Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of Brugia malayi

Abstract

Ivermectin (IVM) is a broad-spectrum anthelmintic used in filariasis control programs. By binding to nematode glutamate-gated chloride channels (GluCls), IVM disrupts neurotransmission processes regulated by GluCl activity. IVM treatment of filarial infections is characterized by an initial dramatic drop in the levels of circulating microfilariae, followed by long-term suppression of their production, but the drug has little direct effect on microfilariae in culture at pharmacologically relevant concentrations. We localized Brugia malayi GluCl expression solely in a muscle structure that surrounds the microfilarial excretory-secretory (ES) vesicle, which suggests that protein release from the ES vesicle is regulated by GluCl activity. Consistent with this hypothesis, exposure to IVM in vitro decreased the amount of protein released from microfilariae. To better understand the scope of IVM effects on protein release by the parasite, three different expression patterns were identified from immunolocalization assays on a representative group of five microfilarial ES products. Patterns of expression suggest that the ES apparatus is the main source of regulated ES product release from microfilariae, as it is the only compartment that appears to be under neuromuscular control. Our results show that IVM treatment of microfilariae results in a marked reduction of protein release from the ES apparatus. Under in vivo conditions, the rapid microfilarial clearance induced by IVM treatment is proposed to result from suppression of the ability of the parasite to secrete proteins that enable evasion of the host immune system.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Boot-strapped (1,000) neighbor-joining tree generated from a ClustalX alignment of the Bma-AVR-14A and -B sequences and sequences of GluCls from other parasitic nematodes and C. elegans. Outgroup sequence for this tree was set with a γ-aminobutyric acid receptor subunit from C. elegans (Cel-UNC-49A). I, nonsusceptible or unknown susceptibility to IVM; II, susceptible to IVM.

Fig. 2.

Immunolocalization of AVR-14 subunits in B. malayi mf by confocal laser scanning microscopy. (A) Bma-AVR-14 specific signal was detected in proximity to the ES apparatus. Counter staining with DAPI and Phalloidin-Rhodamine distinguishes major anatomical features (A, anus; ES, excretory-secretory apparatus; IB, inner body; M, mouth; NR, nerve ring). (B) A closer examination of the signal reveals colocalization of Bma-AVR-14 subunits with a muscle structure surrounding the ES vesicle (ESV).

Fig. 3.

Immunolocalization studies of ES proteins in B.malayi mf reveal three different expression patterns. (A) Protein localization exclusively in the ES apparatus. Representative staining is shown for CPI-2. Also exhibiting the same pattern, VAL-1, TPI, and a fraction of mf stained with anti-TCTP (Fig. S3). (B) Protein localization in either the ES apparatus or the mf inner sheath. This pattern was exhibited in another fraction of specimens stained for TCTP. (C) The third pattern, observed for MIF-1, consisted of prominent expression through the midbody. Images on the Right of B and C are presented as merged images of single planes showing signal presence in the ES-vesicle and differences in compartmentalization of TCTP and MIF-1 toward the parasite surface.

Fig. 4.

Effects of two anthelmintic drugs on protein release from B.malayi mf. (A) IVM reduces protein release in vitro. Mf (2.5 × 105 mf/mL) were incubated from 0 to 72 h in RPMI 1640 with or without IVM. Media exchange was performed each 24 h (n = 3) at 24, 48, and 72 h. (B) Protein release in vitro at 24 h from mf incubated with or without ABZ (10 μM). Mean ± SD; *P < 0.05; **P < 0.01, control vs. treatment; °P < 0.05, 1.0 μM IVM vs. treatment.