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Variant antigen diversity in Trypanosoma vivax is not driven by recombination

Sara Silva Pereira, Kayo J. G. de Almeida Castilho Neto, Craig W. Duffy, Peter Richards, Harry Noyes, Moses Ogugo, Marcos Rogério André, Zakaria Bengaly, Steve Kemp, Marta M. G. Teixeira, Rosangela Z. Machado, Andrew P. Jackson

Preprint posted on August 20, 2019 https://www.biorxiv.org/content/biorxiv/early/2019/08/20/733998

Do we know enough about Trypanosome spp. mechanisms of antigenic variation? T. vivax challenges our current understanding of variant antigen diversity.

Selected by Mariana De Niz

Background

African trypanosomes are vector-borne, extracellular and unicellular hemoparasites that cause African trypanosomiasis in humans and animals. One of the most studied pathogens (and therefore one of the best understood) among the Trypanosoma species is Trypanosoma brucei. In humans, T. brucei infection causes severe pathology including immune and neurological dysregulation, the latter manifesting in the disease known as ‘sleeping sickness’. A combination of successful public health measures for human African trypanosomiasis control, particularly vector control, has led to a decrease in cases by 96-97% over the last decade (1).

Despite this relative success for human health, various trypanosome species besides T. brucei, such as T. vivax and T. congolense, still cause widespread infection in animals including many livestock species (2) with devastating economic consequences. T. vivax is a livestock parasite found throughout sub-Saharan Africa and South America. Although to some extent similar to T. brucei and T. congolense, T. vivax has distinct morphological, cellular and genetic features.

A widely studied topic in Trypanosoma research is antigenic variation. T. brucei expresses one variant surface glycoprotein (VSG) gene at a time from a genomic repertoire of around 2000, and is densely coated at any one time with over 107 VSGs (3-6). Trypanosoma species evade the host’s adaptive immune response by repeatedly replacing their dense VSG coat, at the cell surface. As VSGs are the target of host antibodies, a small fraction of parasites can evade antibody-mediated clearance by switching their VSG coat, and expand to produce another wave of parasites with different VSGs. This ultimately results in a host/pathogen molecular ‘arms race’ resulting in persistent host failure to achieve parasite clearance. In T. brucei, directed gene conversion is fundamental for VSG switching and for generating antigenic diversity during infections.

Like other trypanosome species, T. vivax is also coated with VSGs and also displays antigenic variation. However, T. vivax VSGs are distinct from those in T. brucei or T. congolense, and lack key structures present in T. brucei or T. congolense which are necessary for gene conversion (7,8). In their work, Silva Pereira et al use bioinformatics and in vivo experimental infections to explore the basis behind diversification of T. vivax VSG sequences (9).

 

Key findings and developments

General findings

  • The variant antigen profile (VAP) the authors established for vivax shows that VSG sequence patterns in T. vivax are incompatible with the current T. brucei-based model for antigenic variation in trypanosomes. The VAP for T. vivax VSG gene repertoire allowed examining antigenic diversity across various strains.
  • The study also shows that the global VSG repertoire is broadly conserved across diverse vivax clinical strains, which explains why T. vivax serodemes can span multiple countries.
  • The authors found that recombination hardly drives antigenic diversity in T. vivax, which has important implications for the current mechanistic model of antigenic variation in African trypanosomes. It also may imply species differences in terms of virulence and transmission strategy.
  • The results from this work allow the authors to hypothesize that T. vivax may have an alternate mechanism for immune evasion or a distinct transmission strategy that reduces its reliance on long-term persistence.
  • Altogether, this has important implications for the understanding and control of animal African trypanosomiasis.

 

Figure 1. Variant antigen diversity in T. vivax is not driven by recombination. (Left) Flow-chart of analysis and key findings. (Right) Global T. vivax VSG repertoire is described by 174 phylotypes. Most phylotypes are cosmopolitan, found in multiple strains and in more than two regions. Only a minority are strain- or location-specific.

Specific findings

T. vivax VAP reflect genealogy

  • Genomes of 28 T. vivax clinical strains isolated from seven countries were sequenced. Using sequence homology with known VSG sequences in the T. vivax Y486 and T. brucei TREU927 reference genomes, between 40 and 436 VSG genes were recovered from assembled genome contigs.
  • Four VSG-like gene sub-families in the T. vivax Y486 reference sequence occurred in all genomes in similar proportions, making them unsuitable for discriminating between strains.
  • To overcome this challenge, the authors generated clusters of orthologs (COGs) for all VSG-like sequences from the reference T. vivax strain (Y486) and the 28 clinical strains, defining a COG as a group of VSG-like sequences with >90% sequence identity. Most COGs (78%) were cosmopolitan (i.e. present in multiple locations).
  • Altogether, the authors showed that T. vivax VSG repertoires diverge in concert with the wider genome, and provide a faithful record of population history.

