Transcriptomics analysis highlights potential ways in human pathogenesis in Leishmania braziliensis infected with the viral endosymbiont LRV1.

The parasite Leishmania (Viannia) braziliensis is widely distributed in Brazil and is one of the main species associated with human cases of different forms of tegumentary leishmaniasis (TL) such as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms underlying the pathogenesis of TL are still not fully understood, but it is known that factors related to the host and the parasite act in a synergistic and relevant way to direct the response to the infection. In the host, macrophages have a central connection with the parasite and play a fundamental role in the defense of the organism due to their ability to destroy intracellular parasites and present antigens. In the parasite, some intrinsic factors related to the species or even the strain analyzed are fundamental for the outcome of the disease. One of them is the presence of Leishmania RNA Virus 1 (LRV1), an endosymbiont virus that parasitizes some species of Leishmania that triggers a cascade of signals leading to a more severe TL phenotype, such as ML. One of the strategies for understanding factors associated with the immune response generated after Leishmania/host interaction is through the analysis of molecular patterns after infection. Thus, the gene expression profile in human monocyte-derived macrophages obtained from healthy donors infected in vitro with L. braziliensis positive (LbLRV1+) and negative (LbLRV1-) for LRV1 was evaluated. For this, the microarray assay was used and 162 differentially expressed genes were identified in the comparison LbLRV1+ vs. LbLRV1-, 126 upregulated genes for the type I and II interferons (IFN) signaling pathway, oligoadenylate synthase OAS/RNAse L, non-genomic actions of vitamin D3 and RIG-I type receptors, and 36 down-regulated. The top 10 downregulated genes along with the top 10 upregulated genes were considered for analysis. Type I interferon (IFNI)- and OAS-related pathways results were validated by RT-qPCR and Th1/Th2/Th17 cytokines were analyzed by Cytometric Bead Array (CBA) and enzyme-linked immunosorbent assay (ELISA). The microarray results validated by RT-qPCR showed differential expression of genes related to IFNI-mediated pathways with overexpression of different genes in cells infected with LbLRV1+ compared to LbLRV1- and to the control. No significant differences were found in cytokine levels between LbLRV1+ vs. LbLRV1- and control. The data suggest the activation of gene signaling pathways associated with the presence of LRV1 has not yet been reported so far. This study demonstrates, for the first time, the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 when comparing infections with LbLRV1+ versus LbLRV1- and the control. This finding emphasizes the role of LRV1 in directing the host's immune response after infection, underlining the importance of identifying LRV1 in patients with TL to assess disease progression.

HRM Accuracy and Limitations as a Species Typing Tool for Leishmania Parasites.

High Resolution Melting Analysis (HRM) has been pointed out as a suitable alternative method to detect and identify Leishmania species. Herein, we aimed to evaluate the sensitivity, specificity, accuracy, and limitations of a HSP70-HRM protocol both as a diagnostic scheme applied in clinical samples and as a species typing tool for laboratory research and reference services. Our data reveal the pronounced species-typing potential of the HSP70-HRM in DNA from cultured parasites. For clinical samples, however, we advise caution due to parasite load-dependent accuracy. In light of these findings and considering the importance of parasite load determination for clinical and research purposes, we recommend the integration of the presented typing scheme and the previously published Leishmania quantifying approach as combined tools for clinicians, surveillance, and research.

Exploring Host-Specificity: Untangling the Relationship between Leishmania (Viannia) Species and Its Endosymbiont Leishmania RNA Virus 1.

A relevant aspect in the epidemiology of Tegumentary Leishmaniasis (TL) are the Leishmania parasites carrying a viral endosymbiont, Leishmania RNA Virus 1 (LRV1), a dsRNA virus. Leishmania parasites carrying LRV1 are prone to causing more severe TL symptoms, increasing the likelihood of unfavorable clinical outcomes. LRV1 has been observed in the cultured strains of five L. (Viannia) species, and host specificity was suggested when studying the LRV1 from L. braziliensis and L. guyanensis strains. The coevolution hypothesis of LRV1 and Leishmania was based on phylogenetic analyses, implying an association between LRV1 genotypes, Leishmania species, and their geographic origins. This study aimed to investigate LRV1 specificity relative to Leishmania (Viannia) species hosts by analyzing LRV1 from L. (Viannia) species. To this end, LRV1 was screened in L. (Viannia) species other than L. braziliensis or L. guyanensis, and it was detected in 11 out of 15 L. naiffi and two out of four L. shawi. Phylogenetic analyses based on partial LRV1 genomic sequencing supported the hypothesis of host specificity, as LRV1 clustered according to their respective Leishmania species' hosts. These findings underscore the importance of investigating Leishmania and LRV1 coevolution and its impact on Leishmania (Viannia) species dispersion and pathogenesis in the American Continent.

