Comparative proteomic analysis of Trypanosoma cruzi TcI lineage epimastigotes unveils metabolic and phenotypic differences between fast- and slow-dividing strains.

Trypanosoma cruzi, the causative agent of Chagas disease, is a genetically and phenotypically diverse species, divided into 5 main phylogenetic lineages (TcI to TcVI). TcI is the most widespread lineage in the Americas. Proteomics is a suitable tool to study the global protein expression dynamics in pathogens. Previous proteomic studies have revealed a link between (i) the genetic variability; (ii) the protein expression; and (iii) the biological characteristics of T. cruzi. Here, two-dimensional electrophoresis (2DE) and mass spectrometry were used to characterize the overall protein expression profiles of epimastigotes from four distinct TcI strains displaying different growth kinetics. Ascending hierarchical clustering analysis based on the global 2DE protein expression profiles grouped the strains under study into two clusters that were congruent with their fast or slow growth kinetics. A subset of proteins differentially expressed by the strains in each group were identified by mass spectrometry. Biological differences between the two groups, including use of glucose as an energy source, flagellum length, and metabolic activity, were predicted by proteomic analysis and confirmed by metabolic tests and microscopic measurements performed on the epimastigotes of each strain. Our results show that protein expression profiles are correlated with parasite phenotypes, which may in turn influence the parasite's virulence and transmission capacity.

Subspecific Nomenclature of Giardia duodenalis in the Light of a Compared Population Genomics of Pathogens.

Genetic and genomic data have long recognized that the species Giardia duodenalis is subdivided into at least eight genetic clusters that have been named "assemblages" by specialists in the field. Some of these assemblages have been given the status of species, with Linnean binames. In the framework of the predominant clonal evolution model (PCE), we have shown that, from an evolutionary point of view, G. duodenalis assemblages are equatable to "near-clades", that is to say: clades whose discreteness is somewhat clouded by occasional genetic exchange, but remain discrete and stable in space and time. The implications of this evolutionary status for the species described within G. duodenalis are discussed in light of the most recent genetic and genomic studies. The pattern of this species' subspecific genetic variability and genetic clustering appears to be very similar to the ones of various parasitic, fungal and bacteria species. This underlines the relevance of a compared population genomics of pathogenic species allowed by the broad framework of the PCE model.

Microevolution and subspecific taxonomy of Trypanosoma cruzi.

Trypanosoma cruzi, the agent of Chagas disease, is a highly polymorphic species, subdivided into 6 main evolutionary lineages or near-clades (formerly discrete typing units or DTUs). An additional near-clade (TC-bat) has recently been evidenced. This pattern is considered to be the result of predominant clonal evolution (PCE). PCE is compatible with occasional mating/hybridization, which do not break the prevalent pattern of clonal evolution, the main trait of it being the presence of Multigene Bifurcating Trees (MGBTs) at all evolutionary levels ("clonal frame"). The development of highly resolutive genetic (microsatellites*) and genomic (sequencing and multi-single nucleotide polymorphism {SNP}* typing) markers shows that PCE also operates at a microevolutionary* level within each of the near-clades ("Russian doll pattern"), in spite of occasional meiosis and hybridization events. Within each near-clade, one can evidence widespread clonal multilocus genotypes*, linkage disequilibrium*, Multigene Bifurcating Trees and lesser near-clades. The within near-clade population structure is like a miniature picture of that of the whole species, suggesting gradual rather than saltatory evolution. Additional data are required to evaluate the stability of these lesser near-clades in the long run and to evaluate the need for an adequate nomenclature for this microevolutionary level.

Leishmania and the Model of Predominant Clonal Evolution.

As it is the case for other pathogenic microorganisms, the respective impact of clonality and genetic exchange on Leishmania natural populations has been the object of lively debates since the early 1980s. The predominant clonal evolution (PCE) model states that genetic exchange in these parasites' natural populations may have a high relevance on an evolutionary scale, but is not sufficient to erase a persistent phylogenetic signal and the existence of bifurcating trees. Recent data based on high-resolution markers and genomic polymorphisms fully confirm the PCE model down to a microevolutionary level.

Models in parasite and pathogen evolution: Genomic analysis reveals predominant clonality and progressive evolution at all evolutionary scales in parasitic protozoa, yeasts and bacteria.

The predominant clonal evolution (PCE) model of pathogenic microorganisms postulates that the impact of genetic recombination in those pathogens' natural populations is not enough to erase a persistent phylogenetic signal at all evolutionary scales from microevolution till geological times in the whole ecogeographical range of the species considered. We have tested this model with a set of representative parasitic protozoa, yeasts and bacteria in the light of the most recent genomic data. All surveyed species, including those that were considered as highly recombining, exhibit similar PCE patterns above and under the species level, from macro- to micro-evolutionary scales (Russian doll pattern), suggesting gradual evolution. To our knowledge, it is the first time that such a strong common evolutionary feature among very diverse pathogens has been evidenced. The implications of this model for basic biology and applied research are exposed. These implications include our knowledge on the pathogens' reproductive mode, their population structure, the possibility to type strain and to follow up epidemics (molecular epidemiology) and to revisit pathogens' taxonomy through a flexible use of the phylogenetic species concept (Cracraft, 1983).

Genomics and High-Resolution Typing Confirm Predominant Clonal Evolution Down to a Microevolutionary Scale in Trypanosoma cruzi.

