scholarly journals Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs

2009 ◽  
Vol 2009 ◽  
pp. 1-19 ◽  
Author(s):  
Wanderley de Souza ◽  
Juliany Cola Fernandes Rodrigues
Keyword(s):  
Mammalian Cells ◽  
Mevalonate Pathway ◽  
Fungal Infections ◽  
Light And Electron Microscopy ◽  
Sterol Biosynthesis ◽  
Host Cells ◽  
Mammalian Host ◽  
Squalene Epoxidase ◽  
Eukaryotic Microorganisms ◽  
Protozoan Cell

Sterols are constituents of the cellular membranes that are essential for their normal structure and function. In mammalian cells, cholesterol is the main sterol found in the various membranes. However, other sterols predominate in eukaryotic microorganisms such as fungi and protozoa. It is now well established that an important metabolic pathway in fungi and in members of the Trypanosomatidae family is one that produces a special class of sterols, including ergosterol, and other 24-methyl sterols, which are required for parasitic growth and viability, but are absent from mammalian host cells. Currently, there are several drugs that interfere with sterol biosynthesis (SB) that are in use to treat diseases such as high cholesterol in humans and fungal infections. In this review, we analyze the effects of drugs such as (a) statins, which act on the mevalonate pathway by inhibiting HMG-CoA reductase, (b) bisphosphonates, which interfere with the isoprenoid pathway in the step catalyzed by farnesyl diphosphate synthase, (c) zaragozic acids and quinuclidines, inhibitors of squalene synthase (SQS), which catalyzes the first committed step in sterol biosynthesis, (d) allylamines, inhibitors of squalene epoxidase, (e) azoles, which inhibit C14α-demethylase, and (f) azasterols, which inhibitΔ24(25)-sterol methyltransferase (SMT). Inhibition of this last step appears to have high selectivity for fungi and trypanosomatids, since this enzyme is not found in mammalian cells. We review here the IC50 values of these various inhibitors, their effects on the growth of trypanosomatids (both in axenic cultures and in cell cultures), and their effects on protozoan structural organization (as evaluted by light and electron microscopy) and lipid composition. The results show that the mitochondrial membrane as well as the membrane lining the protozoan cell body and flagellum are the main targets. Probably as a consequence of these primary effects, other important changes take place in the organization of the kinetoplast DNA network and on the protozoan cell cycle. In addition, apoptosis-like and autophagic processes induced by several of the inhibitors tested led to parasite death.

scholarly journals Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

2005 ◽  
Vol 77 (1) ◽  
pp. 77-94 ◽  
Author(s):  
Renato A. Mortara ◽  
Walter K. Andreoli ◽  
Noemi N. Taniwaki ◽  
Adriana B. Fernandes ◽  
Claudio V. da Silva ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Mammalian Cells ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Mammalian Host ◽  
Target Cells ◽  
Sylvatic Cycle ◽  
Final Destination ◽  
Different Strains

Trypanosoma cruzi, the etiological agent of Chagas’ disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

scholarly journals PB2 Residue 271 Plays a Key Role in Enhanced Polymerase Activity of Influenza A Viruses in Mammalian Host Cells

2010 ◽  
Vol 84 (9) ◽  
pp. 4395-4406 ◽  
Author(s):  
Kendra A. Bussey ◽  
Tatiana L. Bousse ◽  
Emily A. Desmet ◽  
Baek Kim ◽  
Toru Takimoto
Keyword(s):  
Mammalian Cells ◽  
Influenza A ◽  
Virus Titer ◽  
Polymerase Activity ◽  
Influenza Viruses ◽  
Host Cells ◽  
Mammalian Host ◽  
Influenza A Viruses ◽  
H5n1 Influenza ◽  
Avian Viruses

