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.

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.

How clonal are Neisseria species? The epidemic clonality model revisited.

The three species Neisseria meningitidis, Neisseria gonorrheae, and Neisseria lactamica are often regarded as highly recombining bacteria. N. meningitidis has been considered a paradigmatic case of the "semiclonal model" or of "epidemic clonality," demonstrating occasional bouts of clonal propagation in an otherwise recombining species. In this model, occasional clonality generates linkage disequilibrium in the short term. In the long run, however, the effects of clonality are countered by recombination. We show that many data are at odds with this proposal and that N. meningitidis fits the criteria that we have proposed for predominant clonal evolution (PCE). We point out that (i) the proposed way to distinguish epidemic clonality from PCE may be faulty and (ii) the evidence of deep phylogenies by microarrays and whole-genome sequencing is at odds with the predictions of the semiclonal model. Last, we revisit the species status of N. meningitidis, N. gonorrheae, and N. lactamica in the light of the PCE model.

Cryptosporidium,Giardia, Cryptococcus, Pneumocystis genetic variability: cryptic biological species or clonal near-clades?

An abundant literature dealing with the population genetics and taxonomy of Giardia duodenalis, Cryptosporidium spp., Pneumocystis spp., and Cryptococcus spp., pathogens of high medical and veterinary relevance, has been produced in recent years. We have analyzed these data in the light of new population genetic concepts dealing with predominant clonal evolution (PCE) recently proposed by us. In spite of the considerable phylogenetic diversity that exists among these pathogens, we have found striking similarities among them. The two main PCE features described by us, namely highly significant linkage disequilibrium and near-clading (stable phylogenetic clustering clouded by occasional recombination), are clearly observed in Cryptococcus and Giardia, and more limited indication of them is also present in Cryptosporidium and Pneumocystis. Moreover, in several cases, these features still obtain when the near-clades that subdivide the species are analyzed separately ("Russian doll pattern"). Lastly, several sets of data undermine the notion that certain microbes form clonal lineages simply owing to a lack of opportunity to outcross due to low transmission rates leading to lack of multiclonal infections ("starving sex hypothesis"). We propose that the divergent taxonomic and population genetic inferences advanced by various authors about these pathogens may not correspond to true evolutionary differences and could be, rather, the reflection of idiosyncratic practices among compartmentalized scientific communities. The PCE model provides an opportunity to revise the taxonomy and applied research dealing with these pathogens and others, such as viruses, bacteria, parasitic protozoa, and fungi.

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.

Reproductive clonality of pathogens: a perspective on pathogenic viruses, bacteria, fungi, and parasitic protozoa.

We propose that clonal evolution in micropathogens be defined as restrained recombination on an evolutionary scale, with genetic exchange scarce enough to not break the prevalent pattern of clonal population structure, a definition already widely used for all kinds of pathogens, although not clearly formulated by many scientists and rejected by others. The two main manifestations of clonal evolution are strong linkage disequilibrium (LD) and widespread genetic clustering ("near-clading"). We hypothesize that this pattern is not mainly due to natural selection, but originates chiefly from in-built genetic properties of pathogens, which could be ancestral and could function as alternative allelic systems to recombination genes ("clonality/sexuality machinery") to escape recombinational load. The clonal framework of species of pathogens should be ascertained before any analysis of biomedical phenotypes (phylogenetic character mapping). In our opinion, this model provides a conceptual framework for the population genetics of any micropathogen.

Phylogenetic character mapping of proteomic diversity shows high correlation with subspecific phylogenetic diversity in Trypanosoma cruzi.

We performed a phylogenetic character mapping on 26 stocks of Trypanosoma cruzi, the parasite responsible for Chagas disease, and 2 stocks of the sister taxon T. cruzi marinkellei to test for possible associations between T. cruzi-subspecific phylogenetic diversity and levels of protein expression, as examined by proteomic analysis and mass spectrometry. We observed a high level of correlation (P < 10(-4)) between genetic distance, as established by multilocus enzyme electrophoresis, and proteomic dissimilarities estimated by proteomic Euclidian distances. Several proteins were found to be specifically associated to T. cruzi phylogenetic subdivisions (discrete typing units). This study explores the previously uncharacterized links between infraspecific phylogenetic diversity and gene expression in a human pathogen. It opens the way to searching for new vaccine and drug targets and for identification of specific biomarkers at the subspecific level of pathogens.

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.

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).

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.

Modelling the transmission of Trypanosoma cruzi: the need for an integrated genetic epidemiological and population genomics approach.

This chapter describes what should be an integrated approach to the genetic epidemiology and population genomics of Chagas disease. Many studies have been conducted on the genetic diversity of Trypanosoma cruzi and the various triatomine bug species able to transmit Chagas disease. Far less research has analyzed the role played by the host's genetic variability on the transmission and severity of the disease. An integrated genetic epidemiology/population genomics approach would analyze these three components of the transmission chain together as well as their possible interactions (co-evolution phenomena). This is facilitated by the recent impressive progress in mega biotechnologies and by the fact that Chagas disease is an ideal model for experimental evolution approaches.

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.

Microbial molecular epidemiology: an overview.

In this introductory chapter, I stress one more time the urgency to better connect molecular epidemiology and evolutionary biology. I show how much population genetics and phylogenetic analyses can confer a considerable added value to all attempts to characterize strains and species of pathogens. The problems dealing with the mere definition of basic concepts, such as species, subspecies, or strains, are briefly summarized. Last, I show the important contribution of molecular epidemiology to our knowledge of the basic biology of pathogens and insist on the necessity not to separate the studies dealing with pathogens from those that concern the hosts and the vectors, in the case of vector-borne diseases.

Multilocus enzyme electrophoresis for parasites and other pathogens.

In this chapter, I expose the main properties and theoretical background of a somewhat out-of-fashion technique, multilocus enzyme electrophoresis (MLEE). I show that the remarkable properties of this marker-clear Mendelian inheritance, codominance, strong phylogenetic signal-are still valid, although of course more modern markers now are able to yield far more refined results. MLEE can still be used in many circumstances when a cheap and reliable marker is required. I summarize what have been the main contributions of MLEE to the study of parasites and other pathogens.

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.

Molecular typing of Trypanosoma cruzi isolates, United States.

Studies have characterized Trypanosoma cruzi from parasite-endemic regions. With new human cases, increasing numbers of veterinary cases, and influx of potentially infected immigrants, understanding the ecology of this organism in the United States is imperative. We used a classic typing scheme to determine the lineage of 107 isolates from various hosts.