The Trypanosoma brucei MISP family of invariant proteins is co-expressed with BARP as triple helical bundle structures on the surface of salivary gland forms, but is dispensable for parasite development within the tsetse vector

Abstract

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.

Beyond the VSG layer: Exploring the role of intrinsic disorder in the invariant surface glycoproteins of African trypanosomes

“…The results presented in this study further expand our understanding of the structure-function relationship of Trypanosoma ISGs, which have been implicated in a wide range of functions (including nutrient uptake, adhesion, environmental sensing, and immune evasion). In agreement with previous reports [ 7 , 28 ], these findings add to a growing body of evidence observed for non-VSG surface antigens in which intrinsic disorder appears to play a prominent, functional role at the host-parasite interface. This enables us to contribute to an updated model of the trypanosome surface coat and its embedded proteins, portraying it as a highly dynamic structure rather than a static barrier.…”

“…Consequently, ISG exposure is likely to occur either through shedding or increased permissiveness of the VSG coat. Recent studies involving ISG65 from BSFs and metacyclic invariant surface proteins (MISP) from metacyclic forms have favored the latter hypothesis [ 7 , 28 ]. For both proteins, intrinsically disordered C-terminal regions (IDRs) were found, which could enable them to operate within as well as beyond the VSG coat.…”

A sticky situation: When trypanosomatids attach to insect tissues

“…TbMISPs contain a large extracellular domain and many of the same functional regions as the T. congolense protein TconCESP (discussed below), suggesting a possible role in attachment. TbMISP isoforms have C-terminal extensions that differ in length, possibly allowing for different types or degrees of attachment [79]. These C-terminal regions also have basic residues that would be susceptible to proteolysis, pointing to a possible mechanism for release of attached cells.…”

“…Efforts to identify new GPI-anchored surface proteins revealed another apparently epimastigote-specific family of proteins called Tb SGE1 [ 77 , 78 ]. Recently, these proteins have been renamed metacyclic invariant surface protein ( Tb MISP) after their expression was detected in all salivary gland stages but most highly in the metacyclic form [ 79 ]. This discrepancy may be due to the inaccessibility of Tb MISP epitopes to antibody-based detection within the dense metacyclic VSG (mVSG) coat, which is acquired in the fly prior to transmission in the mammal.…”