Leishmaniinae: Evolutionary inferences based on protein expression profiles (PhyloQuant) congruent with phylogenetic relationships among Leishmania, Endotrypanum, Porcisia, Zelonia, Crithidia, and Leptomonas.

Evolutionary relationships among parasites of the subfamily Leishmaniinae, which comprises pathogen agents of leishmaniasis, were inferred based on differential protein expression profiles from mass spectrometry-based quantitative data using the PhyloQuant method. Evolutionary distances following identification and quantification of protein and peptide abundances using Proteome Discoverer and MaxQuant software were estimated for 11 species from six Leishmaniinae genera. Results clustered all dixenous species of the genus Leishmania, subgenera L. (Leishmania), L. (Viannia), and L. (Mundinia), sister to the dixenous species of genera Endotrypanum and Porcisia. Placed basal to the assemblage formed by all these parasites were the species of genera Zelonia, Crithidia, and Leptomonas, so far described as monoxenous of insects although eventually reported from humans. Inferences based on protein expression profiles were congruent with currently established phylogeny using DNA sequences. Our results reinforce PhyloQuant as a valuable approach to infer evolutionary relationships within Leishmaniinae, which is comprised of very tightly related trypanosomatids that are just beginning to be phylogenetically unraveled. In addition to evolutionary history, mapping of species-specific protein expression is paramount to understand differences in infection processes, tissue tropisms, potential to jump from insects to vertebrates including humans, and targets for species-specific diagnostic and drug development.

A Tribute to a Visionary Scientist-John R. David-Richard Pearson Strong, Professor of Tropical Public Health, Emeritus, Harvard.

There are rare individuals whose insatiable curiosity and boundless intellect propel them into multiple frontiers of science, leaving an indelible mark on the fields that they venture into [...].

Genomes of Endotrypanum monterogeii from Panama and Zelonia costaricensis from Brazil: Expansion of Multigene Families in Leishmaniinae Parasites That Are Close Relatives of Leishmania spp.

The Leishmaniinae subfamily of the Trypanosomatidae contains both genus Zelonia (monoxenous) and Endotrypanum (dixenous). They are amongst the nearest known relatives of Leishmania, which comprises many human pathogens widespread in the developing world. These closely related lineages are models for the genomic biology of monoxenous and dixenous parasites. Herein, we used comparative genomics to identify the orthologous groups (OGs) shared among 26 Leishmaniinae species to investigate gene family expansion/contraction and applied two phylogenomic approaches to confirm relationships within the subfamily. The Endotrypanum monterogeii and Zelonia costaricensis genomes were assembled, with sizes of 29.9 Mb and 38.0 Mb and 9.711 and 12.201 predicted protein-coding genes, respectively. The genome of E. monterogeii displayed a higher number of multicopy cell surface protein families, including glycoprotein 63 and glycoprotein 46, compared to Leishmania spp. The genome of Z. costaricensis presents expansions of BT1 and amino acid transporters and proteins containing leucine-rich repeat domains, as well as a loss of ABC-type transporters. In total, 415 and 85 lineage-specific OGs were identified in Z. costaricensis and E. monterogeii. The evolutionary relationships within the subfamily were confirmed using the supermatrix (3384 protein-coding genes) and supertree methods. Overall, this study showed new expansions of multigene families in monoxenous and dixenous parasites of the subfamily Leishmaniinae.

Assessing reservoir host status in leishmaniasis with special reference to the infectiousness of Leishmania (Viannia) braziliensis infections in wild rodents.

Molecular methods have been responsible for a notable increase in the detection of Leishmaniinae infections in wild animals. Determining their infectiousness is of paramount importance in evaluating their epidemiological significance. One of the most efficient ways of determining infectiousness for vector borne diseases is xenodiagnosis with the appropriate vector. However, this is logistically very difficult to accomplish in the field, and an ideal solution is to find a molecular surrogate for xenodiagnosis. In this review we discuss different approaches to the problem by focusing on the infectiousness of Leishmania (Viannia) braziliensis in rodents under laboratory and field conditions. Comparisons with similar studies for other Leishmania species emphasizes that there are pivotal differences in the infectiousness and the importance of asymptomatic infections in different hosts. Potentially the most promising surrogate is the real time quantitative PCR (qPCR). However, its success depends on choosing a tissue that relates to the vector's feeding location and the parasite's tissue tropism. This requires detailed knowledge of the infection of each species in its wild hosts. We conclude that for L. (V.) braziliensis infections in wild rodents the tissue of choice for a molecular xenodiagnostic test, based on the qPCR is blood, providing that a significant number of samples must be examined.