 

Global T. vivax VSG repertoire comprises 174 phylotypes

  • The VSG gene complements in the strain genome sequences were incomplete. One of the effects of this incompleteness is that a COG-based VAP will include too many false ‘absences’ to reliably profile individual strains.
  • To overcome this, the authors devised a VAP analysis based on phylotypes, each consisting of multiple, related COGs with >70% sequence identity. Using this approach, 174 VSG phylotypes were observed. The approach and findings supported the idea that the T. vivax VSG repertoire is relatively conserved continent-wide. Population variation exists, though, and appears to originate through differential patterns of lineage loss rather than population-specific gene family expressions.

 

Minimal signature of recombination in T. vivax VSG sequences

  • The authors explored multiple approaches to test the hypothesis that vivax VSG recombine less than T. brucei and T. congolense VSG.
  • Segmental mapping showed that the mean proportion of Y486 VSG that are mosaics of strain genes is significantly lower in T. vivax than in T. congolense and T. brucei, while the number that are orthologous is significantly greater.
  • The authors modelled the history of recombination within fully coupled (orthologous) or multi-coupled (mosaic) sequence quartets using ancestral recombination graphs (ARG) and inferred the time to most recent common ancestor for each quartet.
  • Together, they concluded that brucei and T. congolense VSGs are routinely mosaics while the coalescence of most T. vivax VSG can be modelled without recombination.
  • The VAPs indicated that T.  vivax VSG typically retain orthology and essentially behave like ‘normal’ genes, while T. brucei or T. congolense VSGs recombine frequently, causing loss of orthology, and the appearance of strain-specific mosaics throughout the population.

 

Strong phylogenetic effects in VSG expression in vivo

  • Broadly conserved VSG phylotypes containing little signature of historical recombination, indicate that VSG mosaics do not contribute to antigenic diversity in vivo.
  • The authors tested this by measuring VSG transcript abundance in goats experimentally infected with T. vivax over a 40-day period, and performed transcriptomic analysis at peaks of infection.
  • Variant antigen profiling of the expressed transcripts characterised the dominant (or co-dominant) VSG phylotypes across successive peaks. Persistent expression of a phylotype across peaks, or re-emergence of a phylotype after decline was often seen.
  • Across all peaks, groups of related transcripts of the same phylotype were commonly co-expressed at the same peak.
  • Altogether, the major pattern emerging from in vivo expression profiles was a strong phylogenetic signal.

 

No mosaics of VSG phylotypes during experimental infections

  • Expressed VSG in T. brucei include sequence mosaics, which is interpreted as evidence for recombination of VSG loci during infections.
  • The authors analysed expressed T. vivax VSG transcript sequence mosaics using multiple approaches using BLASTn and GARD to identify potential recombination breakpoints.
  • Altogether, while most transcript alignments contained breakpoints, these only implicated very closely related sequences, and the scale of genetic admixture is comparable with other tandemly arrayed gene families (such as adenylate cyclases), rather than VSG mosaics.

What I like about this paper

The authors focused their work on T. vivax, and found extremely interesting and relevant molecular biology, which differs considerably from our current knowledge and assumptions regarding Trypanosoma spp. antigenic variation. Although T. brucei and T. cruzi are the most studied trypanosome species amongst neglected tropical diseases, other trypanosome species such as T. vivax, are rather understudied. Studying these species is important both from an epidemiological, veterinary, and economic point of view, and from a basic biology point of view.

Finally, I liked a lot that the authors were very thorough in generating and testing multiple hypotheses to explain the observed differences between T. brucei and T. vivax, and that they went as far as testing their hypothesis in vivo in a veterinary relevant host (i.e. goats).

