scholarly journals Prediction pipeline for discovery of regulatory motifs associated with Brugia malayi molting

2019 ◽  
Author(s):  
Alexandra Grote ◽  
Yichao Li ◽  
Canhui Liu ◽  
Denis Voronin ◽  
Adam Geber ◽  
...  
Keyword(s):  
Motif Discovery ◽  
Statistical Tests ◽  
Brugia Malayi ◽  
Life Cycles ◽  
Mammalian Host ◽  
Insect Vector ◽  
Motif Prediction ◽  
Filarial Nematodes ◽  
Free Living Nematode

AbstractFilarial nematodes can cause debilitating diseases in humans. They have complicated life cycles involving an insect vector and mammalian hosts, and they go through a number of developmental molts. While whole genome sequences of parasitic worms are now available, very little is known about transcription factor (TF) binding sites and their cognate transcription factors that play a role in regulating development. To address this gap, we developed a novel motif prediction pipeline, Emotif Alpha, that integrates ten different motif discovery algorithms, multiple statistical tests, and a comparative analysis of conserved elements between the filarial worms Brugia malayi and Onchocerca volvulus, and the free-living nematode Caenorhabditis elegans. We identified stage-specific TF binding motifs in B. malayi, with a particular focus on those potentially involved in the L3-L4 molt, a stage important for the establishment of infection in the mammalian host. Using an in vitro molting system, we tested and validated three of these motifs demonstrating the accuracy of the motif prediction pipeline.

scholarly journals Biofilm Formation by Pneumocystis spp.

2008 ◽  
Vol 8 (2) ◽  
pp. 197-206 ◽  
Author(s):  
Melanie T. Cushion ◽  
Margaret S. Collins ◽  
Michael J. Linke
Keyword(s):  
Biofilm Formation ◽  
Fungal Infections ◽  
Morphological Changes ◽  
Continuous Cultivation ◽  
Life Cycles ◽  
Mammalian Host ◽  
Intense Staining ◽  
In Vitro System ◽  
Major Surface

ABSTRACT Pneumocystis spp. can cause a lethal pneumonia in hosts with debilitated immune systems. The manner in which these fungal infections spread throughout the lung, the life cycles of the organisms, and their strategies used for survival within the mammalian host are largely unknown, due in part to the lack of a continuous cultivation method. Biofilm formation is one strategy used by microbes for protection against environmental assaults, for communication and differentiation, and as foci for dissemination. We posited that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system comprised of insert wells suspended in multiwell plates containing supplemented RPMI 1640 medium supported biofilm formation by P. carinii (from rat) and P. murina (from mouse).Dramatic morphological changes accompanied the transition to a biofilm. Cyst and trophic forms became highly refractile and produced branching formations that anastomosed into large macroscopic clusters that spread across the insert. Confocal microscopy revealed stacking of viable organisms enmeshed in concanavalin A-staining extracellular matrix. Biofilms matured over a 3-week time period and could be passaged. These passaged organisms were able to cause infection in immunosuppressed rodents. Biofilm formation was inhibited by farnesol, a quorum-sensing molecule in Candida spp., suggesting that a similar communication system may be operational in the Pneumocystis biofilms. Intense staining with a monoclonal antibody to the major surface glycoproteins and an increase in (1,3)-β-d-glucan content suggest that these components contributed to the refractile properties. Identification of this biofilm process provides a tractable in vitro system that should fundamentally advance the study of Pneumocystis.

Localization, turnover and conservation of gp15/400 in different stages ofBrugia malayi

Parasitology ◽  
1993 ◽  
Vol 107 (4) ◽  
pp. 449-457 ◽  
Author(s):  
M. E. Selkirk ◽  
W. F. Gregory ◽  
R. E. Jenkins ◽  
R. M. Maizels
Keyword(s):  
Protein Complex ◽  
Cdna Probe ◽  
The Body ◽  
Mammalian Host ◽  
Major Protein ◽  
Homologous Genes ◽  
Acanthocheilonema Viteae ◽  
Filarial Nematodes ◽  
The Matrix

