Glycosomal Glyceraldehyde 3-phosphate Dehydrogenase Research Articles

Diversity of trypanosomes in humans and cattle in the HAT foci Mandoul and Maro, Southern Chad-A matter of concern for zoonotic potential?

African trypanosomes are parasites mainly transmitted by tsetse flies. They cause trypanosomiasis in humans (HAT) and animals (AAT). In Chad, HAT/AAT are endemic. This study investigates the diversity and distribution of trypanosomes in Mandoul, an isolated area where a tsetse control campaign is ongoing, and Maro, an area bordering the Central African Republic (CAR) where the control had not started. 717 human and 540 cattle blood samples were collected, and 177 tsetse flies were caught. Trypanosomal DNA was detected using PCR targeting internal transcribed spacer 1 (ITS1) and glycosomal glyceraldehyde-3 phosphate dehydrogenase (gGAPDH), followed by amplicon sequencing. Trypanosomal DNA was identified in 14 human samples, 227 cattle samples, and in tsetse. Besides T. b. gambiense, T. congolense was detected in human in Maro. In Mandoul, DNA from an unknown Trypanosoma sp.-129-H was detected in a human with a history of a cured HAT infection and persisting symptoms. In cattle and tsetse samples from Maro, T. godfreyi and T. grayi were detected besides the known animal pathogens, in addition to T. theileri (in cattle) and T. simiae (in tsetse). Furthermore, in Maro, evidence for additional unknown trypanosomes was obtained in tsetse. In contrast, in the Mandoul area, only T. theileri, T. simiae, and T. vivax DNA was identified in cattle. Genetic diversity was most prominent in T. vivax and T. theileri. Tsetse control activities in Mandoul reduced the tsetse population and thus the pathogenic parasites. Nevertheless, T. theileri, T. vivax, and T. simiae are frequent in cattle suggesting transmission by other insect vectors. In contrast, in Maro, transhumance to/from Central African Republic and no tsetse control may have led to the high diversity and frequency of trypanosomes observed including HAT/AAT pathogenic species. Active HAT infections stress the need to enforce monitoring and control campaigns. Additionally, the diverse trypanosome species in humans and cattle indicate the necessity to investigate the infectivity of the unknown trypanosomes regarding their zoonotic potential. Finally, this study should be widened to other trypanosome hosts to capture the whole diversity of circulating trypanosomes.

Biological and Genetic Heterogeneity in Trypanosoma dionisii Isolates from Hematophagous and Insectivorous Bats.

This study describes the morphological, biochemical, and molecular differences among Trypanosoma dionisii isolates from hemocultures of hematophagous (Desmodus rotundus; n = 2) and insectivorous (Lonchorhina aurita; n = 1) bats from the Atlantic Rainforest of Rio de Janeiro, Brazil. Fusiform epimastigotes from the hematophagous isolates were elongated, whereas those of the insectivorous isolate were stumpy, reflected in statistically evident differences in the cell body and flagellum lengths. In the hemocultures, a higher percentage of trypomastigote forms (60%) was observed in the hematophagous bat isolates than that in the isolate from the insectivorous bat (4%), which demonstrated globular morphology. Three molecular DNA regions were analyzed: V7V8 (18S rDNA), glycosomal glyceraldehyde 3-phosphate dehydrogenase gene, and mitochondrial cytochrome b gene. The samples were also subjected to multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis. All isolates were identified as T. dionisii by phylogenetic analysis. These sequences were clustered into two separate subgroups with high bootstrap values according to the feeding habits of the bats from which the parasites were isolated. However, other T. dionisii samples from bats with different feeding habits were found in the same branch. These results support the separation of the three isolates into two subgroups, demonstrating that different subpopulations of T. dionisii circulate among bats.

Molecular characterization and phylogenetic analysis of Trypanosoma spp. detected from striped leaf-nosed bats (Hipposideros vittatus) in Zambia.

Bat trypanosomes consist of more than 30 trypanosome species from over 70 species of bats. Recent studies suggest that bats play a role in disseminating trypanosomes from African continent to the terrestrial mammals both in the Afrotropic-Palearctic Ecozones and Nearctic Ecozone. However, the diversity, distribution, and evolution of bat trypanosomes are still unclear. To better understand their evolution, more genetic data of bat trypanosomes from a variety of locations are required. During a survey of Borrelia spp. of bats inhabiting a cave in Zambia, we observed flagellate parasites from 5 of 43 hemocultures. Sequence and phylogenetic analyses of the glycosomal glyceraldehyde 3-phosphate dehydrogenase gene (gGAPDH; 572 bp) and the 18S ribosomal RNA gene (18S rRNA gene; 1,079–1,091 bp) revealed that all were Trypanosoma spp. belonged to the Trypanosoma cruzi clade. Three and two of them exhibited the similarity with T. conorhini and T. dionisii, respectively. The present study provides the first genetic data on Trypanosoma spp. of bats inhabiting Zambia.

