Microevolution of Trypanosoma cruzi reveals hybridization and clonal mechanisms driving rapid genome diversification
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Evaluation Summary:
In this paper the authors dissect the across-the-genome consequences of sexual recombination in Trypanosoma cruzi. It summarises an extensive piece of work, which includes a 5-year in vitro growing of parasites and relatively rigorous genome analyses. It will be of interest to microbiologists working on microbes with cryptic or parasexual modes of genetic exchange.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Protozoa and fungi are known to have extraordinarily diverse mechanisms of genetic exchange. However, the presence and epidemiological relevance of genetic exchange in Trypanosoma cruzi , the agent of Chagas disease, has been controversial and debated for many years. Field studies have identified both predominantly clonal and sexually recombining natural populations. Two of six natural T. cruzi lineages (TcV and TcVI) show hybrid mosaicism, using analysis of single-gene locus markers. The formation of hybrid strains in vitro has been achieved and this provides a framework to study the mechanisms and adaptive significance of genetic exchange. Using whole genome sequencing of a set of experimental hybrids strains, we have confirmed that hybrid formation initially results in tetraploid parasites. The hybrid progeny showed novel mutations that were not attributable to either (diploid) parent showing an increase in amino acid changes. In long-term culture, up to 800 generations, there was a variable but gradual erosion of progeny genomes towards triploidy, yet retention of elevated copy number was observed at several core housekeeping loci. Our findings indicate hybrid formation by fusion of diploid T. cruzi , followed by sporadic genome erosion, but with substantial potential for adaptive evolution, as has been described as a genetic feature of other organisms, such as some fungi.
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Evaluation Summary:
In this paper the authors dissect the across-the-genome consequences of sexual recombination in Trypanosoma cruzi. It summarises an extensive piece of work, which includes a 5-year in vitro growing of parasites and relatively rigorous genome analyses. It will be of interest to microbiologists working on microbes with cryptic or parasexual modes of genetic exchange.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
In this paper the authors dissect the across-the-genome consequences of sexual recombination in Trypanosoma cruzi, a serious human pathogen, which some of the co-authors study for decades. They also did the seminal discovery of hybrid formation in this species almost 20 years ago and this is a follow-up enabled by whole genome sequencing and analyses. The data is novel, with importance outside of the field.
Major strength is the dissection of set of hybrid strains, kept for several years and 800 generations, nothing trivial with a pathogen with a potential to infect the staff. While the conclusions are not revolutionary and have to some extent been made in yeast, it is surprising how different the outcome of sex is in T. brucei and T. cruzi. Also the data on ploisy is certainly novel for all trypanosomatid …
Reviewer #1 (Public Review):
In this paper the authors dissect the across-the-genome consequences of sexual recombination in Trypanosoma cruzi, a serious human pathogen, which some of the co-authors study for decades. They also did the seminal discovery of hybrid formation in this species almost 20 years ago and this is a follow-up enabled by whole genome sequencing and analyses. The data is novel, with importance outside of the field.
Major strength is the dissection of set of hybrid strains, kept for several years and 800 generations, nothing trivial with a pathogen with a potential to infect the staff. While the conclusions are not revolutionary and have to some extent been made in yeast, it is surprising how different the outcome of sex is in T. brucei and T. cruzi. Also the data on ploisy is certainly novel for all trypanosomatid parasites. The same applies for the dramatic loss of up to 23 kb per generation, the increased presence of SNPs after the hybridization etc.
I don't have suggestions for additional analyses, as those presented seem to me comprehensive and justifying the conclusions. I have some mostly minor comments that should help the authorst to improve the moanuscript.
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Reviewer #2 (Public Review):
By following T. cruzi cultures for 5 years (~ 800 generations) the authors were trying to observe the mechanisms by which the parasite re-shapes its genome under laboratory conditions (long-term passage in culture). By analyzing clones that arose from a genetic cross, the authors were also trying to characterize in detail the process of hybridization (genetic exchange), which results in an increase in DNA content (ploidy) and the subsequent events that reduce DNA content by chromosome/gene loss that lead to mosaic aneuploid cells. The authors achieved their aims and the results support their conclusions.
Major strengths:
The major strengths of the methods are the chosen parasite clones used for the experiment, following up on the descendant clones from the experimental cross described originally by Gaunt MW …
Reviewer #2 (Public Review):
By following T. cruzi cultures for 5 years (~ 800 generations) the authors were trying to observe the mechanisms by which the parasite re-shapes its genome under laboratory conditions (long-term passage in culture). By analyzing clones that arose from a genetic cross, the authors were also trying to characterize in detail the process of hybridization (genetic exchange), which results in an increase in DNA content (ploidy) and the subsequent events that reduce DNA content by chromosome/gene loss that lead to mosaic aneuploid cells. The authors achieved their aims and the results support their conclusions.
Major strengths:
The major strengths of the methods are the chosen parasite clones used for the experiment, following up on the descendant clones from the experimental cross described originally by Gaunt MW et al (2003). And the analysis of these clones over long-term passage in culture. By focusing on this particular set of T. cruzi clones, they maximized the chances of observing drastic changes on the ploidy and mutational landscape of these genomes. This is something difficult to achieve for T. cruzi under standard conditions.
The major strengths of the results presented lie in the validation of the mechanism of genetic exchange proposed by Gaunt et al in 2003 (now observed and inspected in greater detail). It also confirms that T. cruzi is a constitutive aneuploid organism, displaying mosaic aneuploidies with a plastic genome.