SUMMARYThe expression of a protein complex designated gp15/400, previously identified via extrinsic iodination of adultBrugia malayi, was examined by labelling all stages found in the mammalian host and immunoprecipitation with a specific antibody raised to a recombinant protein. In this way, gp15/400 could be detected in L3, L4, adult worms and microfilariae recovered from jirds and labelled with Bolton–Hunter reagent. Metabolic labelling indicated that gp15/400 was released into culture medium when adult worms were maintainedin vitro, but at a rate slower than that of gp29, the major soluble cuticular glycoprotein. Immuno-electron microscopy showed that the protein complex was broadly distributed in different tissues, although it was not detectable in the cuticle of adult worms. Dense labelling was observed in the matrix of the basal laminae bordering the hypodermis, somatic musculature and oesophagus, and lower but significant labelling was seen in the cells overlying these extracellular matrices. Hybridization of genomic DNA with a cDNA probe encoding gp15/400 indicated that homologous genes were present inDirofilaria immitisandAcanthocheilonema viteae. The failure to detect related genes in non-filarial nematodes was presumed to be due to divergence beyond the practical limits of detection by nucleic acid probes, as antibody reagents showed that the protein cross-reacted immunologically with ABA-1, a major protein allergen from the body fluid ofAscaris.

Detection of thiol-based redox switch processes in parasites – facts and future

2015 ◽  
Vol 396 (5) ◽  
pp. 445-463 ◽  
Author(s):  
Mahsa Rahbari ◽  
Kathrin Diederich ◽  
Katja Becker ◽  
R. Luise Krauth-Siegel ◽  
Esther Jortzik
Keyword(s):  
Fluorescent Dyes ◽  
Redox Homeostasis ◽  
Life Cycles ◽  
Detection Methods ◽  
Mammalian Host ◽  
Host Immune System ◽  
Insect Vector ◽  
Comprehensive Overview ◽  
Specificity And Sensitivity ◽  
Antiparasitic Drugs

Abstract Malaria and African trypanosomiasis are tropical diseases caused by the protozoa Plasmodium and Trypanosoma, respectively. The parasites undergo complex life cycles in the mammalian host and insect vector, during which they are exposed to oxidative and nitrosative challenges induced by the host immune system and endogenous processes. Attacking the parasite’s redox metabolism is a target mechanism of several known antiparasitic drugs and a promising approach to novel drug development. Apart from this aspect, oxidation of cysteine residues plays a key role in protein-protein interaction, metabolic responses to redox events, and signaling. Understanding the role and dynamics of reactive oxygen species and thiol switches in regulating cellular redox homeostasis is crucial for both basic and applied biomedical approaches. Numerous techniques have therefore been established to detect redox changes in parasites including biochemical methods, fluorescent dyes, and genetically encoded probes. In this review, we aim to give an insight into the characteristics of redox networks in the pathogens Plasmodium and Trypanosoma, including a comprehensive overview of the consequences of specific deletions of redox-associated genes. Furthermore, we summarize mechanisms and detection methods of thiol switches in both parasites and discuss their specificity and sensitivity.

Secreted antigens of filarial nematodes: a survey and characterization of in vitro excreted/secreted products of adult Brugia malayi

1989 ◽  
Vol 11 (6) ◽  
pp. 629-654 ◽  
Author(s):  
G. E. KWAN–LIM ◽  
W. F. GREGORY ◽  
M. E. SELKIRK ◽  
F. PARTONO ◽  
R. M. MAIZELS
Keyword(s):  
Brugia Malayi ◽  
Filarial Nematodes ◽  
Secreted Antigens

scholarly journals Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei.

1989 ◽  
Vol 108 (2) ◽  
pp. 737-746 ◽  
Author(s):  
I Roditi ◽  
H Schwarz ◽  
T W Pearson ◽  
R P Beecroft ◽  
M K Liu ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Tsetse Fly ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Mammalian Host ◽  
Cylindrical Shape ◽  
Surface Coat ◽  
Insect Vector ◽  
Molecular Arrangement

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.

scholarly journals AMP-activated protein kinase: A key enzyme to manage nutritional stress responses in parasites with complex life cycles

2020 ◽  
Author(s):  
Tamara Sternlieb ◽  
Alejandra C. Schoijet ◽  
Patricio D. Genta ◽  
Guillermo D. Alonso
Keyword(s):  
Life Cycle ◽  
Protein Kinase ◽  
Stress Responses ◽  
Nutritional Stress ◽  
Life Cycles ◽  
Nutrient Sensing ◽  
Mammalian Host ◽  
Insect Vector ◽  
Compound C ◽  
Amp Activated Protein Kinase

