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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.

Rhipicephalus (Boophilus) microplus is constantly challenged during its life cycle by microorganisms present in their hosts or in the environment. Tick eggs may be especially vulnerable to environmental conditions because they are exposed to a rich and diverse microflora in the soil. Despite being oviposited in such hostile sites, tick eggs remain viable, suggesting that the egg surface has defense mechanisms against opportunistic and/or pathogenic organisms. R. microplus engorged females deposit a superficial wax layer onto their eggs during oviposition. This egg wax is essential for preventing desiccation as well as acting as a barrier against attack by microorganisms. In this study, we report the detection of anti-biofilm activity of R. microplus egg wax against Pseudomonas aeruginosa PA14. Genes involved in the functions of production and maintenance of the biofilm extracellular matrix, pelA and cdrA, respectively, were markedly downregulated by a tick egg-wax extract. Moreover, this extract strongly inhibited fliC gene expression. Instead of a compact extracellular matrix, P. aeruginosa PA14 treated with egg-wax extract produces a fragile one. Also, the colony morphology of cells treated with egg-wax extract appears much paler and brownish, instead of the bright purple characteristic of normal colonies. Swarming motility was also inhibited by treatment with the egg-wax extract. The inhibition of P. aeruginosa biofilm does not seem to depend on inhibition of the quorum sensing system since mRNA levels of the 3 regulators of this system were not inhibited by egg-wax extract., (Copyright © 2013 Elsevier GmbH. All rights reserved.)