Differential expression and activity of arginine kinase between the American trypanosomatids Trypanosoma rangeli and Trypanosoma cruzi.

Trypanosoma rangeli is a non-virulent hemoflagellate parasite infecting humans, wild and domestic mammals in Central and Latin America. The share of genotypic, phenotypic, and biological similarities with the virulent, human-infective T. cruzi and T. brucei, allows comparative studies on mechanisms of pathogenesis. In this study, investigation of the T. rangeli Arginine Kinase (TrAK) revealed two highly similar copies of the AK gene in this taxon, and a distinct expression profile and activity between replicative and infective forms. Although TrAK expression seems stable during epimastigotes growth, the enzymatic activity increases during the exponential growth phase and decreases from the stationary phase onwards. No differences were observed in activity or expression levels of TrAK during in vitro differentiation from epimastigotes to infective forms, and no detectable AK expression was observed for blood trypomastigotes. Overexpression of TrAK by T. rangeli showed no effects on the in vitro growth pattern, differentiation to infective forms, or infectivity to mice and triatomines. Although differences in TrAK expression and activity were observed among T. rangeli strains from distinct genetic lineages, our results indicate an up-regulation during parasite replication and putative post-translational myristoylation of this enzyme. We conclude that up-regulation of TrAK activity in epimastigotes appears to improve proliferation fitness, while reduced TrAK expression in blood trypomastigotes may be related to short-term and subpatent parasitemia in mammalian hosts.

Messenger RNA levels of the Polo-like kinase gene (PLK) correlate with cytokinesis in the Trypanosoma rangeli cell cycle.

BACKGROUND: Trypanosoma rangeli is a protozoan parasite that is non-virulent to the mammalian host and is morphologically and genomically related to Trypanosoma cruzi, whose proliferation within the mammalian host is controversially discussed. OBJECTIVES: We aimed to investigate the T. rangeli cell cycle in vitro and in vivo by characterizing the timespan of the parasite life cycle and by proposing a molecular marker to assess cytokinesis. METHODOLOGY: The morphological events and their timing during the cell cycle of T. rangeli epimastigotes were assessed using DNA staining, flagellum labelling and bromodeoxyuridine incorporation. Messenger RNA levels of four genes previously associated with the cell cycle of trypanosomatids (AUK1, PLK, MOB1 and TRACK) were evaluated in the different T. rangeli forms. FINDINGS: T. rangeli epimastigotes completed the cell cycle in vitro in 20.8 h. PLK emerged as a potential molecular marker for cell division, as its mRNA levels were significantly increased in exponentially growing epimastigotes compared with growth-arrested parasites or in vitro-differentiated trypomastigotes. PLK expression in T. rangeli can be detected near the flagellum protrusion site, reinforcing its role in the cell cycle. Interestingly, T. rangeli bloodstream trypomastigotes exhibited very low mRNA levels of PLK and were almost entirely composed of parasites in G1 phase. MAIN CONCLUSIONS: Our work is the first to describe the T. rangeli cell cycle in vitro and proposes that PLK mRNA levels could be a useful tool to investigate the T. rangeli ability to proliferate within the mammalian host bloodstream.

Limit of detection of PCR/RFLP analysis of cytochrome oxidase II for the identification of genetic groups of Trypanosoma cruzi and Trypanosoma rangeli in biological material from vertebrate hosts.

Mixed infections with Trypanosoma cruzi and Trypanosoma rangeli and their different genetic groups occur frequently in vertebrate hosts and are difficult to detect by serology. In the present study, we evaluated the limit of detection of polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of cytochrome oxidase II (COII) for the identification of genetic groups of these two parasites in blood and tissue from vertebrate hosts. Reconstitution experiments were performed using human blood (TcI/TcII and KP1+/KP1-) and mouse tissue (TcI/TcII). We tested blood from patients who were in the chronic phase of Chagas disease and tissue from animals that were experimentally infected with all possible combinations of six discrete typing units. In blood samples, T. cruzi and T. rangeli were detected when 5 parasites (pa) were present in the sample, and genetic groups were identified when at least 50 pa were present in the sample. T. cruzi alone could be detected with 1 pa and genotyped (TcI/TcII) with 2 pa. T. rangeli was detected with 2 pa and genotyped (KP+/KP1-) with 25 pa. The present method more readily detected TcII and KP1- in both admixtures and alone. In mouse tissue, TcI and TcII were detected with at least 25 pa. The analysis of blood samples from patients and tissue from animals that were experimentally infected revealed low parasite loads in these hosts, which were below the limit of detection of the present method and could not be genotyped. Our findings indicate that the performance of PCR/RFLP analysis of COII is directly related to the amount and proportion of parasites that are present in the sample and the genetic groups to which the parasites belong.

