Microbes and Infection 16 (2014) 2e5www.elsevier.com/locate/micinf

Highlight

The Leishmania chromosome lottery*

Many items come in pairs: socks, mittens, chopsticks,earrings, and in the case of the majority of eukaryotic organ-isms, chromosomes. This diploid state is considered thecommon standard of the eurkaryotic karyotype, althoughmany plants and fungi may switch between a diploid state anda haploid state depending on the stage in their life cycle.Polyploidy, which involves duplication of the whole set ofchromosomes is relatively well tolerated across a number ofspecies and is believed to be an important driving force ofevolution and diversification in many organisms [2]. However,alterations in chromosome number whereby chromosomepairs behave differently from their comrades usually havedetrimental consequences on organism development in manyeukaryotes [3]. Such gains or losses in chromosomes is termedaneuploidy and is the leading cause of miscarriage in humans[4]. Playing with chromosome numbers results in changes togene dosage leading to stoichiometric imbalances in essentialgene products and reduced cellular fitness [5]. Surely suchchromosomal imbalances cannot be the norm for any organ-ism? In this issue of Microbes and Infection, Sterkers andcolleagues show that aneuploidy is a widespread feature of theeukaryotic Leishmania genus [1] and suggest that far fromspelling developmental disaster, chaotically organised chro-mosomes may offer certain advantages to this unconventionalmicro-organism.

Leishmania is a genus of single-celled parasites transmittedto human hosts via sand flies that cause the disease leishma-niasis, which shows a broad clinical spectrum ranging fromparticularly unpleasant, disfiguring cutaneous lesions topotentially fatal visceral leishmaniasis [6]. Around 20 speciesof Leishmania are thought to cause the disease in humans.Cutaneous Leishmania is caused by species such as the OldWorld Leishmania major and the New World Leishmaniamexicana whereas the more severe visceral leishmaniasis iscaused by species of the Leishmania donovani complex.Despite an estimated divergence of 20e100 million years,gene content amongst species is extremely well conserved,with Old and New World species possessing similar numbersof chromosomes (36 and 35 or 34 respectively), and genomesequencing revealing very few species specific genes [7].Instead, diversity within the realm of these parasites comes

* Article highlight of “Constitutive mosaic aneuploidy is a unique genetic

feature widespread in the Leishmania genus.” by Lachaud, L. et al. [1].

1286-4579/$ - see front matter � 2013 Institut Pasteur. Published by Elsevier Ma

http://dx.doi.org/10.1016/j.micinf.2013.11.008

from gene amplification and deletion events giving rise tohomologous chromosomes of varying sizes [8]. It was longsuspected that further alteration to gene dosage in Leishmaniawas also achieved by natural aneuploidy. The debate overthese parasites employment of what should be considered adevelopmentally insane strategy has largely been put to rest bymicroscopy experiments in L. major [9], and by sequencingacross a range of other species [7,10]. Analysis of chromo-some copy number by sequencing read depth revealed largedifferences for eight strains and species of Leishmania, withtrisomic read depth observed for up to nine chromosomes [7](Note that earlier clues as to the presence of trisomy werepreviously provided by the frustrations of scientists trying tocreate double-knockout mutants for the presumed disomicchromosomes).

However, the story of Leishmania’s dealings with aneu-ploidy does not end here. Sequencing analysis further showedthe peculiar presence of chromosomes with ‘intermediate’read depth that were neither disomic nor trisomic. Theproblem with this type of analysis however is that it givesinformation on a population of cells pooled together into onebig pot that may be (wrongfully) considered as geneticallyidentical. Sterkers and colleagues used DNA FISH toexamine single cell genomic organisation [9]. Of sevenanalysed chromosomes of L. major derived clones, none wereexclusively disomic; they were all monosomic, disomic, ortrisomic. In other words, aneuploidy in the population ofcells was mosaic. Sterkers and colleagues now demonstratethis ‘mosaic aneuploidy’ using single cell resolution techni-ques for six different chromosomes in three Old World spe-cies (Leishmania infantum, L. donovani, and Leishmaniatropica) and one New World species (Leishmania ama-zonensis) [1]. All chromosomes examined were monosomic,disomic, or trisomic, and less frequently tetrasomic in pro-portions that varied depending on the chromosome and spe-cies examined.

To abate the outcry of anyone who has ever worked withHeLa, or indeed with any extensively cultivated cell line, whowould rightly plead that genetic divergence in cell culture isunremarkable, Downing and colleagues measured geneticdiversity in Leishmania derived from clinical samples [10].They found extensive aneuploidy in the genomes of 17 phy-logenetically close strains of L. donovani. Each isolate had aunique karyotype. Vive la (Leishmania) revolution.

sson SAS. All rights reserved.

