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1.  Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity 
Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection.
Author Summary
Within the non-oxidative branch of the pentose phosphate pathway, ribose 5-phosphate isomerase catalyzes the inter-conversion of ribose 5-phosphate and ribulose 5-phosphate. There are two types of ribose 5-phosphate isomerase, namely A and B. The presence of type B in Trypanosoma brucei, and its absence in humans, make this protein a promising drug target. African sleeping sickness is a serious parasitic disease that relies on limited chemotherapeutic options for control. In our study, a functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B is reported. Biochemical studies confirmed enzyme isomerase activity and its downregulation by RNAi affected mainly parasites infectivity in vivo. Overall this study shows that ribose 5-phosphate isomerase depletion is detrimental for parasites infectivity under host pressure.
doi:10.1371/journal.pntd.0003430
PMCID: PMC4287489  PMID: 25568941
2.  Correction: Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine 
PLoS Neglected Tropical Diseases  2013;7(12):10.1371/annotation/eb4faa32-fc8d-43ae-ba92-f34c0c4e5052.
doi:10.1371/annotation/eb4faa32-fc8d-43ae-ba92-f34c0c4e5052
PMCID: PMC3862843
3.  Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine 
Asparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A) and Trypanosoma brucei (TbAS-A): the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg2+. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn't affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.
Author Summary
The amino acid asparagine is important not only for protein biosynthesis, but also for nitrogen homeostasis. Asparagine synthetase catalyzes the synthesis of this amino acid. There are two forms of asparagine synthetase, A and B. The presence of type A in trypanosomes, and its absence in humans, makes this protein a potential drug target. Trypanosomes are responsible for serious parasitic diseases that rely on limited drug therapeutic options for control. In our study we present a functional characterization of trypanosomes asparagine synthetase A. We describe that Trypanosoma brucei and Trypanosoma cruzi type A enzymes are able to use either ammonia or glutamine as a nitrogen donor, within the conversion of aspartate into asparagine. Furthermore, we show that asparagine synthetase A knockdown renders Trypanosoma brucei auxotrophic to asparagine. Overall, this study demonstrates that interfering with asparagine metabolism represents a way to control parasite growth and infectivity.
doi:10.1371/journal.pntd.0002578
PMCID: PMC3854871  PMID: 24340117
4.  Mitochondria-mediated hormetic response in life span extension of calorie-restricted Saccharomyces cerevisiae 
Age  2010;33(2):143-154.
Calorie restriction (CR) is the only proven regimen, which confers lifespan extension benefits across the various phyla right from unicellular organisms like yeast to primates. In a bid to elucidate the mechanism of calorie-restriction-mediated life span extension, the role of mitochondria in the process was investigated. In this study, we found that the mitochondrial content in CR cells remains unaltered as compared to cells grown on nonrestricted media. However, mitochondria isolated from CR cells showed increased respiration and elevated reactive oxygen species levels without augmenting adenosine triphosphate (ATP) generation. The antioxidant defense system was amplified in CR mitochondria, and in CR cells a cross protection to hydrogen-peroxide-induced stress was also observed. Moreover, we also documented that a functional electron transport chain was vital for the life span extension benefits of calorie restriction. Altogether, our results indicate that calorie restriction elicits mitohormetic effect, which ultimately leads to longevity benefit.
doi:10.1007/s11357-010-9169-1
PMCID: PMC3127463  PMID: 20640543
Calorie restriction; Mitochondria; ROS; Stress resistance; Hormesis; atp2
5.  Functional Dissection of the Catalytic Carboxyl-Terminal Domain of Origin Recognition Complex Subunit 1 (PfORC1) of the Human Malaria Parasite Plasmodium falciparum▿ † 
Eukaryotic Cell  2009;8(9):1341-1351.
Origin recognition complex subunit 1 (ORC1) is essential for DNA replication in eukaryotes. The deadly human malaria parasite Plasmodium falciparum contains an ORC1/CDC6 homolog with several interesting domains at the catalytic carboxyl-terminal region that include a putative nucleoside triphosphate-binding and hydrolysis domain, a putative PCNA-interacting-protein (PIP) motif, and an extreme C-terminal region that shows poor homology with other ORC1 homologs. Due to the unavailability of a dependable inducible gene expression system, it is difficult to study the structure and function of essential genes in Plasmodium. Using a genetic yeast complementation system and biochemical experiments, here we show that the putative PIP domain in ORC1 that facilitates in vitro physical interaction with PCNA is functional in both yeast (Saccharomyces cerevisiae) and Plasmodium in vivo, confirming its essential biological role in eukaryotes. Furthermore, despite having less sequence homology, the extreme C-terminal region can be swapped between S. cerevisiae and P. falciparum and it binds to DNA directly, suggesting a conserved role of this region in DNA replication. These results not only provide us a useful system to study the function of the essential genes in Plasmodium, they help us to identify the previously undiscovered unique features of replication proteins in general.
doi:10.1128/EC.00170-09
PMCID: PMC2747822  PMID: 19633266
6.  Correction: CaZF, a Plant Transcription Factor Functions through and Parallel to HOG and Calcineurin Pathways in Saccharomyces cerevisiae to Provide Osmotolerance 
PLoS ONE  2009;4(6):10.1371/annotation/da3ad6f8-bc52-494d-9472-dd96c387c8fd.
doi:10.1371/annotation/da3ad6f8-bc52-494d-9472-dd96c387c8fd
PMCID: PMC2704090
7.  CaZF, a Plant Transcription Factor Functions through and Parallel to HOG and Calcineurin Pathways in Saccharomyces cerevisiae to Provide Osmotolerance 
PLoS ONE  2009;4(4):e5154.
Salt-sensitive yeast mutants were deployed to characterize a gene encoding a C2H2 zinc finger protein (CaZF) that is differentially expressed in a drought-tolerant variety of chickpea (Cicer arietinum) and provides salinity-tolerance in transgenic tobacco. In Saccharomyces cerevisiae most of the cellular responses to hyper-osmotic stress is regulated by two interconnected pathways involving high osmolarity glycerol mitogen-activated protein kinase (Hog1p) and Calcineurin (CAN), a Ca2+/calmodulin-regulated protein phosphatase 2B. In this study, we report that heterologous expression of CaZF provides osmotolerance in S. cerevisiae through Hog1p and Calcineurin dependent as well as independent pathways. CaZF partially suppresses salt-hypersensitive phenotypes of hog1, can and hog1can mutants and in conjunction, stimulates HOG and CAN pathway genes with subsequent accumulation of glycerol in absence of Hog1p and CAN. CaZF directly binds to stress response element (STRE) to activate STRE-containing promoter in yeast. Transactivation and salt tolerance assays of CaZF deletion mutants showed that other than the transactivation domain a C-terminal domain composed of acidic and basic amino acids is also required for its function. Altogether, results from this study suggests that CaZF is a potential plant salt-tolerance determinant and also provide evidence that in budding yeast expression of HOG and CAN pathway genes can be stimulated in absence of their regulatory enzymes to provide osmotolerance.
doi:10.1371/journal.pone.0005154
PMCID: PMC2664467  PMID: 19365545

Results 1-7 (7)