Leishmania (Viannia) naiffi Lainson & Shaw 1989.

Just over 30 years ago, a new species of Leishmania of the subgenus Leishmania (Viannia) was described infecting the armadillo Dasypus novemcinctus; then, a report of human infection followed. From the Brazilian Amazon and apparently restricted to this region and its close borders, Leishmania (Viannia) naiffi has been characterized as a species that grows easily in axenic culture medium and causes few to no lesions after inoculation in experimental animal models. Results in the last decade indicate the occurrence of L. naiffi in vectors and human infections, including a report of therapeutic failure possibly associated with Leishmania RNA virus 1. Overall, such accounts suggest that the parasite is more dispersed and the disease less self-healing than previously expected.

First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare.

BACKGROUND: Epidemiological data related to leishmaniases or Leishmania infection in horses are scarce. However, studies carried out in different regions in the world showed equids parasitised by Leishmania braziliensis, L. infantum and L. martiniquensis. OBJECTIVES: Identify the Leishmania species causing cutaneous leishmaniasis in a mare, living in Rio de Janeiro State (Brazil), and search the presence of Leishmania viruses in the isolated parasite. METHODS: Isoenzymes and polymerase chain reaction (PCR) targeting ITSrDNA region followed by sequencing were conducted for typing the isolated parasite. A search for Leishmania virus infection was also performed. FINDINGS: The mare presented skin nodules and ulcers in the left pinna caused by Leishmania spp. that was detected by culture and PCR. The parasite was identified as Leishmania (Mundinia) martiniquensis, infected by Leishbunyavirus (LBV), representing the first description of this species in South America. The animal travelled to different Brazilian regions, but not to outside the country. MAIN CONCLUSIONS: The worldwide distribution of L. martiniquensis and its infection by LBV were confirmed in this study, indicating the autochthonous transmission cycle in Brazil. The clinical profile of the disease in the mare, showing fast spontaneous healing of cutaneous lesions, may indicate that skin lesions related to L. martiniquensis infection in horses might be underdiagnosed.

Genome Analysis of Triploid Hybrid Leishmania Parasite from the Neotropics.

We discovered a hybrid Leishmania parasite in Costa Rica that is genetically similar to hybrids from Panama. Genome analyses demonstrated the hybrid is triploid and identified L. braziliensis and L. guyanensis-related strains as parents. Our findings highlight the existence of poorly sampled Leishmania (Viannia) variants infectious to humans.

Raising the suspicion of a non-autochthonous infection: identification of Leishmania guyanensis from Costa Rica exhibits a Leishmaniavirus related to Brazilian north-east and French Guiana viral genotypes.

BACKGROUND: Costa Rica has a history of neglecting prevention, control and research of leishmaniasis, including limited understanding on Leishmania species causing human disease across the country and a complete lack of knowledge on the Leishmania RNA virus, described as a factor linked to the worsening and metastasis of leishmanial lesions. OBJECTIVES: The aim of this work was to describe a case of cutaneous leishmaniasis by Leishmania (Viannia) guyanensis, bearing infection with Leishmaniavirus 1 (LRV1) in Costa Rica, raising the suspicion of imported parasites in the region. METHODS: The Leishmania strain was previously identified by routine hsp70 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in Costa Rica and subsequently characterised by isoenzyme electrophoresis and Sanger sequencing in Brazil. Screening for LRV1 was conducted with a dual RT-PCR approach and sequencing of the fragment obtained. FINDINGS: Since 2016 Costa Rica performs Leishmania isolation and typing as part of its epidemiological surveillance activities. Amongst 113 strains typed until 2019, only one was characterised as a L. (V.) guyanensis, corresponding to the first confirmed report of this species in the country. Interestingly, the same strain tested positive for LRV1. Sequencing of the viral orf1 and 2, clustered this sample with other LRV1 genotypes of South American origin, from the Northeast of Brazil and French Guiana. MAIN CONCLUSION: The unique characteristics of this finding raised the suspicion that it was not an autochthonous strain. Notwithstanding its presumed origin, this report points to the occurrence of said endosymbiont in Central American Leishmania strains. The possibility of its local dispersion represents one more challenge faced by regional health authorities in preventing and controlling leishmaniasis.