3Highlight / Microbes and Infection 16 (2014) 2e5

So how does mosaic aneuploidy arise and why is ittolerated? Possible mechanisms include defective segrega-tion of sister chromatids during cell division or chromo-some replication error. DNA FISH experiments on mitoticcells support the latter possibility [9]. Gain or losses inchromosomes may be authorised since the Leishmania thehaploid genome is small (approx. 32 Mb) and is distributedover a large number of chromosomes such that altering thecopy number of one chromosome does not affect manygenes.

Perhaps the most compelling question however is howmight the parasite benefit from such unorthodox chromosomebiology? In Leishmania, genes are organised in large poly-cistronic transcription units made of functionally unrelatedgenes containing no promoter for RNA polymerase II. Thisorganisation precludes typical mechanisms of transcriptionalregulation and instead the parasite relies largely on post-transcriptional control [11]. Increasing or decreasing genecopy number could be a way of regulating gene expression.Indeed, control of genomic output is predicted to be a robustfeature of the parasite’s biology since it has to cope with twovery different host environments: the fly gut, and mammalianmacrophages. These have very different pH, temperature, andnutrients and thus require substantial changes in geneexpression during the parasite’s life cycle. Furthermore,adaptivity to changing environments may be greatly aided bythe peculiar feature of mosaic aneuploidy. In haploids, detri-mental mutations may spell disaster for an individual cell, butthis ensures that such unfit mutants can be quickly weeded outto the benefit of the population as a whole ensuring the rapidselection of appropriate surviving mutants. Hence, deleteriousmutations are more effectively eliminated and beneficialmutations are more easily propagated in haploid rather thandiploid populations.

Leishmaniasis represents a substantial global burden witharound 350 million people at risk of contracting the diseaseand around 59,000 deaths due to visceral leishmaniasis eachyear [12]. There is currently no available vaccine. Mosaicaneuploidy has serious implications on strategies of diseaseprevention and control since any potential vaccine or drugtarget needs to be on a stable chromosome. Sterkers and col-leagues calculated that in the L. major Friedlin strain therecould be up to 2000 different genotypes, with the most fre-quent being present in 10% of the cells and the rarest beingabsent from a population of 1011 cells. This endless possibilityof combinations paints a worrying picture for attempts toconquer this parasite. Perhaps mechanisms creating aneu-ploidy themselves could be a viable drug target. In this sense,the study of a little-known protozoan may be highly relevant toone of nature’s darkest demons, cancer, since despite theseemingly detrimental effects of a fluctuating karyotype,aneuploidy is a hallmark of cancer and several lines of evi-dence suggest that aneuploidy has a casual role intumourgenesis.

Model organisms taught us that two is company, but three’sa crowd. Then along came the Leishmania to get the partystarted.

1. Biosketch e Yvon Sterkers

Dr. Yvon Sterkers completed a medical degree between1989 and 1995 in the Faculty of Medicine Necker (Paris V)and became a resident (Interne des Hopitaux de Paris) spe-cialising in clinical hematology. In 2001, he joined the Scherflaboratory in the Pasteur Institute for a PhD entitled “Plas-modium falciparum parasitized red blood cells surface mole-cules and their role in malaria pathogenesis”. He defended histhesis in 2005, and in 2006 he joined the department of Par-asitology Mycology and Prof. Patrick Bastien in Montpellier,where he obtained a permanent position as a lecturer in 2010.Today he shares his time amongst (i) the molecular diagnosisof toxoplasmosis both in routine and in the molecular pole ofthe reference national center for toxoplasmosis (coordinatedby Prof. P. Bastien; http://cnrtoxoplasmose.chu-reims.fr/); (ii)lectures for the medical school and for the master of science(http://www.masterbs.univ-montp2.fr/); (iii) basic science oncore molecular and cellular processes of Leishmania in UMRMIVEGEC (CNRS5290-IRD224-UM1) together with Lau-rence Lachaud (MCU-PH) and Michel Pages (CR CNRS).

2. Interview with Yvon Sterkers

1. What triggered your interest in mosaic aneuploidy inLeishmania sp?

We are interested in core cellular processes, and work onLeishmania sp., a protozoan parasite responsible for a largespectrum of diseases over four continents. We use molecularmethods with a particular emphasis on single cell methods(Fluorescent in situ hybridization (FISH), Immuno-fluorescence assay). If core cellular processes are heavilystudied in higher eukaryotes, they remain ill-known inLeishmania, considered as ‘ancestral’ or ‘divergent’

4 Highlight / Microbes and Infection 16 (2014) 2e5

eukaryotes. We feel that the peculiarities observed inLeishmania in comparison with model organisms are ofinterest. Indeed, based on these peculiarities, our researchmay open new avenues for the treatment of leishmaniases butin a larger extend, it may also help revisiting some dogma incell biology obtained in classical eukaryotic models (yeast,humans, etc.).

