scholarly journals Coiling Phagocytosis of Trypanosomatids and Fungal Cells

1998 ◽  
Vol 66 (9) ◽  
pp. 4331-4339 ◽  
Author(s):  
M. G. Rittig ◽  
K. Schröppel ◽  
K.-H. Seack ◽  
U. Sander ◽  
E.-N. N’Diaye ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Legionella Pneumophila ◽  
Leishmania Major ◽  
Posterior Pole ◽  
Host Cells ◽  
Human Monocytes ◽  
Eukaryotic Microorganisms ◽  
Microscopic Studies

ABSTRACT Coiling phagocytosis has previously been studied only with the bacteria Legionella pneumophila and Borrelia burgdorferi, and the results were inconsistent. To learn more about this unconventional phagocytic mechanism, the uptake of various eukaryotic microorganisms by human monocytes, murine macrophages, and murine dendritic cells was investigated in vitro by video and electron microscopy. Unconventional phagocytosis of Leishmania spp. promastigotes, Trypanosoma cruzi trypomastigotes,Candida albicans hyphae, and zymosan particles fromSaccharomyces cerevisiae differed in (i) morphology (rotating unilateral pseudopods with the trypanosomatids, overlapping bilateral pseudopods with the fungi), (ii) frequency (high withLeishmania; occasional with the fungi; rare with T. cruzi), (iii) duration (rapid with zymosan; moderate with the trypanosomatids; slow with C. albicans), (iv) localization along the promastigotes (flagellum of Leishmania major andL. aethiopica; flagellum or posterior pole of L. donovani), and (v) dependence on complement (strong with L. major and L. donovani; moderate with the fungi; none with L. aethiopica). All of these various types of unconventional phagocytosis gave rise to similar pseudopod stacks which eventually transformed to a regular phagosome. Further video microscopic studies with L. major provided evidence for a cytosolic localization, synchronized replication, and exocytic release of the parasites, extending traditional concepts about leishmanial infection of host cells. It is concluded that coiling phagocytosis comprises phenotypically similar consequences of various disturbances in conventional phagocytosis rather than representing a single separate mechanism.

scholarly journals Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector of Legionella pneumophila

2010 ◽  
Vol 207 (8) ◽  
pp. 1713-1726 ◽  
Author(s):  
Christopher T.D. Price ◽  
Tasneem Al-Quadan ◽  
Marina Santic ◽  
Snake C. Jones ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Biological Function ◽  
Host Cells ◽  
Dependent Manner ◽  
Protein Farnesyltransferase ◽  
Type Iv ◽  
Eukaryotic Proteins ◽  
Bacterial Effectors

Farnesylation involves covalent linkage of eukaryotic proteins to a lipid moiety to anchor them into membranes, which is essential for the biological function of Ras and other proteins. A large cadre of bacterial effectors is injected into host cells by intravacuolar pathogens through elaborate type III–VII translocation machineries, and many of these effectors are incorporated into the pathogen-containing vacuolar membrane by unknown mechanisms. The Dot/Icm type IV secretion system of Legionella pneumophila injects into host cells the F-box effector Ankyrin B (AnkB), which functions as platforms for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) to enable intravacuolar proliferation in macrophages and amoeba. We show that farnesylation of AnkB is indispensable for its anchoring to the cytosolic face of the LCV membrane, for its biological function within macrophages and Dictyostelium discoideum, and for intrapulmonary proliferation in mice. Remarkably, the protein farnesyltransferase, RCE-1 (Ras-converting enzyme-1), and isoprenyl cysteine carboxyl methyltransferase host farnesylation enzymes are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB. In conclusion, this study shows novel localized recruitment of the host farnesylation machinery and its anchoring of an F-box effector to the LCV membrane, and this is essential for biological function in vitro and in vivo.

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

scholarly journals Antibacterial Effects of Levofloxacin, Erythromycin, and Rifampin in a Human Monocyte System againstLegionella pneumophila

1998 ◽  
Vol 42 (12) ◽  
pp. 3153-3156 ◽  
Author(s):  
Aldona L. Baltch ◽  
Raymond P. Smith ◽  
Mary A. Franke ◽  
Phyllis B. Michelsen
Keyword(s):  
Antibacterial Activity ◽  
Legionella Pneumophila ◽  
Human Monocyte ◽  
Antibacterial Activities ◽  
Human Monocytes ◽  
Antibacterial Effects ◽  
In Vitro System ◽  
Time Period ◽  
Time Periods