First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare

BACKGROUND Epidemiological data related to leishmaniases or Leishmania infection in horses are scarce. However, studies carried out in different regions in the world showed equids parasitised by Leishmania braziliensis, L. infantum and L. martiniquensis. OBJECTIVES Identify the Leishmania species causing cutaneous leishmaniasis in a mare, living in Rio de Janeiro State (Brazil), and search the presence of Leishmania viruses in the isolated parasite. METHODS Isoenzymes and polymerase chain reaction (PCR) targeting ITSrDNA region followed by sequencing were conducted for typing the isolated parasite. A search for Leishmania virus infection was also performed. FINDINGS The mare presented skin nodules and ulcers in the left pinna caused by Leishmania spp. that was detected by culture and PCR. The parasite was identified as Leishmania (Mundinia) martiniquensis, infected by Leishbunyavirus (LBV), representing the first description of this species in South America. The animal travelled to different Brazilian regions, but not to outside the country. MAIN CONCLUSIONS The worldwide distribution of L. martiniquensis and its infection by LBV were confirmed in this study, indicating the autochthonous transmission cycle in Brazil. The clinical profile of the disease in the mare, showing fast spontaneous healing of cutaneous lesions, may indicate that skin lesions related to L. martiniquensis infection in horses might be underdiagnosed.

Interspecies and Intrastrain Interplay among Leishmania spp. Parasites.

Leishmania parasites present astonishing adaptative abilities that represent a matter of life or death within disparate environments during the heteroxenous parasite life cycle. From an evolutionary perspective, organisms develop methods of overcoming such challenges. Strategies that extend beyond the genetic diversity have been discussed and include variability between parasite cells during the infections of their hosts. The occurrence of Leishmania subpopulation fluctuations with variable structural genomic contents demonstrates that a single strain might shelter the variability required to overcome inconsistent environments. Such intrastrain variability provides parasites with an extraordinary ability to adapt and thus survive and propagate. However, different perspectives on this evolution have been proposed. Strains or species living in the same environment can cooperate but also compete. These interactions might increase the replication rate of some parasites but cause the loss of more aggressive competitors for others. Adaptive responses to intra- and interspecific competition can evolve as a fixed strategy (replication is adapted to the average genetic complexity of infections) or an optional strategy (replication varies according to the genetic complexity of the current infection). This review highlights the complexity of interspecies and intrastrain interactions among Leishmania parasites as well as the different factors that influence this interplay.

Nitric Oxide Resistance in Leishmania (Viannia) braziliensis Involves Regulation of Glucose Consumption, Glutathione Metabolism and Abundance of Pentose Phosphate Pathway Enzymes.

In American Tegumentary Leishmaniasis production of cytokines, reactive oxygen species and nitric oxide (NO) by host macrophages normally lead to parasite death. However, some Leishmania braziliensis strains exhibit natural NO resistance. NO-resistant strains cause more lesions and are frequently more resistant to antimonial treatment than NO-susceptible ones, suggesting that NO-resistant parasites are endowed with specific mechanisms of survival and persistence. To tests this, we analyzed the effect of pro- and antioxidant molecules on the infectivity in vitro of L. braziliensis strains exhibiting polar phenotypes of resistance or susceptibility to NO. In addition, we conducted a comprehensive quantitative mass spectrometry-based proteomics analysis of those parasites. NO-resistant parasites were more infective to peritoneal macrophages, even in the presence of high levels of reactive species. Principal component analysis of protein concentration values clearly differentiated NO-resistant from NO-susceptible parasites, suggesting that there are natural intrinsic differences at molecular level among those strains. Upon NO exposure, NO-resistant parasites rapidly modulated their proteome, increasing their total protein content and glutathione (GSH) metabolism. Furthermore, NO-resistant parasites showed increased glucose analogue uptake, and increased abundance of phosphotransferase and G6PDH after nitrosative challenge, which can contribute to NADPH pool maintenance and fuel the reducing conditions for the recovery of GSH upon NO exposure. Thus, increased glucose consumption and GSH-mediated redox capability may explain the natural resistance of L. braziliensis against NO.

