scholarly journals Leishmania donovani Parasites Are Inhibited by the Benzoxaborole AN2690 Targeting Leucyl-tRNA Synthetase

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Reetika Manhas ◽  
Smriti Tandon ◽  
Shib Sankar Sen ◽  
Neha Tiwari ◽  
Manoj Munde ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Drug Targets ◽  
Fungal Pathogens ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Gene Replacement ◽  
Trna Synthetase ◽  
Content Type

ABSTRACT Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Leishmania donovani. Currently available drugs manifest severe side effects. Hence, there is a need to identify new drug targets and drugs. Aminoacyl-tRNA synthetases, required for protein synthesis, are known drug targets for bacterial and fungal pathogens. The aim of the present study was to obtain essentiality data for Leishmania donovani leucyl-tRNA synthetase (LdLRS) by gene replacement. Gene replacement studies indicate that this enzyme plays an essential role in the viability of this pathogenic organism and appears to be indispensable for its survival in vitro. The heterozygous mutant parasites demonstrated a growth deficit and reduced infectivity in mouse macrophages compared to the wild-type cells. We also report that Leishmania donovani recombinant LRS displayed aminoacylation activity and that the protein localized to both the cytosol and the mitochondrion. A broad-spectrum antifungal, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), was found to inhibit parasite growth in both the promastigote and amastigote stages in vitro as well as in vivo in BALB/c mice. This compound exhibited low toxicity to mammalian cells. AN2690 was effective in inhibiting the aminoacylation activity of the recombinant LdLRS. We provide preliminary chemical validation of LdLRS as a drug target by showing that AN2690 is an inhibitor both of L. donovani LRS and of L. donovani cell growth.

scholarly journals TCP1γ Subunit Is Indispensable for Growth and Infectivity of Leishmania donovani

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Shailendra Yadav ◽  
Jitendra Kuldeep ◽  
Mohammad I. Siddiqi ◽  
Neena Goyal
Keyword(s):  
Cell Cycle Progression ◽  
Leishmania Donovani ◽  
Gene Replacement ◽  
Titration Calorimetry ◽  
Content Type ◽  
T Complex ◽  
Complex Protein ◽  
Oligomeric Complex

ABSTRACT T-complex protein-1 (TCP1) is a ubiquitous group II chaperonin and is known to fold various proteins, such as actin and tubulin. In Leishmania donovani, the γ subunit of TCP1 (LdTCP1γ) has been cloned and characterized. It forms a high-molecular-weight homo-oligomeric complex that performs ATP-dependent protein folding. In the present study, we evaluated the essentiality of the LdTCP1γ gene. Gene replacement studies indicated that LdTCP1γ is essential for parasite survival. The LdTCP1γ single-allele-replacement mutants exhibited slowed growth and decreased infectivity in mouse macrophages compared to the growth and infectivity of the wild-type parasites. Modulation of LdTCP1γ expression in promastigotes also modulated cell cycle progression. Suramin, an antitrypanosomal drug, not only inhibited the luciferase refolding activity of the recombinant LdTCP1γ (rLdTCP1γ) homo-oligomeric complex but also exhibited potential antileishmanial efficacy both in vitro and in vivo. The interaction of suramin and LdTCP1γ was further validated by isothermal titration calorimetry. The study suggests LdTCP1γ as a potential drug target and also provides a framework for the development of a new class of drugs.

scholarly journals Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Jixu Li ◽  
Huanping Guo ◽  
Eloiza May Galon ◽  
Yang Gao ◽  
Seung-Hun Lee ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Lytic Cycle ◽  
Type I ◽  
Content Type ◽  
Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

scholarly journals New Method for the Orthogonal Labeling and Purification of Toxoplasma gondii Proteins While Inside the Host Cell

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Gregory M. Wier ◽  
Erica M. McGreevy ◽  
Mark J. Brown ◽  
Jon P. Boyle
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Click Reaction ◽  
Severe Disease ◽  
Protozoan Parasite ◽  
Trna Synthetase ◽  
Content Type ◽  
Fluorescent Tags

ABSTRACTToxoplasma gondiiis an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRSNLL) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed “click” reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRSNLL-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRSNLL-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRSNLL-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS.IMPORTANCEStudying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins duringin vitrogrowth. It should also be applicable duringin vivoinfections and in other apicomplexan parasites, including Plasmodium spp.

scholarly journals A ReplicativeIn VitroAssay for Drug Discovery against Leishmania donovani

2016 ◽  
Vol 60 (6) ◽  
pp. 3524-3532 ◽  
Author(s):  
Diana Tegazzini ◽  
Rosario Díaz ◽  
Fernando Aguilar ◽  
Imanol Peña ◽  
Jesús L. Presa ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Horse Serum ◽  
Protozoan Parasite ◽  
Infection Process ◽  
Infection Model ◽  
Content Type ◽  
Fetal Bovine

The protozoan parasiteLeishmania donovaniis the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identifiedin vitrodid not translate their activities when tested inin vivomodels, highlighting the need to develop more predictivein vitroassays. In the present work, we describe the development of a robust replicative, high-content,in vitrointracellularL. donovaniassay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypicin vitroinfection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation.In vitroandin vivoresults for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.

scholarly journals Neurotrophin Receptor TrkC Is an Entry Receptor for Trypanosoma cruzi in Neural, Glial, and Epithelial Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 4081-4087 ◽  
Author(s):  
Craig Weinkauf ◽  
Ryan Salvador ◽  
Mercio PereiraPerrin
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Neuronal Cell ◽  
Host Cells ◽  
Neurotrophin Receptor ◽  
Content Type

