scholarly journals Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the Parasite Toxoplasma gondii

2006 ◽  
Vol 17 (2) ◽  
pp. 895-906 ◽  
Author(s):  
Nivedita Sahoo ◽  
Wandy Beatty ◽  
John Heuser ◽  
David Sept ◽  
L. David Sibley
Keyword(s):  
Critical Concentration ◽  
Microscopic Analysis ◽  
Protozoan Parasite ◽  
Gliding Motility ◽  
Host Cells ◽  
Filamentous Actin ◽  
Unusual Behavior ◽  
Electron Microscopic ◽  
Unusual Form ◽  
Kinetics Of

Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans.

scholarly journals The Bacteriophage Pf-10—A Component of the Biopesticide “Multiphage” Used to Control Agricultural Crop Diseases Caused by Pseudomonas syringae

Viruses ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 42
Author(s):  
Olesya A. Kazantseva ◽  
Rustam M. Buzikov ◽  
Tatsiana A. Pilipchuk ◽  
Leonid N. Valentovich ◽  
Andrey N. Kazantsev ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Gc Content ◽  
Microscopic Analysis ◽  
Plant Diseases ◽  
Host Cells ◽  
Agricultural Crop ◽  
Electron Microscopic ◽  
Bacterial Diseases ◽  
Dsdna Molecule ◽  
Wide Range

Phytopathogenic pseudomonads are widespread in the world and cause a wide range of plant diseases. In this work, we describe the Pseudomonas phage Pf-10, which is a part of the biopesticide “Multiphage” used for bacterial diseases of agricultural crops caused by Pseudomonas syringae. The Pf-10 chromosome is a dsDNA molecule with two direct terminal repeats (DTRs). The phage genomic DNA is 39,424 bp long with a GC-content of 56.5%. The Pf-10 phage uses a packaging mechanism based on T7-like short DTRs, and the length of each terminal repeat is 257 bp. Electron microscopic analysis has shown that phage Pf-10 has the podovirus morphotype. Phage Pf-10 is highly stable at pH values from 5 to 10 and temperatures from 4 to 60 °C and has a lytic activity against Pseudomonas strains. Phage Pf-10 is characterized by fast adsorption rate (80% of virions attach to the host cells in 10 min), but has a relatively small number of progeny (37 ± 8.5 phage particles per infected cell). According to the phylogenetic analysis, phage Pf-10 can be classified as a new phage species belonging to the genus Pifdecavirus, subfamily Studiervirinae, family Autographiviridae, order Caudovirales.

scholarly journals Purification and molecular characterization of the NAD+-dependent acetaldehyde/alcohol dehydrogenase from Entamoeba histolytica

1994 ◽  
Vol 303 (3) ◽  
pp. 743-748 ◽  
Author(s):  
I Bruchhaus ◽  
E Tannich
Keyword(s):  
Alcohol Dehydrogenase ◽  
Entamoeba Histolytica ◽  
Dna Sequences ◽  
Microscopic Analysis ◽  
Kinetic Studies ◽  
Protozoan Parasite ◽  
Striking Similarity ◽  
Electron Microscopic ◽  
Catalytic Functions

A bifunctional 95 kDa polypeptide (EhADH2) harbouring acetaldehyde dehydrogenase and alcohol dehydrogenase activities was purified to homogeneity from trophozoite extracts of the protozoan parasite Entamoeba histolytica. Kinetic studies revealed that the enzyme utilizes NAD+ rather than NADP+ as cofactor. Km values for acetyl-CoA, acetaldehyde and ethanol were found to be 0.015, 0.15 and 80 mM respectively in the presence of 0.2 mM NAD+. The primary structure of EhADH2 as deduced from respective amoebic DNA sequences showed striking similarity to the trifunctional AdhE protein of Escherichia coli and the bifunctional AAD protein of Clostridium acetobutylicum. Alignment with a number of aldehyde dehydrogenases and alcohol dehydrogenases from various species suggested that the two catalytic functions of EhADH2 are located on separate parts of the molecule. By cross-linking experiments and electron-microscopic analysis, native EhADH2 was found to be organized in a homopolymeric fashion consisting of more than 20 associated promoters which form rods about 50-120 nm in length.

