Functional analysis of a novel cysteine-rich antimicrobial peptide from the salivary glands of the tick Rhipicephalus haemaphysaloides

Abstract

Ticks encounter various microbes while sucking blood from an infected host and carrying these pathogens in themselves. Ticks can then transmit these pathogens to vertebrate hosts. The immune system of ticks can be stimulated to produce many bioactive molecules during feeding and pathogen invasion. Antimicrobial peptides (AMPs) are key effector molecules of a tick's immune response, as they can kill invading pathogenic microorganisms. In this study, we identified a novel cysteine-rich AMP, designated Rhamp1, in the salivary glands of unfed and fed female ticks (Rhipicephalus haemaphysaloides). Rhamp1 is encoded by a gene with an open reading frame of 333 bp, which in turn encodes a peptide of 12 kDa with a 22 amino acid residue signal peptide. The Rhamp1 protein had a pI of 8.6 and contained six conserved cysteine residues at the C-terminus. Rhamp1 shared 43% amino acid identity with a secreted cysteine-rich protein of another tick species, Ixodes scapularis. We cloned the Rhamp1 gene and attempted to express a recombinant protein using prokaryotic and eukaryotic systems, to determine its biological significance. Recombinant Rhamp1 was successfully expressed in both systems, yielding a glutathione S-transferase (GST)-tagged protein (36 kDa) from the prokaryotic system, and a polyhistidine-tagged Rhamp1 protein (14 kDa) from the eukaryotic system. Rhamp1 inhibited the activities of chymotrypsin (16%) and elastase (22%) and exerted low hemolytic activity. It also inhibited the growth of Gram-negative bacteria, including Pseudomonas aeruginosa (49%), Salmonella typhimurium (50%), and Escherichia coli (52%). Our findings suggest that Rhamp1 is a novel AMP in R. haemaphysaloides with the ability to inhibit proteinase activity.

Virome and Blood Meal-Associated Host Responses in Ixodes persulcatus Naturally Fed on Patients

“…Recently, carbohydrate metabolic compensation and peroxiredoxin production were reported to be necessary to maintain the redox balance in ticks ( Kusakisako et al, 2016 ; Della Noce et al, 2019 ). It has been proposed that tick microbiome would be influenced by oxidative stress ( Narasimhan and Fikrig, 2015 ), and different AMP would have different inhibitory consequence on microbial growth ( Saito et al, 2009 ; Zhang et al, 2015 ). These immune responses in ticks may work together and eventually influence the viral and bacterial composition of a specific host blood meal through a complicated pathway.…”

“…PIXR might influence the tick gut microbiome composition by regulating the ability of gram-positive bacteria to form biofilms in the gut when the tick takes a blood meal ( Narasimhan et al, 2017 ). These alterations may influence B. burgdorferi entering the tick gut in multiple ways ( Zhang et al, 2015 ). We identified that the gene encoding a protein similar to PIXR was upregulated in the fully fed I. persulcatus ticks; however, its role in the transmission of Borrelia and other tick-borne pathogens deserves further study.…”