Incidence of Human and Free-Ranging Wild Rodent Infections with Leishmania (Viannia) braziliensis, Aetiological Agent of Cutaneous Leishmaniasis.

BACKGROUND: Human and wild rodent infection rates with Leishmania (Viannia) braziliensis are needed to differentiate transmission pathways in anthropogenically altered habitats. METHODS: Human participants in northeast Brazil were tested by the leishmanin skin test (LST) and inspected for lesions/scars characteristic of American clinical leishmaniasis (ACL). Molecular (PCR/qPCR) test records of free-ranging rodents were available from a concurrent capture-mark-recapture study. Force of Infection (λ) and recovery (ρ) rates were estimated from cross-sectional and longitudinal datasets. RESULTS: Cumulative prevalences of human LST+ves and ACL scar+ves were 0.343-0.563 (n = 503 participants) and 0.122-0.475 (n = 503), respectively. Active ACL lesions were not detected. Annual rates of LST conversions were λ = 0.03-0.15 and ρ = 0.02-0.07. The probability of infection was independent of sex and associated with increasing age in addition to the period of exposure. Rodents (n = 596 individuals of 6 species) showed high rates of exclusively asymptomatic infection (λ = 0.222/month) and potential infectiousness to the sand fly vector. Spatially concurrent rodent and household human infection prevalences were correlated. CONCLUSIONS: Human exposure to L. (V.) braziliensis continues to be high despite the substantial drop in reported ACL cases in recent years. Spill-over transmission risk to humans from rodents in peridomestic habitats is likely supported by a rodent infection/transmission corridor linking houses, plantations, and the Atlantic Forest.

Assessing reservoir host status in leishmaniasis with special reference to the infectiousness of Leishmania (Viannia) braziliensis infections in wild rodents

ABSTRACT Molecular methods have been responsible for a notable increase in the detection of Leishmaniinae infections in wild animals. Determining their infectiousness is of paramount importance in evaluating their epidemiological significance. One of the most efficient ways of determining infectiousness for vector borne diseases is xenodiagnosis with the appropriate vector. However, this is logistically very difficult to accomplish in the field, and an ideal solution is to find a molecular surrogate for xenodiagnosis. In this review we discuss different approaches to the problem by focusing on the infectiousness of Leishmania (Viannia) braziliensis in rodents under laboratory and field conditions. Comparisons with similar studies for other Leishmania species emphasizes that there are pivotal differences in the infectiousness and the importance of asymptomatic infections in different hosts. Potentially the most promising surrogate is the real time quantitative PCR (qPCR). However, its success depends on choosing a tissue that relates to the vector's feeding location and the parasite's tissue tropism. This requires detailed knowledge of the infection of each species in its wild hosts. We conclude that for L. (V.) braziliensis infections in wild rodents the tissue of choice for a molecular xenodiagnostic test, based on the qPCR is blood, providing that a significant number of samples must be examined.

Genome Analysis of Endotrypanum and Porcisia spp., Closest Phylogenetic Relatives of Leishmania, Highlights the Role of Amastins in Shaping Pathogenicity.

While numerous genomes of Leishmania spp. have been sequenced and analyzed, an understanding of the evolutionary history of these organisms remains limited due to the unavailability of the sequence data for their closest known relatives, Endotrypanum and Porcisia spp., infecting sloths and porcupines. We have sequenced and analyzed genomes of three members of this clade in order to fill this gap. Their comparative analyses revealed only minute differences from Leishmaniamajor genome in terms of metabolic capacities. We also documented that the number of genes under positive selection on the Endotrypanum/Porcisia branch is rather small, with the flagellum-related group of genes being over-represented. Most significantly, the analysis of gene family evolution revealed a substantially reduced repertoire of surface proteins, such as amastins and biopterin transporters BT1 in the Endotrypanum/Porcisia species when compared to amastigote-dwelling Leishmania. This reduction was especially pronounced for δ-amastins, a subfamily of cell surface proteins crucial in the propagation of Leishmania amastigotes inside vertebrate macrophages and, apparently, dispensable for Endotrypanum/Porcisia, which do not infect such cells.

