Location: Cattle Fever Tick Research Unit
Project Number: 3094-32000-042-069-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2021
End Date: Dec 31, 2024
Objective:
Pathogen transmission by ticks is facilitated by a phenomenon called saliva-assisted transmission (SAT), which is defined as the ability of tick saliva to increase pathogen establishment. Tick saliva contains several immunomodulatory, anti-hemostatic, and anti-inflammatory molecules that allow ticks to diminish host rejection. Pathogens take advantage of these salivary molecules to avoid host immune responses and establish an infection niche. These molecules include proteins, lipids, and microRNAs. Recent studies have demonstrated that immunomodulatory tick proteins and miRNAs are secreted within Ixodes scapularis, Amblyomma maculatum, and Haemaphysalis longicornis extracellular vesicles. Furthermore, these vesicles appear to act on specific skin cells, affecting or delaying wound repair mechanisms. It is highly likely that the secretion of immunomodulatory molecules within extracellular vesicles is a conserved mechanism in ticks. Moreover, our cooperator’s preliminary data with Amblyomma americanum extracellular vesicles indicates that the cargo within tick extracellular vesicles is very dynamic and changes over time.
Rhipicephalus (Boophilus) microplus is a one-host tick that infests and completes its life cycle in a single host. As such, it has evolved different mechanisms to avoid immune detection by hosts at different life stages and throughout their feeding. Studies looking at the morphological changes that occur in the salivary glands of adult R. microplus over time demonstrate the accumulation of secretory granules and cellular alterations at 24 hr and 74 hr post attachment and during final engorgement. Moreover, these studies reported cellular differences between the acini II and III found in larvae and nymphs when compared to adults. Later, studies showed proteomic variations in R. microplus salivary secretion at 17- and 22-days post attachment, further indicating that this tick species adapts its salivary secretion over time. Moreover, the gene expression within tick salivary immunomodulators changes in response to the host immune response. Nevertheless, what molecular alterations occur in the salivary glands and extracellular vesicles of R. microplus have not been well defined. We hypothesize that the protein and miRNA profiles of Rhipicephalus microplus salivary glands and extracellular vesicles change depending on the feeding time and developmental stage of the tick.
Our objective is to characterize the temporal and intrastadial changes in the molecular cargo of R. microplus salivary glands and extracellular vesicles.
By defining the temporal and interstadial modifications that occur in tick salivary secretions during R. microplus feeding on a single host, we can determine specific adaptations that for this tick to complete their life cycle in a single host. Further, we can identify potential vaccine and therapeutic targets for the control of R. microplus in livestock.
Approach:
This project is divided in two aims:
1) Characterize temporal changes in the proteomic cargo of R. microplus extracellular vesicles.
The feeding period of adult R. microplus females averages 7 days (5- 10 days), according to Senbill et al., 2018, Syst. Appl. Acarol. We will characterize the proteomic cargo of adult R.microplus females at early feeding (24 hours), intermediate or slow feeding (3 - 5 days), and late feeding (7 days). We will feed a total of 700 adult R. microplus and recover ticks at each time point. We know from our collaborator’s experience working with other tick species that they need around 20 ticks per replicate for proteomic analysis (60 ticks for 3 replicates). From their results, they also know that ticks recovered at early feeding (3 days) secrete less vesicles. Therefore, we will adjust the number of ticks to 50 ticks per replicate at 24 hr and 3 days (150 ticks for three replicates). From the remaining ticks, 40 will be used to produce vesicles for Nanoparticle Tracking Analysis (NTA) and western blotting at each time point (120 tick total). This will allow us to determine differences in vesicular secretion between time points and the expression of extracellular markers, respectively. The ticks will be dissected and the salivary glands will be placed in vesicle-free media for 24 hr to allow vesicle secretion. Vesicles will be isolated from the media using a series of centrifugations followed by ultracentrifugation at 100,000xg. Parallel Accumulation Serial Fragmentation (PASEF) MS/MS spectrometry using a timsTOF pro at the Charles W. Gehrke Proteomics Center at the University of Missouri will be used to characterize the proteomic cargo from the extracellular vesicles at each time point. Later experiments will look at changes between life stages.
2) Determine the differences in the microRNA profile of R. microplus salivary glands versus those packed within extracellular vesicles.
microRNAs are regulatory non-coding small RNAs that function in the post-transcriptional regulation of gene expression by binding the untranslated (UTR) 3’ region of mRNAs, leading to their degradation. microRNAs have been reported in R. microplus ticks and salivary glands. miRNAs are expressed in all life stages and show high diversity in salivary glands. It is likely that R. microplus microRNAs function in the regulation of tick salivary proteins and in the modulation of host responses to tick feeding. To investigate the changes in salivary microRNA expression at different R. microplus life stages, we will infest a stanchioned calf with patches of ~1200 larvae on different days. We will recover ~100 larvae at day 5 and ~100 nymphs post-ecdysis at day 10. Adults (~100) will be recovered at day 16. The salivary glands will be dissected immediately after collection and put in vesicle free-media. Extracellular vesicles will be produced and isolated as described in Objective 1. The vesicles and salivary glands from 200 adults and 600 nymphs will be combined. The small RNAs will be isolated and sequenced. miRNA expression will be validated by qRT-PCR. The protein from the vesicles will be used for western blotting to confirm sample purity.
