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Structural similarities between the metacyclic and bloodstream form variant surface glycoproteins of the African trypanosome [1]

['Monica Chandra', 'Division Of Structural Biology Of Infection', 'Immunity', 'German Cancer Research Center', 'Heidelberg', 'Division Of Immune Diversity', 'Sara Đaković', 'Konstantina Foti', 'Johan P. Zeelen', 'Monique Van Straaten']

Date: 2023-02 

During infection of mammalian hosts, African trypanosomes thwart immunity using antigenic variation of the dense Variant Surface Glycoprotein (VSG) coat, accessing a large repertoire of several thousand genes and pseudogenes, and switching to antigenically distinct copies. The parasite is transferred to mammalian hosts by the tsetse fly. In the salivary glands of the fly, the pathogen adopts the metacyclic form and expresses a limited repertoire of VSG genes specific to that developmental stage. It has remained unknown whether the metacyclic VSGs possess distinct properties associated with this particular and discrete phase of the parasite life cycle. We present here three novel metacyclic form VSG N-terminal domain crystal structures (mVSG397, mVSG531, and mVSG1954) and show that they mirror closely in architecture, oligomerization, and surface diversity the known classes of bloodstream form VSGs. These data suggest that the mVSGs are unlikely to be a specialized subclass of VSG proteins, and thus could be poor candidates as the major components of prophylactic vaccines against trypanosomiasis.

The African trypanosome is a single-celled parasite causing African Sleeping Sickness in humans and related diseases in animals. It has a unique protein coat made up mostly of a single protein, called the V ariant S urface G lycoprotein, or VSG . The African trypanosome possesses several thousand different VSG genes. As the immune system of an infected host learns to recognize one coat protein and kill the trypanosomes, the parasite switches to a different VSG protein that is not recognized by the immune system. This allows the parasite to keep thriving in the host as this process repeats. This paper presents molecular images of a subset of VSG proteins that are on the surface of the organism when it is transferred to hosts by the bite of the tsetse fly—the first step in infection. We show that these so-called metacyclic VSGs are neither special nor unique compared to those VSGs that take over in the bloodstream during the course of infection. These results clarify the nature of these first VSG coats the host is exposed to and have important implications for trypanosome infection and vaccine development.

Funding: L.G. was supported by French Government Agence Nationale de la Recherche, ANR-17-CE12-0012 VSGREG and the salary of E. Tihon was paid by this award. The work was also supported by salary funds and resources from the German Cancer Research Center (DKFZ) to C.E.S. and F.N.P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Introduction

T. brucei is a unicellular pathogen and the cause of African trypanosomiasis in both human and cattle in sub-Saharan Africa [1,2]. Transmitted by the tsetse fly (Glossina sp), T. brucei is an extracellular pathogen, which swims freely in the bloodstream and is continuously exposed to immune system surveillance [3]. The bloodstream form (BSF) of T. brucei is covered by approximately ten million copies of the Variant Surface Glycoprotein (VSG) molecule, which comprises over 90% of the surface protein of the pathogen [4,5]. While there are over two thousand VSG-encoding genes and pseudogenes in the trypanosome genome, only one VSG is expressed at a given time from one of numerous bloodstream expression sites (BES) [6]. During the course of infection in the mammalian host, the pathogen undergoes antigenic variation, a phenomenon by which cells expressing one particular VSG are cleared by the humoral arm of the host immune system while a sub-population of trypanosomes switch their coat protein to an antigenically distinct variant and thus can no longer be identified and cleared [7]. The parasites with the “new” coat escape the host immune system and proliferate. This results in waves of parasitemia that characterize trypanosome infection[8].