Upregulation of Cysteine Synthase and Cystathionine ß-Synthase Contributes to Leishmania braziliensis Survival under Oxidative Stress.

Cysteine metabolism is considered essential for the crucial maintenance of a reducing environment in trypanosomatids due to its importance as a precursor of trypanothione biosynthesis. Expression, activity, functional rescue, and overexpression of cysteine synthase (CS) and cystathionine ß-synthase (CßS) were evaluated in Leishmania braziliensis promastigotes and intracellular amastigotes under in vitro stress conditions induced by hydrogen peroxide (H2O2), S-nitroso-N-acetylpenicillamine, or antimonial compounds. Our results demonstrate a stage-specific increase in the levels of protein expression and activity of L. braziliensis CS (LbrCS) and L. braziliensis CßS (LbrCßS), resulting in an increment of total thiol levels in response to both oxidative and nitrosative stress. The rescue of the CS activity in Trypanosoma rangeli, a trypanosome that does not perform cysteine biosynthesis de novo, resulted in increased rates of survival of epimastigotes expressing the LbrCS under stress conditions compared to those of wild-type parasites. We also found that the ability of L. braziliensis promastigotes and amastigotes overexpressing LbrCS and LbrCßS to resist oxidative stress was significantly enhanced compared to that of nontransfected cells, resulting in a phenotype far more resistant to treatment with the pentavalent form of Sb in vitro. In conclusion, the upregulation of protein expression and increment of the levels of LbrCS and LbrCßS activity alter parasite resistance to antimonials and may influence the efficacy of antimony treatment of New World leishmaniasis.

The genome of Anopheles darlingi, the main neotropical malaria vector.

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector-human and vector-parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.

Trypanosoma rangeli expresses a ß-galactofuranosyl transferase.

Glycoconjugates play essential roles in cell recognition, infectivity and survival of protozoan parasites within their insect vectors and mammalian hosts. ß-galactofuranose is a component of several glycoconjugates in many organisms, including a variety of trypanosomatids, but is absent in mammalian and African trypanosomes. Herein, we describe the presence of a ß(1-3) galactofuranosyl transferase (GALFT), an important enzyme of the galactofuranose biosynthetic pathway, in Trypanosoma rangeli. The T. rangeli GALFT gene (TrGALFT) has an ORF of 1.2 Kb and is organized in two copies in the T. rangeli genome. Antibodies raised against an internal fragment of the transferase demonstrated a 45 kDa protein coded by TrGALFT was localized in the whole cytoplasm, mainly in the Golgi apparatus and equally expressed in epimastigotes and trypomastigotes from T. rangeli. Despite the high sequence similarity with Trypanosoma cruzi and Leishmania spp. orthologous TrGALFT showed a substitution of the metal-binding DXD motif, conserved amongst glycosyltransferases, for a DXE functionally analogous motif. Moreover, a reduced number of GALFT genes were present in T. rangeli when compared with other pathogenic kinetoplastid species.

Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum.

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

AnnotaPipeline: An integrated tool to annotate eukaryotic proteins using multi-omics data.