ABSTRACT The antibacterial activities of levofloxacin, erythromycin, and rifampin against intracellular Legionella pneumophilaL-1033, serogroup 1, were studied. In an in vitro system utilizing adherent human monocytes, L. pneumophila L-1033, a phagocytosis time period of 1 h, and antibiotic (levofloxacin, erythromycin, and/or rifampin) at 1 to 10 times the MIC, the CFU/ml values for the monocyte lysate were determined during 0- to 4-day time periods. The decrease in CFU/ml with levofloxacin at pH 7.4 was rapid, occurring within 24 h, and was drug concentration dependent (P < 0.01). The decrease in CFU with rifampin was first observed at 48 h (P < 0.01), while only a minimal decrease in CFU/ml was observed with erythromycin. Combination of levofloxacin and rifampin and of levofloxacin and erythromycin at ten times their MICs significantly decreased the CFU/ml value (P < 0.01), to the value attained by levofloxacin alone, while combination of rifampin and erythromycin did not. Removal of levofloxacin after 24 h of incubation resulted in regrowth ofL. pneumophila L-1033, while a continued slow decrease in CFU/ml was seen following rifampin removal; CFU/ml values were unaffected by the removal of erythromycin. At 4 days, and even in assays performed following antibiotic removal, the CFU/ml value continued to be lower in the levofloxacin and rifampin assays than in the assays with erythromycin. Levofloxacin had a significantly higher bactericidal activity against L. pneumophila L-1033 than erythromycin or rifampin. In these assays, the addition of erythromycin or rifampin did not affect the antibacterial activity of levofloxacin.

scholarly journals Flagellum of Legionella pneumophilaPositively Affects the Early Phase of Infection of Eukaryotic Host Cells

2001 ◽  
Vol 69 (4) ◽  
pp. 2116-2122 ◽  
Author(s):  
Claudia Dietrich ◽  
Klaus Heuner ◽  
Bettina C. Brand ◽  
Jörg Hacker ◽  
Michael Steinert
Keyword(s):  
Electron Microscopy ◽  
Legionella Pneumophila ◽  
Kanamycin Resistance ◽  
Etiologic Agent ◽  
Polyclonal Antiserum ◽  
Host Cells ◽  
Kanamycin Resistance Cassette ◽  
Legionnaires Disease ◽  
Invasion Capacity

ABSTRACT Legionella pneumophila, the etiologic agent of Legionnaires' disease, contains a single, monopolar flagellum which is composed of one major subunit, the FlaA protein. To evaluate the role of the flagellum in the pathogenesis and ecology ofLegionella, the flaA gene of L. pneumophila Corby was mutagenized by introduction of a kanamycin resistance cassette. Immunoblots with antiflagellin-specific polyclonal antiserum, electron microscopy, and motility assays confirmed that the specific flagellar mutant L. pneumophila Corby KH3 was nonflagellated. The redelivery of the intact flaA gene into the chromosome (L. pneumophila Corby CD10) completely restored flagellation and motility. Coculture studies showed that the invasion efficiency of the flaA mutant was moderately reduced in amoebae and severely reduced in HL-60 cells. In contrast, adhesion and the intracellular rate of replication remained unaffected. Taking these results together, we have demonstrated that the flagellum of L. pneumophila positively affects the establishment of infection by facilitating the encounter of the host cell as well as by enhancing the invasion capacity.

scholarly journals Fibroblasts as Host Cells in Latent Leishmaniosis

2000 ◽  
Vol 191 (12) ◽  
pp. 2121-2130 ◽  
Author(s):  
Christian Bogdan ◽  
Norbert Donhauser ◽  
Reinhard Döring ◽  
Martin Röllinghoff ◽  
Andreas Diefenbach ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Leishmania Major ◽  
Chronic Phase ◽  
Host Cells ◽  
Confocal Laser ◽  
Intracellular Parasites ◽  
Important Host ◽  
Mechanisms Of Persistence ◽  
Activated Fibroblasts

Intracellular parasites are known to persist lifelong in mammalian hosts after the clinical cure of the disease, but the mechanisms of persistence are poorly understood. Here, we show by confocal laser microscopy that in the draining lymph nodes of mice that had healed a cutaneous infection with Leishmania major, 40% of the persisting parasites were associated with fibroblasts forming the reticular meshwork of the lymph nodes. In vitro, both promastigotes and amastigotes of L. major infected primary skin or lymph node fibroblasts. Compared with macrophages, cytokine-activated fibroblasts had a reduced ability to express type 2 nitric oxide synthase and to kill intracellular L. major. These data identify fibroblasts as an important host cell for Leishmania during the chronic phase of infection and suggest that they might serve as safe targets for the parasites in clinically latent disease.

