Multilocus Microsatellite Typing (MLMT) of Strains from Turkey and Cyprus Reveals a Novel Monophyletic L. donovani Sensu Lato Group

In eastern Mediterranean, leishmaniasis represents a major public health problem with considerable impact on morbidity and potential to spread. Cutaneous leishmaniasis (CL) caused by L. major or L. tropica accounts for most cases in this region although visceral leishmaniasis (VL) caused by L. infantum is also common. New foci of human CL caused by L. donovani complex strains were recently described in Cyprus and Turkey. Herein we analyzed Turkish strains from human CL foci in Çukurova region (north of Cyprus) and a human VL case in Kuşadasi. These were compared to Cypriot strains that were previously typed by Multilocus Enzyme Electrophoresis (MLEE) as L. donovani MON-37. Nevertheless, they were found genetically distinct from MON-37 strains of other regions and therefore their origin remained enigmatic. A population study was performed by Multilocus Microsatellite Typing (MLMT) and the profile of the Turkish strains was compared to previously analyzed L. donovani complex strains. Our results revealed close genetic relationship between Turkish and Cypriot strains, which form a genetically distinct L. infantum monophyletic group, suggesting that Cypriot strains may originate from Turkey. Our analysis indicates that the epidemiology of leishmaniasis in this region is more complicated than originally thought

Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain.IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery

The paraphyletic composition of Leishmania donovani zymodeme MON-37 revealed by multilocus microsatellite typing.

International audienceMultilocus microsatellite typing (MLMT) was employed to compare strains of Leishmania donovani belonging to the MON-37 zymodeme (MON-37 strains) from Cyprus and Israel to MON-37 strains from the Indian subcontinent, the Middle East, China and East Africa as well as strains of other zymodemes. The MLMT data were processed with a distance-based method for construction of phylogenetic trees, factorial correspondence analysis and a Bayesian model-based clustering algorithm. All three approaches assigned the MON-37 strains to different distantly related genetically defined subgroups, corresponding to their geographical origin. Specifically, the Kenyan, Sri Lankan and Indian MON-37 strains were genetically closer to strains of other zymodemes from the same regions than to MON-37 strains from other areas. MON-37 strains from Cyprus and Israel were clearly different not only among themselves, but also compared to all the other MON-37 strains studied and could, therefore, be autochthonous. This study showed that the zymodeme MON-37 is paraphyletic and does not reflect the genetic relationship between strains of different geographical origin

Genetic diversity and structure in Leishmania infantum populations from southeastern Europe revealed by microsatellite analysis

WOS: 000328837200003PubMed ID: 24308691Background: The dynamic re-emergence of visceral leishmaniasis (VL) in south Europe and the northward shift to Leishmania-free European countries are well-documented. However, the epidemiology of VL due to Leishmania infantum in southeastern (SE) Europe and the Balkans is inadequately examined. Herein, we aim to re-evaluate and compare the population structure of L. infantum in SE and southwestern (SW) Europe. Methods: Leishmania strains collected from humans and canines in Turkey, Cyprus, Bulgaria, Greece, Albania and Croatia, were characterized by the K26-PCR assay and multilocus enzyme electrophoresis (MLEE). Genetic diversity was assessed by multilocus microsatellite typing (MLMT) and MLM Types were analyzed by model- and distance-based algorithms to infer the population structure of 128 L. infantum strains. Results: L. infantum MON-1 was found predominant in SE Europe, whilst 16.8% of strains were MON-98. Distinct genetic populations revealed clear differentiation between SE and SW European strains. Interestingly, Cypriot canine isolates were genetically isolated and formed a monophyletic group, suggesting the constitution of a clonal MON-1 population circulating among dogs. In contrast, two highly heterogeneous populations enclosed all MON-1 and MON-98 strains from the other SE European countries. Structure sub-clustering, phylogenetic and Splitstree analysis also revealed two distinct Croatian subpopulations. A mosaic of evolutionary effects resulted in consecutive sub-structuring, which indicated substantial differentiation and gene flow among strains of both zymodemes. Conclusions: This is the first population genetic study of L. infantum in SE Europe and the Balkans. Our findings demonstrate the differentiation between SE and SW European strains; revealing the partition of Croatian strains between these populations and the genetic isolation of Cypriot strains. This mirrors the geographic position of Croatia located in central Europe and the natural isolation of the island of Cyprus. We have analysed the largest number of MON-98 strains so far. Our results indicate extensive gene flow, recombination and no differentiation between MON-1 and MON-98 zymodemes. No correlation either to host specificity or place and year of strain isolation was identified. Our findings may be associated with intensive host migration and common eco-epidemiological characteristics in these countries and give valuable insight into the dynamics of VL.GSRT-TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [SBAG-GSRT-23 (109S448)]This study was supported by the GSRT-TUBITAK grant [joint Research and Technology programmes 2010-2011, Greece-Turkey, Project no: SBAG-GSRT-23 (109S448)]. The authors also thank Dr. E. Dotsika (Laboratory of Cellular Immunology, Hellenic Pasteur Institute) for kindly providing strain BG1 and Dr. Teita Myrseli and Dr. Silva Bino (Control of Infectious Diseases Department, Institute of Public Health, Tirana, Albania) for providing Leishmania DNA for the strains AH2, AH4 and AD16. This work was in partial fulfillment of the requirements of Charite Universitatsmedizin in Berlin (Germany) toward the PhD of Ms Evi Gouzelou

Factorial correspondence analysis (FCA) of the 76 <i>L. donovani</i> complex strains studied.