Trypanosoma cruzi, the agent of Chagas disease, is a paradigmatic case of the predominant clonal evolution (PCE) model, which states that the impact of genetic recombination in pathogens' natural populations is not sufficient to suppress a persistent phylogenetic signal at all evolutionary scales. In spite of indications for occasional recombination and meiosis, recent genomics and high-resolution typing data in T. cruzi reject the counterproposal that PCE does not operate at lower evolutionary scales, within the evolutionary units (=near-clades) that subdivide the species. Evolutionary patterns in the agent of Chagas disease at micro- and macroevolutionary scales are strikingly similar ("Russian doll pattern"), suggesting gradual, rather than saltatory evolution.

In Vitro Benznidazole and Nifurtimox Susceptibility Profile of Trypanosoma cruzi Strains Belonging to Discrete Typing Units TcI, TcII, and TcV.

We ascertain the in vitro Benznidazole (BZN) and Nifurtimox (NFX) susceptibility pattern of epimastigotes, trypomastigotes, and amastigotes of 21 T. cruzi strains, from patients, reservoir, and triatomine bugs of various geographic origins. Using this panel of isolates, we compute the Epidemiological cut off value (COwt). Then, the frequency of the susceptible phenotype (Wild type) towards benznidazole (BZN) and nifurtimox (NFX) within this set of strains belonging to three discrete typing units (DTUs), TcI, TcII, and TcV, was deduced. We observed that the susceptibility status of individual T. cruzi isolates toward BZN and NFX is related to the genetic background and underlying factors that are probably related to the individual life trait history of each strain. Analyzing drug susceptibility in this conceptual framework would offer the possibility to evidence a link between isolates expressing a low susceptibility level (not wild-type) as defined by the COwt value and none-curative treatment. It will also permit us to track drug-resistant parasites in the T. cruzi population.

Hybridization in Trypanosoma congolense does not challenge the predominant clonal evolution model. A comment on Tihon et al., 2017, Mol. Ecol.

Tihon et al. have just published in Mol. Ecol. a fine genomic study on Trypanosoma congolense, agent of Animal African Trypanosomiasis. They present very convincing evidence that T. congolense underwent several hybridization events between distinct genetic lines in Zambia. They claim that their data challenge our predominant clonal evolution model (PCE) of micropathogens. We point out the main tenets of our model and show that Tihon et al.'s claim is based on a misinterpretation of the PCE model. Actually, their data strongly support PCE in T. congolense at a microevolutionary level.

Pioneer study of population genetics of Rhodnius ecuadoriensis (Hemiptera: Reduviidae) from the central coastand southern Andean regions of Ecuador.

Effective control of Chagas disease vector populations requires a good understanding of the epidemiological components, including a reliable analysis of the genetic structure of vector populations. Rhodnius ecuadoriensis is the most widespread vector of Chagas disease in Ecuador, occupying domestic, peridomestic and sylvatic habitats. It is widely distributed in the central coast and southern highlands regions of Ecuador, two very different regions in terms of bio-geographical characteristics. To evaluate the genetic relationship among R. ecuadoriensis populations in these two regions, we analyzed genetic variability at two microsatellite loci for 326 specimens (n=122 in Manabí and n=204 in Loja) and the mitochondrial cytochrome b gene (Cyt b) sequences for 174 individuals collected in the two provinces (n=73 and=101 in Manabí and Loja respectively). The individual samples were grouped in populations according to their community of origin. A few populations presented positive FIS, possible due to Wahlund effect. Significant pairwise differentiation was detected between populations within each province for both genetic markers, and the isolation by distance model was significant for these populations. Microsatellite markers showed significant genetic differentiation between the populations of the two provinces. The partial sequences of the Cyt b gene (578bp) identified a total of 34 haplotypes among 174 specimens sequenced, which translated into high haplotype diversity (Hd=0.929). The haplotype distribution differed among provinces (significant Fisher's exact test). Overall, the genetic differentiation of R. ecuadoriensis between provinces detected in this study is consistent with the biological and phenotypic differences previously observed between Manabí and Loja populations. The current phylogenetic analysis evidenced the monophyly of the populations of R. ecuadoriensis within the R. pallescens species complex; R. pallescens and R. colombiensis were more closely related than they were to R. ecuadoriensis.

Relevant units of analysis for applied and basic research dealing with neglected transmissible diseases: The predominant clonal evolution model of pathogenic microorganisms.

The predominant clonal evolution (PCE) model seeks to formulate a common population genetics framework for all micropathogens (namely, parasitic protozoa, fungi and yeasts, bacteria, and viruses). It relies on a definition of clonality that is only based on population structure features (namely, strongly restrained genetic recombination). Its clear-cut properties make it of strong interest for applied and basic research, since it permits the definition of stable, clearly delimited units of analysis below the species level: clonal genotypes and discrete genetic subdivisions ("near-clades"). These units of analysis can be used for clinical and epidemiological studies, vaccine and drug design, species description, and evolutionary studies on natural and experimental populations. In this review, the evolutionary and population genetics background of the model will be only briefly mentioned, while considerable emphasis will be given to its practical significance for the study and control of neglected tropical diseases. The goal of the paper is to make this practical usefulness accessible to a broad audience of readers, including scientists who are not evolution specialists, such as epidemiologists, field scientists, and clinicians. For extensive developments about the evolutionary background of the model, see our previous papers [1-9]. Citations of these former articles lead to the many references quoted in them, which cannot be listed again here.

Reproductive clonality in protozoan pathogens--truth or artifact? A comment on Ramírez and Llewellyn.

The predominant clonal evolution (PCE) model of micropathogens proposed by us has been challenged by a recent paper in Molecular Ecology. We review the main tenets of our model and show that the criticisms raised by the paper's authors are based on papers that are either misunderstood or misquoted. We argue that the PCE model and its recent developments (in particular the 'Russian doll model' dealing with micro-clonal evolution) are supported in most cases when adequate data are available.