Lisa M. Harrison

A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylanicum

Crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) are harmless to vertebrates, but they are highly toxic to insects and nematodes. Their value in controlling insects that destroy crops and transmit human diseases is well established. Although it has recently been demonstrated that a few individual Bt Cry proteins, such as Cry5B, are toxic to a wide range of free-living nematodes, the potential activity of purified Cry proteins against parasitic nematodes remains largely unknown. We report here studies aimed at characterizing in vitro and in vivo anthelminthic activities of purified recombinant Cry5B against the hookworm parasite Ancylostoma ceylanicum, a bloodfeeding gastrointestinal nematode for which humans are permissive hosts. By using in vitro larval development assays, Cry5B was found to be highly toxic to early stage hookworm larvae. Exposure of adult A. ceylanicum to Cry5B was also associated with significant toxicity, including a substantial reduction in egg excretion by adult female worms. To demonstrate therapeutic efficacy in vivo, hamsters infected with A. ceylanicum were treated with three daily oral doses of purified Cry5B, the benzimidazole anthelminthic mebendazole, or buffer. Compared with control (buffer-treated) animals, infected hamsters that received Cry5B showed statistically significant improvements in growth and blood hemoglobin levels as well as reduced worm burdens that were comparable to the mebendazole-treated animals. These data demonstrate that Cry5B is highly active in vitro and in vivo against a globally significant nematode parasite and that Cry5B warrants further clinical development for human and veterinary use.

Isolation and molecular cloning of a secreted hookworm platelet inhibitor from adult Ancylostoma caninum.

Hookworms, bloodfeeding intestinal nematodes, are a leading cause of iron deficiency anemia in the developing world. These parasites have evolved potent mechanisms of interfering with mammalian hemostasis, presumably for the purpose of facilitating bloodfeeding. Adult Ancylostoma caninum worm extracts contain an activity that inhibits platelet aggregation and adhesion by blocking the function of two cell surface integrin receptors, Glycoprotein IIb/IIIa and GPIa/IIa. Using rpHPLC, the hookworm platelet inhibitor activities have been purified from protein extracts of A. caninum. Because the two inhibitory activities co-purified through multiple chromatographic steps, have similar molecular masses and share identical N-terminal as well as internal amino acid sequence homology, it is likely that they represent a single gene product. A cDNA corresponding to the purified hookworm platelet inhibitor (HPI) protein has been cloned from adult A. caninum RNA, and the translated amino acid sequence shows significant homology to Neutrophil Inhibitory Factor and Ancylostoma Secreted Proteins, suggesting that these related hookworm proteins represent a novel class of integrin receptor antagonists. Polyclonal antibodies raised against the recombinant HPI protein recognize corresponding native proteins in A. caninum extracts and excretory/secretory products, and immunohistochemistry data have identified the cephalic glands as the major source of the inhibitor within the adult hookworm. These data suggest that HPI is secreted by the adult stage of the parasite at the site of intestinal attachment. As such, it may represent a viable target for a vaccine-based strategy aimed at interfering with hookworm-induced gastrointestinal hemorrhage and iron deficiency anemia.

Structural and Functional Characterization of a Secreted Hookworm Macrophage Migration Inhibitory Factor (MIF) That Interacts with the Human MIF Receptor CD74

Hookworms, parasitic nematodes that infect nearly one billion people worldwide, are a major cause of anemia and malnutrition. We hypothesize that hookworms actively manipulate the host immune response through the production of specific molecules designed to facilitate infection by larval stages and adult worm survival within the intestine. A full-length cDNA encoding a secreted orthologue of the human cytokine, Macrophage Migration Inhibitory Factor (MIF) has been cloned from the hookworm Ancylostoma ceylanicum. Elucidation of the three-dimensional crystal structure of recombinant AceMIF (rAceMIF) revealed an overall structural homology with significant differences in the tautomerase sites of the human and hookworm proteins. The relative bioactivities of human and hookworm MIF proteins were compared using in vitro assays of tautomerase activity, macrophage migration, and binding to MIF receptor CD74. The activity of rAceMIF was not inhibited by the ligand ISO-1, which was previously determined to be an inhibitor of the catalytic site of human MIF. These data define unique immunological, structural, and functional characteristics of AceMIF, thereby establishing the potential for selectively inhibiting the hookworm cytokine as a means of reducing parasite survival and disease pathogenesis.