Scanning electron-microscopic study of the uptake of Leishmania parasites by macrophages

1979 ◽  
Vol 39 (1) ◽  
pp. 187-199 ◽  
Author(s):  
A. Zenian ◽  
P. Rowles ◽  
D. Gingell
Keyword(s):  
Protozoan Parasite ◽  
Peritoneal Macrophages ◽  
The Body ◽  
Host Cells ◽  
Electron Microscopic ◽  
Scanning Electron Microscopic Study ◽  
Steady Rate ◽  
Mouse Peritoneal Macrophages ◽  
Scanning Electron

The interaction of promastigotes of the protozoan parasite Leishmania tropica with mouse peritoneal macrophages in vitro was studied by scanning electron microscopy. Motile promastigotes attached to host cells by their flagellar tips to which the macrophages responded by producing rather closely fitting lamellar sheaths and progressively enveloping first the flagellum and then the body of the parasite. Lamellar advance during engulfment was rapid in the first 10 min but much slower later on. Fully engulfed parasites could be seen after 1 h but most parasites associated with host cells remained extracellular even after 4 h. On the other hand, parasites immobilized by fixation adhered by either their flagellar or somatic ends. Engulfment proceeded at a steady rate, and by 4 h most of them were completely engulfed. Both the attachment and engulfment stages of parasite uptake were inhibited by low temperature, cytochalasin D and mild fixation of macrophages. The rheological features of the host cells' response to parasite adherence indicate that invasion by parasites is through phagocytosis rather than penetration.

scholarly journals Direct measurement of cortical force generation and polarization in a living parasite

2017 ◽  
Vol 28 (14) ◽  
pp. 1912-1923 ◽  
Author(s):  
Rachel V. Stadler ◽  
Lauren A. White ◽  
Ke Hu ◽  
Brian P. Helmke ◽  
William H. Guilford
Keyword(s):  
Toxoplasma Gondii ◽  
Life Cycles ◽  
Gliding Motility ◽  
Host Cells ◽  
Force Generation ◽  
Cytoplasmic Calcium ◽  
Laser Trap ◽  
Intracellular Parasites ◽  
Lysine Methyltransferase ◽  
Kinetics Of

Apicomplexa is a large phylum of intracellular parasites that are notable for the diseases they cause, including toxoplasmosis, malaria, and cryptosporidiosis. A conserved motile system is critical to their life cycles and drives directional gliding motility between cells, as well as invasion of and egress from host cells. However, our understanding of this system is limited by a lack of measurements of the forces driving parasite motion. We used a laser trap to measure the function of the motility apparatus of living Toxoplasma gondii by adhering a microsphere to the surface of an immobilized parasite. Motion of the microsphere reflected underlying forces exerted by the motile apparatus. We found that force generated at the parasite surface begins with no preferential directionality but becomes directed toward the rear of the cell after a period of time. The transition from nondirectional to directional force generation occurs on spatial intervals consistent with the lateral periodicity of structures associated with the membrane pellicle and is influenced by the kinetics of actin filament polymerization and cytoplasmic calcium. A lysine methyltransferase regulates both the magnitude and polarization of the force. Our work provides a novel means to dissect the motile mechanisms of these pathogens.

scholarly journals Isolation and characterization of a new type of chlorovirus that infects an endosymbiotic Chlorella strain of the heliozoon Acanthocystis turfacea

2005 ◽  
Vol 86 (10) ◽  
pp. 2871-2877 ◽  
Author(s):  
Julia A. Bubeck ◽  
Artur J. P. Pfitzner
Keyword(s):  
Genome Structure ◽  
Microscopic Analysis ◽  
Host Cells ◽  
Natural Environments ◽  
Electron Microscopic ◽  
Chlorella Virus ◽  
Isolation And Characterization ◽  
The Family ◽  
New Type