<p>CY, Cyprus; ET, Ethiopia; GR, Greece; IN, India; KE, Kenya; LK, Sri Lanka; PT, Portugal; SD, Sudan; SP, Spain; TR, Turkey; Populations designated as MON-1 (red squares) and non MON-1 (orange and brown squares) include respectively <i>L. infantum</i> MON-1 strains from Greece, Turkey, Spain and Portugal and <i>L. infantum</i> non MON-1 strains from Spain, Portugal, France and Italy, corresponding to previous analyses <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd.0001507-Kuhls2" target="_blank">[18]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd.0001507-Kuhls3" target="_blank">[21]</a>; populations designated as IN1 (India 1), IN3 (India 3) and LK (Sri Lanka) (yellow squares); SD/ET (Sudan/Ethiopia) (pink squares); KE/IN2 (Kenya/India 2) (blue squares) compose the <i>L. donovani</i> genetic group, as detected previously <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd.0001507-Alam1" target="_blank">[16]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd.0001507-Alam2" target="_blank">[17]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd.0001507-Kuhls2" target="_blank">[18]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd.0001507-Kuhls3" target="_blank">[21]</a>; <i>L. infantum</i> non MON-1 strains from Turkey and Cyprus are designated as TR (dark purple squares) and CY (light purple squares), respectively. The MON-37 clone of CD44 strain from Cyprus groups with the other CY non MON-1 strains. The TR/CY strains are placed between the <i>L. infantum</i> MON-1 and non MON-1 populations and the hybrid strain EP59 from Turkey between the TR non MON-1 and MON-1 populations. The BUCK strain is placed very close to the TR non MON-1 strains, as previously described <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd.0001507-Kuhls2" target="_blank">[18]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd.0001507-Kuhls3" target="_blank">[21]</a>.</p

Midpoint rooted Neighbor-joining tree for the 76 <i>L. donovani</i> complex strains analysed.

<p>This midpoint rooted tree was inferred from Dps-distances calculated for the MLMT data (14 microsatellite markers) of our sample set. The tree leaves are coloured according to the populations defined by structure analysis (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd-0001507-g002" target="_blank">Fig. 2</a>). Bootstrap values (1000 re-samplings) above 50% are indicated at key nodes. The EP59 strain was excluded from this analysis due to its hybrid profile.</p

Estimated population structure of the 76 <i>L. donovani s.l.</i> strains as inferred by STRUCTURE.

<p>MLMT profiles are based on variations in 14 microsatellite markers. Each strain is represented by a single vertical line divided into K colours, where K is the number of populations assumed. Each colour represents one population and the length of the coloured segments shows the strain's estimated proportion of membership in that population. Strains with mixed memberships to the different populations are represented by different coloured segments in the vertical bar, which are proportional to the membership coefficient. <b>A.</b> When the likelihood of population number is calculated according to Evanno et al. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd.0001507-Evanno1" target="_blank">[28]</a>, the derived graph for ΔK shows a peak at K = 2 indicating the existence of two main populations in the studied strain set. However, eight populations are observed based on a log-likelihood plot, which plateaus at K = 8. At K = 8, all Turkish non MON-1 strains group with MON-37 strains from Cyprus, forming the TR/CY non MON-1 population. Bar plots for K = 3 and K = 4 are also shown to help determine ancestral populations. <b>B.</b> When the <i>L. infantum</i> non MON-1 & TR/CY non MON-1 strains are re-analysed separately the log-likelihood plot plateaus at K = 5, while the ΔΚ graph shows a major peak at K = 2 and a minor one at K = 5. At K = 5 a split of the TR/CY non MON-1 population was observed.</p

<i>K26</i>-PCR confirms that CUK1, CUK2 and EP59 strains belong to the <i>L. donovani</i> complex.

<p>Lanes: M, 100 bp DNA ladder; CH35 (MON-37, 700 bp); PM1 (MON-1, 626 bp); SC23 (MON-38, 515 bp); CUK1 (MON-309, 480 bp); CUK2 (MON-309, 480 bp); HUSSEN (MON-31, 430 bp); EP59 (MON-308, 385 bp); BUCK (MON-78, 385 bp). Strains CUK3, CUK4, CUK7 and CUK 10 gave identical results to CUK1 and CUK2 (not shown).</p

Characteristics of the 76 <i>Leishmania</i> strains used in this study.

<p>The non MON-1 strains from Turkey as well as the Cyprian canine isolate clone 1 analysed herein are presented in bold.</p><p>VL, visceral leishmaniasis; CL, cutaneous leishmaniasis; PKDL, post Kala Azar dermal leishmaniasis; CanL, canine leishmaniasis.</p><p>*Only one of the three MON-37 clones (cl.1) isolated from the parent CD44 strain was further analyzed;</p><p>**Hybrid strain; CY, Cyprus; ET, Ethiopia; GR, Greece; IN, India; KE, Kenya; LK, Sri Lanka; PT, Portugal; SD, Sudan; SP, Spain; TR, Turkey; n.d., not defined.</p

Genetic variability parameters of the strain populations under study.

<p>N, number of strains; P, proportion of polymorphic loci; MNA, mean number of alleles; H_e, expected heterozygosity; H_o, observed heterozygosity; F_IS, inbreeding coefficient; CY, Cyprus; GR, Greece; PT, Portugal; SP, Spain; TR, Turkey.</p><p>*When all 76 strains are analyzed 8 populations are identified by STRUCTURE (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd-0001507-g002" target="_blank">Fig. 2A</a>). Here the genetic variability parameters of four of these populations are shown;</p>a<p>The two <i>L. infantum</i> non MON-1 populations are analyzed together;</p><p>**At a subsequent STRUCTURE analysis the TR/CY non MON-1 group splits into two subpopulations (TR non MON-1 and CY non MON-1, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001507#pntd-0001507-g002" target="_blank">Fig. 2B</a>), which are analyzed here;</p>b<p>The respective values when the hybrid EP59 strain is included in the respective population are given in parenthesis.</p