Research Proteomics and Phylogenetic Analysis of the Cathepsin L Protease Family of the Helminth Pathogen Fasciola hepatica EXPANSION OF A REPERTOIRE OF VIRULENCE-ASSOCIATED FACTORS* Mark W. Robinson‡§¶, Jose F. Tort‡ʈ, Jonathan Lowther‡, Sheila M. Donnelly‡, Emily Wong‡, Weibo Xu‡, Colin M. Stack‡**, Matthew Padula‡‡, Ben Herbert‡‡, and John P. Dalton‡§§ Cathepsin L proteases secreted by the helminth pathogen zymes. The prosegment region was highly conserved Fasciola hepatica have functions in parasite virulence in- between the clades except at the boundary of prosegment cluding tissue invasion and suppression of host immune and mature enzyme. Despite the lack of conservation at this Downloaded from responses. Using proteomics methods alongside phylo- section, sites for exogenous cleavage by asparaginyl en- genetic studies we characterized the profile of cathepsin dopeptidases and a Leu-Ser2His motif for autocatalytic L proteases secreted by adult F. hepatica and hence iden- cleavage by cathepsin Ls were preserved. Molecular & tified those involved in host-pathogen interaction. Phylo- Cellular Proteomics 7:1111–1123, 2008. genetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications https://www.mcponline.org followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) The helminth pathogens Fasciola hepatica and Fasciola and two clades specific to infective juvenile stages gigantica are the causative agents of liver fluke disease (fas- (Clades 3 and 4). Consistent with these observations our ciolosis) in sheep and cattle. Although infections of F. hepat- proteomics studies identified representatives from ica occur predominantly in regions with temperate climates, Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. the parasite has been reported on all continents (except Ant- hepatica secretory products. Clades 1 and 2 account for arctica) as a result of introduction by European settlers. In at UQ Library on April 27, 2020 67.39 and 27.63% of total secreted cathepsin Ls, respec- contrast, F. gigantica infections are largely restricted to trop- tively, suggesting that their expansion was positively driven and that these proteases are most critical for par- ical regions (1). Fasciolosis is also an important food-borne asite survival and adaptation. Sequence comparison stud- zoonotic disease of humans with estimates of 2.4–17 million ies revealed that the expansion of cathepsin Ls by gene people infected worldwide; a further 91.1 million people are duplication was followed by residue changes in the S2 currently living at risk of infection (2–4). Human disease is pocket of the active site. Our biochemical studies showed particularly prevalent in the Andean countries of South Amer- that these changes result in alterations in substrate bind- ica, Egypt, Iran, and Vietnam where farming practices allow ing and suggested that the divergence of the cathepsin L infected animals to roam among plants used for consumption family produced a repertoire of enzymes with overlapping (3, 4). Following ingestion of contaminated vegetation infec- and complementary substrate specificities that could tive parasite larvae migrate from the intestine into the liver cleave host macromolecules more efficiently. Although where they cause significant tissue injury and induce immu- the cathepsin Ls are produced as zymogens containing a nologically related damage before they move into the bile prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active en- ducts. The parasites can remain for up to 1–2 years in the bile ducts of cattle and as long as 20 years in sheep (1). Studies in our laboratory have shown that the most pre- From the ‡Institute for the Biotechnology of Infectious Diseases dominant molecules secreted by F. hepatica parasites in vitro and ‡‡Proteomics Technology Centre of Expertise, University of are cathepsin L cysteine proteases (5, 6), and a recent anal- Technology Sydney, Level 6, Building 4, corner of Thomas and Harris Streets, Ultimo, Sydney, New South Wales 2007, Australia, §School of ysis of bile taken from animals harboring adult parasites con- Medical Sciences, Institute of Medical Sciences, University of Aber- firmed that these enzymes also represent the majority of deen, Aberdeen AB25 2ZD, Scotland, United Kingdom, and ʈDeparta- protein produced in situ (7). The secretion of proteases facil- mento de Genetica, Facultad de Medicina, Universidad de la Repu´blica, itates migration of the parasite through host tissue and the General Flores 2125, CP 11800 Montevideo, Uruguay degradation of host macromolecules to provide essential free Received, November 26, 2007, and in revised form, February 20, 2008 amino acids for the