Jason Mulvenna

The tumorigenic liver fluke Opisthorchis viverrini – multiple pathways to cancer

Liver fluke infection caused by Opisthorchis viverrini is a major public health problem in Thailand and adjacent countries. In addition to infection-associated morbidity, infection with O. viverrini and the related Clonorchis sinensis are unarguable risk factors for cholangiocarcinoma (CAA, bile-duct cancer). Here we review the pathogenesis of opisthorchiasis and the association between O. viverrini infection and bile-duct cancer, focusing on the molecular parallels between wound healing, chronic inflammation, and cancer development. We review a schema for human disease progression from fluke infection, chronic opisthorchiasis, advanced periductal fibrosis, and cholangiocarcinogenesis, and present a rationale for biomarker discovery to facilitate early intervention. We conclude by addressing post-genomic advances with a view to developing new control strategies to combat this infectious cancer.

Opisthorchiasis and Opisthorchis-associated cholangiocarcinoma in Thailand and Laos

Liver fluke infection caused by Opisthorchis viverrini is a major public health problem in Thailand and the Lao Peoples Democratic Republic (Lao PDR; Laos). Currently, more than 600 million people are at risk of infection with these fish-borne trematodes and/or their close relatives. Opisthorchiasis has been studied extensively in Thailand, where about 8 million people are infected with the liver fluke. Here we review the pathogenesis, control and re-emergence of O. viverrini infection, in particular in Thailand and, to a lesser extent in Lao PDR given the contiguous geographical range of O. viverrini through these two regions. We also review the association of O. viverrini infection and cholangiocarcinoma, bile duct cancer, and highlight new findings on pathogenesis of liver fluke-induced cholangiocarcinogenesis. Last, we comment on national control strategies in Thailand for the control of O. viverrini infection aimed at reduction in the prevalence of O. viverrini-associated liver cancer in the longer term.

Proteomics Analysis of the Excretory/Secretory Component of the Blood-feeding Stage of the Hookworm, Ancylostoma caninum

Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting ∼600 million people worldwide. These parasites form an intimate association with the host and are able to avoid vigorous immune responses in many ways including skewing of the response phenotype to promote parasite survival and longevity. The primary interface between the parasite and the host is the excretory/secretory component, a complex mixture of proteins, carbohydrates, and lipids secreted from the surface or oral openings of the parasite. The composition of this complex mixture is for the most part unknown but is likely to contain proteins important for the parasitic lifestyle and hence suitable as drug or vaccine targets. Using a strategy combining the traditional technology of one-dimensional SDS-PAGE and the newer fractionation technology of OFFGEL electrophoresis we identified 105 proteins from the excretory/secretory products of the blood-feeding stage of the dog hookworm, Ancylostoma caninum. Highly represented among the identified proteins were lectins, including three C-type lectins and three β-galactoside-specific S-type galectins, as well as a number of proteases belonging to the three major classes found in nematodes, aspartic, cysteine, and metalloproteases. Interestingly 28% of the identified proteins were homologous to activation-associated secreted proteins, a family of cysteine-rich secreted proteins belonging to the sterol carrier protein/Tpx-1/Ag5/PR-1/Sc-7 (TAPS) superfamily. Thirty-four of these proteins were identified suggesting an important role in host-parasite interactions. Other protein families identified included hyaluronidases, lysozyme-like proteins, and transthyretin-like proteins. This work identified a suite of proteins important for the parasitic lifestyle and provides new insight into the biology of hookworm infection.

A Granulin-Like Growth Factor Secreted by the Carcinogenic Liver Fluke, Opisthorchis viverrini, Promotes Proliferation of Host Cells

The human liver fluke, Opisthorchis viverrini, infects millions of people throughout south-east Asia and is a major cause of cholangiocarcinoma, or cancer of the bile ducts. The mechanisms by which chronic infection with O. viverrini results in cholangiocarcinogenesis are multi-factorial, but one such mechanism is the secretion of parasite proteins with mitogenic properties into the bile ducts, driving cell proliferation and creating a tumorigenic environment. Using a proteomic approach, we identified a homologue of human granulin, a potent growth factor involved in cell proliferation and wound healing, in the excretory/secretory (ES) products of the parasite. O. viverrini granulin, termed Ov-GRN-1, was expressed in most parasite tissues, particularly the gut and tegument. Furthermore, Ov-GRN-1 was detected in situ on the surface of biliary epithelial cells of hamsters experimentally infected with O. viverrini. Recombinant Ov-GRN-1 was expressed in E. coli and refolded from inclusion bodies. Refolded protein stimulated proliferation of murine fibroblasts at nanomolar concentrations, and proliferation was inhibited by the MAPK kinase inhibitor, U0126. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of murine fibroblasts and a human cholangiocarcinoma cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumorigenic environment that may ultimately manifest as cholangiocarcinoma.

Exposed proteins of the Schistosoma japonicum tegument.

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.

The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini

Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products and, utilizing selective labeling and sequential solubilization, from the host‐exposed tegument. The excretory/secretory included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti‐apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno‐modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin‐2. Novel roles in pathogenesis were suggested for the tegument–host interface since more than ten surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke.