A Broad Spectrum Kunitz Type Serine Protease Inhibitor Secreted by the Hookworm Ancylostoma ceylanicum

Although blood-feeding hookworms infect over a billion people worldwide, little is known about the molecular mechanisms through which these parasitic nematodes cause gastrointestinal hemorrhage and iron deficiency anemia. A cDNA corresponding to a secreted Kunitz type serine protease inhibitor has been cloned from adult Ancylostoma ceylanicum hookworm RNA. The translated sequence of the A. ceylanicum Kunitz type inhibitor 1 (AceKI-1) cDNA predicts a 16-amino acid secretory signal sequence, followed by a 68-amino acid mature protein with a molecular mass of 7889 daltons. Recombinant protein (rAceKI-1) was purified from induced lysates of Escherichia colitransformed with the rAceKI-1/pET 28a plasmid, and in vitrostudies demonstrate that rAceKI-1 is a tight binding inhibitor of the serine proteases chymotrypsin, pancreatic elastase, neutrophil elastase, and trypsin. AceKI-1 inhibitory activity is present in soluble protein extracts and excretory/secretory products of adult hookworms but not the infective third stage larvae. The native AceKI-1 inhibitor has been purified to homogeneity from soluble extracts of adult A. ceylanicum using size exclusion and reverse-phase high pressure liquid chromatography. As a potent inhibitor of mammalian intestinal proteases, AceKI-1 may play a role in parasite survival and the pathogenesis of hookworm anemia.

Epidemiology of Hookworm Infection in Kintampo North Municipality, Ghana: Patterns of Malaria Coinfection, Anemia, and Albendazole Treatment Failure

A cross-sectional pilot study of hookworm infection was carried out among 292 subjects from 62 households in Kintampo North, Ghana. The overall prevalence of hookworm infection was 45%, peaking in those 11-20 years old (58.5%). In children, risk factors for hookworm infection included coinfection with malaria and increased serum immunoglobulin G reactivity to hookworm secretory antigens. Risk factors for infection in adults included poor nutritional status, not using a latrine, not wearing shoes, and occupation (farming). Although albendazole therapy was associated with an overall egg reduction rate of 82%, 37 subjects (39%) remained infected. Among those who failed therapy, treatment was not associated with a significant reduction in egg excretion, and nearly one-third had higher counts on repeat examination. These data confirm a high prevalence of low-intensity hookworm infection in central Ghana and its association with poor nutritional status. The high rate of albendazole failure raises concern about emerging resistance.

Regulation of Proinflammatory Cytokine Expression by Shiga Toxin 1 and/or Lipopolysaccharides in the Human Monocytic Cell Line THP-1

ABSTRACT Infection with Shiga toxin (Stx)-producing bacteria and the subsequent release of Stxs and endotoxins into the bloodstream may damage blood vessels in the colon, kidneys, and central nervous system, leading to bloody diarrhea, acute renal failure, and neurological complications. The proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) may contribute to the pathogenesis of Stx-induced vascular lesions by up-regulating toxin receptor expression on endothelial cells. We previously showed that macrophages treated with purified Shiga toxin 1 (Stx1) or lipopolysaccharides (LPS) secrete TNF-α and IL-1β. Northern blot analysis revealed that treatment of the human monocytic cell line THP-1 with LPS induced a rapid and transient increase in steady-state TNF-α and IL-1β transcripts. In contrast, Stx1 induced slower but prolonged elevations in cytokine transcripts. The presence of both stimulants resulted in optimal cytokine mRNA induction in terms of kinetics and prolonged expression. Compared to LPS, Stx1 was a poor inducer of IL-1β protein expression, although levels of soluble IL-1β induced by all treatments continually increased over 72 h. IL-1β transcripts were not induced by Stx1 B-subunits. Using the transcriptional inhibitor actinomycin D, we determined that treatment with Stx1 or Stx1 plus LPS induced cytokine transcripts with increased stability compared to transcripts induced by LPS alone. For all treatments, IL-1β mRNA decay was slower than TNF-α. Collectively, our data suggest that Stxs affect cytokine expression, in part, at the posttranscriptional level by stabilizing mRNAs. Optimal TNF-α expression occurs when both Stxs and LPS are present.

Chemokine Expression in the Monocytic Cell Line THP-1 in Response to Purified Shiga Toxin 1 and/or Lipopolysaccharides

ABSTRACT Infections with Shiga toxin (Stx)-producing bacteria are associated with bloody diarrhea and postdiarrheal sequelae, including hemolytic uremic syndrome and central nervous system (CNS) abnormalities. Stx-induced intestinal, renal, and CNS vascular lesions may involve a localized production of proinflammatory cytokines in target organs, as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) up-regulate Stx receptor globotriaosylceramide (Gb3) expression on vascular endothelial cells. However, leukocyte recruitment to injured sites may also exacerbate vascular damage. A cytokine macroarray analysis of transcripts derived from macrophage-like THP-1 cells treated with Stx1, lipopolysaccharides (LPS), or both demonstrated a consistent up-regulation of TNF-α, IL-1β, and four genes encoding the chemokines interleukin-8 (IL-8), macrophage inflammatory protein-1α (MIP-1α), MIP-1β, and growth-related oncogene beta (GRO-β). Real-time PCR analysis verified the macroarray results. Northern blot analyses after the addition of the transcriptional inhibitor actinomycin D revealed increased IL-8 mRNA stability in THP-1 cells treated with Stx1 or Stx1 plus LPS. Finally, enzyme-linked immunosorbent assay data for Stx1- plus LPS-treated cells demonstrated a poor correlation between IL-8, MIP-1α, MIP-1β, and GRO-β mRNA levels and protein production, indicating a posttranscriptional regulatory effect. Our data suggest that in response to Stx1 and LPS, macrophages may be a source of chemokines that promote tissue damage through leukocyte recruitment and activation.