Open questions

  1. In your discussion, you mention that while the number of T. vivax VSG genes is comparable to T. brucei and T. congolense, these provide fewer unique antigens because they are extremely similar, expressed simultaneously, and cannot recombine. A baseline arising from T. brucei is that immune evasion is achieved through VSGs. Could it be that immune evasion by T. vivax is linked to a different gene family which works together with, or is even alternative to, VSGs?
  2. Following from the previous question, there are now various studies using T. brucei exploring the in vivo effects on pathology and survival in mammalian hosts, upon simultaneous expression of more than one VSG (eg. Refs. 10, 11). How do these pathologies compare with what you observe in T. vivax?
  3. You mention in different parts of your paper, the idea of tropism. Other parasites, such as Plasmodium compromise specific organs important for antibody generation, such as the spleen and the bone marrow. This is linked to poor parasite control during infection, and to poor establishment of memory. Is there a chance that T. vivax tropism compromises adaptive immunity in a way that avoiding specific antibodies is not a priority? If so, from your data, are you interested in exploring which gene families those might be?
  4. In your work, you used mostly African clinical samples. While your comparison to the Brazilian strain shows high conservation, do you expect a similar finding if you compared strains across Latin America?
  5. Following from the previous question, in the Plasmodium field, one hypothesis is that given the geographical coincidence during evolution of multiple Plasmodium species with different degrees of virulence (eg. P. falciparum and P. vivax), the least virulent strain (P. vivax) has developed alternative methods of immune evasion (including dormancy), which may provide it with a survival advantage in the case of co-infections. If T. vivax and other trypanosome species, including T. brucei, are geographically co-related, what is known about co-infections, and might your findings hereby discussed give any advantage to T. vivax upon a co-infection?
  6. In terms of methodology, could you go explain further how you determine the clusters of orthologues, and how you define phylotypes?
  7. I personally like open science, and so was interested in the tool you developed, VAPPER (12). Could you explain in general, what VAPPER allows you to do, and why it is an important and valuable advance/contribution for Trypanosoma research?

References

  1. WHO, Global Health Observatory data (Accessed October 2019) https://www.who.int/gho/neglected_diseases/human_african_trypanosomiasis/en/
  2. Silva-Pereira S, Trindade S, De Niz M, Figueiredo LM, Tissue tropism in parasitic diseases, Open Biology, 9(5):190036 (2019)
  3. Cross GAM, Kim HS, Wickstead B, Capturing the variant surface glycoprotein reperotoire (the VSGnome) of brucei Lister 427. Mol. Biochem. Parasitol. 195, 59-73 (2014).
  4. Cross GAM, Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei, Parasitology, 71, 393 (1975).
  5. Mugnier, MR, Cross GAM, Papavasiliou FN, The in vivo dynamics of antigenic variation in brucei, Science 347, 1470-1473 (2015).
  6. Pinger J, Chowdhury S, Papavasiliou FN, Variant surface glycoprotein density defines an immune evasion threshold for African trypanosomes undergoing antigenic variation. Nat Comm. 8(1):828 (2017)
  7. Jackson AP, et al, Antigenic diversity is generated by distinct evolutionary mechanisms in African trypanosome species, Natl. Acad. Sci, 109, 3416-3421 (2012).
  8. Jackson AP, et al, A cell-surface phylome for African Trypanosomes, PLoS Negl Trop Dis, 7, (2013).
  9. Silva Pereira S, et al, Variant antigen diversity in Trypanosoma vivax is not driven by recombination, bioRxiv, (2019).
  10. Faria J, Glover L, Hutchinson S, Boehm C, Field MC, Horn D, Monoallelic expression and epigenetic inheritance sustained by a Trypanosoma brucei variant surface glycoprotein exclusion complex, Nat. Comm. 10(1):3023, (2019)
  11. Aresta-Branco F, Sanches-Vaz M, Bento F, Rodrigues JA, Figueiredo LM, African trypanosomes expressing multiple VSGs are rapidly eliminated by the immune system, Natl. Acad. Sci, 116(41):20725-20735, (2019).
  12. Silva Pereira S, Heap J, Jones AR, Jackson AP, VAPPER: high throughput variant antigen profiling in African trypanosomes of livestock, Gigascience, 8(9), 2019.

Acknowledgements

I am very grateful to Sara Silva Pereira and Andrew P. Jackson for the engaging discussions, and to Mate Palfy for his helpful comments on this highlight.

 

Posted on: 21st October 2019

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  • Author's response

    Sara Silva Pereira and Andrew P. Jackson shared

    Open questions

    1. In your discussion, you mention that while the number of T. vivax VSG genes is comparable to T. brucei and T. congolense, these provide fewer unique antigens because they are extremely similar, expressed simultaneously, and cannot recombine. A baseline arising from T. brucei is that immune evasion is achieved through VSGs. Could it be that immune evasion by T. vivax is linked to a different gene family which works together with, or is even alternative to, VSGs?

    T. vivax has several species-specific surface gene families whose function has yet to be discovered. As such, it is possible that they may contribute to antigenic variation somehow. In fact, it has been known for a while that the surface coat of T. vivax is less dense, perhaps because diverse proteins are present, besides the VSG. However, given their individual size, degree of conservation, and selection pressures, we think it is unlikely any of those would completely replace VSG as the major surface protein.