scholarly journals Prion Filament Networks in [Ure3] Cells of Saccharomyces cerevisiae

2001 ◽  
Vol 153 (6) ◽  
pp. 1327-1336 ◽  
Author(s):  
Vladislav V. Speransky ◽  
Kimberly L. Taylor ◽  
Herman K. Edskes ◽  
Reed B. Wickner ◽  
Alasdair C. Steven
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Normal Function ◽  
Nitrogen Regulation ◽  
Wild Type ◽  
Cell Extracts ◽  
Cytoplasmic Filaments ◽  
Altered Form ◽  
Filament Networks

The [URE3] prion (infectious protein) of yeast is a self-propagating, altered form of Ure2p that cannot carry out its normal function in nitrogen regulation. Previous data have shown that Ure2p can form protease-resistant amyloid filaments in vitro, and that it is aggregated in cells carrying the [URE3] prion. Here we show by electron microscopy that [URE3] cells overexpressing Ure2p contain distinctive, filamentous networks in their cytoplasm, and demonstrate by immunolabeling that these networks contain Ure2p. In contrast, overexpressing wild-type cells show a variety of Ure2p distributions: usually, the protein is dispersed sparsely throughout the cytoplasm, although occasionally it is found in multiple small, focal aggregates. However, these distributions do not resemble the single, large networks seen in [URE3] cells, nor do the control cells exhibit cytoplasmic filaments. In [URE3] cell extracts, Ure2p is present in aggregates that are only partially solubilized by boiling in SDS and urea. In these aggregates, the NH2-terminal prion domain is inaccessible to antibodies, whereas the COOH-terminal nitrogen regulation domain is accessible. This finding is consistent with the proposal that the prion domains stack to form the filament backbone, which is surrounded by the COOH-terminal domains. These observations support and further specify the concept of the [URE3] prion as a self-propagating amyloid.

scholarly journals Aziridine-2,3-Dicarboxylate-Based Cysteine Cathepsin Inhibitors Induce Cell Death in Leishmania major Associated with Accumulation of Debris in Autophagy-Related Lysosome-Like Vacuoles

2010 ◽  
Vol 54 (12) ◽  
pp. 5028-5041 ◽  
Author(s):  
Uta Schurigt ◽  
Caroline Schad ◽  
Christin Glowa ◽  
Ulrike Baum ◽  
Katja Thomale ◽  
...  
Keyword(s):  
Cell Death ◽  
Leishmania Major ◽  
Cysteine Proteinase ◽  
Membrane Integrity ◽  
New Drugs ◽  
Host Cells ◽  
Electron Microscopic ◽  
Cysteine Cathepsins ◽  
Cysteine Cathepsin ◽  
Microscopic Studies

ABSTRACT The papain-like cysteine cathepsins expressed by Leishmania play a key role in the life cycle of these parasites, turning them into attractive targets for the development of new drugs. We previously demonstrated that two compounds of a series of peptidomimetic aziridine-2,3-dicarboxylate [Azi(OBn)2]-based inhibitors, Boc-(S)-Leu-(R)-Pro-(S,S)-Azi(OBn)2 (compound 13b) and Boc-(R)-Leu-(S)-Pro-(S,S)-Azi(OBn)2 (compound 13e), reduced the growth and viability of Leishmania major and the infection rate of macrophages while not showing cytotoxicity against host cells. In the present study, we characterized the mode of action of inhibitors 13b and 13e in L. major. Both compounds targeted leishmanial cathepsin B-like cysteine cathepsin cysteine proteinase C, as shown by fluorescence proteinase activity assays and active-site labeling with biotin-tagged inhibitors. Furthermore, compounds 13b and 13e were potent inducers of cell death in promastigotes, characterized by cell shrinkage, reduction of mitochondrial transmembrane potential, and increased DNA fragmentation. Transmission electron microscopic studies revealed the enrichment of undigested debris in lysosome-like organelles participating in micro- and macroautophagy-like processes. The release of digestive enzymes into the cytoplasm after rupture of membranes of lysosome-like vacuoles resulted in the significant digestion of intracellular compartments. However, the plasma membrane integrity of compound-treated promastigotes was maintained for several hours. Taken together, our results suggest that the induction of cell death in Leishmania by cysteine cathepsin inhibitors 13b and 13e is different from mammalian apoptosis and is caused by incomplete digestion in autophagy-related lysosome-like vacuoles.

scholarly journals Intracellular Survival of Leishmania major in Neutrophil Granulocytes after Uptake in the Absence of Heat-Labile Serum Factors

