Evolution of the tissue factor pathway inhibitor-like Kunitz domain-containing protein family in Rhipicephalus microplus

Abstract

One of the principle mechanisms utilised by ticks to obtain a blood meal is the subversion of the host's haemostatic response. This is achieved through the secretion of saliva containing antihaemostatic proteins into the feeding lesion. Lineage-specific expansion of predicted secretory protein families have been observed in all previously studied ticks and occurred in response to adaptation to a blood-feeding environment. Of these, the predominant families are common between both hard and soft ticks. One of these families, namely the Kunitz domain-containing protein family, includes proven tissue factor pathway inhibitor-like (TFPI-like) anti-haemostatics such as ixolaris and penthalaris that play a crucial role during tick feeding. Although Kunitz-type proteins have been found in Rhipicephalus microplus, the TFPI-like Kunitz protein family has not yet been studied. We report a comprehensive search for TFPI-like Kunitz domain-containing proteins in R. microplus expressed sequence tag libraries, resulting in the identification of 42 Page 2 of 36 homologues. The homologues were bioinformatically and phylogenetically studied, including the application of an intensive Bayesian Markov Chain Monte Carlo (MCMC) analysis of the individual Kunitz domain nucleotide sequences. We show that the R. microplus TFPI-like Kunitz protein family groups into two main clades that presumably underwent ancient duplication, which indicates that a whole genome duplication event occurred at least 150 million years ago. Evidence for recent and ancient gene and domain duplication events was also found. Furthermore, the divergence times of the various tick lineages estimated in this paper correspond with those presented in previous studies. The elucidation of this large protein family's evolution within R. microplus adds to current knowledge of this economically important tick.

Are ticks venomous animals?

“…Such protein families are found in the venoms of several other Metazoan species [2]. Examples of such venomous protein families found in tick saliva are defensins [65], lectins [66], cystatins [67], lipocalins [21,68-71], hyaluronidase [72], phospholipase A2 [73], Kunitz-like peptides [56,74,75], metalloproteases [76], AVIT [77], CAP proteins (Cysteine-Rich Secretion Proteins, Antigen 5, and Pathogenesis-Related) [2] and sphingomyelinase D [2]. …”

Understanding the evolutionary structural variability and target specificity of tick salivary Kunitz peptides using next generation transcriptome data

“…With the exception of the annotated I. scapularis sialome [6,9], both protein BLAST [43] and PSI-BLAST [44] searches do not reveal any known or functionally described single Kunitz-domain peptide with the Cys motif of G3, G6 and G11. The proteins that slightly resemble the Cys motif of G6, and lack the archetypal Cys2 and Cys4 disulfide bridge, are the I. scapularis salivary Kunitz proteins Ixolaris [38] and Penthalaris [39], both possessing multiple Kunitz domains, and some members of the Kunitz family found in R. microplus [45]. An alignment using the classical Kunitz serine protease inhibitor BPTI (UniProt: P00974) with G1 and G7 shows the conserved positively charged arginine (Arg or R) or lysine (Lys or K), known as the P1 site that interacts with the negatively charged active site of serine proteases (highlighted blue in Figure 1C).…”

Ancestral reconstruction of tick lineages

“…This included studies of the BTSP family , the Kunitz/BPTI family (Dai et al 2012;Louw et al 2013;Mans and Neitz, 2004b;Mans et al , 2003b2002a;Schwarz et al 2014b), the lipocalin family (Beaufays et al 2008b;Mans, 2005;Mans and Neitz, 2004c;Ribeiro, 2008a, 2008b;Mans et al , 2008cMans et al , 2003a) and the apyrases (Stutzer et al 2009). These studies suggested that argasid BPTI anti-hemostatic factors share a common evolutionary pathway; that biogenic amine binding lipocalins and TXA 2 /LTB 4 scavenging lipocalins are closely related; that the biogenic amine binding site is probably ancestral; that tick lipocalins share a monophyletic origin with lipocalins from other metazoan and that apyrase activity probably evolved a single time in the ancestral tick lineage.…”

“…A few studies have attempted to date the divergences of the protein families using molecular clock approaches (Louw et al 2013;Schwarz et al 2014b). However, due to low sequence similarity among family members, the trees obtained give contradictory results and general low branch support.…”

“…Analysis restricted to BPTI sequences from a single species (R. microplus), yielded estimates for different clades between 1-11 MYA (Louw et al 2013). These were interpreted as recent duplications, but are almost as old as the estimates for the origin of the Rhipicephalinae ~14 MYA (Barker and Murrel, 2004), suggesting that most of these duplications did not originate in R. microplus, but before diversification of a number of lineages.…”