부산대학교 도서관

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T. brucei-infected flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of GPI-anchored surface proteins herein named as Metacyclic Invariant Surface Proteins (MISP) because of its predominant expression on the surface of metacyclic trypomastigotes. The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are potentially extended above the metacyclic VSG coat, and thus could be tested as a transmission-blocking vaccine target. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both CRISPR-Cas9-driven knock out and RNAi knock down of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We suggest MISP may be relevant during trypanosome transmission or establishment in the vertebrate's skin. Author summary: The Trypanosoma brucei group of parasites are exclusively transmitted to the vertebrate host by the tsetse vector alongside insect saliva. To better understand trypanosome transmission, we investigated the protein composition of T. brucei-infected tsetse saliva using a mass spectrometry proteomics approach. We found that, in addition to proteins from tsetse saliva and Sodalis glossinidius (a bacterial tsetse symbiont), trypanosome-infected saliva contains several parasite surface glycoproteins, including a partially characterized family of invariant proteins herein named Metacyclic Invariant Surface Proteins (MISP). We show that MISP is primarily expressed, together with mVSG and BARP, on the surface of the infectious metacyclic stage of T. brucei. Its triple helix bundle architecture appears tethered to the outer membrane by an extended GPI-anchored C-terminal tail that putatively projects MISP above the VSG coat. Our findings provide new insights into the surface architecture of the T. brucei metacyclic stage and describes the challenges associated with developing transmission-blocking vaccines against tsetse-transmitted trypanosomes. [ABSTRACT FROM AUTHOR]