Multilocus Genotyping of Giardia duodenalis in Mostly Asymptomatic Indigenous People from the Tapirapé Tribe, Brazilian Amazon.

Little information is available on the occurrence and genetic variability of the diarrhoea-causing enteric protozoan parasite Giardia duodenalis in indigenous communities in Brazil. This cross-sectional epidemiological survey describes the frequency, genotypes, and risk associations for this pathogen in Tapirapé people (Brazilian Amazon) at four sampling campaigns during 2008-2009. Microscopy was used as a screening test, and molecular (PCR and Sanger sequencing) assays targeting the small subunit ribosomal RNA, the glutamate dehydrogenase, the beta-giardin, and the triosephosphate isomerase genes as confirmatory/genotyping methods. Associations between G. duodenalis and sociodemographic and clinical variables were investigated using Chi-squared test and univariable/multivariable logistic regression models. Overall, 574 individuals belonging to six tribes participated in the study, with G. duodenalis prevalence rates varying from 13.5-21.7%. The infection was positively linked to younger age and tribe. Infected children <15 years old reported more frequent gastrointestinal symptoms compared to adults. Assemblage B accounted for three out of four G. duodenalis infections and showed a high genetic diversity. No association between assemblage and age or occurrence of diarrhoea was demonstrated. These data indicate that the most likely source of infection was anthropic and that different pathways (e.g., drinking water) may be involved in the transmission of the parasite.

Identification of Leishmania (Viannia) species and clinical isolates of Leishmania (Leishmania) amazonensis from Brazil using PCR-RFLP of the heat-shock protein 70 gene reveals some unexpected observations.

Hsp70 is a cytoplasmic heat-shock protein, encoded by a multicopy tandemly repeated gene that has recently been gaining popularity as a valuable marker for typing Leishmania species. In this study, we used a previously described hsp70 PCR-RFLP method for identifying Brazilian Leishmania isolates. We identified two distinct L. (L.) amazonensis hsp70 alleles that resulted in two different RFLP patterns. Also, we found RFLP polymorphisms amongst L. (Viannia) naiffi strains. The profiles of both L. (V.) shawi and L. (V.) lindenbergi were very similar to those of other L. (Viannia) species. The observations described herein reflect the polymorphism found within species of Leishmania and indicate that results from this hsp70 PCR-RFLP method should be used with caution when typing isolates from clinical cases of leishmaniasis and Leishmania species from Brazil.

Influences of climate change on the potential distribution of Lutzomyia longipalpis sensu lato (Psychodidae: Phlebotominae).

This study explores the present day distribution of Lutzomyia longipalpis in relation to climate, and transfers the knowledge gained to likely future climatic conditions to predict changes in the species' potential distribution. We used ecological niche models calibrated based on occurrences of the species complex from across its known geographic range. Anticipated distributional changes varied by region, from stability to expansion or decline. Overall, models indicated no significant north-south expansion beyond present boundaries. However, some areas suitable both at present and in the future (e.g., Pacific coast of Ecuador and Peru) may offer opportunities for distributional expansion. Our models anticipated potential range expansion in southern Brazil and Argentina, but were variably successful in anticipating specific cases. The most significant climate-related change anticipated in the species' range was with regard to range continuity in the Amazon Basin, which is likely to increase in coming decades. Rather than making detailed forecasts of actual locations where Lu. longipalpis will appear in coming years, our models make interesting and potentially important predictions of broader-scale distributional tendencies that can inform heath policy and mitigation efforts.

Genome of Diaporthe sp. provides insights into the potential inter-phylum transfer of a fungal sesquiterpenoid biosynthetic pathway.