A novel virus, named Acanthocystis turfacea Chlorella virus (ATCV), that infects endosymbiotic Chlorella algae of the heliozoon Acanthocystis turfacea was isolated from freshwater samples. Electron microscopic analysis of ATCV revealed that the viral capsid has a distinct icosahedral shape with a diameter of 140–190 nm. Filamentous structures extending from some of the virus vertices, which may aid attachment of the virus to host cells, were also observed. The capsid is made up of one major coat protein of about 50 kDa and contains a large dsDNA genome. ATCV is a member of the genus Chlorovirus, which belongs to the family Phycodnaviridae, a group of large, icosahedral, dsDNA-containing viruses that infect algae and are ubiquitous in natural environments. However, ATCV is clearly distinct from the prototype Chlorovirus, Paramecium bursaria Chlorella virus (PBCV-1), in some aspects of its genome structure and gene content and therefore must be regarded as a member of a new group of Chlorella viruses.

scholarly journals Viroplasm matrix protein Pns9 from rice gall dwarf virus forms an octameric cylindrical structure

2011 ◽  
Vol 92 (9) ◽  
pp. 2214-2221 ◽  
Author(s):  
Fusamichi Akita ◽  
Naoyuki Miyazaki ◽  
Hiroyuki Hibino ◽  
Takumi Shimizu ◽  
Akifumi Higashiura ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Matrix Protein ◽  
Three Dimensional ◽  
Microscopic Analysis ◽  
Host Cells ◽  
Size Exclusion ◽  
Dwarf Virus ◽  
Electron Microscopic ◽  
The Family ◽  
Rice Gall Dwarf Virus

The non-structural Pns9 protein of rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. Immunofluorescence and immunoelectron microscopy of RGDV-infected vector cells in monolayers, using antibodies against Pns9 of RGDV and expression of Pns9 in Spodoptera frugiperda cells, demonstrated that Pns9 is the minimal viral factor necessary for formation of viroplasm inclusion during infection by RGDV. When Pns9 in solution was observed under a conventional electron microscope, it appeared as ring-like aggregates of approximately 100 Å in diameter. Cryo-electron microscopic analysis of these aggregates revealed cylinders of octameric Pns9, whose dimensions were similar to those observed under the conventional electron microscope. Octamerization of Pns9 in solution was confirmed by the results of size-exclusion chromatography. Among proteins of viruses that belong to the family Reoviridae whose three-dimensional structures are available, a matrix protein of the viroplasm of rotavirus, NSP2, forms similar octamers, an observation that suggests similar roles for Pns9 and NSP2 in morphogenesis in animal-infecting and in plant-infecting reoviruses.

scholarly journals Serine Protease Inhibitors Block Invasion of Host Cells by Toxoplasma gondii

1999 ◽  
Vol 43 (6) ◽  
pp. 1358-1361 ◽  
Author(s):  
V. Conseil ◽  
M. Soête ◽  
J. F. Dubremetz
Keyword(s):  
Toxoplasma Gondii ◽  
Serine Protease ◽  
Protease Inhibitors ◽  
Early Stage ◽  
Protozoan Parasite ◽  
Gliding Motility ◽  
Host Cells ◽  
Dependent Manner ◽  
Serine Protease Inhibitors ◽  
Experimental Conditions

ABSTRACT We investigated the effect of protease inhibitors on the asexual development of the protozoan parasite Toxoplasma gondii. Among the inhibitors tested only two irreversible serine protease inhibitors, 3,4-dichloroisocoumarin and 4-(2-aminoethyl)-benzenesulfonyl fluoride, clearly prevented invasion of the host cells by specifically affecting parasite targets in a dose-dependent manner, with 50% inhibitory concentrations between 1 and 5 and 50 and 100 μM, respectively. Neither compound significantly affected parasite morphology, basic metabolism, or gliding motility within the range of the experimental conditions in which inhibition of invasion was demonstrated. No partial invasion was observed, meaning that inhibition occurred at an early stage of the interaction. These results suggest that at least one serine protease of the parasite is involved in the invasive process of T. gondii.

scholarly journals Calcium-dependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii

2014 ◽  
Vol 25 (17) ◽  
pp. 2579-2591 ◽  
Author(s):  
Qing Tang ◽  
Nicole Andenmatten ◽  
Miryam A. Hortua Triana ◽  
Bin Deng ◽  
Markus Meissner ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Calcium Ionophore ◽  
Protozoan Parasite ◽  
Gliding Motility ◽  
Lytic Cycle ◽  
Host Cells ◽  
Response To Treatment ◽  
Apicomplexan Parasites ◽  
Calcium Dependent