The VSG protein can be roughly divided into three functional units: the signal peptide for delivery to the cell surface (and which is cleaved during translocation), a large N-terminal domain (NTD) that forms the bulk of the molecule, and the smaller C-terminal domain (CTD) that is tethered to the NTD by a flexible linker that can often be removed by endogenous and exogenous proteases [5,9]. The CTD bears the GPI-anchor, which attaches the VSG to the membrane. The N- and C-terminal domains of the VSGs are divided into different sub-categories based on sequence similarity and the positions of the cysteine residues. Five different classifications of VSG NTDs and six types of the CTD have been suggested amongst other classifications from sequence analysis [10–12]. Despite the wide arrays of antigenically distinct VSGs, there are only six atomic-resolution structures of the NTD that have been published: from T. brucei brucei VSG1 (MITat 1.1), VSG2 (MITat 1.2 or VSG221), VSG3 (MITat 1.3 or VSG224), VSG13 (MITat 1.13), ILTat1.24, as well as VSGsur from T. brucei rhodesiense, with three of these determined in the last four years [13–17]. Structures of the smaller, buried, and more flexible CTDs from VSG2 and IlTat1.24 have been determined separately by NMR [18,19].

The published NTD structures show that the tertiary folds of the VSGs share overall conservation, each resembling a dumbbell-shaped entity with upper and lower globular elements (“lobes”) connected by a three-helix bundle core [9]. These lobes are found as inserted sequences that connect the helices, replacing short linkers seen in more minimal bundle architectures. The conservation of this overall tertiary fold has suggested that the distinct antigenicity of the VSGs is produced primarily by the divergence in amino acid sequence, although the recent structure of VSG3 has broadened the structural “diversity space” of these coat proteins [16]. The presence of a post-translational modification (PTM) on VSG3 (an O-glycan at the top-surface of VSG NTD) added another layer of complexity to VSG antigenicity, as the glycan was shown to modulate the host immune response. In addition, the more recent structures of VSG13 and VSGsur [17] have shown that many architectural assumptions need reassessment, as these VSGs show significant deviation from the folds of older structures. Finally, the structure of VSG3 and recent biochemical work [20] have suggested that the NTDs of VSGs of this class can adopt monomeric (lower concentration) and trimeric (higher concentration) oligomeric states, in contrast to the strict dimers of other VSGs.

T. brucei has a complex life cycle which alternates between the mammalian host and its vector, the tsetse fly. The infectious metacyclic form of T. brucei inhabits the salivary gland of the tsetse fly and is injected into the mammalian host when the vector takes a bloodmeal. Metacyclic cells differentiate further to the rapidly growing bloodstream form soon after delivery into the host. In contrast to bloodstream VSGs, metacyclic VSGs (mVSGs) are expressed from a dedicated monocistronic metacyclic expression site (MES) that is shorter than the expression sites associated genes (ESAGs) containing bloodstream expression sites (BES)[21,22]. A specific subset of VSGs is expressed in the metacyclic stage, identified as mVSGs 397, 531, 559, 636, 639, 653, 1954, and 3591 (for the Lister 427 strain, [23,24]). These mVSGs represent the first and primary antigenic surface that is presented to the mammalian host immune system, an interaction that initially is focused on Th1 (pro-inflammatory type 1) cytokines, neutrophils and NK cell reactions to pathogen-associated molecular patterns (PAMPs), followed by T-cell independent IgM antibody activity. [25–29].

Because of these distinct stages of development and pathogenesis, it has been hypothesized that the mVSGs could manifest differences with bloodstream form VSGs in some manner related to their use in the initial stage of mammalian infection [30]. These could include structural divergence as well as differences in post-translational modifications (PTMs), such as are found in VSG3 [16]. VSG3 is topologically distinct from all previously characterized VSGs, potentially differing in oligomerization states, and as noted above, harbors O-linked hexose chains that are potent immune-modulators. This expansion of the “diversity space” of the VSGs raised the intriguing question: Would the mVSGs show additional structural or chemical diversity that might serve the pathogen in the first stages of entry into the host organism from the tsetse fly?

To begin to address these questions, we have solved the high-resolution crystal structures of mVSG397, mVSG531, and mVSG1954 from T. brucei brucei, strain Lister 427. Our results lead us to conclude that, as a whole, the mVSGs closely resemble the bloodstream form surface coat proteins both in structure and function.

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[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0011093

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