Fungi have highly active secondary metabolic pathways which enable them to produce a wealth of sesquiterpenoids that are bioactive. One example is Δ6-protoilludene, the precursor to the cytotoxic illudins, which are pharmaceutically relevant as anticancer therapeutics. To date, this valuable sesquiterpene has only been identified in members of the fungal division Basidiomycota. To explore the untapped potential of fungi belonging to the division Ascomycota in producing Δ6-protoilludene, we isolated a fungal endophyte Diaporthe sp. BR109 and show that it produces a diversity of terpenoids including Δ6-protoilludene. Using a genome sequencing and mining approach 17 putative novel sesquiterpene synthases were identified in Diaporthe sp. BR109. A phylogenetic approach was used to predict which gene encodes Δ6-protoilludene synthase, which was then confirmed experimentally. These analyses reveal that the sesquiterpene synthase and its putative sesquiterpene scaffold modifying cytochrome P450(s) may have been acquired by inter-phylum horizontal gene transfer from Basidiomycota to Ascomycota. Bioinformatic analyses indicate that inter-phylum transfer of these minimal sequiterpenoid secondary metabolic pathways may have occurred in other fungi. This work provides insights into the evolution of fungal sesquiterpenoid secondary metabolic pathways in the production of pharmaceutically relevant bioactive natural products.

Infectiousness of Sylvatic and Synanthropic Small Rodents Implicates a Multi-host Reservoir of Leishmania (Viannia) braziliensis.

BACKGROUND: The possibility that a multi-host wildlife reservoir is responsible for maintaining transmission of Leishmania (Viannia) braziliensis causing human cutaneous and mucocutaneous leishmaniasis is tested by comparative analysis of infection progression and infectiousness to sandflies in rodent host species previously shown to have high natural infection prevalences in both sylvatic or/and peridomestic habitats in close proximity to humans in northeast Brazil. METHODS: The clinical and parasitological outcomes, and infectiousness to sandflies, were observed in 54 colonized animals of three species (18 Necromys lasiurus, 18 Nectomys squamipes and 18 Rattus rattus) experimentally infected with high (5.5 × 10(6)/ml) or low (2.8 × 10(5)/ml) dose L. (V.) braziliensis (MBOL/BR/2000/CPqAM95) inoculum. Clinical signs of infection were monitored daily. Whole animal xenodiagnoses were performed 6 months post inoculation using Lutzomyia longipalpis originating from flies caught in Passira, Pernambuco, after this parasite evaluation was performed at necropsy. Heterogeneities in Leishmania parasite loads were measured by quantitative PCR in ear skin, liver and spleen tissues. RESULTS: All three rodent species proved to establish infection characterized by short-term self-resolving skin lesions, located on ears and tail but not on footpads (one site of inoculation), and variable parasite loads detected in all three tissues with maximum burdens of 8.1 × 10(3) (skin), 2.8 × 10(3) (spleen), and 8.9 × 10(2) (liver). All three host species, 18/18 N. lasiurus, 10/18 N. squamipes and 6/18 R. rattus, also proved infectious to sandflies in cross-sectional study. R. rattus supported significantly lower tissue parasite loads compared to those in N. lasiurus and N. squamipes, and N. lasiurus appeared to be more infectious, on average, than either N. squamipes or R. rattus. CONCLUSIONS: A multi-host reservoir of cutaneous leishmaniasis is indicated in this region of Brazil, though with apparent differences in the competence between the rodent species. The results provide preliminary insights into links between sylvatic and peri-domestic transmission cycles associated with overlaps in the rodent species' ecological niches.

Recent advances in phlebotomine sand fly research related to leishmaniasis control.

Phlebotomine sand flies are the subject of much research because of the role of their females as the only proven natural vectors of Leishmania species, the parasitic protozoans that are the causative agents of the neglected tropical disease leishmaniasis. Activity in this field was highlighted by the eighth International Symposium on Phlebotomine Sand flies (ISOPS) held in September 2014, which prompted this review focusing on vector control. Topics reviewed include: Taxonomy and phylogenetics, Vector competence, Genetics, genomics and transcriptomics, Eco-epidemiology, and Vector control. Research on sand flies as leishmaniasis vectors has revealed a diverse array of zoonotic and anthroponotic transmission cycles, mostly in subtropical and tropical regions of Africa, Asia and Latin America, but also in Mediterranean Europe. The challenge is to progress beyond descriptive eco-epidemiology, in order to separate vectors of biomedical importance from the sand fly species that are competent vectors but lack the vectorial capacity to cause much human disease. Transmission modelling is required to identify the vectors that are a public health priority, the ones that must be controlled as part of the integrated control of leishmaniasis. Effective modelling of transmission will require the use of entomological indices more precise than those usually reported in the leishmaniasis literature.