Assignment of gene function has been a crucial, laborious, and time-consuming step in genomics. Due to a variety of sequencing platforms that generates increasing amounts of data, manual annotation is no longer feasible. Thus, the need for an integrated, automated pipeline allowing the use of experimental data towards validation of in silico prediction of gene function is of utmost relevance. Here, we present a computational workflow named AnnotaPipeline that integrates distinct software and data types on a proteogenomic approach to annotate and validate predicted features in genomic sequences. Based on FASTA (i) nucleotide or (ii) protein sequences or (iii) structural annotation files (GFF3), users can input FASTQ RNA-seq data, MS/MS data from mzXML or similar formats, as the pipeline uses both transcriptomic and proteomic information to corroborate annotations and validate gene prediction, providing transcription and expression evidence for functional annotation. Reannotation of the available Arabidopsis thaliana, Caenorhabditis elegans, Candida albicans, Trypanosoma cruzi, and Trypanosoma rangeli genomes was performed using the AnnotaPipeline, resulting in a higher proportion of annotated proteins and a reduced proportion of hypothetical proteins when compared to the annotations publicly available for these organisms. AnnotaPipeline is a Unix-based pipeline developed using Python and is available at: https://github.com/bioinformatics-ufsc/AnnotaPipeline.

Emergence of Two Distinct SARS-CoV-2 Gamma Variants and the Rapid Spread of P.1-like-II SARS-CoV-2 during the Second Wave of COVID-19 in Santa Catarina, Southern Brazil.

The western mesoregion of the state of Santa Catarina (SC), Southern Brazil, was heavily affected as a whole by the COVID-19 pandemic in early 2021. This study aimed to evaluate the dynamics of the SARS-CoV-2 virus spreading patterns in the SC state from March 2020 to April 2021 using genomic surveillance. During this period, there were 23 distinct variants, including Beta and Gamma, among which the Gamma and related lineages were predominant in the second pandemic wave within SC. A regionalization of P.1-like-II in the Western SC region was observed, concomitant to the increase in cases, mortality, and the case fatality rate (CFR) index. This is the first evidence of the regionalization of the SARS-CoV-2 transmission in SC and it highlights the importance of tracking the variants, dispersion, and impact of SARS-CoV-2 on the public health systems.

Swab pooling: A new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution.

To minimize sample dilution effect on SARS-CoV-2 pool testing, we assessed analytical and diagnostic performance of a new methodology, namely swab pooling. In this method, swabs are pooled at the time of collection, as opposed to pooling of equal volumes from individually collected samples. Paired analysis of pooled and individual samples from 613 patients revealed 94 positive individuals. Having individual testing as reference, no false-positives or false-negatives were observed for swab pooling. In additional 18,922 patients screened with swab pooling (1,344 pools), mean Cq differences between individual and pool samples ranged from 0.1 (Cr.I. -0.98 to 1.17) to 2.09 (Cr.I. 1.24 to 2.94). Overall, 19,535 asymptomatic patients were screened using 4,400 RT-qPCR assays. This corresponds to an increase of 4.4 times in laboratory capacity and a reduction of 77% in required tests. Therefore, swab pooling represents a major alternative for reliable and large-scale screening of SARS-CoV-2 in low prevalence populations.

A recombinant protein (MyxoTLm) for the serological diagnosis of acute and chronic Trypanosoma vivax infection in cattle.

Human trypanosomiases and animal trypanosomoses are caused by distinct protozoan parasites of the genus Trypanosoma. The etiological agents of bovine trypanosomosis (BT) are T. vivax, T. congolense, or T. brucei, whose acute infections are initially characterized by hyperthermia, following moderate to severe anemia, subcutaneous edema, lethargy, reduced milk production, progressive weight loss, enlarged lymph nodes, reproductive disorders and death. Animals that survive the acute phase might recover and progress to the chronic, often asymptomatic, phase of infection. Despite their low sensitivity due to the characteristic low parasitemia, simple and costless direct parasitological examinations are the preferred diagnostic methods for animals. Thus, most of the epidemiological studies of BT are based on serological techniques using crude antigen. In this study, we describe the use of the MyxoTLm recombinant protein as an antigen on serological assays. Anti-T. vivax IgM and anti-T. vivax IgG ELISA assays using purified MyxoTLm revealed specificity rates of 91.30 % and 95.65 % and sensitivity rates of 82.35 % and 88.23 %, respectively, being higher than reported for crude antigens. Also, MyxoTLm demonstrated a good performance to detect IgM (ROC curve area = 0.8568) and excellent performance to detect IgG (ROC curve area = 0.9565) when compared to a crude antigen. T. evansi crude antigen used in the indirect anti-T. vivax IgM ELISA reached 70.58 % sensitivity and 78.26 % specificity, and had a lower test performance (ROC curve area = 0.7363). When applied to the anti-T. vivax IgG ELISA, the crude antigen reached 82.35 % sensitivity and 69.56 % specificity, also presenting a low performance with area under the ROC curve of 0.7570. Therefore, the use of MyxoTLm as an antigen on serological diagnosis of BT revealed to increase the sensitivity and the specificity if compared to crude antigens.