2. What was your first reaction when you faced the results?Did you expect them?

For FISH analysis, having good probes is the mostchallenging part of the technique and at the very beginning,the data were very puzzling. Nevertheless, the ploidy ofLeishmania has been debated over the last 20 years; but fora long time, the debate was fed only with indirect argu-ments. Until 2011, Leishmania was generally considered tobe diploid, although a few chromosomes were described asaneuploid. In that sense, the data we obtained did not sur-prise us, but we underestimated the plasticity of thisgenome. We could not foresee that mosaic aneuploidy inLeishmania was a widespread, constitutive feature andcorresponded to a dynamic equilibrium as we have descri-bed now.

3. How will the project go on?

The project needs to take two directions. (i) Until now weworked on parasites that were grown in vitro; we have todescribe the phenomenon in a population of parasites derivedfrom a natural infection. But since Montpellier is located in aregion endemic for visceral leishmaniasis due to L. infantum,we have a project on the ploidy of the amastigotes, the parasitestage in the mammalian host, derived from an infected dog.(ii) Our data, in particular the preliminary insights we obtainedon the mechanisms that underlie mosaic aneuploidy, questionthe regulation of DNA replication in Leishmania; therefore wehave begun to study DNA replication using molecular comb-ing and ChIP-seq. In addition, our data lead us to revisit thedata obtained from population studies and the consequenceson the biology of the parasite. In that sense, the question of theexistence of a “classical” sexuality which include the alter-nation of haploid and diploid stages versus a parasexual cyclehas to be raised.

4. What is the take-home message of the article?

Mitosis is an extremely accurate event in most eukaryoticcells, the main ‘objective’ for a cell is to reproduce an iden-tical copy of itself. This feature is largely found from yeast toman and plants, as a paradigm termed the “reproducibleinvariants” by Monod in ‘Chance and necessity’. We high-lighted that with the mosaic aneuploidy, a divergent eukaryotefollows a distinct ‘objective’ and a distinct ‘logic’ than theclassical eukaryotic models (yeast, humans, .). With mosaic

aneuploidy, the parasite, depending on the circumstances, maybenefit from haploidy, diploidy and triploidy.

5. Do you have a personal motto, quote or leading sentence?

Louis Pasteur used to say “Theory guides, experimentsdecide”.

6. What is your favourite hang-out method after a tough dayat the lab?

After a tough week, I like spending time with my wife andmy four kids, going either to the country side or sailing. Seaand beaches is a very positive aspect of working in Mont-pellier which makes the quality of life very high. After a toughday, beaches are less than twenty minutes by car from thelaboratory and you can swim from May to the end of Octoberin Montpellier.

7. In your opinion, what is the most important (scientific)discovery of the last decade?

Biology remains very complex and mostly undeciphered;sequencing of genomes had raised lots of hope. It has changedour daily lives as scientists and allows many technical devel-opments but was partly disappointing, because in the end,sequencing did not solve problems. We are now witnessing thedevelopment of Epigenetics that underlies a great deal of thevariation that occurs in all living beings and will allow us toobtain a much more precise picture of the core cellularprocesses.

8. If you could travel back in time, what historical person-ality would you like to meet and what scientific discoveryto assist to?

I have two answers; first, the pleasure in science comesfrom the discovery and the interpretation of unexpected/newdata. Many scientists have had this pleasure but in that sense, itcannot reoccur. Regarding my second answer, Louis Pasteur isthe scientist I would like to meet, and, since I am both a MDand a scientist, I could imagine myself, being Jacques-JosephGrancher and participating in the first successful vaccinationagainst rabies on Joseph Meister, a 9-year-old boy who hadbeen bitten by a rabid dog.

9. If you could travel forward in time what invention wouldyou like to check out?

In the fight against some infectious disease like AIDS andmalaria for example, I really feel that we need an eventualinvention. There is something we haven’t yet understood in thebiology of these organisms, in the way they bypass the defenceof the host. My only hope, for the best of the patients, is that Idon’t have to travel too far in the future to check out that.