ABSTRACT The direct infection of humans with highly pathogenic avian H5N1 influenza viruses has suggested viral mutation as one mechanism for the emergence of novel human influenza A viruses. Although the polymerase complex is known to be a key component in host adaptation, mutations that enhance the polymerase activity of avian viruses in mammalian hosts are not fully characterized. The genomic comparison of influenza A virus isolates has identified highly conserved residues in influenza proteins that are specific to either human or avian viruses, including 10 residues in PB2. We characterized the activity of avian polymerase complexes containing avian-to-human mutations at these conserved PB2 residues and found that, in addition to the E627K mutation, the PB2 mutation T271A enhances polymerase activity in human cells. We confirmed the effects of the T271A mutation using recombinant WSN viruses containing avian NP and polymerase genes with wild-type (WT) or mutant PB2. The 271A virus showed enhanced growth compared to that of the WT in mammalian cells in vitro. The 271A mutant did not increase viral pathogenicity significantly in mice compared to that of the 627K mutant, but it did enhance the lung virus titer. Also, cell infiltration was more evident in lungs of 271A-infected mice than in those of the WT. Interestingly, the avian-derived PB2 of the 2009 pandemic H1N1 influenza virus has 271A. The characterization of the polymerase activity of A/California/04/2009 (H1N1) and corresponding PB2 mutants indicates that the high polymerase activity of the pandemic strain in mammalian cells is, in part, dependent on 271A. Our results clearly indicate the contribution of PB2 amino acid 271 to enhanced polymerase activity and viral growth in mammalian hosts.

Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor inTrypanosoma cruzi

2001 ◽  
Vol 114 (21) ◽  
pp. 3933-3942 ◽  
Author(s):  
Ana C. S. Monteiro ◽  
Magnus Abrahamson ◽  
Ana P. C. A. Lima ◽  
Marcos A. Vannier-Santos ◽  
Julio Scharfstein
Keyword(s):  
Cell Surface ◽  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Mammalian Cells ◽  
Tight Binding ◽  
Cysteine Proteases ◽  
Host Cells ◽  
Cysteine Protease Inhibitor ◽  
Reversible Inhibitor ◽  
Mammalian Host

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas’ heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.

scholarly journals Inhibition of Trypanosoma cruzi growth in mammalian cells by purine and pyrimidine analogs.

1996 ◽  
Vol 40 (11) ◽  
pp. 2455-2458 ◽  
Author(s):  
J Nakajima-Shimada ◽  
Y Hirota ◽  
T Aoki
Keyword(s):  
Trypanosoma Cruzi ◽  
Growth Inhibition ◽  
Host Cell ◽  
Culture System ◽  
Mammalian Cells ◽  
Screening Method ◽  
Host Cells ◽  
Mammalian Host ◽  
Dependent Manner ◽  
Pyrimidine Analogs

Trypanosoma cruzi, the causative agent of Chagas' disease, exhibits two different developmental stages in mammals, the amastigote, an intracellular form that proliferates in the cytoplasm of host cells, and the trypomastigote, an extracellular form that circulates in the bloodstream. We have already established an in vitro culture system using mammalian host cells (HeLa) infected with T. cruzi in which the time course of parasite growth is determined quantitatively. We adopted this system for the screening of anti-T. cruzi agents that would ideally prove to be effective against trypanosomes with no toxicity to the host cell. Of the purine analogs tested, allopurinol markedly inhibited the growth of amastigotes in a dose-dependent manner, with no lethal effect on trypomastigotes. 3'-Deoxyinosine and 3'-deoxyadenosine also suppressed T. cruzi growth inside the host cell, with the concentrations causing 50% growth inhibition being 10 and 5 microM, respectively, in contrast to a concentration causing 50% growth inhibition of 3 microM for allopurinol. Among the pyrimidine analogs examined, 3'-azido-3'-deoxythymidine (zidovudine) significantly reduced the growth of the parasite at concentrations as low as 1 microM. The anti-human immunodeficiency virus agents 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine caused a decrease in amastigote growth, while 2',3'-dideoxycytidine and 2',3'-dideoxyuridine had no inhibitory effect. When Swiss 3T3 fibroblasts were used as host cells, allopurinol, 3'-deoxyinosine, 3'-deoxyadenosine, and 3'-azid-3'-deoxythymidine also markedly inhibited T. cruzi proliferation. These results indicate that our culture system is useful as a primary screening method for candidate compounds against T. cruzi on the basis of two criteria, namely, intracellular replication by the parasite and host-cell infection rate.