Class XIVa myosins comprise a unique group of myosin motor proteins found in apicomplexan parasites, including those that cause malaria and toxoplasmosis. The founding member of the class XIVa family, Toxoplasma gondii myosin A (TgMyoA), is a monomeric unconventional myosin that functions at the parasite periphery to control gliding motility, host cell invasion, and host cell egress. How the motor activity of TgMyoA is regulated during these critical steps in the parasite's lytic cycle is unknown. We show here that a small-molecule enhancer of T. gondii motility and invasion (compound 130038) causes an increase in parasite intracellular calcium levels, leading to a calcium-dependent increase in TgMyoA phosphorylation. Mutation of the major sites of phosphorylation altered parasite motile behavior upon compound 130038 treatment, and parasites expressing a nonphosphorylatable mutant myosin egressed from host cells more slowly in response to treatment with calcium ionophore. These data demonstrate that TgMyoA undergoes calcium-dependent phosphorylation, which modulates myosin-driven processes in this important human pathogen.

scholarly journals Mediation of Cryptosporidium parvumInfection In Vitro by Mucin-Like Glycoproteins Defined by a Neutralizing Monoclonal Antibody

2000 ◽  
Vol 68 (9) ◽  
pp. 5167-5175 ◽  
Author(s):  
Ana María Cevallos ◽  
Najma Bhat ◽  
Renaud Verdon ◽  
Davidson H. Hamer ◽  
Barry Stein ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Diarrheal Disease ◽  
Intestinal Epithelial Cells ◽  
Protozoan Parasite ◽  
Gliding Motility ◽  
Host Cells ◽  
Apical Region ◽  
Intestinal Epithelial ◽  
Neutralizing Monoclonal Antibody

ABSTRACT The protozoan parasite Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. Attachment to and invasion of host intestinal epithelial cells by C. parvumsporozoites are crucial steps in the pathogenesis of cryptosporidiosis. The molecular basis of these initial interactions is unknown. In order to identify putative C. parvum adhesion- and invasion-specific proteins, we raised monoclonal antibodies (MAbs) to sporozoites and evaluated them for inhibition of attachment and invasion in vitro. Using this approach, we identified two glycoproteins recognized by 4E9, a MAb which neutralized C. parvuminfection and inhibited sporozoite attachment to intestinal epithelial cells in vitro. 4E9 recognized a 40-kDa glycoprotein named gp40 and a second, >220-kDa protein which was identified as GP900, a previously described mucin-like glycoprotein. Glycoproteins recognized by 4E9 are localized to the surface and apical region of invasive stages and are shed in trails from the parasite during gliding motility. The epitope recognized by 4E9 contains α-N-acetylgalactosamine residues, which are present in a mucin-type O-glycosidic linkage. Lectins specific for these glycans bind to the surface and apical region of sporozoites and block attachment to host cells. The surface and apical localization of these glycoproteins and the neutralizing effect of the MAb and α-N-acetylgalactosamine-specific lectins strongly implicate these proteins and their glycotopes as playing a role in C. parvum-host cell interactions.

Preparatory Procedures for Electron Microscopic Analysis at the Molecular Level of Resolution

1978 ◽  
Vol 36 (3) ◽  
pp. 516-520
Author(s):  
F.J. Sjostrand
Keyword(s):  
Electron Microscope ◽  
Molecular Level ◽  
Microscopic Analysis ◽  
Resolving Power ◽  
Cellular Structures ◽  
Electron Microscopic ◽  
Systematic Analysis ◽  
Technical Developments ◽  
Thin Sectioning ◽  
Obvious Reason

In the 1940's and 1950's electron microscopy conferences were attended with everybody interested in learning about the latest technical developments for one very obvious reason. There was the electron microscope with its outstanding performance but nobody could make very much use of it because we were lacking proper techniques to prepare biological specimens. The development of the thin sectioning technique with its perfectioning in 1952 changed the situation and systematic analysis of the structure of cells could now be pursued. Since then electron microscopists have in general become satisfied with the level of resolution at which cellular structures can be analyzed when applying this technique. There has been little interest in trying to push the limit of resolution closer to that determined by the resolving power of the electron microscope.

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