Background

Aneuploidy, which involves the gain or loss of

chromosomes leading to imbalance in gene

dosage, is usually a disadvantage to organism

and cell fitness. Although under specific cir-

cumstances cells may benefit from an irregular

karyotype, most notably in cancer, ‘natural

aneuploidy’ is observed in only a handful of

organisms. These include certain yeast, some

common fungal pathogens, and parasites of the

Leishmania genus. Leishmania is transmitted to

human hosts via the bite of an infected sand fly

and cause the potentially fatal disease leishma-

niasis. Emerging evidence suggests not only the

presence of natural aneuploidy in Leishmania,but also strikingly that cells within the same

population may differ in karyotype. New evi-

dence presented in this issue of Microbes and

Infection show that this ‘mosaic aneuploidy’ is a

common feature of the Leishmania genus,

which could facilitate rapid evolution of the

parasite in response to changing environmental

conditions. With no vaccines and limited treat-

ment available, leishmaniasis is a major global

health problem. The discovery of mosaic aneu-

ploidy has important implications for drug

design and highlights Leishmania as a good

model organism in which to study aneuploidy

with relevance to other human diseases such as

cancer.

In a Nutshell

� ‘Mosasic aneuploidy’, defined as a mixed cell pop-ulation of with chromosomes of various somies, waspreviously suspected by genome sequencing of anumber of Leishmania species, but to date has onlybeen definitely shown by single cell resolution inLeishmania major.

� Using fluorescence in situ hybridisation, Sterkers andcolleagues demonstrate mosaic aneuploidy at thesingle cell level for six different chromosomes inthree Old World species (L. infantum, L. donovani,and L. tropica) and one New World species (L.amazonensis) of Leishmania. All chromosomes testedwere monosomic, disomic, or trisomic, and less fre-quently tetrasomic in proportions that varieddepending on the chromosome and species examined.

� These data show definitively for the first time thatmosaic aneuploidy is a widespread feature of theLeishmania genus.

5Highlight / Microbes and Infection 16 (2014) 2e5

References

[1] Lachaud L, Bourgeois N, Kuk N, Morelle C, Crobu L, Merlin G, et al.

Constitutive mosaic aneuploidy is a unique genetic feature widespread in

the Leishmania genus. Microbes Infect 2014;16:61e6.

[2] Otto SP, Whitton J. Polyploid incidence and evolution. Annu Rev Genet

2000;34:401e37. http://dx.doi.org/10.1146/annurev.genet.34.1.401.[3] Sheltzer JM, Amon A. The aneuploidy paradox: costs and benefits of an

incorrect karyotype. Trends Genet 2011;27:446e53. http://dx.doi.org/

10.1016/j.tig.2011.07.003.

[4] Brown S. Miscarriage and its associations. Semin Reprod Med

2008;26:391e400. http://dx.doi.org/10.1055/s-0028-1087105.

[5] Williams BR, Prabhu VR, Hunter KE, Glazier CM, Whittaker CA,

Housman DE, et al. Aneuploidy affects proliferation and spontaneous

immortalization in mammalian cells. Science 2008;322:703e9. http://

dx.doi.org/10.1126/science.1160058.

[6] Pearson RD, Sousa AQ. Clinical spectrum of Leishmaniasis. Clin Infect

Dis Off Publ Infect Dis Soc Am 1996;22:1e13.[7] Rogers MB, Hilley JD, Dickens NJ, Wilkes J, Bates PA, Depledge DP, et al.

Chromosome and gene copynumber variation allowmajor structural change

between species and strains of Leishmania. GenomeRes 2011;21:2129e42.http://dx.doi.org/10.1101/gr.122945.111.

[8] Pages M, Bastien P, Veas F, Rossi V, Bellis M, Wincker P, et al. Chro-

mosome size and number polymorphisms in Leishmania infantum sug-

gest amplification/deletion and possible genetic exchange. Mol Biochem

Parasitol 1989;36:161e8.

[9] Sterkers Y, Lachaud L, Crobu L, Bastien P, Pages M. FISH analysis

reveals aneuploidy and continual generation of chromosomal mosaicism

in Leishmania major. Cell Microbiol 2011;13:274e83. http://dx.doi.org/

10.1111/j.1462-5822.2010.01534.x.

[10] Downing T, Imamura H, Decuypere S, Clark TG, Coombs GH,

Cotton JA, et al. Whole genome sequencing of multiple Leishmania

donovani clinical isolates provides insights into population structure and

mechanisms of drug resistance. Genome Res 2011;21:2143e56. http://

dx.doi.org/10.1101/gr.123430.111.

[11] Clayton CE. Life without transcriptional control? From fly to man and

back again. EMBO J 2002;21:1881e8. http://dx.doi.org/10.1093/emboj/

21.8.1881.

[12] Sterkers Y, Lachaud L, Bourgeois N, Crobu L, Bastien P, Pages M. Novel

insights into genome plasticity in eukaryotes: mosaic aneuploidy in

Leishmania. Mol Microbiol 2012;86:15e23. http://dx.doi.org/10.1111/

j.1365-2958.2012.08185.x.

Emma Louise WaltonE-mail address: ewalton86@gmail.com

13 November 2013