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

Abstract

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.

Fig. 1.

Analysis of F. hepatica somatic and secretory proteins by 1-DE. Shown is the typical 1-D profile of somatic proteins expressed by dormant F. hepatica larvae as well as proteins secreted by F. hepatica NEJs (NEJ), 21-day-old immature flukes (Immature), and adult parasites (Adult). Proteins (10 μg) were separated using NuPAGE Novex 4–12% Bis-Tris gels (Invitrogen) and stained with colloidal Coomassie Blue G-250. Following trypsin digests, peptides were extracted from gel sections 1–6 and analysed by mass spectrometry.

Fig. 2.

Expression of F. hepatica proteases. A, the expression levels of the major proteolytic enzymes used by F. hepatica during its mammalian life cycle. The label-free emPAI (22) derived from Fasciola MS/MS CID data was used to determine the abundance of the various cathepsin Ls (FhCL), cathepsin Bs (FhCB), and asparaginyl endopeptidases (FhAE) that are expressed by juvenile flukes (dormant larvae) and secreted by the immature (liver stage) flukes and adult (bile duct stage) parasites. B, identification of F. hepatica proteases by nano-LC-ESI-MS/MS. Dormant larvae somatic proteins and those secreted by NEJs, 21-day old immature flukes and adult F. hepatica were separated by 1-DE and analyzed by nano-LC-ESI-MS/MS. Mass spectrometry data were submitted to either the MSDB or a custom-made database composed of all F. hepatica ESTs (14,031 entries) currently available from the Wellcome Trust Sanger Centre using MASCOT and PEAKS software, respectively. Adult F. hepatica ESTs that matched with MS/MS data were submitted as queries to BLASTn (18) or conceptually translated and submitted to InterProScan (19) to detect conserved domains and motifs. Footnotes are as follows. 1, MASCOT scores are given for matches to F. hepatica cDNAs. 2, number of unique peptide matches. 3, raw emPAI values provided by the MASCOT search engine give an approximate quantification of the protein (22). These values were converted to a percentage of total protease expression within each Fasciola developmental stage (see “Experimental Procedures”) and were used to generate the pie charts shown in A. 4, section of the 1-D gel from which the peptides were identified. 5, PEAKS scores (expressed as a percentage) are given for matches to peptides encoded by F. hepatica ESTs. 6, single peptide-based identification (see supplemental Fig. 4).

Fig. 3.

Expression of F. hepatica antioxidants. A, the expression levels of the major antioxidant molecules used by F. hepatica during its mammalian life cycle. The emPAI values (22) derived from Fasciola CID data were used to determine the abundance of the various antioxidants that are expressed and/or secreted by juvenile flukes (dormant larvae and NEJs) and secreted by the immature (liver stage) flukes and adult (bile duct stage) parasites. B, identification of F. hepatica antioxidants by nano-LC-ESI-MS/MS. Dormant larvae somatic proteins and those secreted by NEJs, 21-day-old immature flukes, and adult F. hepatica were separated by 1-DE and analyzed by nano-LC-ESI-MS/MS. MASCOT searches were performed against the MSDB. Footnotes are as follows. 1, number of unique peptide matches. 2, raw emPAI values provided by the MASCOT search engine give an approximate quantification of the protein (22). These values were converted to a percentage of total antioxidant expression within each Fasciola developmental stage (see “Experimental Procedures”) and were used to generate the pie charts shown in A. 3, section of the 1-D gel from which the peptides were identified.