Observations on the feeding behaviour of parasitic third-stage hookworm larvae

Hawdon, John M.; Volk, Susan W.; Rose, Richard A.; Pritchard, David; Behnke, Jerzy M.; Schad, Gerhard A.

doi:10.1017/s0031182000074953

Cited by 29 publications

(27 citation statements)

References 17 publications

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“…RNA was also isolated from 5,000 L3 that had been activated by incubating them in 50% fetal bovine serum for 2 h at 37°C. This process has been shown to induce feeding of hookworm L3 and upregulate the expression of selected genes (27)(28)(29)(30)(31)(32). The primers used for the RT-PCR corresponded to a 200-bp fragment of the AceKI cDNA.…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA was isolated from A. ceylanicum L3 cultured from the feces of infected hamsters (21,22), as well as from adult worms. Additional RNA was isolated from A. ceylanicum L3 that had been activated by incubation in 50% fetal calf serum, which has been shown to stimulate feeding of hookworm larvae and upregulate expression of certain genes (27)(28)(29)(30)(31)(32). As shown in Fig.…”

Section: Vol 72 2004 Hookworm Kunitz Inhibitor 2215mentioning

confidence: 99%

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Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

Chu¹,

Bungiro²,

Ibanez³

et al. 2004

Infect Immun

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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 Met 26 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.Hookworm infection remains a major global health problem, and over one billion people are reportedly infected in developing countries (9, 14). Hookworms, which are bloodfeeding intestinal nematodes, are a major cause of iron deficiency anemia and malnutrition (15,20,(59)(60)(61)64). While the anemia is presumably due to the cumulative effect of chronic intestinal blood loss, the molecular mechanisms underlying the pathogenesis of hookworm malnutrition remain unknown. Although it has been suggested that hookworm malnutrition and growth delay occur secondary to chronic iron deficiency, particularly in children, evidence from prior clinical studies suggests that hookworm infection is also associated with various degrees of intestinal malabsorption (18,35,54,57,62). It has been hypothesized that this hookworm malabsorption syndrome might occur secondary to mucosal inflammation triggered by the adult worm attached to the intestinal epithelium or might be a result of secretion of parasite inhibitors of host digestive enzymes (18).As part of a series of ongoing studies aimed at characterizing adult hookworm secretory proteins, a cDNA corresponding to the gene encoding a putative Kunitz-type serine protease inhibitor was previously identified from adult Ancylostoma ceylanicum RNA by using a PCR-based approach (48). The A. ceylanicum Kunitz-type inhibitor (AceKI) cDNA was expressed in Escherichia coli, and the recombinant protein was found to inhibit the pancreatic enzymes chymotrypsin, pancreatic elastase, and trypsin in vitro, with equilibrium inhibitory dissociation constant (K i ) values ranging from picomolar levels to low nanomolar levels. The native AceKI prote...

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Section: Methodsmentioning

confidence: 99%

Section: Vol 72 2004 Hookworm Kunitz Inhibitor 2215mentioning

confidence: 99%

Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

Chu¹,

Bungiro²,

Ibanez³

et al. 2004

Infect Immun

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“…ceylanicum larvae (22,23) were generously provided by John Hawdon and Peter Hotez at The Yale University School of Medicine, and the parasite life cycle was maintained as described previously (24). Three-week-old Syrian hamsters (LVG strain) were infected orally by gavage with 100 -150 L 3 per animal.…”

Section: Hookworms--infective Third Stage (L 3 )mentioning

confidence: 99%

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

Milstone

Harrison

Bungiro

et al. 2000

Journal of Biological Chemistry

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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 coli transformed with the rAceKI-1/pET 28a plasmid, and in vitro studies 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.

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“…Blood loss resulting from A. ceylanicum infection can cause severe anemia. Eosinophilic enteritis is presumed to be an allergic response to hookworm secretions (Hawdon et al 1993;Hotez et al 1995;Harrop et al 1996). Infections with this nematode are very common in tropical regions, especially in Asia (Hotez et al 1995;Chan 1997).…”

Section: Introductionmentioning

confidence: 99%

“…Proteinases of larval and adult stages of A. caninum, the closest relative of A. ceylanicum, have been extensively studied. Apart from cysteine proteinases, metalloproteinases (Dowd et al 1994;Hawdon et al 1993) and aspartic proteinases (Harrop et al 1995a, b;Brown et al 1999) have been found. By contrast, little is known about proteinases of A. ceylanicum and the role of these enzymes in host-parasite interactions.…”

Section: Introductionmentioning

confidence: 99%

Molecular cloning of a cysteine proteinase cDNA from adult Ancylostoma ceylanicum by the method of rapid amplification of cDNA ends using polymerase chain reaction

Mieszczanek¹,

Kofta²,

Wędrychowicz³

2000

Parasitol Res

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The hookworm Ancylostoma ceylanicum is a parasite of great importance in human and veterinary medicine. The most promising vaccination trials against hookworm infections are based on antigens belonging to the proteinase family. The aim of the present research was to isolate a cysteine proteinase gene from A. ceylanicum. This was achieved by rapid amplification of cDNA ends using polymerase chain reaction (RACE-PCR). A set of consensus oligonucleotide primers was designed to anneal to the conserved coding regions of cysteine proteinase. The PCR products were cloned and sequenced. The novel sequence displayed a high degree of homology with genes of cysteine proteinases known from other hookworm species. In the coding region the nucleotide identity with accp-1, the cysteine proteinase gene of A. caninum, reaches 84.3%. Analysis of the expression of acey-1. the cysteine proteinase gene of A. ceylanicum, suggests that it is produced exclusively in the gland cells of either adult worms or blood-feeding stages of A. ceylanicum.

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Observations on the feeding behaviour of parasitic third-stage hookworm larvae

Cited by 29 publications

References 17 publications

Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

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

Molecular cloning of a cysteine proteinase cDNA from adult Ancylostoma ceylanicum by the method of rapid amplification of cDNA ends using polymerase chain reaction

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