Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes

Background

Hematophagous insects have evolved special adaptations to ensure a successful bloodmeal from vertebrate hosts, key among which is their saliva. Salivary components have been studied in a range of contexts including interactions with the host immune system. However, few studies have addressed the importance of the post-translational modifications in these proteins. N-glycosylation is a highly common post- and co-translational modification that can affect protein folding, protein stability, ligand binding, and protein antigenicity. N-glycans have a wide variety of functions, encompassing structural and modulatory properties to the binding of other proteins and cell-cell interactions.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of the tsetse genes that encode for salivary proteins, affecting its anti-hemostatic and anti-clotting properties (1). To better understand trypanosome transmission and the potential role of gylcans in insect blood-feeding, in the present work, Kozak et al (2) characterize the salivary glycome of T. brucei-infected and uninfected tsetse flies (Glossina spp.).

 

Key findings and developments

Bioinformatic analysis identified that 72% of Glossina proteins have at least one potential glycosylation site. Further confirmation was performed by molecular methods, and salivary proteins were identified using mass spectrometry. This was followed by identification of the type of glycosylation (N– or O-linked), and proteomic analysis, which showed that suggesting that the main type of sugars linked to tsetse salivary glycoproteins are N-glycans. Characterization of the type of N-glycosylation present indicated the presence of several high mannose or hybrid type N-glycans.

A full structural characterization of G. morsitans salivary N-glycans was then performed. Hydrophilic interaction lipid chromatography (HILIC)- ultra-high pressure liquid chromatography (UHPLC) analysis revealed 13 peaks that correspond to a variety of potential high mannose and hybrid N-glycan structures. The peak of highest intensity corresponds to the core structure Man3GlcNAc2-2AB (paucimmanose). Secondary confirmation of the salivary N-glycan structures was performed by positive-ion ESI-MS and ESI-MS/MS. Overall, these results confirm the findings by HILIC-UHPLC, and suggest that tsetse salivary glycans consist mainly of highly processed Man3GlcNAc2 in addition to several other paucimannose, oligomannose, and three hybrid-type glycans: Man3GlcNAc3, Man4GlcNAc3 and Man5GlcNAc3 .

Since T. brucei infection affects the composition of tsetse saliva (1), the authors investigated if infection changes salivary glycosylation as well. For this, they first compared the salivary profiles of uninfected flies with those that had either a salivary gland or a midgut infection with T. b. brucei. They found no major changes in the profile of salivary proteins in the different physiological states. They then went on to determine whether T. b. brucei infection or fly age (teneral vs. adult) alters the structure of salivary N-glycans, and found no significant quantitative differences between all conditions. They then determined whether trypanosome infection alters the immune reactivity of tsetse salivary glycoproteins. Recognition of control G. morsitans saliva before and after cleavage of the glycans appears unaffected; however, during salivary gland infection the polyclonal serum only detected the high molecular weight proteins after glycans were cleaved. The effect is more readily seen during salivary gland infections, possibly due to the downregulation of other salivary proteins during infection, and seems to be concealed both in the saliva of naïve flies and those with midgut infection. The authors were also able to identify proteolytic products of salivary proteins that are formed as a result of the trypanosome infection in the gland.

Next, they investigated whether glycosylated salivary proteins bind onto metacyclic trypanosomes. Molecular and mass-spectrometry based assays identified components that likely correspond to 5’Nucleotidase-related protein sgp3, TSGF and the Tsal glycoproteins, plus two small additional bands identified using anti-saliva IgGs.  Interestingly, the abundant, non-glycosylated TAg5 protein in saliva was found not to bind to the metacylic trypanosome surface. Knowing that N-glycans from G. morsitans salivary glycoproteins are recognised by the mannose receptor and DC-SIGN, the authors performed overlay assays of recombinant mannose receptor and DC-SIGN with tsetse saliva. They found that the mannose receptor recognizes saliva glycoproteins better than DC-SIGN; that recognition is fully linked to N-linked mannosylated glycans, as binding disappears upon treatment with N-glycanase.

Altogether, the authors suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

What I like about this preprint

I think the work is very relevant to the field of host-pathogen interactions, and covers a relatively unknown and understudied field in vector biology, which is nonetheless important for our understanding of pathogen transmission. And also on its own, to know more about the differences in saliva among insects!

 

Questions to authors

1. You mention along your work that trypanosomes causes a profound transcriptional downregulation of most tsetse salivary proteins. At what point of infection does this occur? Is this specific to Trypanosoma brucei? If not, can you expand on why this is the case?
2. Is it known if fly gender affects their brucei transmission ability, and saliva composition?
3. Is it known if salivary content recognition is equal among mammalian species, suggesting perhaps a more successful adaptation of protein contents to one host species but not others?
4. You found that only glycosylated salivary proteins associate with the trypanosome surface. Could you expand further on the significance of this? Has something similar been observed in other hosts/pathogens?

References

1. Van Den Abbeele J et al: The Glossina morsitans tsetse fly saliva: general characteristics and identification of novel salivary proteins. Insect Biochem Mol Biol, 2007.
2. Kozak RP, et al, Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognized by C-type lectins and bind to trypanosomes, bioRxiv,2020.

 

doi: https://doi.org/10.1242/prelights.22976

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