Distribution of Lutzomyia longipalpis chemotype populations in São Paulo state, Brazil.

BACKGROUND: American visceral leishmaniasis (AVL) is an emerging disease in the state of São Paulo, Brazil. Its geographical expansion and the increase in the number of human cases has been linked to dispersion of Lutzomyia longipalpis into urban areas. To produce more accurate risk maps we investigated the geographic distribution and routes of expansion of the disease as well as chemotype populations of the vector. METHODOLOGY/PRINCIPAL FINDINGS: A database, containing the annual records of municipalities which had notified human and canine AVL cases as well as the presence of the vector, was compiled. The chemotypes of L. longipalpis populations from municipalities in different regions of São Paulo State were determined by Coupled Gas Chromatography - Mass Spectrometry. From 1997 to June 2014, L. longipalpis has been reported in 166 municipalities, 148 of them in the Western region. A total of 106 municipalities were identified with transmission and 99 were located in the Western region, where all 2,204 autochthonous human cases occurred. Both the vector and the occurrence of human cases have expanded in a South-easterly direction, from the Western to central region, and from there, a further expansion to the North and the South. The (S)-9-methylgermacrene-B population of L. longipalpis is widely distributed in the Western region and the cembrene-1 population is restricted to the Eastern region. CONCLUSION/SIGNIFICANCE: The maps in the present study show that there are two distinct epidemiological patterns of AVL in São Paulo State and that the expansion of human and canine AVL cases through the Western region has followed the same dispersion route of only one of the two species of the L. longipalpis complex, (S)-9-methylgermacrene-B. Entomological vigilance based on the routes of dispersion and identification of the chemotype population could be used to identify at-risk areas and consequently define the priorities for control measures.

Identification of a fungal 1,8-cineole synthase from Hypoxylon sp. with specificity determinants in common with the plant synthases.

Terpenes are an important and diverse class of secondary metabolites widely produced by fungi. Volatile compound screening of a fungal endophyte collection revealed a number of isolates in the family Xylariaceae, producing a series of terpene molecules, including 1,8-cineole. This compound is a commercially important component of eucalyptus oil used in pharmaceutical applications and has been explored as a potential biofuel additive. The genes that produce terpene molecules, such as 1,8-cineole, have been little explored in fungi, providing an opportunity to explore the biosynthetic origin of these compounds. Through genome sequencing of cineole-producing isolate E7406B, we were able to identify 11 new terpene synthase genes. Expressing a subset of these genes in Escherichia coli allowed identification of the hyp3 gene, responsible for 1,8-cineole biosynthesis, the first monoterpene synthase discovered in fungi. In a striking example of convergent evolution, mutational analysis of this terpene synthase revealed an active site asparagine critical for water capture and specificity during cineole synthesis, the same mechanism used in an unrelated plant homologue. These studies have provided insight into the evolutionary relationship of fungal terpene synthases to those in plants and bacteria and further established fungi as a relatively untapped source of this important and diverse class of compounds.

Phylogenetic relationships of Leishmania species based on trypanosomatid barcode (SSU rDNA) and gGAPDH genes: Taxonomic revision of Leishmania (L.) infantum chagasi in South America.