The presence of SARS-CoV-2 RNA in human sewage in Santa Catarina, Brazil, November 2019.

Human sewage from Florianopolis (Santa Catarina, Brazil) was analyzed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) from October 2019 until March 2020. Twenty five ml of sewage samples were clarified and viruses concentrated using a glycine buffer method coupled with polyethylene glycol precipitation, and viral RNA extracted using a commercial kit. SARS-CoV-2 RNA was detected by RT-qPCR using oligonucleotides targeting N1, S and two RdRp regions. The results of all positive samples were further confirmed by a different RT-qPCR system in an independent laboratory. S and RdRp amplicons were sequenced to confirm identity with SARS-CoV-2. Genome sequencing was performed using two strategies; a sequence-independent single-primer amplification (SISPA) approach, and by direct metagenomics using Illumina's NGS. SARS-CoV-2 RNA was detected on 27th November 2019 (5.49 ± 0.02 log10 SARS-CoV-2 genome copies (GC) L-1), detection being confirmed by an independent laboratory and genome sequencing analysis. The samples in the subsequent three events were positive by all RT-qPCR assays; these positive results were also confirmed by an independent laboratory. The average load was 5.83 ± 0.12 log10 SARS-CoV-2 GC L-1, ranging from 5.49 ± 0.02 log10 GC L-1 (27th November 2019) to 6.68 ± 0.02 log10 GC L-1 (4th March 2020). Our findings demonstrate that SARS-CoV-2 was likely circulating undetected in the community in Brazil since November 2019, earlier than the first reported case in the Americas (21st January 2020).

Healthcare-Associated Infections-Related Bacteriome and Antimicrobial Resistance Profiling: Assessing Contamination Hotspots in a Developing Country Public Hospital.

Hospital-built environment colonization by healthcare-associated infections-related bacteria (HAIrB) and the interaction with their occupants have been studied to support more effective tools for HAI control. To investigate HAIrB dynamics and antimicrobial resistance (AMR) profile we carried out a 6-month surveillance program in a developing country public hospital, targeting patients, hospital environment, and healthcare workers, using culture-dependent and culture-independent 16S rRNA gene sequencing methods. The bacterial abundance in both approaches shows that the HAIrB group has important representativeness, with the taxa Enterobacteriaceae, Pseudomonas, Staphylococcus, E. coli, and A. baumannii widely dispersed and abundant over the time at the five different hospital units included in the survey. We observed a high abundance of HAIrB in the patient rectum, hands, and nasal sites. In the healthcare workers, the HAIrB distribution was similar for the hands, protective clothing, and mobile phones. In the hospital environment, the healthcare workers resting areas, bathrooms, and bed equipment presented a wide distribution of HAIrB and AMR, being classified as contamination hotspots. AMR is highest in patients, followed by the environment and healthcare workers. The most frequently detected beta-lactamases genes were, bla SHV-like, bla OXA- 23 -like, bla OXA- 51 -like, bla KPC-like, bla CTX-M- 1, bla CTX-M- 8, and bla CTX-M- 9 groups. Our results demonstrate that there is a wide spread of antimicrobial resistance due to HAIrB in the hospital environment, circulating among patients and healthcare workers. The contamination hotspots identified proved to be constant over time. In the fight for patient safety, these findings can reorient practices and help to set up new guidelines for HAI control.