AbstractTrypanosoma cruzi, the etiological agent of Chagas disease, has a digenetic life cycle. In its passage from the insect vector to the mammalian host, and vice versa, it must be prepared to cope with abrupt changes in environmental conditions in order to survive. Sensing and signaling pathways that allow the parasite to adapt, have unique characteristics with respect to their hosts and other free-living organisms. Many of the canonical proteins involved in these transduction pathways have not yet been found in the genomes of these parasites, because they present divergences either at the functional, structural and/or protein sequence level. All of this makes these pathways promising targets for therapeutic drugs.The AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by environmental stresses that results in reduction of ATP and increase of AMP levels. Thus, AMPK is regarded as a fuel gauge, functioning both as a nutrient and an energy sensor, to maintain energy homeostasis and, eventually, to protect cells from death by nutrient starvation.In the present study, we report the characterization of AMPK complexes for the first time in T. cruzi and describe the function of TcAMPK as a novel regulator of nutritional stress in epimastigote forms. We demonstrate that this complex possesses specific AMPK kinase activity in epimastigotes, which is inhibited by Compound C and is modulated by carbon source availability. In addition, TcAMPKα2 subunit has an unprecedented functional substitution (Ser x Thr) at the activation loop and its overexpression in epimastigotes led to higher autophagic activity during prolonged nutritional stress. Moreover, the over-expression of the catalytic subunits resulted in antagonistic phenotypes associated with proliferation. Together, these results point to a role of TcAMPK in autophagy and nutrient sensing, key processes for the survival of trypanosomatids and for its life cycle progression.

scholarly journals Homology Modeling of NAD+-Dependent DNA Ligase of the Wolbachia Endosymbiont of Brugia malayi and Its Drug Target Potential Using Dispiro-Cycloalkanones

2015 ◽  
Vol 59 (7) ◽  
pp. 3736-3747 ◽  
Author(s):  
Nidhi Shrivastava ◽  
Jeetendra K. Nag ◽  
Jyoti Pandey ◽  
Rama Pati Tripathi ◽  
Priyanka Shah ◽  
...  
Keyword(s):  
Adverse Effects ◽  
Drug Target ◽  
Brugia Malayi ◽  
Great Promise ◽  
Dna Ligase ◽  
Content Type ◽  
T4 Dna Ligase ◽  
Filarial Nematodes

ABSTRACTLymphatic filarial nematodes maintain a mutualistic relationship with the endosymbiontWolbachia. Depletion ofWolbachiaproduces profound defects in nematode development, fertility, and viability and thus has great promise as a novel approach for treating filarial diseases. NAD+-dependent DNA ligase is an essential enzyme of DNA replication, repair, and recombination. Therefore, in the present study, the antifilarial drug target potential of the NAD+-dependent DNA ligase of theWolbachiasymbiont ofBrugia malayi(wBm-LigA) was investigated using dispiro-cycloalkanone compounds. Dispiro-cycloalkanone specifically inhibited the nick-closing and cohesive-end ligation activities of the enzyme without inhibiting human or T4 DNA ligase. The mode of inhibition was competitive with the NAD+cofactor. Docking studies also revealed the interaction of these compounds with the active site of the target enzyme. The adverse effects of these inhibitors were observed on adult and microfilarial stages ofB. malayiin vitro, and the most active compounds were further monitoredin vivoin jirds and mastomys rodent models. Compounds 1, 2, and 5 had severe adverse effectsin vitroon the motility of both adult worms and microfilariae at low concentrations. Compound 2 was the best inhibitor, with the lowest 50% inhibitory concentration (IC50) (1.02 μM), followed by compound 5 (IC50, 2.3 μM) and compound 1 (IC50, 2.9 μM). These compounds also exhibited the same adverse effect on adult worms and microfilariaein vivo(P< 0.05). These compounds also tremendously reduced the wolbachial load, as evident by quantitative real-time PCR (P< 0.05).wBm-LigA thus shows great promise as an antifilarial drug target, and dispiro-cycloalkanone compounds show great promise as antifilarial lead candidates.

Molecular biology of Leishmania

1989 ◽  
Vol 67 (9) ◽  
pp. 516-524 ◽  
Author(s):  
Enzo Bard
Keyword(s):  
Regulation Of Gene Expression ◽  
Mammalian Host ◽  
Insect Vector ◽  
Free Living ◽  
Regulatory Motifs ◽  
Trans Splicing ◽  
Obligatory Intracellular Parasite ◽  
Two Stages ◽  
Physiological And Biochemical

