In Vitro Screening Test Using Leishmania Promastigotes Stably Expressing mCherry Protein

Transgenic Leishmania major and Leishmania donovani axenic promastigotes constitutively expressing mCherry were used for in vitro antileishmanial drug screening. This method requires minimal sample manipulation and can be easily adapted to automatic drug tests, allowing primary high-throughput screenings without the need for expensive and sophisticated instrumentsFil: Vacchina, Paola. University Of Notre Dame-Indiana; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Miguel A. Morales. University Of Notre Dame-Indiana; Estados Unido

LmaPA2G4, a Homolog of Human Ebp1, Is an Essential Gene and Inhibits Cell Proliferation in L. major

We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in aphosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidasemember of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. Anull mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene inLeishmaniamajor. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigotestages. Interestingly, the over-expression affected cell proliferation and virulence in mouse footpad analysis. LmaPA2G4 binds asynthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] as shown in an electrophoretic mobility shiftassay (EMSA). Quantitative proteomics revealed that the over-expression of LmaPA2G4 led to accumulation of factors involved intranslation initiation and elongation. Significantly, we found a strong reduction ofde novoprotein biosynthesis in transgenicparasites using a non-radioactive metabolic labeling assay. In conclusion, LmaPA2G4 is an essential gene and is potentiallyimplicated in fundamental biological mechanisms, such as translation, making it an attractive target for therapeutic intervention.Fil: Norris Mullins, Brianna. University Of Notre Dame-Indiana; Estados UnidosFil: VanderKolk, Kaitlin. University Of Notre Dame-Indiana; Estados UnidosFil: Vacchina, Paola. University Of Notre Dame-Indiana; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Joyce, Michelle V.. University Of Notre Dame-Indiana; Estados UnidosFil: Morales, Miguel A.. University Of Notre Dame-Indiana; Estados Unido

Catalytic activity of a novel serine/threonine protein phosphatase PP5 from

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs) and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5) in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention

Catalytic activity of a novel serine/threonine protein phosphatase PP5 from Leishmania major

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs) and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5) in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention

Lipoic acid metabolism in Trypanosoma cruzi as putative target for chemotherapy

Lipoic acid (LA) is a cofactor of relevant enzymatic complexes including the glycine cleave system and 2-ketoacid dehydrogenases. Intervention on LA de novo synthesis or salvage could have pleiotropic deleterious effect in cells, making both pathways attractive for chemotherapy. We show that Trypanosoma cruzi was susceptible to treatment with LA analogues. 8-Bromo-octanic acid (BrO) inhibited the growth of epimastigote forms of both Dm28c and CL Brener strains, although only at high (chemotherapeutically irrelevant) concentrations. The methyl ester derivative MBrO, was much more effective, with EC50 values one order of magnitude lower (62–66 μM). LA did not bypass the toxic effect of its analogues. Small monocarboxylic acids appear to be poorly internalized by T. cruzi: [14C]-octanoic acid was taken up 12 fold less efficiently than [14C]-palmitic acid. Western blot analysis of lipoylated proteins allowed the detection of the E2 subunits of pyruvate dehydrogenase (PDH), branched chain 2-ketoacid dehydrogenase and 2-ketoglutarate dehydrogenase complexes. Growth of parasites in medium with 10 fold lower glucose content, notably increased PDH activity and the level of its lipoylated E2 subunit. Treatment with BrO (1 mM) and MBrO (0.1 mM) completely inhibited E2 lipoylation and all three dehydrogenases activities. These observations indicate the lack of specific transporters for octanoic acid and most probably also for BrO and LA, which is in agreement with the lack of a LA salvage pathway, as previously suggested for T. brucei. They also indicate that the LA synthesis/protein lipoylation pathway could be a valid target for drug intervention. Moreover, the free LA available in the host would not interfere with such chemotherapeutic treatments.Fil: Vacchina, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Lambruschi, Daniel Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Uttaro, Antonio Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