Trypanosoma cruzi and Leishmania infantum chagasi Infection in Wild Mammals from Maranhão State, Brazil.

Trypanosoma and Leishmania are obligate parasites that cause important diseases in human and domestic animals. Wild mammals are the natural reservoirs of these parasites, which are transmitted by hematophagous arthropods. The present study aimed to detect the natural occurrence of trypanosomatids through serological diagnosis, PCR of whole blood and blood culture (hemoculture), and phylogenetic relationships using small subunit ribosomal DNA (SSU rDNA), cytochrome b, and glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) genes. Samples from 131 wild animals, including rodents, marsupials, and bats, were sampled in six areas in the state of Maranhão, in a transition zone of semiarid climates northeast of the equatorial humid Amazon. Serological analysis for Leishmania (Leishmania) infantum chagasi was performed in opossums by indirect fluorescent antibody test (IFAT), and all animals were serologically negative. Nine positive hemocultures (6.77%) were isolated and cryopreserved and from mammals of the Didelphimorphia and Chiroptera orders and positioned in phylogenies on the basis of sequences from different genes with reference strains of Trypanosoma cruzi marinkellei and T. cruzi. From primary samples (blood and tissues) only one bat, Pteronotus parnellii, was positive to SSU rDNA and gGAPDH genes and grouped with the L. infantum chagasi branch. The studies conducted in Maranhão State provide knowledge of parasite diversity. It is important to determine the presence of trypanosomatids in wild mammals with synanthropic habits.

Genetic diversity of Trypanosoma cruzi in bats, and multilocus phylogenetic and phylogeographical analyses supporting Tcbat as an independent DTU (discrete typing unit)

Trypanosoma cruzi is a complex of phenotypically and genetically diverse isolates distributed in six discrete typing units (DTUs) designated as TcI–TcVI. Five years ago, T. cruzi isolates from Brazilian bats showing unique patterns of traditional ribosomal and spliced leader PCRs not clustering into any of the six DTUs were designated as the Tcbat genotype. In the present study, phylogenies inferred using SSU rRNA (small subunit of ribosomal rRNA), gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cytb (cytochrome b) genes strongly supported Tcbat as a monophyletic lineage prevalent in Brazil, Panama and Colombia. Providing strong support for Tcbat, sequences from 37 of 47 nuclear and 12 mitochondrial genes (retrieved from a draft genome of Tcbat) and reference strains of all DTUs available in databanks corroborated Tcbat as an independent DTU. Consistent with previous studies, multilocus analysis of most nuclear genes corroborated the evolution of T. cruzi from bat trypanosomes its divergence into two main phylogenetic lineages: the basal TcII; and the lineage clustering TcIV, the clade comprising TcIII and the sister groups TcI–Tcbat. Most likely, the common ancestor of Tcbat and TcI was a bat trypanosome. However, the results of the present analysis did not support Tcbat as the ancestor of all DTUs. Despite the insights provided by reports of TcIII, TcIV and TcII in bats, including Amazonian bats harbouring TcII, further studies are necessary to understand the roles played by bats in the diversification of all DTUs. We also demonstrated that in addition to value as molecular markers for DTU assignment, Cytb, ITS rDNA and the spliced leader (SL) polymorphic sequences suggest spatially structured populations of Tcbat. Phylogenetic and phylogeographical analyses, multiple molecular markers specific to Tcbat, and the degrees of sequence divergence between Tcbat and the accepted DTUs strongly support the definitive classification of Tcbat as a new DTU.