Characterization of Trypanosoma cruzi isolated from humans, vectors, and animal reservoirs following an outbreak of acute human Chagas disease in Santa Catarina State, Brazil.

During March 2005, 24 cases of acute human Chagas disease were detected in Santa Catarina State, southern Brazil, all of them related to the ingestion of Trypanosoma cruzi-contaminated sugar cane juice. Following field studies allowed the isolation of 13 T. cruzi strains from humans, opossums (Didelphis aurita and Didelphis albiventris), and vectors (Triatoma tibiamaculata). The isolated strains were characterized by multilocus enzyme electrophoresis (MLEE) and analysis of the spliced-leader and 24Salpha rRNA genes. The assays revealed that all strains isolated from humans belong to the TcII group but revealed a TcII variant pattern for the phosphoglucomutase enzyme. Strains isolated from opossums also showed a TcI profile in all analysis, but strains isolated from triatomines revealed a mixed TcI/TcII profile by MLEE. No indication of the presence of Trypanosoma rangeli was observed in any assay. Considering that mixed strains (TcI/TcII) were isolated from triatomines in an area without active vectorial transmission to humans and that all strains isolated from humans belong to the TcII group, our results show that T. cruzi TcI and TcII groups are circulating among reservoirs and vectors in southern Brazil and indicate that selection toward TcII group in humans may occur after ingestion of a mixed (TcI/TcII) T. cruzi population.

A Comparative In Silico Study of the Antioxidant Defense Gene Repertoire of Distinct Lifestyle Trypanosomatid Species.

Kinetoplastids are an ancestral group of protists that contains free-living species and parasites with distinct mechanisms in response to stress. Here, we compared genes involved in antioxidant defense (AD), proposing an evolution model among trypanosomatids. All genes were identified in Bodo saltans, suggesting that AD mechanisms have evolved prior to adaptation for parasitic lifestyles. While most of the monoxenous and dixenous parasites revealed minor differences from B. saltans, the endosymbiont-bearing species have an increased number of genes. The absence of these genes was mainly observed in the extracellular parasites of the genera Phytomonas and Trypanosoma. In trypanosomes, a distinction was observed between stercorarian and salivarian parasites, except for Trypanosoma rangeli. Our analyses indicate that the variability of AD among trypanosomatids at the genomic level is not solely due to the geographical isolation, being mainly related to specific adaptations of their distinct biological cycles within insect vectors and to a parasitism of a wide range of hosts.

Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1(-) strains and the ancestry of the Amazonian group.

Assessment of the genetic variability and population structure of Trypanosoma rangeli, a non-pathogenic American trypanosome, was carried out through microsatellite and single-nucleotide polymorphism analyses. Two approaches were used for microsatellite typing: data mining in expressed sequence tag /open reading frame expressed sequence tags libraries and PCR-based Isolation of Microsatellite Arrays from genomic libraries. All microsatellites found were evaluated for their abundance, frequency and usefulness as markers. Genotyping of T. rangeli strains and clones was performed for 18 loci amplified by PCR from expressed sequence tag/open reading frame expressed sequence tags libraries. The presence of single-nucleotide polymorphisms in the nuclear, multi-copy, spliced leader gene was assessed in 18 T. rangeli strains, and the results show that T. rangeli has a predominantly clonal population structure, allowing a robust phylogenetic analysis. Microsatellite typing revealed a subdivision of the KP1(-) genetic group, which may be influenced by geographical location and/or by the co-evolution of parasite and vectors occurring within the same geographical areas. The hypothesis of parasite-vector co-evolution was corroborated by single-nucleotide polymorphism analysis of the spliced leader gene. Taken together, the results suggest three T. rangeli groups: (i) the T. rangeli Amazonian group; (ii) the T. rangeli KP1(-) group; and (iii) the T. rangeli KP1(+) group. The latter two groups possibly evolved from the Amazonian group to produce KP1(+) and KP1(-) strains.

Parity between kinetoplast DNA and mini-exon gene sequences supports either clonal evolution or speciation in Trypanosoma rangeli strains isolated from Rhodnius colombiensis, R. pallescens and R. prolixus in Colombia.