scholarly journals Characterization of Sec-Translocon-Dependent Extracytoplasmic Proteins of Rickettsia typhi

2008 ◽  
Vol 190 (18) ◽  
pp. 6234-6242 ◽  
Author(s):  
Nicole C. Ammerman ◽  
M. Sayeedur Rahman ◽  
Abdu F. Azad
Keyword(s):  
Host Cell ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Mammalian Host ◽  
Signal Peptides ◽  
Cell Infection ◽  
Rickettsia Typhi ◽  
Sec Translocon

ABSTRACT As obligate intracellular, vector-borne bacteria, rickettsiae must adapt to both mammalian and arthropod host cell environments. Deciphering the molecular mechanisms of the interactions between rickettsiae and their host cells has largely been hindered by the genetic intractability of these organisms; however, research in other gram-negative pathogens has demonstrated that many bacterial determinants of attachment, entry, and pathogenesis are extracytoplasmic proteins. The annotations of several rickettsial genomes indicate the presence of homologs of the Sec translocon, the major route for bacterial protein secretion from the cytoplasm. For Rickettsia typhi, the etiologic agent of murine typhus, homologs of the Sec-translocon-associated proteins LepB, SecA, and LspA have been functionally characterized; therefore, the R. typhi Sec apparatus represents a mechanism for the secretion of rickettsial proteins, including virulence factors, into the extracytoplasmic environment. Our objective was to characterize such Sec-dependent R. typhi proteins in the context of a mammalian host cell infection. By using the web-based programs LipoP, SignalP, and Phobius, a total of 191 R. typhi proteins were predicted to contain signal peptides targeting them to the Sec translocon. Of these putative signal peptides, 102 were tested in an Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system. Eighty-four of these candidates exhibited signal peptide activity in E. coli, and transcriptional analysis indicated that at least 54 of the R. typhi extracytoplasmic proteins undergo active gene expression during infections of HeLa cells. This work highlights a number of interesting proteins possibly involved in rickettsial growth and virulence in mammalian cells.

scholarly journals Molecular, functional and structural properties of the prolyl oligopeptidase of Trypanosoma cruzi (POP Tc80), which is required for parasite entry into mammalian cells

2005 ◽  
Vol 388 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Izabela M. D. BASTOS ◽  
Philippe GRELLIER ◽  
Natalia F. MARTINS ◽  
Gloria CADAVID-RESTREPO ◽  
Marian R. de SOUZA-AULT ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Single Copy ◽  
Catalytic Triad ◽  
Host Cells ◽  
Mammalian Host ◽  
Kinetic Properties ◽  
Prolyl Oligopeptidase ◽  
Single Chromosome

We have demonstrated that the 80 kDa POP Tc80 (prolyl oligopeptidase of Trypanosoma cruzi) is involved in the process of cell invasion, since specific inhibitors block parasite entry into non-phagocytic mammalian host cells. In contrast with other POPs, POP Tc80 is capable of hydrolysing large substrates, such as fibronectin and native collagen. In this study, we present the cloning of the POPTc80 gene, whose deduced amino acid sequence shares considerable identity with other members of the POP family, mainly within its C-terminal portion that forms the catalytic domain. Southern-blot analysis indicated that POPTc80 is present as a single copy in the genome of the parasite. These results are consistent with mapping of POPTc80 to a single chromosome. The active recombinant protein (rPOP Tc80) displayed kinetic properties comparable with those of the native enzyme. Novel inhibitors were assayed with rPOP Tc80, and the most efficient ones presented values of inhibition coefficient Ki≤1.52 nM. Infective parasites treated with these specific POP Tc80 inhibitors attached to the surface of mammalian host cells, but were incapable of infecting them. Structural modelling of POP Tc80, based on the crystallized porcine POP, suggested that POP Tc80 is composed of an α/β-hydrolase domain containing the catalytic triad Ser548–Asp631–His667 and a seven-bladed β-propeller non-catalytic domain. Docking analysis suggests that triple-helical collagen access to the catalytic site of POP Tc80 occurs in the vicinity of the interface between the two domains.