parasite (6). Furthermore although it has Published, MCP Papers in Press, February 21, 2008, DOI 10.1074/ been known for several decades that fasciolid parasites se- mcp.M700560-MCP200 crete a variety of molecules that suppress the immune re- © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics 7.6 1111 This paper is available on line at http://www.mcponline.org Proteomics Analysis of F. hepatica Virulence Factors sponses of their host (8–10), cathepsin L proteases are con- the secretory products of the F. hepatica parasite, supporting sidered the principle participants; the parasite enzymes the suggestion of Irving et al. (12) that this subclade expanded cleave host antibodies specifically in the hinge region to pre- after the separation of the two species. Comparative bio- vent antibody-mediated cell damage (5) and alter the function chemistry and sequence alignments showed that the F. he- of cells of the innate and adaptive cellular immune systems patica repertoire of virulence-associated cathepsin Ls was to suppress the development of protective Th1-driven re- established by a process of gene duplication followed by sponses (11). refinement of the active site residues that influence substrate The F. hepatica cathepsin L proteases are represented by a specificity. Clade-specific variations also took place at the large gene family that expanded within the genus Fasciola by boundary between prosegment and mature enzyme, but spe- a series of gene duplications that resulted in a monophyletic cific cleavage sites required for activation of the cathepsin L group consisting of several discreet clades (12). The func- zymogens were preserved. tional diversity of the various members of the gene family and their relationship to pathogen virulence and host adaptation EXPERIMENTAL PROCEDURES are of particular interest (6, 12). Using molecular clock anal- Alignments and Phylogenetic Analysis—Phylogenetic trees were ysis, Irving et al. (12) estimated that the duplications and created using 32 selected F. hepatica and F. gigantica cathepsin L TM divergence of the family occurred over the last 135 million DNA sequences. Carica papaya papain (GenBank accession num- ber M15203) was used to root the fasciolid sequences. All of the years, and the timing of duplications correlates with the evo- Downloaded from nucleotide sequences used for tree construction encoded the full- lution of rodents, ruminants, and higher mammals. However, length cathepsin L precursor protein including the prosegment region most of the duplications took place ϳ25 million years ago at but excluding the signal peptide. The DNA sequences were initially about the time climatic conditions favored the development of aligned using ClustalW (14), and the trees were created using the grasslands and the expansion of common hosts of F. hepat- bootstrapped (1000 trials) neighbor-joining method of MEGA version 4.0 (15) using the Kimura 2 parameter model with uniform rates for all ica, suggesting that the divergence of the cathepsin L prote- https://www.mcponline.org sites. The GenBank accession numbers of the cathepsin L sequences ase family was important in the evolution and adaptation of used for alignment and phylogenetic analyses are as follows: the parasite to a wider host range (12). At the molecular FhCL1A_ie1 (U62288), FhCL1A_pe (AF490984), FhCL1A_au3 level, this divergence involved changes in residues within (L33771), FhCL1A_pl (AY277628), FhCL1A_pt1 (AY519971), the active site of the enzymes in particular at positions that FhCL1A_tr (AY573569), FhCL1B_nl1 (AY279092), FhCL1B_ar (AY029229), FgCL1C_id (AF510856), FhCL1C_jpA (AB010923), are known to occupy the S2 subsite and are critical to FhCL1C_jpB (AB009306), FgCL1C_cn (EF36899), FgCL1C_thA determining the substrate specificities of the proteases (6, (AF112566), FgCL1C_thB (AF239264), FhCL2_jn3 (AB010924), 12, 13). It was suggested that these changes gave rise to FhCL2_ie2 (U62289), FhCL2_chC (Z22765), FgCL2_thD at UQ Library on April 27, 2020 proteases with overlapping and complementary specificities (AF239266), FhCL3_nl64 (AJ279093), FgCL3_thG (AF419329), that allowed the parasite to degrade a wider variety of FhCL3_nl22 (AJ279091), FhCL3_uy5 (EU287914), FhCL3_uy9 (EU287915), FhCL3_pl1 (EU191984), FhCL3_pl2 (EU195859), macromolecules (6, 12). FhCL4_uy7 (EU287916), FhCL4_uy18 (EU287917), FgCL4_thH In the present study, we analyzed and characterized the (AY428949), FgCL5_thC (AF239265), FhCL5_au4 (L33772), and profile of cathepsin L proteases secreted by adult F. hepatica FhCL5_au5 (AF271385). The naming scheme reflects the different by two-dimensional gel electrophoresis (2-DE)1 and MS to clades identified and