2002 ◽  
Vol 70 (2) ◽  
pp. 826-835 ◽  
Author(s):  
Helmut Laufs ◽  
Kerstin Müller ◽  
Jens Fleischer ◽  
Norbert Reiling ◽  
Nicole Jahnke ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Intracellular Survival ◽  
Host Cells ◽  
Polymorphonuclear Neutrophil ◽  
Heat Inactivation ◽  
Neutrophil Granulocytes ◽  
Complement Components ◽  
Serum Factors

ABSTRACT The role of polymorphonuclear neutrophil granulocytes (PMN) in defense against the intracellular parasite Leishmania is poorly understood. In the present study, the interaction of human PMN with Leishmania major promastigotes was investigated in vitro. In the presence of fresh human serum, about 50% of PMN phagocytosed the parasites within 10 min and the parasite uptake led to PMN activation, resulting in the killing of most ingested parasites. Heat inactivation of the serum markedly reduced the rate of early parasite phagocytosis, suggesting a role of complement components in the early uptake of Leishmania. However, over 50% of PMN were able to ingest parasites in the presence of heat-inactivated serum if the coincubation was extended to 3 h. After 3 h, 10% of the PMN were found to internalize Leishmania even under serum-free conditions. These findings indicate that PMN possess mechanisms for both opsonin/complement-dependent and -independent uptake of Leishmania. Both pathways of uptake could be partially blocked by anti-CR3 antibody. Mannan-binding lectin was found not to be involved in this process. When phagocytosed in the absence of opsonin, the majority of Leishmania parasites survived intracellularly in PMN for at least 1 day. These data suggest a dual role of PMN in the early response to L. major infection. On the one hand, PMN can rapidly eliminate the intracellular parasites, and on the other hand, Leishmania can survive intracellularly in PMN. These data, together with the finding that intact parasites were seen in PMN isolated from the skin of infected mice, suggest that PMN can serve as host cells for the intracellular survival of Leishmania within the first hours or days after infection.

Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system

1985 ◽  
Vol 5 (9) ◽  
pp. 2361-2368
Author(s):  
L S Symington ◽  
P Morrison ◽  
R Kolodner
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Electrophoretic Mobility ◽  
Electrophoretic Analysis ◽  
Individual Band ◽  
Agarose Gels ◽  
Cell Extracts ◽  
Recombination System ◽  
Plasmid Recombination

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.

scholarly journals Development of a New Antileishmanial Aziridine-2,3-Dicarboxylate-Based Inhibitor with High Selectivity for Parasite Cysteine Proteases

2015 ◽  
Vol 60 (2) ◽  
pp. 797-805 ◽  
Author(s):  
Caroline Schad ◽  
Ulrike Baum ◽  
Benjamin Frank ◽  
Uwe Dietzel ◽  
Felix Mattern ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Neglected Tropical Diseases ◽  
Cathepsin L ◽  
Selective Inhibition ◽  
Cysteine Proteases ◽  
Host Cells ◽  
Th2 Response ◽  
Content Type

ABSTRACTLeishmaniasis is one of the major neglected tropical diseases of the world. Druggable targets are the parasite cysteine proteases (CPs) of clan CA, family C1 (CAC1). In previous studies, we identified two peptidomimetic compounds, the aziridine-2,3-dicarboxylate compounds 13b and 13e, in a series of inhibitors of the cathepsin L (CL) subfamily of the papain clan CAC1. Both displayed antileishmanial activityin vitrowhile not showing cytotoxicity against host cells. In further investigations, the mode of action was characterized inLeishmania major. It was demonstrated that aziridines 13b and 13e mainly inhibited the parasitic cathepsin B (CB)-like CPC enzyme and, additionally, mammalian CL. Although these compounds induced cell death ofLeishmaniapromastigotes and amastigotesin vitro, the induction of a proleishmanial T helper type 2 (Th2) response caused by host CL inhibition was observedin vivo. Therefore, we describe here the synthesis of a new library of more selective peptidomimetic aziridine-2,3-dicarboxylates discriminating between host and parasite CPs. The new compounds are based on 13b and 13e as lead structures. One of the most promising compounds of this series is compound s9, showing selective inhibition of the parasite CPsLmaCatB (a CB-like enzyme ofL. major; also namedL. majorCPC) andLmCPB2.8 (a CL-like enzyme ofLeishmania mexicana) while not affecting mammalian CL and CB. It displayed excellent leishmanicidal activities againstL. majorpromastigotes (50% inhibitory concentration [IC50] = 37.4 μM) and amastigotes (IC50= 2.3 μM). In summary, we demonstrate a new selective aziridine-2,3-dicarboxylate, compound s9, which might be a good candidate for futurein vivostudies.

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