Leishmania is a trypanosomatid protozoa with a digenetic life cycle. Sandflies inject promastigotes, the free living form present in their salivary glands, into mammals where the parasite colonizes macrophages, transforming into intracellular amastigotes. The cycle is completed when during a blood meal the insect ingests infected macrophages, the amastigotes are released in the gut where they transform back into promastigotes. Leishmania has to adapt to the changing life conditions, from free-living forms in the poikilothermic insect vector to obligatory intracellular parasite in the homeothermic mammalian host. It also has to adapt to the acidic pH of the macrophage's phagolysosome where amastigotes multiply. The adaptative response of Leishmania includes morphological, physiological, and biochemical changes. Promastigotes can be grown in culture medium. Studies of changes taking place during adaptation have been facilitated by the establishment of in vitro conditions that allow the transformation of amastigotes into promastigotes and vice versa. The system is well suited for studying regulation of gene expression during adaptative differentiation. Some mechanisms of mRNA processing are unique to these protozoa: trans-splicing and RNA editing. Several genes that are differentially expressed in the two stages have been studied. No obvious cis regulatory motifs have been found in the DNA.Key words: Leishmania, genes, differentiation, regulation.

scholarly journals Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

2020 ◽  
Author(s):  
Rebecca J. Burge ◽  
Andreas Damianou ◽  
Anthony J. Wilkinson ◽  
Boris Rodenko ◽  
Jeremy C. Mottram
Keyword(s):  
Life Cycle ◽  
Crystal Structure Analysis ◽  
Structural Basis ◽  
Mammalian Host ◽  
Insect Vector ◽  
Post Translational Modification ◽  
Mouse Infection ◽  
Null Mutants

AbstractPost-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved interface, highlighting the importance of stable UBC2-UEV1 interaction in the function of this complex across diverse eukaryotes. Furthermore, recombinant L. mexicana E1 UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can also cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.Author summaryThe post-translational modification of proteins is key for allowing Leishmania parasites to transition between the different life cycle stages that exist in its insect vector and mammalian host. In particular, components of the ubiquitin system are important for the transformation of Leishmania from its insect (promastigote) to mammalian (amastigote) stage and normal infection in mice. However, little is known about the role of the enzymes that generate ubiquitin modifications in Leishmania. Here we characterise 28 enzymes of the ubiquitination pathway and show that many are required for life cycle progression or mouse infection by this parasite. Two proteins, UBC2 and UEV1, were selected for further study based on their importance in the promastigote to amastigote transition. We demonstrate that UBC2 and UEV1 form a heterodimer capable of carrying out ubiquitination and that the structural basis for this activity is conserved between Leishmania, Saccharomyces cerevisiae and humans. We also show that the interaction of UBC2 with UEV1 alters the nature of the ubiquitination activity performed by UBC2. Overall, we demonstrate the important role that ubiquitination enzymes play in the life cycle and infection process of Leishmania and explore the biochemistry underlying UBC2 and UEV1 function.

Polyamines and Related Nitrogen Compounds in the Chemotherapy of Neglected Diseases Caused by Kinetoplastids

2018 ◽  
Vol 18 (5) ◽  
pp. 321-368 ◽  
Author(s):  
Juan A. Bisceglia ◽  
Maria C. Mollo ◽  
Nadia Gruber ◽  
Liliana R. Orelli
Keyword(s):  
Drug Targets ◽  
Redox Homeostasis ◽  
Cost Effective ◽  
Structural Features ◽  
Mammalian Host ◽  
Neglected Diseases ◽  
Nitrogen Compounds ◽  
Chemical Structures

Neglected diseases due to the parasitic protozoa Leishmania and Trypanosoma (kinetoplastids) affect millions of people worldwide, and the lack of suitable treatments has promoted an ongoing drug discovery effort to identify novel nontoxic and cost-effective chemotherapies. Polyamines are ubiquitous small organic molecules that play key roles in kinetoplastid parasites metabolism, redox homeostasis and in the normal progression of cell cycles, which differ from those found in the mammalian host. These features make polyamines attractive in terms of antiparasitic drug development. The present work provides a comprehensive insight on the use of polyamine derivatives and related nitrogen compounds in the chemotherapy of kinetoplastid diseases. The amount of literature on this subject is considerable, and a classification considering drug targets and chemical structures were made. Polyamines, aminoalcohols and basic heterocycles designed to target the relevant parasitic enzyme trypanothione reductase are discussed in the first section, followed by compounds directed to less common targets, like parasite SOD and the aminopurine P2 transporter. Finally, the third section comprises nitrogen compounds structurally derived from antimalaric agents. References on the chemical synthesis of the selected compounds are reported together with their in vivo and/or in vitro IC50 values, and structureactivity relationships within each group are analyzed. Some favourable structural features were identified from the SAR analyses comprising protonable sites, hydrophobic groups and optimum distances between them. The importance of certain pharmacophoric groups or amino acid residues in the bioactivity of polyamine derived compounds is also discussed.

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