‘No, we would never say that we now know how ticks stick’, says Wageningen researcher Siddharth Deshpande. ‘At most, that is what SpringerNature has made of it.’ What he and his Maastricht colleague Ingrid Dijkgraaf have published in Nature Chemistry, however, is a study showing that at least one abundant protein in tick saliva can divide into two liquid phases. The most concentrated phase can then solidify into a gel. Deshpande readily admits that this does not prove that this is the mechanism that makes a tick stick for days – but it can hardly be anything else.

In evolutionary terms, the mechanism must be pretty clever. Ticks use their mouthparts to bite into a host, but immediately afterwards the saliva forms a ‘cement cone’ that takes over the coupling so that the creature can suck blood in peace. To do this, the saliva contains thousands of different proteins, all of which undoubtedly have a purpose. But what is this purpose?

Coacervates

Deshpande’s interest arose by chance. Since 2019, he has led the EmBioSys lab in Wageningen, a group that mainly focuses on the internal organisation of the cytoplasm in living cells. Coacervates are colloidal droplets that contain the same molecules as the surrounding liquid, but in a much higher concentration. This form of liquid-liquid phase separation is known from intrinsically disordered proteins (IDPs), which cannot fold stably.

‘GRPs are the major component of tick saliva, they form coacervates and they harden’

Siddarth Deshpande

When Deshpande came across a publication about the presence of glycine-rich proteins (GRPs) in tick saliva, he immediately recognised a possible family of IDPs in the amino acid sequences. There seemed to be a good chance that they would form coacervates, and this process could very well be the start of the cement cone – something similar has been observed in other animal species, such as mussels.

To test his hypothesis, Deshpande sought out biochemical tick experts. There are not many in the Netherlands, and he almost inevitably came across Ingrid Dijkgraaf, professor of biomimetic chemistry at Maastricht University, who has been working with these creatures since 2011. Her approach is completely different: ‘We want to visualise unstable plaques in human blood vessels before they can lead to heart attacks or strokes. This can be done using chemokines, a type of cytokine signalling protein that is expressed around inflammation and attracts white blood cells. A colleague showed me a recently published paper showing that ticks have proteins in their saliva that bind to chemokines. These tick proteins are not found in humans and we thought we might be able to use them as a tag. That’s how we started the whole tick research project.’

Since then, she has broadened her focus. By binding to chemokines, you neutralise them and may also be able to inhibit inflammation. ‘Ticks also have proteins that destroy platelets and the whole blood-clotting process. This could be fantastic for thrombosis patients.’

The challenge of synthesis

Synthesising proteins is an important part of the research, says Dijkgraaf: ‘You can isolate proteins from the cement of a tick that has fed on a rabbit or a mouse, but this cement is always contaminated with cells from the host. And so it was that when Deshpande came to see her, she had just assigned a master’s student to work on the synthesis of a GRP protein chosen more or less at random. ‘She wanted to do an internship in my group. I didn’t really have any vacancies, but I said: if you can make this protein, that would be great. I knew it was going to be difficult. You never know with proteins, but with so many identical amino acids in the chain, especially glycine, the chances of failure are very high. She was very motivated, so she tried anyway, and in the end she succeeded.’

Deshpande obtained almost half a milligram of GRP, enough to run experiments for over a year. His group was able to demonstrate the expected coacervation by fluorescently labelling the proteins and filming the process. Evaporation of the diluted liquid phase showed that the coacervates did indeed form a gel with some adhesive strength. Two members of the group went tick hunting in the Veluwe, removed the salivary glands of the animals they caught and also found protein-rich droplets in the saliva. At a later stage, EnzyTag from Nuth in Limburg, a start-up company specialising in protein synthesis with which Dijkgraaf has close contacts, was brought in to carry out preliminary tests with slightly different GRP proteins to get an idea of the influence of details in the sequence.

‘It is fascinating that the saliva is liquid in the animal itself and only becomes hard and solid when it comes out’

Ingrid Dijkgraaf

Separation

Deshpande is pretty sure he is on the right track. ‘GRPs are the main component of salivary secretions, they form coacervates and these harden.’ But exactly how this hardening takes place and why the adhesive strength is so great remains unclear. It is possible that the GRP proteins form cross-links or that other structural changes occur. A follow-up publication on the influence of pH is in preparation.

Dijkgraaf suspects that disulfide bridges between two cysteine building blocks may be involved. The protein currently under investigation contains only one cysteine, but this is not necessarily the case for all GRPs. She is very curious to see what happens when you try not just one, but several of the thousands of proteins from saliva at the same time. ‘It is also fascinating that saliva is liquid inside the animal and only becomes hard and solid when it comes out. You can see the same thing in spider webs, for example.’ It’s also unclear how the tick detaches itself after a blood meal: ’It’s not gradual, it’s very sudden. I think it uses enzymes for this phenomenon. I would like to do proteomics on the cement or the saliva.

All in all, she finds the research sufficiently interesting to continue, even if it does not fit in with her main focus on cardiovascular disease. The same goes for Deshpande and his enthusiastic group. ‘We have enjoyed this project so much…’