Weaknesses:
The weaknesses of the study lie on the use of a single sequencing technology (short-reads) that certainly limited the ability of authors to observe other genome shaping events (structural variants, translocations, complex rearrangements, breakpoints). The lack of a phased diploid genome assembly for the hybrids (or some other means of taking advantage of long range information for constructing haplotypes) also prevented observing recombination between parental chromosomes.
Impact:
The paper is a very important contribution that builds up on previous important findings (Gaunt MW et al 2003), and provide a comprehensive look at events that ensue after genetic exchange by hybridization (cell fusion) in T. cruzi. The data would be extremely useful for the community of scientists doing research on this important pathogen.
Additional context:
Readers should be familiar with "parasexuality" or "cryptic sexuality" as described for other unicellular organisms such as Candida albicans (fungal pathogen). On top of a parasexual genetic exchange process achieved by hybridization (fusion of diploid cells to produce tetraploid ) kinetoplastids such as Leishmanias and Trypanosoma cruzi (as shown herein), are very plastic genomes showing varying degrees of "mosaic aneuploidies".
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Reviewer #3 (Public Review):
The authors cultivated Trypanosoma cruzi parents and their hybrids in vitro for 800 generations and investigated changes in DNA content and genome diversity over time. Hybrid clones were generated in vitro from a cross between two TcI strains; this cross was done within the context of a research study published in 2003 (Gaunt et al. 2003; https://doi.org/10.1038/nature01438). Here, the authors sequenced the parents and their hybrids, cultured them for 5 years and then re-sequenced all parasite clones. Using extensive genome analyses, the authors demonstrated that hybrids were tetraploid and that there is a reduction in DNA/genome content following hybridisation. This pattern resembles the parasexual mechanism, rather than canonical meiotic recombination, as has been observed in other microorganisms such as …
Reviewer #3 (Public Review):
The authors cultivated Trypanosoma cruzi parents and their hybrids in vitro for 800 generations and investigated changes in DNA content and genome diversity over time. Hybrid clones were generated in vitro from a cross between two TcI strains; this cross was done within the context of a research study published in 2003 (Gaunt et al. 2003; https://doi.org/10.1038/nature01438). Here, the authors sequenced the parents and their hybrids, cultured them for 5 years and then re-sequenced all parasite clones. Using extensive genome analyses, the authors demonstrated that hybrids were tetraploid and that there is a reduction in DNA/genome content following hybridisation. This pattern resembles the parasexual mechanism, rather than canonical meiotic recombination, as has been observed in other microorganisms such as Candida albicans.
Strengths:
The main strength of this paper is that the authors investigated the genetic consequences of hybridisation and clonal reproduction experimentally. Virtually all studies investigated these processes using "natural" isolates, where many confounding factors could complicate the correct interpretation of the data at hand (see for instance the review of Ramirez and Llewellyn, 2014; https://doi.org/10.1111/mec.12872). Here, the authors sequenced clones from the only genetic exchange event observed experimentally in T. cruzi (Gaunt et al. 2003). In addition, the authors went at length by culturing parental and hybrid clones for 5 years, which is a relatively laborious and time-consuming process, allowing them to follow the evolution of hybrid genomes under long-term in vitro culturing. This effort provided unique data demonstrating that tetraploid T cruzi hybrids undergo genome erosion.
Weaknesses:
One of the main limitations of the study is that it investigates hybrids from one single cross. We now know that T cruzi parasites - like many other protozoans - display disparate reproductive strategies and rates of genetic exchange, even within single disease foci (e.g. Schwabl et al. 2019; https://doi.org/10.1038/s41467-019-11771-z). Here, the study investigates the impact of hybridisation from one single cross that was done decades ago, and for which it was already postulated that hybridisation followed a parasexual process (Gaunt et al. 2003). Hence, the aim of this paper is essentially restricted to investigating the genetic consequences of parasexual hybridisation followed by clonal evolution in vitro. This should be clearly explained when outlining the aims of this paper, highlighting that it investigates one of many possible reproductive strategies that T cruzi employs.
Insufficient analyses are performed to support the key claims in the manuscript by the data presented. In particular:
a) One of the main claims is that tetraploid Tcruzi hybrids undergo gradual genome erosion. I agree with the authors that there is genome erosion, but this is not gradual for all hybrid clones. The largest reduction in DNA content occured at the beginning of the culturing experiment, and three of six hybrid clones show a similar DNA content after 800 generations of culturing. This is also apparent from the microsatellite data where alleles were mainly lost in only one of the three hybrid strains. These results are completely ignored by the authors. Within this context, it is unfortunate that the authors sequenced the parental and hybrid clones at only two time points, and not at intermediary time points, which would allow pinpointing the genomic loss events during the first tens of generations of culturing.
b) Another main claim of the study is that surface molecule genes represent regions of the genome with higher genetic diversity and more rapid evolution. However, these surface molecule genes are often highly repetitive, and such regions are generally masked before variant calling because variant calling within such regions often lead to false-positive SNPs. During culturing, it is evident that mitotic recombination events may reshuffle such repetitive regions, which will also lead to the identification of new (but false) variants by the end of the experiment. The bio-informatic analyses are not sufficient to convince me that the authors have adequately dealt with the repetitive nature of the T cruzi genome to allow such claims.
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