RESEARCH ARTICLE OPEN ACCESS Catalytic activity of a novel serine/threonine protein

Abstract – Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs) and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5) in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed autoinhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention. Key words: signaling, phosphatases, mutagenesis, activity, drug target. Résumé – Activité catalytique d’une nouvelle phosphatase de protéine sérine/thréonine PP5 de Leishmania major. La leishmaniose est une maladie transmise par un vecteur, due à des parasites protozoaires appartenant au genre Leishmania. Notre connaissance des phosphatases de protéines (PPs) et leur implication dans la signalisation est très limitée. Nous rapportons ici l’expression, la caractérisation et les analyses de mutagenèse d’une nouvelle PP5 chez Leishmania major. La PP5 recombinante est une phosphatase authentique, enzymatiquement active. La mutagenèse dirigée a identifié les rôles auto-inhibiteurs de la région N-terminale de la PP5. Il s’agit d’une premièr

A mitochondrial HSP70 (HSPA9B) is linked to miltefosine resistance and stress response in Leishmania donovani

Background: Protozoan parasites of the genus Leishmania are responsible for leishmaniasis, a neglected tropical disease affecting millions worldwide. Visceral leishmaniasis (VL), caused by Leishmania donovani, is the most severe form of leishmaniasis with high rates of mortality if left untreated. Current treatments include pentavalent antimonials and amphotericin B. However, high toxicity and emergence of resistance hinder the success of these options. Miltefosine (HePC) is the first oral treatment available for leishmaniasis. While treatment with HePC has proven effective, higher tolerance to the drug has been observed, and experimental resistance is easily developed in an in vitro environment. Several studies, including ours, have revealed that HePC resistance has a multi-factorial origin and this work aims to shed light on this complex mechanism. Methods: 2D-DIGE quantitative proteomics comparing the soluble proteomes of sensitive and HePC resistant L. donovani lines identified a protein of interest tentatively involved in drug resistance. To test this link, we employed a gain-of-function approach followed by mutagenesis analysis. Functional studies were complemented with flow cytometry to measure HePC incorporation and cell death. Results: We identified a mitochondrial HSP70 (HSPA9B) downregulated in HePC-resistant L. donovani promastigotes. The overexpression of HSPA9B in WT lines confers an increased sensitivity to HePC, regardless of whether the expression is ectopic or integrative. Moreover, the increased sensitivity to HePC is specific to the HSPA9B overexpression since dominant negative mutant lines were able to restore HePC susceptibility to WT values. Interestingly, the augmented susceptibility to HePC did not correlate with an increased HePC uptake. Leishmania donovani promastigotes overexpressing HSPA9B were subjected to different environmental stimuli. Our data suggest that HSPA9B is capable of protecting cells from stressful conditions such as low pH and high temperature. This phenotype was further corroborated in axenic amastigotes overexpressing HSPA9B. Conclusions: The results from this study provide evidence to support the involvement of a mitochondrial HSP70 (HSPA9B) in experimental HePC resistance, a mechanism that is not yet fully understood, and reveal potential fundamental roles of HSPA9B in the biology of Leishmania. Overall, our findings are relevant for current and future antileishmanial chemotherapy strategies.Fil: Vacchina, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. University of Notre Dame; Estados UnidosFil: Norris Mullins B.. University of Notre Dame; Estados UnidosFil: Carlson, E. S.. University of Notre Dame; Estados UnidosFil: Morales, M. A.. University of Notre Dame; Estados Unido

Functional characterization of the first lipoyl-relay pathway from a parasitic protozoan