Ultrastructure and molecular phylogeny of four new species of monoxenous trypanosomatids from flies (Diptera: Brachycera) with redefinition of the genus Wallaceina

Four new species of monoxenous kinetoplastid parasites are described from Brachycera flies, namely Wallaceina raviniae Votýpka et Lukes, 2014 and Crithidia otongatchiensis Votýpka et Lukes, 2014 from Ecuador, Leptomonas moramango Votypka et Lukes, 2014 from Madagascar, and Crithidia pragensis Votýpka, Klepetková et Lukes, 2014 from the Czech Republic. The new species are described here based on sequence analysis of their spliced leader (SL) RNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and small subunit (SSU) rRNA genes, as well as their morphology and ultrastructure. High-pressure freezing and Bernhard's EDTA regressive staining, used for the first time for monoxenous (one host) trypanosomatids, revealed the presence of viral particles with cytosolic localization in one and unique mitochondrial localization in another species. In accordance with previous observations, our results emphasize a discrepancy between morphology and molecular taxonomy of the family Trypanosomatidae. All four newly described species are represented by typical morphotypes (mainly choano- and promastigotes) and are virtually indistinguishable from other monoxenous trypanosomatids by morphology. Nevertheless, they all differ in their phylogenetic affinities. Whereas three of them grouped within the recently defined subfamily Leishmaniinae, which includes numerous representatives of the genera Leishmania Ross, 1903, Crithidia Léger, 1902 and Leptomonas Kent, 1880, the fourth species clusters together with the 'collosoma' clade (named after 'Leptomonas' collosoma Wallace, Clark, Dyer et Collins, 1960). Here we demonstrate that the 'collosoma' group represents the elusive genus Wallaceina Podlipaev, Frolov et Kolesnikov, 1999. We redefine this genus in molecular terms based on similarities of the respective molecular markers and propose to use this taxon name for the group of species of the 'collosoma' clade.

Molecular dynamics simulation of the gGAPDH–NAD+ complex from Trypanosoma cruzi

A 50-ns molecular dynamics simulation has been used to study the homotetramer of the enzyme glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) complexes, from Trypanosoma cruzi, with nicotinamide adenine dinucleotide (NAD+) cofactors in aqueous solution. The root mean square deviation indicates that the overall structure of the homotetramer does not undergo significant change. The largest structural change observed was in the NAD+ binding domain of subunit (chain) D; as a consequence, the NAD+ cofactor was dislocated from its initial position. However, the other subunits were not affected, suggesting that the gGAPDH enzyme exhibits non-cooperative behaviour. Our simulation estimates that the NAD+ binding domain rotates about 4.8° relative to the catalytic domain in the apo–holo form transition. The hydrogen bond analysis reveals that the residues R12, I13, D38 and M39 are essential for gGAPDH–NAD+ interaction. Furthermore, two promising cavities to be explored in drug design were found: one formed by residues I13, R12, T197, T199, E336 and Y339, and the other by residues C166, H194, R249, I13, R12, T197, T199, E336 and Y339. The results presented in this paper offer new insight into the search for inhibitors of the gGAPDH enzyme of T. cruzi protozoan.

Evolutionary Insights from Bat Trypanosomes: Morphological, Developmental and Phylogenetic Evidence of a New Species, Trypanosoma (Schizotrypanum) erneyi sp. nov., in African Bats Closely Related to Trypanosoma (Schizotrypanum) cruzi and Allied Species

Parasites of the genus Trypanosoma are common in bats and those of the subgenus Schizotrypanum are restricted to bats throughout the world, with the exception of Trypanosoma (Schizotrypanum) cruzi that also infects other mammals and is restricted to the American Continent. We have characterized trypanosome isolates from Molossidae bats captured in Mozambique, Africa. Morphology and behaviour in culture, supported by phylogenetic inferences using SSU (small subunit) rRNA, gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cyt b (cytochrome b) genes, allowed to classify the isolates as a new Schizotrypanum species named Trypanosoma (Schizotrypanum) erneyi sp. nov. This is the first report of a Schizotrypanum species from African bats cultured, characterized morphologically and biologically, and positioned in phylogenetic trees. The unprecedented finding of a new species of the subgenus Schizotrypanum from Africa that is closest related to the America-restricted Trypanosoma (Schizotrypanum) cruzi marinkellei and T. cruzi provides new insights into the origin and evolutionary history of T. cruzi and closely related bat trypanosomes. Altogether, data from our study support the hypothesis of an ancestor trypanosome parasite of bats evolving to infect other mammals, even humans, and adapted to transmission by triatomine bugs in the evolutionary history of T. cruzi in the New World.