Movement of spotted fever rickettsiae through tick host cells in vitro

1996 ◽  
Vol 54 ◽  
pp. 810-811
Author(s):  
U. G. Munderloh ◽  
S. F. Hayes ◽  
J. Cummings ◽  
T. J. Kurtti
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Spotted Fever ◽  
Shigella Flexneri ◽  
Light And Electron Microscopy ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Symbiotic Association ◽  
Obligate Intracellular

Spotted fever group (SFG) rickettsiae are obligate intracellular prokaryotes that include tick-borne pathogens of animals and man as well as organisms that live in symbiotic association with their tick hosts. A striking feature of the behavior of pathogenic rickettsiae in the vertebrate is their ability to quickly disseminate between cells from the original site of entry shortly after infection, and before severe lesions are detected. Similarly, ticks become systemically infected with SFG rickettsiae, indicating that an efficient mechanism of dispersal also exists in the vector. This is accomplished despite the fact that rickettsiae are not motile.Kadurugamuwa et al. (1991) have used light and electron microscopy to show that Shigella flexneri utilize host cytoskeletal components to travel through cytoplasmic extensions and penetrate into neighboring cells. Using mammalian cells cultured in vitro, Heinzen et al. (1993) have demonstrated that SFG rickettsiae cause host cell actin polymerization at one rickettsial pole causing them to be propelled through the cytoplasm, and to transfer rapidly from cell to cell.

scholarly journals Amastigotes of Trypanosoma cruzi sustain an infective cycle in mammalian cells.

1988 ◽  
Vol 168 (2) ◽  
pp. 649-659 ◽  
Author(s):  
V Ley ◽  
N W Andrews ◽  
E S Robbins ◽  
V Nussenzweig
Keyword(s):  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Protozoan Parasite ◽  
Acute Stage ◽  
Host Cells ◽  
Mammalian Host ◽  
Human Monocytes ◽  
Lower Efficiency ◽  
Cultured Fibroblasts

The two main stages of development of the protozoan parasite Trypanosoma cruzi found in the vertebrate host are the trypomastigote and the amastigote. It has been generally assumed that only trypomastigotes are capable of entering cells and that amastigotes are the intracellular replicative form of the parasite. We show here that after incubation for 4 h with human monocytes in vitro 90% or more of extracellularly derived (24 h) amastigotes of T. cruzi are taken up by the cells. Within 2 h they escape the phagocytic vacuole and enter the cytoplasm, where they divide and after 4-5 d transform into trypomastigotes. Trypomastigotes also invade cultured human monocytes. However, they show a lag of several hours between invasion and the start of DNA duplication, while amastigotes commence replication without an apparent lag. Amastigotes also infect cultured fibroblasts, albeit with lower efficiency. When injected intraperitoneally into mice, amastigotes are as infective as trypomastigotes. Based on these results, and on prior findings that amastigotes are found free in the circulation of mice during the acute stage of the disease (3), it seems likely that the cellular uptake of amastigotes can initiate an alternative subcycle within the life cycle of this parasite in the mammalian host. Also, because trypomastigotes and amastigotes have diverse surface antigens, they may use different strategies to invade host cells.