Lipoic acid (LA) is a sulfur-containing cofactor covalently attached to key enzymes of central metabolism in prokaryotes and eukaryotes. LA can be acquired by scavenging, mediated by a lipoate ligase, or de novo synthesized by a pathway requiring an octanoyltransferase and a lipoate synthase. A more complex pathway, referred to as “lipoyl-relay”, requires two additional proteins, GcvH, the glycine cleavage system H subunit, and an amidotransferase. This route was described so far in Bacillus subtilis and related Gram-positive bacteria, Saccharomyces cerevisiae, Homo sapiens, and Caenorhabditis elegans. Using collections of S. cerevisiae and B. subtilis mutants, defective in LA metabolism, we gathered evidence that allows us to propose for the first time that lipoyl-relay pathways are also present in parasitic protozoa. By a reverse genetic approach, we assigned octanoyltransferase and amidotransferase activity to the products of Tb927.11.9390 (TblipT) and Tb927.8.630 (TblipL) genes of Trypanosoma brucei, respectively. The B. subtilis model allowed us to identify the parasite amidotransferase as the target of lipoate analogs like 8-bromo-octanoic acid, explaining the complete loss of protein lipoylation and growth impairment caused by this compound in T. cruzi. This model could be instrumental for the screening of selective and more efficient chemotherapies against trypanosomiases.M.C.M and A.D.U. are members of the Carrera del Investigador Científico del Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. A.S. is a doctoral fellow from CONICET, A.L and D.A.L were doctoral fellows and P.V. was a career member from the same institution. This work was supported by Fondo para la Investigación Científica y Tecnológica (Grant PICT 2016-1853); Ministerio de Ciencia Tecnología e Innovación Productiva (Grant EULACH 16/T02-0161); and Consejo Nacional de Investigaciones Científicas y Técnicas (Grant P-UE-0039 2016).Peer reviewe

Fitness and phenotypic characterization of miltefosine-resistant Leishmania major

Trypanosomatid parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease with several clinical manifestations. Leishmania major is the causative agent of cutaneous leishmaniasis (CL), which is largely characterized by ulcerative lesions appearing on the skin. Current treatments of leishmaniasis include pentavalent antimonials and amphotericin B, however, the toxic side effects of these drugs and difficulty with distribution makes these options less than ideal. Miltefosine (MIL) is the first oral treatment available for leishmaniasis. Originally developed for cancer chemotherapy, the mechanism of action of MIL in Leishmania spp. is largely unknown. While treatment with MIL has proven effective, higher tolerance to the drug has been observed, and resistance is easily developed in an in vitro environment. Utilizing stepwise selection we generated MIL-resistant cultures of L. major and characterized the fitness of MIL-resistant L. major. Resistant parasites proliferate at a comparable rate to the wild-type (WT) and exhibit similar apoptotic responses. As expected, MIL-resistant parasites demonstrate decreased susceptibility to MIL, which reduces after the drug is withdrawn from culture. Our data demonstrate metacyclogenesis is elevated in MIL-resistant L. major, albeit these parasites display attenuated in vitro and in vivo virulence and standard survival rates in the natural sandfly vector, indicating that development of experimental resistance to miltefosine does not lead to an increased competitive fitness in L. major.Fil: Turner, Kimbra G.. University of Notre Dame-Indiana; Estados UnidosFil: Vacchina, Paola. University of Notre Dame-Indiana; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Robles Murguia, Maricela. University of Notre Dame-Indiana; Estados UnidosFil: Wadsworth, Mariha. University of Notre Dame-Indiana; Estados UnidosFil: McDowell, Mary Ann. University of Notre Dame-Indiana; Estados UnidosFil: Morales, Miguel A.. University of Notre Dame-Indiana; Estados Unido

2D-DIGE quantitative phosphoproteomics analysis of GFPK7.

<p>An enlarged region of the 2D-DIGE gels showing Cy3-labeled WT stationary promastigotes and Cy5-labeled stationary GFPK7 promastigotes is presented. Spot ID 207 (white arrow) was over-represented in GFPK7. In the lower panel, a graphical representation of the BVA (Biological Variation Analysis) module of Decyder software (GE Healthcare) with statistics for spot ID 207 (2.97 fold and p = 0.00056). The spot was analyzed by mass spectrometry and identified as LmjF19.0160, a putative aminopeptidase.</p