Proteases in trematode biology.

Molecular & Cellular Proteomics

Menon

Donnelly

et al. 2009

248

249

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...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Relative Expression Levels Of Fasciola Proteases and Relatiomentioning

confidence: 99%

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An Integrated Transcriptomics and Proteomics Analysis of the Secretome of the Helminth Pathogen Fasciola hepatica

Molecular & Cellular Proteomics

Menon

Donnelly

et al. 2009

248

249

“…The prosegment is removed by catalytic cleavage following secretion into the parasite intestine to reveal an active mature protease (28). Our MS studies showed that all cathepsin L proteases secreted by adult F. hepatica lack the prosegment and are thus fully processed mature enzymes.…”

Section: Identification Of Adult F Hepatica Es Proteins By Nano-lc-ementioning

confidence: 99%

“…3. Despite the lack of conservation in this signature section, each still reserves an Asn residue and thereby retains a highly specific cleavage site for the trans-processing enzyme asparaginyl endopeptidase that initiates or "kick-starts" the enzyme activation process (28). Another highly conserved feature within the non-conserved C-terminal region of the prosegment is a Leu-Ser2His motif that we have shown is essential for autocatalytic cleavage between cathepsin L proteases once activation is initiated by the asparaginyl endopeptidase (20).…”

Section: Identification Of Adult F Hepatica Es Proteins By Nano-lc-ementioning

confidence: 99%

Proteomics and Phylogenetic Analysis of the Cathepsin L Protease Family of the Helminth Pathogen Fasciola hepatica

Molecular & Cellular Proteomics

Tort

Lowther

et al. 2008

117

131

Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-Ser2His motif for autocatalytic cleavage by cathepsin Ls were preserved.

The Phylogeny, Structure and Function of Trematode Cysteine Proteases, with Particular Emphasis on the Fasciola hepatica Cathepsin L Family

Stack

Dalton

Advances in Experimental Medicine and Biology

2011

Helminth parasites (nematodes, flatworms and cestodes) infect over 1 billion of the world's population causing high morbidity and mortality. The large tissue-dwelling worms express papain-like cysteine peptidases, termed cathepsins that play important roles in virulence including host entry, tissue migration and the suppression of host immune responses. Much of our knowledge of helminth cathepsins comes from studies using flatworms or trematode (fluke) parasites. The developmentally-regulated expression of these proteases correlates with the passage of parasites through host tissues and their encounters with different host macromolecules. Recent phylogenetic, biochemical and structural studies indicate that trematode cathepsins exhibit overlapping but distinct substrate specificities due to divergence within the protease active site. Here we provide an overview of the evolution, biochemistry and structure of these important enzymes and highlight how recent advances in proteomics and gene silencing techniques are allowing researchers to probe their biological functions. We focus mainly on members of the cathepsin L gene family of the animal and human pathogen, Fasciola hepatica, because of our deep understanding of their function, biochemistry and structure.