Raising the suspicion of a non-autochthonous infection: identification of Leishmania guyanensis from Costa Rica exhibits a Leishmaniavirus related to Brazilian north-east and French Guiana viral genotypes

BACKGROUND Costa Rica has a history of neglecting prevention, control and research of leishmaniasis, including limited understanding on Leishmania species causing human disease across the country and a complete lack of knowledge on the Leishmania RNA virus, described as a factor linked to the worsening and metastasis of leishmanial lesions. OBJECTIVES The aim of this work was to describe a case of cutaneous leishmaniasis by Leishmania (Viannia) guyanensis, bearing infection with Leishmaniavirus 1 (LRV1) in Costa Rica, raising the suspicion of imported parasites in the region. METHODS The Leishmania strain was previously identified by routine hsp70 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in Costa Rica and subsequently characterised by isoenzyme electrophoresis and Sanger sequencing in Brazil. Screening for LRV1 was conducted with a dual RT-PCR approach and sequencing of the fragment obtained. FINDINGS Since 2016 Costa Rica performs Leishmania isolation and typing as part of its epidemiological surveillance activities. Amongst 113 strains typed until 2019, only one was characterised as a L. (V.) guyanensis, corresponding to the first confirmed report of this species in the country. Interestingly, the same strain tested positive for LRV1. Sequencing of the viral orf1 and 2, clustered this sample with other LRV1 genotypes of South American origin, from the Northeast of Brazil and French Guiana. MAIN CONCLUSION The unique characteristics of this finding raised the suspicion that it was not an autochthonous strain. Notwithstanding its presumed origin, this report points to the occurrence of said endosymbiont in Central American Leishmania strains. The possibility of its local dispersion represents one more challenge faced by regional health authorities in preventing and controlling leishmaniasis.

Insights from Leishmania (Viannia) guyanensis in vitro behavior and intercellular communication.

BACKGROUND: Pentavalent antimonial-based chemotherapy is the first-line approach for leishmaniasis treatment and disease control. Nevertheless antimony-resistant parasites have been reported in some endemic regions. Treatment refractoriness is complex and is associated with patient- and parasite-related variables. Although amastigotes are the parasite stage in the vertebrate host and, thus, exposed to the drug, the stress caused by trivalent antimony in promastigotes has been shown to promote significant modification in expression of several genes involved in various biological processes, which will ultimately affect parasite behavior. Leishmania (Viannia) guyanensis is one of the main etiological agents in the Amazon Basin region, with a high relapse rate (approximately 25%). METHODS: Herein, we conducted several in vitro analyses with L. (V.) guyanensis strains derived from cured and refractory patients after treatment with standardized antimonial therapeutic schemes, in addition to a drug-resistant in vitro-selected strain. Drug sensitivity assessed through Sb(III) half-maximal inhibitory concentration (IC50) assays, growth patterns (with and without drug pressure) and metacyclic-like percentages were determined for all strains and compared to treatment outcomes. Finally, co-cultivation without intercellular contact was followed by parasitic density and Sb(III) IC50 measurements. RESULTS: Poor treatment response was correlated with increased Sb(III) IC50 values. The decrease in drug sensitivity was associated with a reduced cell replication rate, increased in vitro growth ability, and higher metacyclic-like proportion. Additionally, in vitro co-cultivation assays demonstrated that intercellular communication enabled lower drug sensitivity and enhanced in vitro growth ability, regardless of direct cell contact. CONCLUSIONS: Data concerning drug sensitivity in the Viannia subgenus are emerging, and L. (V.) guyanensis plays a pivotal epidemiological role in Latin America. Therefore, investigating the parasitic features potentially related to relapses is urgent. Altogether, the data presented here indicate that all tested strains of L. (V.) guyanensis displayed an association between treatment outcome and in vitro parameters, especially the drug sensitivity. Remarkably, sharing enhanced growth ability and decreased drug sensitivity, without intercellular communication, were demonstrated.

Overcoming the Negligence in Laboratory Diagnosis of Mucosal Leishmaniasis.

