A novel nucleotide incorporation activity implicated in the editing of mitochondrial transfer RNAs in Acanthamoeba castellanii.
(1/587)
In Acanthamoeba castellanii, most of the mtDNA-encoded tRNAs are edited by a process that replaces one or more of the first three nucleotides at their 5' ends. As a result, base pairing potential is restored at acceptor stem positions (1:72, 2:71, and/or 3:70, in standard tRNA nomenclature) that are mismatched according to the corresponding tRNA gene sequence. Here we describe a novel nucleotide incorporation activity, partially purified from A. castellanii mitochondria, that has properties implicating it in mitochondrial tRNA editing in this organism. This activity is able to replace nucleotides at the first three positions of a tRNA (positions 1, 2, and 3), matching the newly incorporated residues through canonical base pairing to the respective partner nucleotide in the 3' half of the acceptor stem. Labeling experiments with natural (Escherichia coli tRNATyr) and synthetic (run-off transcripts corresponding to A. castellanii mitochondrial tRNALeu1) substrates suggest that the nucleotide incorporation activity consists of at least two components, a 5' exonuclease or endonuclease and a template-directed 3'-to-5' nucleotidyltransferase. The nucleotidyltransferase component displays an ATP requirement and generates 5' pppN... termini in vitro. The development of an accurate and efficient in vitro system opens the way for detailed studies of the biochemical properties of this novel activity and its relationship to mitochondrial tRNA editing in A. castellanii. In addition, the system will allow delineation of the structural features in a tRNA that identify it as a substrate for the labeling activity. (+info)
Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex.
(2/587)
The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament-a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex. (+info)
In vivo tandem scanning confocal microscopy in acanthamoeba keratitis.
(3/587)
The in vivo confocal microscopy technique provides us with a real-time, non-invasive way of examining the human cornea. The most important advantage of this type of microscopy is to reveal the etiologic agents in infectious keratitis such as Acanthamoeba keratitis. We present several representative cases of Acanthamoeba keratitis, which were diagnosed in their early stages using in vivo confocal microscopy and managed based on that diagnosis. In our Acanthamoeba keratitis cases, highly-reflective round or ovoid organisms with a diameter of about 10-25 um were visualized distinctly against relatively-dark normal parenchymal structures, such as epithelial cells or keratocyte nuclei. Double-walled structures of Acanthamoeba cysts were clearly demonstrated in some cases. We can confirm that in vivo tandem scanning confocal microscopy is a powerful diagnostic tool for identifying the infecting organisms in Acanthamoeba keratitis. (+info)
Serum antibodies to Balamuthia mandrillaris, a free-living amoeba recently demonstrated to cause granulomatous amoebic encephalitis.
(4/587)
Free-living amoebae cause three well-defined disease entities: a rapidly fatal primary meningoencephalitis, a chronic granulomatous amoebic encephalitis (GAE), and a chronic amoebic keratitis. GAE occurs in immunocompromised persons. Recently, another type of free-living amoeba, Balamuthia mandrillaris, has been shown to cause GAE. The finding that this amoeba has caused infection in some healthy children has raised the possibility that humans may lack immunity to B. mandrillaris. Human serum was examined for the presence of surface antibodies specific for this amoeba by immunofluorescence. Sera from adults contained titers of 1/64-1/256 of anti-B. mandrillaris antibodies (IgM and IgG classes), which did not cross-react with other amoebae. Cord blood contained very low antibody levels, but levels similar to those in adults were seen in serum of 1- to 5-year-old children. (+info)
Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages.
(5/587)
In previous reports we described a 22-kb Legionella pneumophila chromosomal locus containing 18 genes. Thirteen of these genes (icmT, -R, -Q, -P, -O, -M, -L, -K, -E, -C, -D, -J, and -B) were found to be completely required for intracellular growth and killing of human macrophages. Three genes (icmS, -G, and -F) were found to be partially required, and two genes (lphA and tphA) were found to be dispensable for intracellular growth and killing of human macrophages. Here, we analyzed the requirement of these genes for intracellular growth in the protozoan host Acanthamoeba castellanii, a well-established important environmental host of L. pneumophila. We found that all the genes that are completely required for intracellular growth in human macrophages are also completely required for intracellular growth in A. castellanii. However, the genes that are partially required for intracellular growth in human macrophages are completely required for intracellular growth in A. castellanii. In addition, the lphA gene, which was shown to be dispensable for intracellular growth in human macrophages, is partially required for intracellular growth in A. castellanii. Our results indicate that L. pneumophila utilizes the same genes to grow intracellularly in both human macrophages and amoebae. (+info)
Rho-family GTPases require the Arp2/3 complex to stimulate actin polymerization in Acanthamoeba extracts.
(6/587)
BACKGROUND: Actin filaments polymerize in vivo primarily from their fast-growing barbed ends. In cells and extracts, GTPgammaS and Rho-family GTPases, including Cdc42, stimulate barbed-end actin polymerization; however, the mechanism responsible for the initiation of polymerization is unknown. There are three formal possibilities for how free barbed ends may be generated in response to cellular signals: uncapping of existing filaments; severing of existing filaments; or de novo nucleation. The Arp2/3 complex localizes to regions of dynamic actin polymerization, including the leading edges of motile cells and motile actin patches in yeast, and in vitro it nucleates the formation of actin filaments with free barbed ends. Here, we investigated actin polymerization in soluble extracts of Acanthamoeba. RESULTS: Addition of actin filaments with free barbed ends to Acanthamoeba extracts is sufficient to induce polymerization of endogenous actin. Addition of activated Cdc42 or activation of Rho-family GTPases in these extracts by the non-hydrolyzable GTP analog GTPgammaS stimulated barbed-end polymerization, whereas immunodepletion of Arp2 or sequestration of Arp2 using solution-binding antibodies blocked Rho-family GTPase-induced actin polymerization. CONCLUSIONS: For this system, we conclude that the accessibility of free barbed ends regulates actin polymerization, that Rho-family GTPases stimulate polymerization catalytically by de novo nucleation of free barbed ends and that the primary nucleation factor in this pathway is the Arp2/3 complex. (+info)
Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.
(7/587)
We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate. (+info)
Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease.
(8/587)
We report the identification of a set of Legionella pneumophila genes that encode products with homology to proteins of the type II general secretion pathway of gram-negative bacteria. A strain containing a deletion-substitution mutation of two of these genes was unable to secrete the Msp protease. This strain was unable to multiply within the free-living amoeba Acanthamoeba castellanii yet was able to kill HL-60-derived macrophages. Because Msp is not required for growth in amoebae, other proteins which are important for growth in amoebae are likely secreted by this pathway. (+info)