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Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses.

ABSTRACT: BACKGROUND: Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized. METHODOLOGY/PRINCIPAL FINDINGS: Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or -170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (K(D) = 0.4 nM) and to the active site of FXa (K(D) = 3.07 nM). We propose the name "Simukunin" for this novel protein. CONCLUSIONS: We conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.

SUBMITTER: Tsujimoto H

PROVIDER: S-EPMC3285612 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses.

Tsujimoto Hitoshi H Kotsyfakis Michail M Francischetti Ivo M B IM Eum Jai Hoon JH Strand Michael R MR Champagne Donald E DE

PloS one 20120223 2

<h4>Background</h4>Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized.<h4>Methodology/principal findings</h4>Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins ...[more]

PMID: 22383955

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Project description:BackgroundSeveral antimicrobial peptides (AMPs) belonging to the cecropin family have been identified from the salivary glands of different black fly species, however, the immunological functions for these molecules were poorly understood.MethodsA novel cecropin-like antimicrobial peptide (SibaCec) was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black fly Simulium bannaense. The amino acid sequence of SibaCec was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-negative bacteria Escherichia coli treated with SibaCec were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the mRNA expression of the inducible NO synthase (iNOS) and pro-inflammatory cytokines. Nitric oxide (NO) generation was examined using a Griess assay and the secretion of pro-inflammatory cytokines was determined by an enzyme-linked immunosorbent assay (ELISA). The activation of extracellular signal-regulated kinase (ERK), p38, and the nuclear translocation of nuclear factor-kappaB (NF-κB) were assessed by Western blotting analysis. Circular dichroism (CD) spectroscopy was performed to evaluate the secondary structure of SibaCec in solvent environment. Interaction of SibaCec with lipopolysaccharide (LPS) was studied using fluorescein isothiocyanate (FITC)- conjugated LPS aggregates. Neutralization of LPS by SibaCec was assayed with the chromogenic limulus amebocyte lysate (LAL) test. qPCR was also used to analyze the expression of SibaCec mRNA in the salivary glands of insects after oral infection with the bacteria E.coli.ResultsSibaCec possessed potent antimicrobial activity against Gram-negative bacteria, and showed low cytotoxicity toward mammalian cells. SEM analysis indicated that SibaCec killed bacteria through the disruption of cell membrane integrity. Furthermore, SibaCec significantly inhibited lipopolysaccharide (LPS)-induced production of NO and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-1β (IL-1β) and interferon-6 (IL-6) by blocking the activation of MAPKs and NF-κB signaling pathways. It mainly adopted an α-helix conformation in membrane-mimetic environments. SibaCec could interact and neutralize LPS. Infection of black flies with bacteria caused an upregulation of the expression of SibaCec.ConclusionsThese results demonstrated that in addition to the bactericidal capacity, SibaCec can function as immune regulator, inhibiting host secretion of inflammatory factors.

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Dataset Information

Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses.

Publications

Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses.

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