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated byT. cruzisurfacetrans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used byT. cruzito enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant toT. cruzibecame highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore,trkCtransfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive toT. cruziafter transfection with thetrkCgene. Additionally, NT-3 specifically blockedT. cruziinfection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blockedT. cruziinfection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected byT. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broadT. cruziinfection bothin vitroandin vivo.

scholarly journals SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

2014 ◽  
Vol 82 (7) ◽  
pp. 2890-2901 ◽  
Author(s):  
Marilena Gallotta ◽  
Giovanni Gancitano ◽  
Giampiero Pietrocola ◽  
Marirosa Mora ◽  
Alfredo Pezzicoli ◽  
...  
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Group A Streptococcus ◽  
Host Cells ◽  
Knockout Mutant ◽  
Content Type ◽  
Moonlighting Protein ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of thespy0269gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interactin vitrowith the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cellsin vitroand thatLactococcus lactisexpressing Spy0269 on its cell surface could adhere to mammalian cellsin vitroand to mice nasal mucosain vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (StreptococcuspyogenesAdhesion andDivision protein).

scholarly journals Functional genetic encoding of sulfotyrosine in mammalian cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinyuan He ◽  
Yan Chen ◽  
Daisy Guiza Beltran ◽  
Maia Kelly ◽  
Bin Ma ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Trna Synthetase ◽  
Functional Verification ◽  
Cellular Processes ◽  
Genetic Encoding ◽  
Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

Leishmania donovani infection of bone marrow stromal macrophages selectively enhances myelopoiesis, by a mechanism involving GM-CSF and TNF-α

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1642-1651 ◽  
Author(s):  
Sara E. J. Cotterell ◽  
Christian R. Engwerda ◽  
Paul M. Kaye
Keyword(s):  
Infectious Disease ◽  
Bone Marrow ◽  
Stromal Cell ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Gm Csf ◽  
Tnf Α ◽  
Macrophage Colony

Alterations in hematopoiesis are common in experimental infectious disease. However, few studies have addressed the mechanisms underlying changes in hematopoietic function or assessed the direct impact of infectious agents on the cells that regulate these processes. In experimental visceral leishmaniasis, caused by infection with the protozoan parasite Leishmania donovani, parasites persist in the spleen and bone marrow, and their expansion in these sites is associated with increases in local hematopoietic activity. The results of this study show that L donovani targets bone marrow stromal macrophages in vivo and can infect and multiply in stromal cell lines of macrophage, but not other lineages in vitro. Infection of stromal macrophages increases their capacity to support myelopoiesis in vitro, an effect mediated mainly through the induction of granulocyte macrophage-colony stimulating factor and tumor necrosis factor-. These data are the first to directly demonstrate that intracellular parasitism of a stromal cell population may modify its capacity to regulate hematopoiesis during infectious disease.

scholarly journals THEORETICAL APPROACH ON TARGETING PLANT FUNGAL PATHOGENIC PROTEINS AGAINST NATURALLY ISOLATED COMPOUNDS FROM CHITINIPHILUS SHINANONENSIS

2019 ◽  
pp. 138-142
Author(s):  
KRITHIKA S ◽  
CHELLARAM C
Keyword(s):  
Drug Targets ◽  
Fungal Pathogens ◽  
Interaction Analysis ◽  
Pathogenic Fungus ◽  
Three Dimensional ◽  
Data Bank ◽  
Asiatic Acid ◽  
Literature Study

Objective: The objective of this study was to find the potency and bioefficacy of Asiatic acid and triterpene against four different plant fungal pathogens using a structure-based drug designing approach. Methods: The pathogenic fungus which causes a dreadful effect on plants is reviewed from literature study, and its three-dimensional structures are retrieved from the protein data bank database. On the other hand, ligands are prepared. Finally, prepared fungal drug targets are docked with naturally isolated compounds using AutoDock tools. Results: Both compounds Asiatic acid and triterpene structures are complementary to bind at the active site of four different drug targets. Comparatively, it is more favorable for Avr2 effector protein from Fusarium oxysporum with Ki value of 126.60 μM, 1.76 μM, and dock score value of −5.32 kcal/mol and −7.85 kcal/mol for Asiatic acid and triterpene, respectively. Thus, interaction analysis was carried out only for these protein-ligand complexes. Conclusion: The computational biology study states that these two compounds can be the lead candidate for treating disease caused by plant fungal pathogen F. oxysporum. However, further study has to be done in vitro and in vivo to prove its same efficacy.

scholarly journals Fenbendazole Controls In Vitro Growth, Virulence Potential, and Animal Infection in the Cryptococcus Model

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Haroldo C. de Oliveira ◽  
Luna S. Joffe ◽  
Karina S. Simon ◽  
Rafael F. Castelli ◽  
Flavia C. G. Reis ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Mammalian Cells ◽  
Fungal Pathogens ◽  
Therapeutic Potential ◽  
Low Cost ◽  
Macrophage Infection ◽  
Content Type ◽  
Virulence Potential ◽  
Cryptococcal Disease

ABSTRACT The human diseases caused by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are associated with high indices of mortality and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anticryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anticryptococcal benzimidazole. Fenbendazole was inhibitory against 17 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anticryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anticryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mouse model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.

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