Multilocus phylogeographical analysis of Trypanosoma ( Megatrypanum) genotypes from sympatric cattle and water buffalo populations supports evolutionary host constraint and close phylogenetic relationships with genotypes found in other ruminants

Species of the subgenus Trypanosoma ( Megatrypanum) have been reported in cattle and other domestic and wild ruminants worldwide. A previous study in Brazil found at least four genotypes infecting cattle ( Bos taurus), but only one in water buffalo ( Bubalus bubalis). However, the small number of isolates examined from buffalo, all inhabiting nearby areas, has precluded evaluation of their diversity, host associations and geographical structure. To address these questions, we evaluated the genetic diversity and phylogeographical patterns of 25 isolates from water buffalo and 28 from cattle from four separate locations in Brazil and Venezuela. Multigene phylogenetic analyses of ssrRNA, internal transcribed spacer of rDNA (ITSrDNA), 5SrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH), mitochondrial cytochrome b (Cyt b), spliced leader (SL) and cathepsin L-like (CATL) sequences positioned all isolates from sympatric and allopatric buffalo populations into the highly homogeneous genotype TthIA, while the cattle isolates were assigned to three different genotypes, all distinct from TthIA. Polymorphisms in all of these sequences separated the trypanosomes infecting water buffalo, cattle, sheep, antelope and deer, and suggested that they correspond to separate species. Congruent phylogenies inferred with all genes indicated a predominant clonal structure of the genotypes. The multilocus analysis revealed one monophyletic assemblage formed exclusively by trypanosomes of ruminants, which corresponds to the subgenus T. ( Megatrypanum). The high degree of host specificity, evidenced by genotypes exclusive to each ruminant species and lack of genotype shared by different host species, suggested that the evolutionary history of trypanosomes of this subgenus was strongly constrained by their ruminant hosts. However, incongruence between ruminant and trypanosome phylogenies did not support host–parasite co-evolution, indicating that host switches have occurred across ruminants followed by divergences, giving rise to new trypanosome genotypes adapted exclusively to one host species.

Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats

The genetic diversity and phylogeographical patterns of Trypanosoma species that infect Brazilian bats were evaluated by examining 1043 bats from 63 species of seven families captured in Amazonia, the Pantanal, Cerrado and the Atlantic Forest biomes of Brazil. The prevalence of trypanosome-infected bats, as estimated by haemoculture, was 12.9%, resulting in 77 cultures of isolates, most morphologically identified as Trypanosoma cf. cruzi, classified by barcoding using partial sequences from ssrRNA gene into the subgenus Schizotrypanum and identified as T. cruzi (15), T. cruzi marinkellei (37) or T. cf. dionisii (25) . Phylogenetic analyses using nuclear ssrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and mitochondrial cytochrome b (Cyt b) gene sequences generated three clades, which clustered together forming the subgenus Schizotrypanum. In addition to vector association, bat trypanosomes were related by the evolutionary history, ecology and phylogeography of the bats. Trypanosoma cf. dionisii trypanosomes (32.4%) infected 12 species from four bat families captured in all biomes, from North to South Brazil, and clustered with T. dionisii from Europe despite being separated by some genetic distance. Trypanosoma cruzi marinkellei (49.3%) was restricted to phyllostomid bats from Amazonia to the Pantanal (North to Central). Trypanosoma cruzi (18.2%) was found mainly in vespertilionid and phyllostomid bats from the Pantanal/Cerrado and the Atlantic Forest (Central to Southeast), with a few isolates from Amazonia.

Crystal structure of Leishmania mexicana glycosomal glyceraldehyde-3-phosphate dehydrogenase in a new crystal form confirms the putative physiological active site structure

The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the trypanosomatid parasite Leishmania mexicana in a new crystal form has been determined by X-ray crystallography. The protein crystallizes in space group P2 1 with one 156 kDa tetramer per asymmetric unit. The model of the protein with bound NAD +s and phosphates has been refined against 81% complete data from 10.0 to 2.8 Å to a crystallographic R factor of 0.217. The present structure confirms two key aspects of the previously reported orthorhombic crystal structure of L. mexicana GAPDH (LmGAPDH): the unusual conformation of a loop in the active site, and the repositioning of the inorganic phosphate binding site compared with crystal structures of GAPDHs from other organisms. As the monoclinic crystals of LmGAPDH were grown at a phosphate concentration and pH that were even closer to physiological conditions than were the orthorhombic LmGAPDH crystals, the present structure reinforces the physiological relevance of the active site structure seen in the previous orthorhombic crystal of LmGAPDH.

The rational design of trypanocidal drugs: selective inhibition of the glyceraldehyde-3-phosphate dehydrogenase in Trypanosomatidae.

