Identification of the Babesia-responsive leucine-rich repeat domain-containing protein from the hard tick Haemaphysalis longicornis

Abstract

Haemaphysalis longicornis is a tick known for transmitting Babesia parasites, including Babesia gibsoni, in East Asian countries. The vector tick must have strategies to control Babesia parasites, while Babesia parasites are also considered to establish an evasive mechanism from the tick's innate immunity. Due to this mutual tolerance, H. longicornis is considered to be a vector of Babesia parasites. Recent studies have shown the important roles of leucine-rich repeat (LRR) domain-containing proteins in innate immunity in many living organisms. Some LRR domain-containing proteins were identified in ticks; however, their functions are still unknown. In this study, a novel LRR domain-containing protein was identified from H. longicornis (HlLRR). HlLRR contains two LRR domains, and the expression levels of mRNA and proteins were upregulated during blood feeding, particularly in the salivary glands and midgut. In addition, recombinant HlLRR (rHlLRR) demonstrated growth inhibition activity against B. gibsoni in vitro without a hemolytic effect at any concentration used. Moreover, the diameters of Babesia merozoites treated with rHlLRR were significantly larger than those of the control group. These results strongly indicate the key roles of HlLRR in the tick's innate immunity against Babesia parasites. Furthermore, HlLRR might be a potential alternative drug to treat babesiosis.

The Dual Prey-Inactivation Strategy of Spiders—In-Depth Venomic Analysis of Cupiennius salei

“…It could be shown that LRR domain-containing proteins were upregulated in the salivary gland and midgut of the infected tick Haemaphysalis longicornis during blood feeding. It is stated that LRRs play a key role in the tick innate immunity in controlling Babesia parasites [78]. Analogous to this, a possible function of LRRs in venom glands could be protection against undesired microbes that might invade spiders after injecting venom into an organism (Supplementary Figure S1.19).…”

Establishment of a novel tick-Babesia experimental infection model

“…H. longicornis is easy to maintain and is widely used as a model tick to study pathophysiology in tick infestation33. To date, a number of bioactive molecules have been found in H. longicornis , and those products might be available for not only tick control but also for novel drug discovery in the veterinary and medical fields78343536. The experimental infection of ticks with pathogens is considered to be an attractive tool for studying tick-pathogen interaction56.…”

Functional analysis of recombinant 2‐Cys peroxiredoxin from the hard tick Haemaphysalis longicornis

“…The WT open reading frame sequence was amplified by PCR using a forward primer (HlPrx2 F‐ Xho I) containing an Xho I recognition site and a reverse primer (HlPrx2 R‐ Eco RI) containing an Eco RI recognition site (Table ). The amplified PCR product was then purified using a GENECLEAN ® II KIT (MP Biomedical, Solon, OH, USA) and subcloned into the frame of the pRSET C. The WT was expressed in the Escherichia coli BL21 (DE3) strain and purified as described previously (Maeda et al ., ).…”

“…The WT open reading frame sequence was amplified by PCR using a forward primer (HlPrx2 F-XhoI) containing an XhoI recognition site and a reverse primer (HlPrx2 R-EcoRI) containing an EcoRI recognition site ( Table 1). The amplified PCR product was then purified using a GENECLEAN V R II KIT (MP Biomedical, Solon, OH, USA) and subcloned into the frame of the pRSET C. The WT was expressed in the Escherichia coli BL21 (DE3) strain and purified as described previously (Maeda et al, 2015). To prepare the mutated proteins, inverse PCR of the plasmid DNA (pRSET C containing HlPrx2 WT) was performed by using mutation primers and KOD-Plus-Neo (TOYOBO, Osaka, Japan) following the manufacturer's protocol for a single nucleotide substitution (Table 1).…”