Ancylostoma caninum: Metalloprotease Release Coincides with Activation of Infective Larvae in Vitro

Hawdon, John M.; Jones, Brian F.; Perregaux, M.; Hotez, Peter J.

doi:10.1006/expr.1995.1025

Cited by 77 publications

(41 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…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%

See 1 more Smart Citation

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

View full text Add to dashboard Cite

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...

show abstract

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

View full text Add to dashboard Cite

show abstract

“…When L3 of the canine hookworm A. caninum are activated in vitro, they release metalloproteases (Hawdon et al, 1995) and other molecules of unknown function referred to as Ancylostoma Secreted Protein 1 and 2 (Ac-ASP-1, Ac-ASP-2; Hawdon et al, 1996;Hawdon et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Necator americanus: The Na-ASP-2 protein secreted by the infective larvae induces neutrophil recruitment in vivo and in vitro

Bower¹,

Constant²,

Méndez³

2008

Experimental Parasitology

View full text Add to dashboard Cite

The L3-secreted Ancylostoma Secreted Protein-2 from the human hookworm Necator americanus (Na-ASP-2) has been selected as a candidate vaccine antigen in anticipation of clinical trials. Its crystal structure revealed that Na-ASP-2 has structural and charge similarities to CC-chemokines, suggesting that it might act as a chemokine mimic when released by the infective larvae during tissue migration. Using the air pouch model of acute inflammation, we found that Na-ASP-2 induced a significant leukocyte influx to the skin pouch, mostly comprised of neutrophils (60%) and monocytes (30%) that was transient and resolved in 24 h. Other hookworm larval proteins did not cause any inflammatory leukocytes to migrate into air pouches. In vitro chemotaxis assays confirmed our results and demonstrated that leukocyte migration was a direct effect of Na-ASP-2 exposure and not caused by other molecules released by host cells in the inflammatory microenvironment or by the expression vector.

show abstract

“…The basic function of these enzymes, according to some authors, may be connected to the penetration of the larvae in host tissues, since they have been found in abundance in the infesting stages of Strongyloides stercoralis (Brindley et al 1995, Gallego et al 2005, Ancylostoma spp. (Hawdon et al 1995), and the E/SP of L1 Dermatobia hominis (Brant et al 2010).…”

Section: Discussionmentioning

confidence: 99%

Proteolytic activity of excretory/secretory products of Cochliomyia hominivorax larvae (Diptera: Calliphoridae)

et al. 2016

View full text Add to dashboard Cite

The protein profiles and proteolytic activity of the excretory secretory products (E/SP) of the first (L1), second (L2) and third (L3) larval stages of Cochliomyia hominivorax were studied in the laboratory. Analysis on the E/SP protein profile was carried out using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). The E/SP of each larval stage (L1, L2 and L3) treated with protease inhibitors, containing 30μg, 40μg and 50μg of protein, was applied to the 10% polyacrylamide gel. The proteolytic activity of the crude E/SP was analyzed in gels copolymerized with gelatin and by colorimetric assays using azocasein as a substrate, with the characterization of the proteases using synthetic inhibitors. Different protein profiles were observed for the larval instars, with L1 presenting the most complex profile. Nevertheless, various protein bands were observed that were common to all the larval instars. The E/SP of all the instars showed proteolytic activity on gelatin, evidenced by proteolysis zones, predominantly with apparently higher molecular masses in L1, while for L2 and L3 the proteolysis zones could also be observed in regions with lower masses. Tests with protease inhibitors using gelatin as substrate showed that the E/SP of larvae were mainly composed of serine proteases. Additionally, inhibition was observed in L2 E/SP treated previously with EDTA, an inhibitor of metalloproteases. The assays with azocasein revealed a gradual increase of proteolytic activity on this substrate with larval development progress, with the strongest inhibitions being observed after treatments with 3,4-dichloroisocoumarin (DCI) for E/SP of L1, L2 and L3. These results suggest that C. hominivorax larvae produce different proteases, a fact that can be related to the parasite's vital processes for survival, such as penetration into the host's tissues and nutrition during the larval stage.

show abstract

Ancylostoma caninum: Metalloprotease Release Coincides with Activation of Infective Larvae in Vitro

Cited by 77 publications

References 0 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

Necator americanus: The Na-ASP-2 protein secreted by the infective larvae induces neutrophil recruitment in vivo and in vitro

Proteolytic activity of excretory/secretory products of Cochliomyia hominivorax larvae (Diptera: Calliphoridae)

Contact Info

Product

Resources

About