The crystal structure and localization ofTrypanosoma bruceiinvariant surface glycoproteins suggest a more permissive VSG coat in the tsetse-transmitted metacyclic stage

Abstract

1 surface glycoproteins suggest a more permissive VSG coat in the tsetse-2 transmitted metacyclic stage 3 4 5 Abstract 23 Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes 24 inside the tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, 25 nothing is known about expression of invariant surface antigens by the metacyclic stage. 26 Proteomic analysis of saliva from T. brucei-infected flies revealed a novel family of 27 hypothetical GPI-anchored surface proteins herein named Metacyclic Invariant Surface 28 Proteins (MISP). MISP are encoded by five homolog genes and share ~80% protein identity. 29The crystal structure of MISP N-terminus at 1.82 Å resolution revealed a triple helical bundle 30 that shares key features with other trypanosome surface proteins. However, molecular 31 modelling combined with live fluorescent microscopy suggest that MISP N-termini are 32 extended above the metacyclic VSG coat, exposing immunogenic epitopes. Collectively, we 33 suggest that the metacyclic cell surface architecture appears more permissive than 34 bloodstream forms in terms of expression of invariant GPI-anchored glycoproteins, which 35 could be exploited for the development of novel vaccines against African trypanosomiases. 36 37 38 39 40 41 42 43 44 45concerted action of major surface metalloproteases and the GPI-phospholipase C (GPI-70 PLC) 12,13 , which is a process that appears to modulate the tsetse's immunity in favor of the 71 parasite infection 14 . Concomitant with VSG disappearance, the parasites express a new coat 72 of GPEET-procyclins, which is later replaced by a family of EP-procyclin isoforms once the 73 infection is established in the MG 15,16,17 . It is well accepted that procyclic trypomastigotes 74 colonize the tsetse ectoperitrophic space, from which they further migrate to the tsetse cardia 75 or proventriculus (PV) 18 . Within the PV, mesocyclic trypomastigotes (MSCs) eventually 76 differentiate into epimastigote forms (EMFs), migrate to the salivary glands (SGs), attach to 77 the epithelial cell microvilli and switch on the expression of the surface-localized Brucei 78Alanine-Rich Proteins (BARP) 19 . The attached EMFs further differentiate into pre-metacyclic 79 trypomastigotes (P-MCFs) 20,21 , during which time BARP expression is lost, a new coat of 80 metacyclic VSG (mVSG) develops 22,23 , and cells detach from the SG epithelium. Finally, 81 mature metacyclic trypomastigote forms (MCFs) 21,24 are inoculated along with saliva into the 82 skin of a vertebrate host in a subsequent feed. Unlike in BSFs, the surface composition of T. 83brucei MCFs is less well characterized primarily due to the lack of an in vitro culture system 84 and to the low yield of MCF cells obtained from infected tsetse. Recently, however, 85 trypanosomes overexpressing the RNA-binding protein 6 have been shown to develop into 86MCFs in vitro 25 , although the yields are low and the environment does not precisely mimic the 87 tsetse SG. 88It is well establish...

Proteomic identification of the UDP-GlcNAc: PI α1–6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei

“…Other T. brucei surface molecules that have been shown experimentally to possess a GPI membrane anchor are the ESAG6-subunit of the BSF transferrin receptor (TfR) [2] and the procyclins, the major surface glycoproteins of the tsetse mid-gut dwelling procyclic form (PCF) of the parasite [3]. In addition, many other surface molecules with N-terminal signal peptides and C-terminal GPI addition signal peptides are predicted to be GPI-anchored in T. brucei, including the BSF haptaglobin-haemaglobin receptor [4] and the factor H receptor [5], the epimastigote BARP glycoprotein [6] and the metacyclic trypomastigote invariant surface protein (MISP) [7]. Thus far, GPI anchor structures have been completely or partially solved for four T. brucei VSGs [8][9][10][11], the TfR [2] and the procyclins [3].…”

“…In addition, many other surface molecules with N-terminal signal peptides and C-terminal GPI addition signal peptides are predicted to be GPI-anchored in T . brucei , including the BSF haptaglobin-haemaglobin receptor [ 4 ] and the factor H receptor [ 5 ], the epimastigote BARP glycoprotein [ 6 ] and the metacyclic trypomastigote invariant surface protein (MISP) [ 7 ]. Thus far, GPI anchor structures have been completely or partially solved for four T .…”

Proteomic identification of the UDP-GlcNAc : PI α1-6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway ofTrypanosoma brucei

“…(C) Relative intensity plot using a new algorithm. The same data as (B) are plotted with a different y-axis, whereby each protein group is assigned an intensity rank from the most abundant protein group (1) to least abundant protein groups (7,125) based on their summed eXtracted Ion Currents (XICs) for the total BSF proteome. (Details of the mass spectrometry and data analysis are provided in in Materials and Methods.…”

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

“…suggest these probably represent proteolytic products of salivary proteins that are formed as a result of the trypanosome infection in the gland [32]. Fig 4C shows that Con A equally recognised salivary glycoproteins from either naïve or trypanosome-infected flies.…”