Phylogenetic studies on trypanosomatid barcode using V7V8 SSU rRNA and gGAPDH gene sequences have provided support for redefining some trypanosomatid species and positioning new isolates. The genus Leishmania is a slow evolving monophyletic group and including important human pathogens. The phylogenetic relationships of this genus have been determined by the natural history of its vertebrate hosts, vector specificity, clinical manifestations, geographical distribution and molecular approaches using different markers. Thus, in an attempt to better understand the phylogenetic relationships of Leishmania species, we performed phylogenetic analysis on trypanosomatid barcode using V7V8 SSU rRNA and gGAPDH gene sequences among a large number of Leishmania species and also several Brazilian visceral Leishmania infantum chagasi isolates obtained from dogs and humans. Our phylogenetic analysis strongly suggested that Leishmania hertigi and Leishmania equatoriensis should be taxonomically revised so as to include them in the genus Endotrypanum; and supported ancient divergence of Leishmania enriettii. This, together with recent data in the literature, throws light on the discussion about the evolutionary southern supercontinent hypothesis for the origin of Leishmania ssp. and validates L. infantum chagasi from Brazil, thus clearly differentiating it from L. infantum, for the first time.

Bilateral anomaly in Evandromyia evandroi (Diptera: Psychodidae: Phlebotominae) captured in Vicência municipality, Northern Rainforest region of Pernambuco State, Brazil.

Approximately 800 species of phlebotomine sand flies, many of which are vectors of Leishmania, have been described. Besides morphological similarities within groups, the occurrence of anomalies within a species may lead to an erroneous description of new species. This paper describes one phlebotomine sand fly, Evandromyia evandroi, with a symmetrical bilateral anomaly in the number of spines on the gonostyle. In this specimen, the anomalous spine is located in the external region of gonostyle, inserted between the upper external and the lower external spines. It is important to document morphological anomalies, so as to avoid erroneous sand fly identifications.

A Role for the 2' OH of peptidyl-tRNA substrate in peptide release on the ribosome revealed through RF-mediated rescue.

The 2' OH of the peptidyl-tRNA substrate is thought to be important for catalysis of both peptide bond formation and peptide release in the ribosomal active site. The release reaction also specifically depends on a release factor protein (RF) to hydrolyze the ester linkage of the peptidyl-tRNA upon recognition of stop codons in the A site. Here, we demonstrate that certain amino acid substitutions (in particular those containing hydroxyl or thiol groups) in the conserved GGQ glutamine of release factor RF1 can rescue defects in the release reaction associated with peptidyl-tRNA substrates lacking a 2' OH. We explored this rescue effect through biochemical and computational approaches that support a model where the 2' OH of the P-site substrate is critical for orienting the nucleophile in a hydrogen-bonding network productive for catalysis.

Microspatial distributional patterns of vectors of cutaneous leishmaniasis in pernambuco, northeastern Brazil.

The purpose of this study is to analyze the spatial distribution and population trends through time of Lutzomyia species in a long-term focus of cutaneous leishmaniasis transmission in an Atlantic Forest area, northeastern Brazil. Sand fly populations of different ecological niches were monitored spatiotemporally in 2009. To summarize vegetation characteristics and phenology, we calculated the Normalized Difference Vegetation Index from Landsat images. Using niche modeling approaches, we assessed suites of environmental factors to identify areas of transmission risk. Although 12 species were detected, L. whitmani was the most abundant and broadly distributed across the area, particularly in peridomiciliary locations, and associated negatively with denser vegetation areas. On the other hand, L. complexa, L. sordelli, and L. tupynambai were found almost exclusively in forested areas (P < 0.05), and associated positively with denser vegetation. Lutzomyia species' occurrences are related to specific environmental combinations (with contrast among species) in the region.

Short report: Molecular characterization of Blastocystis obtained from members of the indigenous Tapirapé ethnic group from the Brazilian Amazon region, Brazil.

A total of 382 stool samples were examined during a survey of intestinal parasites in members of the Tapirapé ethnic group, who live in the Brazilian Amazon region of Mato Grosso. Fecal DNAs from Blastocystis-positive samples were extracted, polymerase chain reaction amplified using Blastocystis-specific primers targeting the small subunit rRNA gene, and sequenced. Three subtypes (STs) were identified: ST1 (41%), ST2 (32%), and ST3 (17%). Seven mixed infections were found (11%). The subtype distribution was markedly different from that reported in Europe in that ST4 was not detected and ST3 was not the most common subtype. This study is the first to include molecular characterization of Blastocystis in Brazil and in indigenous communities from Latin America.