The northern region of Brazil, which has the largest number of cases of tegumentary leishmaniasis (TL) in the country, is also the region that has the highest diversity of species of vectors and Leishmania parasites. In this region, cases of mucosal leishmaniasis (ML), a clinical form of TL, exceed the national average of cases, reaching up to 12% of the total annual TL notifications. ML is associated with multiple factors, such as the parasite species and the viral endosymbiont Leishmania RNA virus 1 (LRV1). Being a chronic parasitological disease, laboratory diagnosis of ML poses a challenge for health services. Here, we evaluated more than 700 clinical samples from patients with clinical suspicion of TL, including patients with cutaneous leishmaniasis (CL) and mucosal leishmaniasis, comparing the results of parasitological tests-direct parasitological examination by microscopy (DP) and conventional PCR (cPCR) targeting of both kDNA and hsp70. The DP was performed by collecting material from lesions through biopsies (mucosal lesions) or scarification (cutaneous lesions); for PCR, a cervical brush was used for sample collection. Blood samples were tested employing standardized real-time PCR (qPCR) protocol targeting the HSP70 gene. PCR tests showed higher sensitivity than DP for both CL and ML samples. Considering ML samples only (N = 89), DP showed a sensitivity of 49.4% (N = 44) against 98.8% (N = 88) for kDNA PCR. The qPCR hsp70 for blood samples from patients with ML (N = 14) resulted in superior sensitivity (50%; N = 7) compared to DP (21.4%; N = 3) for samples from the same patients. Our results reinforced the need to implement a molecular test for the diagnosis of ML, in addition to proposing methods less invasive for collecting material from TL patients. Sample collection using a cervical brush in lesions observed in CL and ML patients is easy to perform and less invasive, compared to scarification and biopsies. Blood samples could be a good source for qPCR diagnosis for ML patients. Thus, we propose here a standardized method for collection and for performing of molecular diagnosis of clinical samples from suspicious ML patients that can be applied in reference services for improving ML diagnosis.

In Vitro Susceptibility to Miltefosine of Leishmania infantum (syn. L. chagasi) Isolates from Different Geographical Areas in Brazil.

Treatment of visceral leishmaniasis in Brazil still relies on meglumine antimoniate, with less than ideal efficacy and safety, making new therapeutic tools an urgent need. The oral drug miltefosine was assayed in a phase II clinical trial in Brazil with cure rates lower than previously demonstrated in India. The present study investigated the susceptibility to miltefosine in 73 Brazilian strains of Leishmania infantum from different geographic regions, using intracellular amastigote and promastigote assays. The EC50 for miltefosine of 13 of these strains evaluated in intracellular amastigotes varied between 1.41 and 4.57 µM. The EC50 of the 73 strains determined in promastigotes varied between 5.89 and 23.7 µM. No correlation between in vitro miltefosine susceptibility and the presence of the miltefosine sensitive locus was detected among the tested strains. The relatively low heterogeneity in miltefosine susceptibility observed for the 73 strains tested in this study suggests the absence of decreased susceptibility to miltefosine in Brazilian L. infantum and does not exclude future clinical evaluation of miltefosine for VL treatment in Brazil.

The Maze Pathway of Coevolution: A Critical Review over the Leishmania and Its Endosymbiotic History.

The description of the genus Leishmania as the causative agent of leishmaniasis occurred in the modern age. However, evolutionary studies suggest that the origin of Leishmania can be traced back to the Mesozoic era. Subsequently, during its evolutionary process, it achieved worldwide dispersion predating the breakup of the Gondwana supercontinent. It is assumed that this parasite evolved from monoxenic Trypanosomatidae. Phylogenetic studies locate dixenous Leishmania in a well-supported clade, in the recently named subfamily Leishmaniinae, which also includes monoxenous trypanosomatids. Virus-like particles have been reported in many species of this family. To date, several Leishmania species have been reported to be infected by Leishmania RNA virus (LRV) and Leishbunyavirus (LBV). Since the first descriptions of LRVs decades ago, differences in their genomic structures have been highlighted, leading to the designation of LRV1 in L. (Viannia) species and LRV2 in L. (Leishmania) species. There are strong indications that viruses that infect Leishmania spp. have the ability to enhance parasitic survival in humans as well as in experimental infections, through highly complex and specialized mechanisms. Phylogenetic analyses of these viruses have shown that their genomic differences correlate with the parasite species infected, suggesting a coevolutionary process. Herein, we will explore what has been described in the literature regarding the relationship between Leishmania and endosymbiotic Leishmania viruses and what is known about this association that could contribute to discussions about the worldwide dispersion of Leishmania.

