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The nematode neuropeptide, AF2 (KHEYLRF-NH2), increases voltage-activated calcium currents in Ascaris suum muscle.

ABSTRACT: Resistance to all the classes of anti-nematodal drugs like the benzimidazoles, cholinergic agonists and avermectins, has now been recorded in parasites of animals and/or humans. The development of novel anthelmintics is an urgent and imperative need. Receptors of nematode neuropeptides have been suggested to be suitable target sites for novel anthelmintic drugs.To investigate the effect of AF2 on calcium-currents in Ascaris suum somatic muscle cells we employed the two-micropipette current-clamp and voltage-clamp techniques and a brief application of AF2.Here we report the isolation of voltage-activated, transient, inward calcium currents. These currents are similar in characteristics to Caenorhabditis elegans UNC-2 type currents, non-L-type calcium currents. Following a 2-minute application of 1 microM AF2 , there was a significant long-lasting increase in the transient inward calcium current; AF2 increased the maximum current (from -84 nA to -158 nA) by shifting the threshold in the hyperpolarising direction (V (50) changed from -7.2 to -12.8 mV) and increasing the maximum conductance change from 1.91 to 2.94 microS.These studies demonstrate a mechanism by which AF2 increased the excitability of the neuromuscular system by modulating calcium currents in nematodes. A selective small molecule agonist of the AF2 receptor is predicted to increase the contraction and act synergistically with cholinergic anthelmintics and could counter resistance to these compounds.

SUBMITTER: Verma S

PROVIDER: S-EPMC2014128 | biostudies-other | 2007 Jul

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Ascaris lumbricoides is one of the three major soil-transmitted gastrointestinal helminths (STHs) that infect more than 440 million people in the world, ranking this neglected tropical disease among the most common afflictions of people living in poverty. Children infected with this roundworm suffer from malnutrition, growth stunting as well as cognitive and intellectual deficits. An effective vaccine is urgently needed to complement anthelmintic deworming as a better approach to control helminth infections. As37 is an immunodominant antigen of Ascaris suum, a pig roundworm closely related to the human A. lumbricoides parasite, recognized by protective immune sera from A. suum infected mice. In this study, the immunogenicity and vaccine efficacy of recombinant As37 were evaluated in a mouse model. METHODOLOGY/PRINCIPAL FINDINGS:As37 was cloned and expressed as a soluble recombinant protein (rAs37) in Escherichia coli. The expressed rAs37 was highly recognized by protective immune sera from A. suum egg-infected mice. Balb/c mice immunized with 25 μg rAs37 formulated with AddaVax™ adjuvant showed significant larval worm reduction after challenge with A. suum infective eggs when compared with a PBS (49.7%) or adjuvant control (48.7%). Protection was associated with mixed Th1/2-type immune responses characterized by high titers of serological IgG1 and IgG2a and stimulation of the production of cytokines IL-4, IL-5, IL-10 and IL-13. In this experiment, the AddaVax™ adjuvant induced better protection than the Th1-type adjuvant MPLA (38.9%) and the Th2-type adjuvant Alhydrogel (40.7%). Sequence analysis revealed that As37 is a member of the immunoglobulin superfamily (IgSF) and highly conserved in other human STHs. Anti-As37 antibodies strongly recognized homologs in hookworms (Necator americanus, Ancylostoma ceylanicum, A. caninum) and in the whipworm Trichuris muris, but there was no cross-reaction with human spleen tissue extracts. These results suggest that the nematode-conserved As37 could serve as a pan-helminth vaccine antigen to prevent all STH infections without cross-reaction with human IgSF molecules. CONCLUSIONS/SIGNIFICANCE:As37 is an A. suum expressed immunodominant antigen that elicited significant protective immunity in mice when formulated with AddaVax™. As37 is highly conserved in other STHs, but not in humans, suggesting it could be further developed as a pan-helminth vaccine against STH co-infections.

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The nematode neuropeptide, AF2 (KHEYLRF-NH2), increases voltage-activated calcium currents in Ascaris suum muscle.

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