Molecular characterization of Ancylostoma ceylanicum Kunitz-type serine protease inhibitor: evidence for a role in hookworm-associated growth delay.

ABSTRACT Hookworm infection is a major cause of iron deficiency anemia and malnutrition in developing countries. The Ancylostoma ceylanicum Kunitz-type inhibitor (AceKI) is a 7.9-kDa broad-spectrum inhibitor of trypsin, chymotrypsin, and pancreatic elastase that has previously been isolated from adult hookworms. Site-directed mutagenesis of the predicted P1 inhibitory reactive site amino acid confirmed the role of Met26 in mediating inhibition of the three target serine proteases. By using reverse transcription-PCR, it was demonstrated that the level of AceKI gene expression increased following activation of third-stage larvae with serum and that the highest level of expression was reached in the adult stage of the parasite. Immunohistochemistry studies performed with polyclonal immunoglobulin G raised against recombinant AceKI showed that the inhibitor localized to the subcuticle of the adult hookworm, suggesting that it has a potential in vivo role in neutralizing intestinal proteases at the surface of the parasite. Immunization with recombinant AceKI was shown to confer partial protection against hookworm-associated growth delay without a measurable effect on anemia. Taken together, the data suggest that AceKI plays a role in the pathogenesis of hookworm-associated malnutrition and growth delay, perhaps through inhibition of nutrient absorption in infected hosts.

Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum.

Hookworms, bloodfeeding intestinal nematodes, infect nearly one billion people in resource limited countries and are a leading cause of anaemia and malnutrition. Like other nematodes, hookworms lack the capacity to synthesise essential fatty acids de novo and therefore must acquire those from exogenous sources. The cDNA corresponding to a putative Ancylostoma ceylanicum fatty acid and retinol binding protein-1 (AceFAR-1) was amplified from adult hookworm mRNA. Studies using quantitative reverse transcriptase real-time PCR demonstrate that AceFAR-1 transcripts are most abundant in the earliest developmental stages of the parasite, and greater in females than males. Using in vitro assays, the recombinant AceFAR-1 (rAceFAR-1) was shown to bind individual fatty acids with equilibrium dissociation constants in the low micromolar range. The pattern of fatty acid uptake by live adult worms cultured ex vivo was similar to the in vitro binding profile of rAceFAR-1, raising the possibility that the native protein may be involved in acquisition of fatty acids by A. ceylanicum. Animals vaccinated orally with rAceFAR-1 and the mucosal adjuvant cholera toxin exhibited a statistically significant (40-47%) reduction in intestinal worm burden compared with controls immunized with antigen or adjuvant alone. Together, these data suggest a potential role for AceFAR-1 in hookworm biology, making it a potentially valuable target for drug and vaccine development.

Role for Nitric Oxide in Hookworm-Associated Immune Suppression

ABSTRACT Hookworm infection is a major cause of anemia and malnutrition in resource-poor countries. Human and animal studies suggest that infection with these intestinal nematodes is associated with impaired cellular immunity, characterized by reduced lymphocyte proliferation in response to both parasite and heterologous antigens. We report here data from studies aimed at defining mechanisms through which hookworms modulate the host cellular immune response. Splenocytes and mesenteric lymph node (MLN) cells from hamsters infected with Ancylostoma ceylanicum showed minimal proliferation in response to mitogen at days 20 and 30 postinfection (p.i.), with partial recovery noted at day 70 p.i. The proliferative capacity of enriched splenocyte T-cell preparations from infected animals following stimulation with hookworm antigens was partially restored in the presence of antigen-presenting cells from uninfected hamsters. Analysis by fluorescence-activated cell sorting revealed that hookworm infection is associated with reduced percentages of both CD4+ and surface immunoglobulin G-positive lymphocytes in the spleen and MLN cells. Splenocytes from infected hamsters also secreted more nitric oxide (NO) in culture than did those from naïve animals. Inhibition of NO secretion was associated with partial restoration of the proliferative capacity of splenocytes from infected animals in response to concanavalin A, suggesting a role for NO in mediating this effect. Together, these data demonstrate that hookworm infection is associated with impaired function of antigen-presenting cells and depletion of important lymphocyte subpopulations and also suggests a role for NO in parasite-induced immunosuppression.