Babesial vector tick defensin against Babesia sp. parasites

Abstract

Antimicrobial peptides are major components of host innate immunity, a well-conserved, evolutionarily ancient defensive mechanism. Infectious disease-bearing vector ticks are thought to possess specific defense molecules against the transmitted pathogens that have been acquired during their evolution. We found in the tick Haemaphysalis longicornis a novel parasiticidal peptide named longicin that may have evolved from a common ancestral peptide resembling spider and scorpion toxins. H. longicornis is the primary vector for Babesia sp. parasites in Japan. Longicin also displayed bactericidal and fungicidal properties that resemble those of defensin homologues from invertebrates and vertebrates. Longicin showed a remarkable ability to inhibit the proliferation of merozoites, an erythrocyte blood stage of equine Babesia equi, by killing the parasites. Longicin was localized at the surface of the Babesia sp. parasites, as demonstrated by confocal microscopic analysis. In an in vivo experiment, longicin induced significant reduction of parasitemia in animals infected with the zoonotic and murine B. microti. Moreover, RNA interference data demonstrated that endogenous longicin is able to directly kill the canine B. gibsoni, thus indicating that it may play a role in regulating the vectorial capacity in the vector tick H. longicornis. Theoretically, longicin may serve as a model for the development of chemotherapeutic compounds against tick-borne disease organisms.

FIG. 1.

Molecular and functional characterization of longicin. (A) cDNA and deduced amino acid sequences and charge density of longicin. Locations of secondary structure elements are shown in the form of cylinders (α-helixes) and an arrow (β-strand). Italics indicate a putative signal sequence, the triangle indicates the mature peptide, the box indicates P4 peptide, and a bold underline marks the polyadenylation signal. (B) Alignment of longicin and the scorpion ion channel blocker (SICB) from Sahara scorpion. Asterisks indicate conserved residues. (C) Coomassie blue-stained recombinant longicins. (D) Endogenous longicin in tick protein extract. Detection was performed by two-dimensional immunoblot analysis with anti-longicin antibody. (E) Longicin expression induced by blood feeding. An equal amount of protein (three ticks) was loaded in each lane. Detection was performed by immunoblot analysis with anti-longicin antibody. Pre, prefeeding. (F) Immunohistochemical localization of endogenous longicin. Note the strong staining of the midgut epithelium (high magnification). Abbreviations: cu, cuticle; mg, midgut; sg, salivary gland; tr, trachea. (G) Bactericidal activity of longicin. Bacterial cells were exposed to recombinant longicin or synthetic peptides for 2 h at 37°C, and then the incubation mixture was spread on TSB agar plates. The results shown are from four plates from different batches. (H) Fungal cells were exposed to recombinant longicin or P4 peptide for 2 h at 37°C, and then the incubation mixture was spread on YTB agar plates. (I) Morphological changes in P. pastoris induced by longicin.

FIG. 2.

Babesiacidal activity of longicin. Longicin or synthetic peptides were incubated with B. equi-infected erythrocytes (1% parasitemia) in culture medium. Parasite-infected erythrocytes were counted as percentages of total erythrocytes. (A) Parasiticidal effect in the presence of longicin. Babesia-infected cells had almost disappeared on day 2 of 1.0 μmol treatment. (B) Parasiticidal effect in the presence of synthetic peptides. The error bars indicate standard errors of the mean (SEM). (C) Detection of longicin at the surface of B. equi merozoites. FITC-conjugated longicin P4 synthetic peptide was used for localization by fluorescent confocal microscopy. The control panel shows that FITC-labeled P1 did not bind to any cells. PC, phase contrast.

FIG. 3.

In vivo babesiacidal activity of longicin. (A) Therapeutic effect of a single treatment dose. Arrows and an arrowhead indicate administration of longicin and 60% reduction of parasitemia, respectively. (B) Representative images of the parasites from mice treated with longicin (3.0 mg/kg). Longicin blocked parasite invasion but not host erythrocyte rupture. (C) Growth inhibition was dependent on the dose of longicin. (D) Growth inhibition by a double treatment with longicin. The error bars indicate SEM.

FIG. 4.

Knockdown of longicin by RNAi facilitates transmission of Babesia parasite through the vector ticks. Unfed adult female ticks were injected with longicin dsRNA (ds longicin) into the hemocoel through the fourth coxae by use of fine-point glass needles. Control ticks were injected with PBS alone. Ticks were collected from the ear of a Babesia gibsoni-infected dog on day 6 after attachment. (A) Microinjection-treated ticks at day 3 on the ear of a dog. Injection of longicin dsRNA inhibited endogenous longicin expression (green) in the midgut. Figures are representative of three independent RNAi experiments. (B) RT-PCR analysis (day 3). Note the reduced expression of longicin mRNA in longicin dsRNA-injected ticks. (C) Immunoblot analysis of endogenous longicin expression (day 3). Results showed the absence of longicin expression, indicating that the effective knockdown of longicin mRNA was achieved by dsRNA injection. (D) Images of the tick midgut. B. gibsoni parasites were visualized by use of mouse anti-B. gibsoni antibody (green). The two right panels highlight the lumen and epithelium from the merge section of the midgut. (E) Ticks on day 6. The body weights of ticks with silencing longicin were significantly lower than those of control ticks at day 6. Suppression of endogenous longicin expression in longicin dsRNA-treated ticks was seen up to engorgement. Preoviposition, oviposition, and egg periods of ticks treated with dsRNA were similar to those of PBS control ticks. Smaller engorged ticks silenced with longicin subsequently transmitted larger numbers of Babesia sp. parasites than did PBS control ticks. One scale, 1 mm. (F) Prevalence and intensity of B. gibsoni infection. The numbers of invaded parasites were evaluated by P18 genes on the B. gibsoni genome DNA by use of a real-time quantitative PCR. (G) Representative image of the migrating Babesia parasite in the tick ovary. (H) Intensity of B. gibsoni infection. Data represent the means ± SEM for three experiments with five ticks. Quantitative results demonstrated that repression of longicin enhanced the B. gibsoni transmission in the vector tick. The error bars indicate SEM for three independent experiments with three ticks.