scholarly journals The Sca2 Autotransporter Protein from Rickettsia conorii Is Sufficient To Mediate Adherence to and Invasion of Cultured Mammalian Cells

2009 ◽  
Vol 77 (12) ◽  
pp. 5272-5280 ◽  
Author(s):  
Marissa M. Cardwell ◽  
Juan J. Martinez
Keyword(s):  
Mammalian Cells ◽  
Spotted Fever ◽  
Critical Role ◽  
Bioinformatic Analysis ◽  
Surface Proteins ◽  
Host Cells ◽  
Mammalian Host ◽  
Rickettsia Conorii ◽  
Spotted Fever Group ◽  
E Coli

ABSTRACT Obligate intracellular bacteria of the genus Rickettsia must adhere to and invade the host endothelium in order to establish an infection. These processes require the interaction of rickettsial surface proteins with mammalian host cell receptors. A previous bioinformatic analysis of sequenced rickettsial species identified a family of at least 17 predicted “surface cell antigen” (sca) genes whose products resemble autotransporter proteins. Two members of this family, rOmpA and rOmpB of spotted fever group (SFG) rickettsiae have been identified as adhesion and invasion factors, respectively; however, little is known about the putative functions of the other sca gene products. An intact sca2 gene is found in the majority of pathogenic SFG rickettsiae and, due to its sequence conservation among these species, we predict that Sca2 may play an important function at the rickettsial surface. Here we have shown that sca2 is transcribed and expressed in Rickettsia conorii and have used a heterologous gain-of-function assay in E. coli to determine the putative role of Sca2. Using this system, we have demonstrated that expression of Sca2 at the outer membrane of nonadherent, noninvasive E. coli is sufficient to mediate adherence to and invasion of a panel of mammalian cells, including endothelial cells. Furthermore, soluble Sca2 protein is capable of diminishing R. conorii invasion of cultured mammalian cells. This is the first evidence that Sca2 participates in the interaction between SFG rickettsiae and host cells and suggests that in addition to other surface proteins, Sca2 may play a critical role in rickettsial pathogenesis.

scholarly journals Trichomonas vaginalis extracellular vesicles are internalized by host cells using proteoglycans and caveolin-dependent endocytosis

2019 ◽  
Vol 116 (43) ◽  
pp. 21354-21360 ◽  
Author(s):  
Anand Kumar Rai ◽  
Patricia J. Johnson
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Extracellular Vesicles ◽  
Mammalian Cells ◽  
Trichomonas Vaginalis ◽  
Host Cells ◽  
Titration Calorimetry ◽  
Mammalian Host ◽  
Target Cells ◽  
Host Cell Surface

Trichomonas vaginalis, a human-infective parasite, causes the most prevalent nonviral sexually transmitted infection worldwide. This pathogen secretes extracellular vesicles (EVs) that mediate its interaction with host cells. Here, we have developed assays to study the interface between parasite EVs and mammalian host cells and to quantify EV internalization by mammalian cells. We show that T. vaginalis EVs interact with glycosaminoglycans on the surface of host cells and specifically bind to heparan sulfate (HS) present on host cell surface proteoglycans. Moreover, competition assays using HS or removal of HS from the host cell surface strongly inhibit EV uptake, directly demonstrating that HS proteoglycans facilitate EV internalization. We identified an abundant protein on the surface of T. vaginalis EVs, 4-α-glucanotransferase (Tv4AGT), and show using isothermal titration calorimetry that this protein binds HS. Tv4AGT also competitively inhibits EV uptake, defining it as an EV ligand critical for EV internalization. Finally, we demonstrate that T. vaginalis EV uptake is dependent on host cell cholesterol and caveolin-1 and that internalization proceeds via clathrin-independent, lipid raft-mediated endocytosis. These studies reveal mechanisms used to drive host:pathogen interactions and further our understanding of how EVs are internalized by target cells to allow cross-talk between different cell types.

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