RNA interference‐mediated depletion of N‐ethylmaleimide Sensitive Fusion Protein and Synaptosomal Associated Protein of 25 kDa results in the inhibition of blood feeding of the Gulf Coast tick, Amblyomma maculatum

Abstract

The signaling pathways in tick salivary glands that control “sialo-secretome” secretion at the tick-host interface remain elusive. The complex processes by which tick sialo-secretome is exocytosed from the salivary gland cells and manipulates host hemostatic responses are essential for successful prolonged blood feeding. Exocytosis of the sialo-secretome in the salivary glands requires a core of Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) and their receptor proteins (SNAREs). SNAREs have been identified as the key components in regulating the secretion of the sialo-secretome in the salivary gland cells. In this study, we investigated the functional role of two Amblyomma maculatum SNARE complex proteins, AmNSF and AmSNAP-25, in the tick salivary glands during extended blood-feeding on the vertebrate host. The qRT-PCR analysis exhibited cyclic regulation of AmNSF transcript, increasing nearly four-fold around 48 hour post infestation in the salivary glands. Similarly, AmSNAP-25 transcript followed cyclic regulation, increasing three-fold around 72 hour post infestation in the midguts. Immunolocalization of AmNSF showed the presence of NSF in secretory granule containing cells of Acini II and Acini III in the unfed stage, with widespread localization in the partially fed glands. Knockdown of AmNSF and AmSNAP-25 transcript resulted in death, impaired feeding on the host, and lack of engorgement in both experimental groups at eleven days post infestation, 13 mg for dsRNA-AmNSF, 191 mg for dsRNA-AmSNAP-25, and 383 mg for control ticks. Depletion also led to important morphological changes in the collapse of the Golgi apparatus in the salivary gland cells. Our results imply a functional significance of AmNSF and AMSNAP-25 in the prolonged tick feeding, and survival on the host. Further characterization of the factors that regulate exocytosis will lead to novel approaches to prevent tick-borne diseases.

Amblyomma maculatum SECIS binding protein 2 and putative selenoprotein P are indispensable for pathogen replication and tick fecundity

“…All the procedures for protein isolation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting were those reported previously by Browning and Karim (2013) and Budachetri et al (2014). Nitrocellulose membranes were incubated with mouse anti-selenoprotein P (an anti-human mouse monoclonal antibody, 1:200) in iBind™ apparatus (ThermoFisher Scientific, Waltham, MA, USA) and anti-mouse HRP secondary IgG (1:2000).…”

An insight into the functional role of thioredoxin reductase, a selenoprotein, in maintaining normal native microbiota in the Gulf Coast tick (Amblyomma maculatum)

“…Total RNA extraction, conversion into complementary DNA and qRT-PCR were performed as previously described (Browning and Karim 2013). Briefly, the tick midguts and salivary gland tissues stored in RNAlater were used for total RNA extraction using Illustra™ RNAspin Mini Isolation Kit (GE Healthcare, Piscataway, NJ, USA) according to manufacturer’s instructions.…”

“…Synthesis of dsRNA for the TrxR gene and tick manipulations were performed according to the methods described previously (Browning and Karim 2013). Briefly, TrxR PCR products were joined to the Block-iT™ T7 TOPO ® linker using a BLOCK-iT™ RNAi TOPO® Transcription Kit (Invitrogen, Carlsbad, CA, USA).…”

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control

“…Soluble N-ethylmaleimide-sensitive factor attachment receptors (SNARE) complex proteins, which mediate exocytosis in secretory pathways of the salivary glands, have been characterized in Amblyomma ticks. These include N-ethylmaleimide-sensitive fusion (NSF) protein, Synaptosomal Associated Protein of 25 kDa (SNAP-25) [77], Ykt6 [65], and vesicle transport through interaction with t-SNAREs (Vti) [78]. Silencing various genes such as Salp14, Salp9pac [39], Neuronal isoform munc18-1 (nSec1) [59], and synaptobrevin [36] affected the secretion of anticoagulant or the anticoagulant activity of salivary gland extracts, indicating that these genes are important in tick anti-hemostatic mechanism.…”