    2. Following from the previous question, there are now various studies using brucei exploring the in vivo effects on pathology and survival in mammalian hosts, upon simultaneous expression of more than one VSG (eg. Refs. 10, 11). How do these pathologies compare with what you observe in T. vivax?

    The T. brucei studies you reference describe the pathology and behaviour of parasite clones that express more than one VSG protein. In our paper, we are looking at the parasite infra-population, rather than individual parasites. We assume that T. vivax, like T. brucei cells, express a single VSG gene at any given moment, and that the transcriptomes we have produced represent the sum of multiple VSG across a whole population of cells. However, only single-cell transcriptomics coupled with single-cell proteomics would allow us to confirm this assumption, and then compare parasite survival and disease progression among clones that expressed different numbers of VSG.

    3. You mention in different parts of your paper, the idea of tropism. Other parasites, such as Plasmodium compromise specific organs important for antibody generation, such as the spleen and the bone marrow. This is linked to poor parasite control during infection, and to poor establishment of memory. Is there a chance that vivax tropism compromises adaptive immunity in a way that avoiding specific antibodies is not a priority? If so, from your data, are you interested in exploring which gene families those might be?

    We think it is not very clear yet to what degree T. vivax shows tissue tropism and what implications that may have on the infection. However, there are clear pathological changes in the spleen, liver, and bone marrow throughout infection, which may compromise parasite clearance and the development of an effective immune response.

    4. In your work, you used mostly African clinical samples. While your comparison to the Brazilian strain shows high conservation, do you expect a similar finding if you compared strains across Latin America?

    It was assumed that all South American strains derived from West Africa. Yet, we show that they do not because our Brazilian strains are genetically closer to Ugandan strains. This means that we may have more diversity in South America than previously anticipated. However, we expect strains in Africa to always be the most diverse due many more years of co-evolution, compared to a recent (less than 200 years) introduction of T. vivax in South America, but also because of biological transmission, which only happens in Africa.

    5. Following from the previous question, in the Plasmodium field, one hypothesis is that given the geographical coincidence during evolution of multiple Plasmodium species with different degrees of virulence (eg. P. falciparum and P. vivax), the least virulent strain (P. vivax) has developed alternative methods of immune evasion (including dormancy), which may provide it with a survival advantage in the case of co-infections. If T. vivax and other trypanosome species, including T. brucei, are geographically co-related, what is known about co-infections, and might your findings hereby discussed give any advantage to T. vivax upon a co-infection?

    The available epidemiological data suggests that co-infections are quite common in Africa. Despite what appears to be a diminished capacity for immune evasion due to the limited VSG repertoire, it is possible that T. vivax has devised a strategy to cause acute infections more often, so that it can increase the transmission potential. On the contrary, T. brucei very often causes prolonged infections, characterised by low parasitaemia and dissemination to tissues, hence the need for a virtually unlimited antigenic repertoire. From a transmission point of view, there are other things that may allow T. vivax to “get away” with lower VSG diversity. For example, T. vivax not only can be mechanically transmitted by biting flies, but also can infect tsetse flies of a wider age range, whereas T. brucei is more successful at infecting teneral flies.

    6. In terms of methodology, could you go explain further how you determine the clusters of orthologues, and how you define phylotypes?

    We defined clusters of orthologues has VSG-like sequences with ≥90% sequence identity to each other. We have created preliminary COGs using Orthofinder and then we manually curated them.  Phylotypes were defined as COGs with ≥70% average sequence identity using BLASTp and manual curation. Sorting sequences into COGs and phylotypes is very dependent on how you sample the population. There are places we were unable to sample, like Ethiopia and Zambia. We would expect the number, and potentially membership, of phylotypes to change as we add more data from different parts of Africa. We can see from the age of the phylotypes, however, that the general pattern of strongly discontinuous variation among T. vivax VSG, and the l;ack of recombination, will not change.

    7. I personally like open science, and so was interested in the tool you developed, VAPPER (12). Could you explain in general, what VAPPER allows you to do, and why it is an important and valuable advance/contribution for Trypanosoma research?

    VAPPER, which is described in another publication, is a tool that allows automated analysis of the variant surface glycoprotein repertoires of T. vivax and T. congolense. It produces variant antigen profiles for any isolate of these veterinary pathogens from genomic and transcriptomic sequencing data. This software facilitates the systematic analysis of VSG diversity and expression, which is necessary if we want to improve our general knowledge of antigenic variation, diversity, and evolution.

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