneal injection of parasite excretorysecretory (ES) products. Administration of a recombinant antioxidant thioredoxin peroxidase (TPx), which is contained within the ES products, also induced the recruitment of AAM 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 E 2 , 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.

During helminth infections, alternatively activated macrophages (AAMacs) are key to promoting Th2 responses and suppressing Th1-driven inflammatory pathology. Th2 cytokines IL-4 and/or IL-13 are believed to be important in the induction and activation of AAMacs. Using murine models for the helminth infections caused by Fasciola hepatica (Fh) and Schistosoma mansoni (Sm), we show that a secreted antioxidant, peroxiredoxin (Prx), induces alternative activation of macrophages. These activated, Ym1-expressing macrophages enhanced the secretion of IL-4, IL-5, and IL-13 from naive CD4(+) T cells. Administration of recombinant FhPrx and SmPrx to wild-type and IL-4(-/-) and IL-13(-/-) mice induced the production of AAMacs. In addition, Prx stimulated the expression of markers of AAMacs (particularly, Ym1) in vitro, and therefore can act independently of IL-4/IL-13 signaling. The immunomodulatory property of Prx is not due to its antioxidant activity, as an inactive recombinant variant with active site Cys residues replaced by Gly could also induce AAMacs and Th2 responses. Immunization of mice with recombinant Prx or passive transfer of anti-Prx antibodies prior to infection with Fh not only blocked the induction of AAMacs but also the development of parasite-specific Th2 responses. We propose that Prx activates macrophages as an initial step in the induction of Th2 responses by helminth parasites and is thereby a novel pathogen-associated molecular pattern.

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. q

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, Pf A-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the x-ray crystal structure of the monomeric Pf A-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the x-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the Pf A-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to Pf A-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in Pf A-M17 and one in Pf A-M1) in both substrate and drug binding. Our detailed understanding of the Pf A-M1 and Pf A-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.drug design | malaria | protease | structural biology | neutral aminopeptidases