DNA damage drives antigen diversification in Trypanosoma brucei | Nature

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Subjects

- DNA damage and repair

- Parasite evolution

- Parasite host response

- Parasite immune evasion

- Pathogens

Abstract

Antigenic variation, using large genomic repertoires of antigen-encoding genes, allows pathogens to evade host antibody. Many pathogens, including the African trypanosome Trypanosoma brucei, extend their antigenic repertoire through genomic diversification. Although evidence suggests that T. brucei depends on the generation of new variant surface glycoprotein (VSG) genes to maintain a chronic infection1,2,3,4, a lack of experimentally tractable tools for studying this process has obscured its underlying mechanisms. Here we present a highly sensitive targeted sequencing approach for measuring VSG diversification. Using this method, we demonstrate that a Cas9-induced DNA double-strand break within the VSG coding sequence can induce RAD51- and BRCA2-dependent VSG recombination with patterns identical to those observed during infection. These newly generated VSGs are antigenically distinct from parental clones and thus capable of facilitating immune evasion. Together, these results provide insight into the mechanisms of VSG diversification and an experimental framework for studying the evolution of antigen repertoires in pathogenic microorganisms.

Main

Pathogen survival in a host depends upon effective and continuous immune evasion. Several bacteria and eukaryotic pathogens have adopted the strategy of antigenic variation to evade host immunity, a process in which the pathogen continuously alters antigenic surface proteins to escape the host’s adaptive immune response. The African trypanosome Trypanosoma brucei, a unicellular eukaryotic parasite and causative agent of human and animal African trypanosomiasis, uses an especially sophisticated system of antigenic variation. The parasite, which remains extracellular throughout infection and thus faces a perpetual onslaught of host antibody, periodically ‘switches’ expression of a surface coat consisting of 107 copies of a single, immunogenic protein known as the variant surface glycoprotein (VSG). This process allows parasites to escape host antibody and maintain a chronic infection.

Although the T. brucei VSG repertoire contains thousands of VSGs, it is probably too small to maintain a chronic infection through VSG switching alone. During an infection, each T. brucei parasite expresses a single VSG at a time from one of about 15 telomeric bloodstream expression sites (BESs) (the ‘active’ BES)5. The remaining VSG-encoding genes are stored in other expression sites, subtelomeric arrays and minichromosomes, all of which remain transcriptionally silenced6. The parasite switches its VSG either by transcriptional activation of a silent BES (in situ switching) or through a gene conversion event in which a new VSG is copied into the active expression site. Although gene conversion-based switching allows for the activation of VSGs outside of a BES, analysis of the T. brucei genome has shown that only around 20% of the VSGs in the parasite genome are full-length genes encoding a functional VSG protein. The remaining approximately 80% of VSGs in the parasite genome consist of pseudogenes or gene fragments6,7,8 and cannot immediately be used for immune evasion through in situ or gene conversion switching. Moreover, the number of VSGs expressed in a population of parasites at a single time during experimental infection sometimes exceeds the total number of intact VSGs in the parasite genome1,9, further indicating that the repertoire of intact VSGs is insufficient to achieve the antigenic diversity required to maintain a chronic infection.

Evidence suggests that T. brucei deals with this shortage of antigens through diversification of the VSG repertoire. Many studies of experimental infections in mice have shown that novel VSGs, generated during infection, predominate at later stages of infection1,2,4, whereas analysis of parasites from natural human infections revealed expressed VSGs that were nearly completely absent from the genomes of contemporary field isolates10. These observations suggest that the generation of new VSGs has a critical role in sustaining T. brucei antigenic variation3.

There are two mechanisms thought to be responsible for extending the VSG repertoire: mosaic formation and de novo point mutation. Mosaic VSGs form when two or more VSG genes combine through segmental gene conversion to form a novel VSG. This mechanism allows parasites to access pseudogenes and VSG fragments within the repertoire. Where mosaic VSGs have been described in the literature, they are often found under strong, antibody-mediated selection11,12,13,14,15 or late during infection1,4,16,17, making it difficult to discern how exactly they arose. VSGs also appear occasionally to acquire de novo point mutations12,18, although these can be difficult to distinguish from small gene conversion events. Ultimately, de novo mutation of VSGs woul