Colonization and genetic diversification processes of Leishmania infantum in the Americas.

Leishmania infantum causes visceral leishmaniasis, a deadly vector-borne disease introduced to the Americas during the colonial era. This non-native trypanosomatid parasite has since established widespread transmission cycles using alternative vectors, and human infection has become a significant concern to public health, especially in Brazil. A multi-kilobase deletion was recently detected in Brazilian L. infantum genomes and is suggested to reduce susceptibility to the anti-leishmanial drug miltefosine. We show that deletion-carrying strains occur in at least 15 Brazilian states and describe diversity patterns suggesting that these derive from common ancestral mutants rather than from recurrent independent mutation events. We also show that the deleted locus and associated enzymatic activity is restored by hybridization with non-deletion type strains. Genetic exchange appears common in areas of secondary contact but also among closely related parasites. We examine demographic and ecological scenarios underlying this complex L. infantum population structure and discuss implications for disease control.

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis.

Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania.

Ecological divergence and hybridization of Neotropical Leishmania parasites.

The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the Leishmania braziliensis species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean Leishmania as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host-parasite interactions. Within the Andean species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock and ecological niche modeling suggested that the history of diversification of the Andean lineages is limited to the Late Pleistocene and intimately associated with habitat contractions driven by climate change. These results suggest that changes in forestation over the past 150,000 y have influenced speciation and diversity of these Neotropical parasites. Second, genome-scale analyses provided evidence of meiotic-like recombination between Andean and Amazonian Leishmania species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species. We further show that mitochondrial minicircles-but not maxicircles-show a similar evolutionary pattern to the nuclear genome, suggesting that compatibility between nuclear-encoded mitochondrial genes and minicircle-encoded guide RNA genes is essential to maintain efficient respiration. By comparing full nuclear and mitochondrial genome ancestries, our data expand our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa.

In-depth quantitative proteomics uncovers specie-specific metabolic programs in Leishmania (Viannia) species.

Leishmania species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic Leishmania spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the Leishmania proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing L. braziliensis, L. panamensis and L. guyanensis species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. "Total Protein Approach" and "Proteomic Ruler" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete Leishmania proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas L. braziliensis relies the more on glycolysis, L. panamensis and L. guyanensis seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and provide rich substrate for explore new molecules for diagnosing purposes. Data are available via ProteomeXchange with identifier PXD017696.

Occurrence of multiple genotype infection caused by Leishmania infantum in naturally infected dogs.

Genetic polymorphisms in natural Leishmania populations have been reported in endemic areas. Microsatellite typing is a useful tool to elucidate the genetic variability of parasite strains, due to its capability for high-resolution mapping of genomic targets. The present study employed multilocus microsatellite typing (MLMT) to explore the genotypic composition of Leishmania infantum in naturally infected dogs by genotyping parasites infecting different tissues with or without in vitro expansion. Eighty-six samples were collected from 46 animals in an endemic region of visceral leishmaniasis (VL). MLMT was performed for 38 spleen samples and 48 L. infantum cultures isolated from different tissues. Of the 86 samples, 23 were effectively genotyped by MLMT, identifying nine multilocus genotypes (MLG; referred to as MLG A-I). MLGs A, B and C were detected in more than one type of tissue and in more than one sample. Conversely, MLG D-I were uniquely detected in one sample each. The results showed that multiple genotype infections occur within a single host and tissue. Paired sample analysis revealed the presence of different MLMT alleles in 14 dogs, while the same MLG allele was present in 15 animals. STRUCTURE analysis demonstrated the presence of two populations; 13 samples displayed a similar admixture of both ancestral populations, and these were not assigned to any population. Only samples for which Q ≥ 0.70 after CLUMPP alignment were considered to be part of Population 1 (POP1) or Population 2 (POP2). POP2 comprised the majority of samples (n = 54) compared to POP1 (n = 19). This study presents evidence of multiple genotype infections (caused by L. infantum) in dogs in an area with high VL transmission. Further investigations must be undertaken to determine the effects of multiple infection on the host immune response and disease dynamics and treatment.