Genome of the human hookworm Necator americanus

The hookworm Necator americanus is the predominant soil-transmitted human parasite. Adult worms feed on blood in the small intestine, causing iron-deficiency anemia, malnutrition, growth and development stunting in children, and severe morbidity and mortality during pregnancy in women. We report sequencing and assembly of the N. americanus genome (244 Mb, 19,151 genes). Characterization of this first hookworm genome sequence identified genes orchestrating the hookworms invasion of the human host, genes involved in blood feeding and development, and genes encoding proteins that represent new potential drug targets against hookworms. N. americanus has undergone a considerable and unique expansion of immunomodulator proteins, some of which we highlight as potential treatments against inflammatory diseases. We also used a protein microarray to demonstrate a postgenomic application of the hookworm genome sequence. This genome provides an invaluable resource to boost ongoing efforts toward fundamental and applied postgenomic research, including the development of new methods to control hookworm and human immunological diseases.

CyBase: a database of cyclic protein sequence and structure

CyBase is a curated database and information source for backbone-cyclized proteins. The database incorporates naturally occurring cyclic proteins as well as synthetic derivatives, grafted analogues and acyclic permutants. The database provides a centralized repository of information on all aspects of cyclic protein biology and addresses issues pertaining to the management and searching of topologically circular sequences. The database is freely available at .

Generalized urticaria induced by the Na-ASP-2 hookworm vaccine: Implications for the development of vaccines against helminths

BACKGROUND Necator americanus Ancylostoma-secreted protein 2 (Na-ASP-2) is secreted by infective hookworm larvae on entry into human hosts. Vaccination of laboratory animals with recombinant Na-ASP-2 provides significant protection against challenge infections. In endemic areas antibodies to Na-ASP-2 are associated with reduced risk of heavy N americanus infections. OBJECTIVE To assess the safety and immunogenicity of recombinant Na-ASP-2 adjuvanted with Alhydrogel in healthy Brazilian adults previously infected with N americanus. METHODS Participants were randomized to receive Na-ASP-2 or hepatitis B vaccine. Major IgG and IgE epitopes of the Na-ASP-2 molecule were mapped by using sera from these same subjects. Seroepidemiologic studies in adults and children residing in hookworm-endemic areas were conducted to assess the prevalence of IgE responses to Na-ASP-2. RESULTS Vaccination with a single dose of Na-ASP-2 resulted in generalized urticarial reactions in several volunteers. These reactions were associated with pre-existing Na-ASP-2-specific IgE likely induced by previous hookworm infection. Surveys revealed that a significant proportion of the population in hookworm-endemic areas had increased levels of IgE to Na-ASP-2. Epitope mapping demonstrated sites on the Na-ASP-2 molecule that are uniquely or jointly recognized by IgG and IgE antibodies. CONCLUSION Infection with N americanus induces increased levels of total and specific IgE to Na-ASP-2 that result in generalized urticaria on vaccination with recombinant Na-ASP-2. These data advance knowledge of vaccine development for helminths given their propensity to induce strong T(H)2 responses. Study data highlight the important differences between the immune responses to natural helminth infection and to vaccination with a recombinant helminth antigen.

An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection

Hookworms digest hemoglobin from erythrocytes via a proteolytic cascade that begins with the aspartic protease, APR‐1. Ac‐APR‐1 from the dog hookworm, Ancylostoma caninum, protects dogs against hookworm infection via antibodies that neutralize enzymatic activity and interrupt blood‐feeding. Toward developing a human hookworm vaccine, we expressed both wild‐type (Na‐APR‐1wt) and mutant (Na‐APR‐1mut—mutagenesis of the catalytic aspartic acids) forms of Na‐APR‐1 from the human hookworm, Necator americanus. Refolded Na‐APR‐1wt was catalytically active, and Na‐APR‐1mut was catalytically inactive but still bound substrates. Vaccination of canines with Na‐APR‐1mut and heterologous challenge with A. caninum resulted in significantly reduced parasite egg burdens (P=0.034) and weight loss (P=0.022). Vaccinated dogs also had less gut pathology, fewer adult worms, and reduced blood loss compared to controls but these did not reach statistical significance. Vaccination with Na‐APR‐1mut induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Na‐APR‐1wt and APR‐1 orthologues from three other hookworm species that infect humans. IgG1 against Na‐APR‐1mut was the most prominently detected antibody in sera from people resident in high‐transmission areas for N. americanus, indicating that natural boosting may occur in exposed humans. Na‐APR‐1mut is now a lead antigen for the development of an antihematophagy vaccine for human hookworm disease.—Pearson, M. S., Bethony, J. M., Pickering, D. A., de Oliveira, L. M., Jariwala, A., Santiago, H., Miles, A. P., Zhan, B., Jiang, D., Ranjit, N., Mulvenna, J., Tribolet, L., Plieskatt, J., Smith, T., Bottazzi, M. E., Jones, K., Keegan, B., Hotez, P. J., Loukas, A. An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection. FASEB J. 23, 3007–3019 (2009). www.fasebj.org