Trypanosoma rangeli are kinetoplastid protozoa which have been largely recognized and defined in several Latin American countries in relation to T. cruzi, because the two trypanosome species are frequently found in mixed infections in triatominae vectors, humans and a variety of wild and domestic mammals. We report the molecular characterization of 18 T. rangeli strains isolated from the salivary glands of naturally infected Rhodnius colombiensis, R. pallescens and R. prolixus by using two independent set of molecular markers. kDNA and mini-exon amplification indicated dimorphism within both DNA sequences: KP1, KP2 and KP3 or KP2 and KP3 products for kDNA mini-circles and 380 or 340bp products for the mini-exon. One of two associations was observed within individual strains: KP1, KP2 and KP3 kDNA products with the 340bp mini-exon product and the KP2 and KP3 kDNA products with the 380bp mini-exon product. Independent mitochondrial and nuclear molecular markers showed a clear division of T. rangeli into two major phylogenetic groups associated with specific vectors in Colombia and in other Latin America countries. These results support either clonal evolution or speciation in T. rangeli populations, probably derived as a secondary adaptation to their parasitic condition in triatomine vectors.

Trypanosoma rangeli Transcriptome Project: Generation and analysis of expressed sequence tags.

Trypanosoma rangeli is an important hemoflagellate parasite of several mammalian species in Central and South America, sharing geographical areas, vectors and reservoirs with T. cruzi, the causative agent of Chagas disease. Thus, the occurrence of single and/or mixed infections, including in humans, must be expected and are of great importance for specific diagnosis and epidemiology. In comparison to several Trypanosomatidae species, the T. rangeli biology and genome are little known, reinforcing the needs of a gene discovery initiative. The T. rangeli transcriptome initiative aims to promote gene discovery through the generation of expressed sequence tags (ESTs) and Orestes (ORF ESTs) from both epimastigote and trypomastigote forms of the parasite, allowing further studies of the parasite biology, taxonomy and phylogeny.

Autochthonous Chagas' disease in Santa Catarina State, Brazil: report of the first case of digestive tract involvement.

We report the first case of digestive tract pathology (megaesophagus) determined by Trypanosoma cruzi infection in Santa Catarina State, southern Brazil. A 63-year-old female had presumptive clinical diagnosis of Chagas' disease, which was confirmed by imaging (endoscopy and esophagogram) and immunological methods. Further molecular diagnosis was carried out with esophagus and blood samples collected during corrective surgery. Polymerase chain reaction tested positive for Trypanosoma cruzi in both esophagus and buffy coat samples.

Transsulfuration is an active pathway for cysteine biosynthesis in Trypanosoma rangeli.

BACKGROUND: Cysteine, a sulfur-containing amino acid, plays an important role in a variety of cellular functions such as protein biosynthesis, methylation, and polyamine and glutathione syntheses. In trypanosomatids, glutathione is conjugated with spermidine to form the specific antioxidant thiol trypanothione (T[SH]2) that plays a central role in maintaining intracellular redox homeostasis and providing defence against oxidative stress. METHODS: We cloned and characterised genes coding for a cystathionine ß-synthase (CßS) and cysteine synthase (CS), key enzymes of the transsulfuration and assimilatory pathways, respectively, from the hemoflagellate protozoan parasite Trypanosoma rangeli. RESULTS: Our results show that T. rangeli CßS (TrCßS), similar to its homologs in T. cruzi, contains the catalytic domain essential for enzymatic activity. Unlike the enzymes in bacteria, plants, and other parasites, T. rangeli CS lacks two of the four lysine residues (Lys26 and Lys184) required for activity. Enzymatic studies using T. rangeli extracts confirmed the absence of CS activity but confirmed the expression of an active CßS. Moreover, CßS biochemical assays revealed that the T. rangeli CßS enzyme also has serine sulfhydrylase activity. CONCLUSION: These findings demonstrate that the RTS pathway is active in T. rangeli, suggesting that this may be the only pathway for cysteine biosynthesis in this parasite. In this sense, the RTS pathway appears to have an important functional role during the insect stage of the life cycle of this protozoan parasite.