Within the framework of a project aimed at the structure-based design of drugs for use against sleeping sickness, selective inhibitors were designed, synthesised and tested. The target protein was glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the adenosine part of the NAD cofactor was chosen as lead. After one design cycle and exploiting the selectivity cleft in trypanosomal GAPDH near the C2 of the adenosine ribose, a selective inhibitor, 2'-deoxy-2'-(3-methoxybenzamido)adenosine, was obtained. This compound inhibits human GAPDH only marginally, whereas the enzymes from Trypanosoma brucei and Leishmania mexicana are inhibited by 50% at 2.2 and 0.3 mM, respectively. Moreover, the inhibition of the parasite enzyme is 45-fold (T. brucei) or 170-fold (L. mexicana) greater with this substituted analogue than that produced with adenosine.

Crystal structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Leishmania mexicana: implications for structure-based drug design and a new position for the inorganic phosphate binding site.

The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the trypanosomatid parasite Leishmania mexicana has been determined by X-ray crystallography. The protein crystallizes in space group P2(1)2(1)2(1) with unit cell parameters a = 99.0 A, b = 126.5 A, and c = 138.9 A. There is one 156,000 Da protein tetramer per asymmetric unit. The model of the protein with bound NAD+s and phosphates has been refined against 86% complete data from 10.0 to 2.8 A to a crystallographic Rfactor of 0.198. Density modification by noncrystallographic symmetry averaging was used during model building. The final model of the L. mexicana GAPDH tetramer shows small deviations of less than 0.5 degrees from ideal 222 molecular symmetry. The structure of L. mexicana GAPDH is very similar to that of glycosomal GAPDH from the related trypanosomatid Trypanosoma brucei. A significant structural difference between L. mexicana GAPDH and most previously determined GAPDH structures occurs in a loop region located at the active site. This unusual loop conformation in L. mexicana GAPDH occludes the inorganic phosphate binding site which has been seen in previous GAPDH structures. A new inorganic phosphate position is observed in the L. mexicana GAPDH structure. Model building studies indicate that this new anion binding site is well situated for nucleophilic attack of the inorganic phosphate on the thioester intermediate in the GAPDH-catalyzed reaction. Since crystals of L. mexicana GAPDH can be grown reproducibly and diffract much better than those of T. brucei GAPDH, L. mexicana GAPDH will be used as a basis for structure-based drug design targeted against trypanosomatid GAPDHs.

Synthesis and structure-activity relationships of analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine, a selective inhibitor of trypanosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase.

In continuation of a project aimed at the structure-based design of drugs against sleeping sickness, analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine (1) were synthesized and tested to establish structure-activity relationships for inhibiting glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compound 1 was recently designed using the NAD:GAPDH complexes of the human enzyme and that of Trypanosoma brucei, the causative agent of sleeping sickness. In an effort to exploit an extra hydrophobic domain due to Val 207 of the parasite enzyme, several new 2'-amido-2'-deoxyadenosines were synthesized. Some of them displayed an interesting improvement in inhibitory activity compared to 1. Carbocyclic or acyclic analogs showed marked loss of activity, illustrating the importance of the typical (C-2'-endo) puckering of the ribose moiety. We also describe the synthesis of a pair of compounds that combine the beneficial effects of a 2- and 8-substituted adenine moiety on potency with the beneficial effect of a 2'-amido moiety on selectivity. Unfortunately, in both cases, IC50 values demonstrate the incompatibility of these combined modifications. Finally, introduction of a hydrophobic 5'-amido group on 5'-deoxyadenosine enhances the inhibition of the protozoan enzyme significantly, although the gain in selectivity is mediocre.

Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: An evolutionary scenario of subcellular compartmentation in Kinetoplastida

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in both the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoenzymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated that the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the trypanosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arranged genes were found which are 95% identical. The two encoded polypeptides differ in 17 residues. Their sequences are 72-77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, specific insertions, and a small carboxy-terminal extension containing the sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has undergone some mutations at positions coding for highly conserved residues of the active site and the NAD(+)-binding domain of GAPDH. Modeling of the protein's three-dimensional structure suggested that several of the substitutions compensate each other, retaining the functional coenzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by horizontal gene transfer during the evolution of the Kinetoplastida, in the lineage leading to the suborder Trypanosomatina (Trypanosoma, Leishmania), after the divergence from the Bodonina (Trypanoplasma). Furthermore, the data clearly suggest that the original GAPDH of the Kinetoplastida has been compartmentalized during evolution.