Search

The cyst wall of the eye pathogen Acanthamoeba castellanii contains cellulose and has ectocyst and endocyst layers connected by conical ostioles. Cyst walls contain families of lectins that localize to the ectocyst layer (Jonah) or the endocyst layer and ostioles (Luke and Leo). How lectins and an abundant laccase bind cellulose and why proteins go to locations in the wall are not known and are the focus of the studies here. Structural predictions identified β-jelly-roll folds (BJRFs) of Luke and sets of four disulfide knots (4DKs) of Leo, each of which contains linear arrays of aromatic amino acids, also present in carbohydrate-binding modules of bacterial and plant endocellulases. Ala mutations showed that these aromatics are necessary for cellulose binding and proper localization of Luke and Leo in the Acanthamoeba cyst wall. BJRFs of Luke, 4DKs of Leo, a single β-helical fold (BHF) of Jonah, and a copper oxidase domain of the laccase each bind to glycopolymers in both layers of deproteinated cyst walls. Promoter swaps showed that ectocyst localization does not just correlate with but is caused by early encystation-specific expression, while localization in the endocyst layer and ostioles is caused by later expression. Evolutionary studies showed distinct modes of assembly of duplicated domains in Luke, Leo, and Jonah lectins and suggested Jonah BHFs originated from bacteria, Luke BJRFs share common ancestry with slime molds, while 4DKs of Leo are unique to Acanthamoeba .IMPORTANCE Acanthamoebae is the only human parasite with cellulose in its cyst wall and conical ostioles that connect its inner and outer layers. Cyst walls are important virulence factors because they make Acanthamoebae resistant to surface disinfectants, hand sanitizers, contact lens sterilizers, and antibiotics applied to the eye. The goal here was to understand better how proteins are targeted to specific locations in the cyst wall. To this end, we identified three new proteins in the outer layer of the cyst wall, which may be targets for diagnostic antibodies in corneal scrapings. We used structural predictions and mutated proteins to show linear arrays of aromatic amino acids of two unrelated wall proteins are necessary for binding cellulose and proper wall localization. We showed early expression during encystation causes proteins to localize to the outer layer, while later expression causes proteins to localize to the inner layer and the ostioles., Competing Interests: The authors declare no conflict of interest.

Acanthamoeba is a free-living pathogenic protozoan that is distributed in different environmental reservoirs, including lakes and soil. Pathogenic Acanthamoeba can cause severe human diseases, such as blinding keratitis and granulomatous encephalitis. Therefore, it is important to understand the pathogenic relationship between humans and Acanthamoeba. By comparison of systemic analysis results for Acanthamoeba isolates, we identified a novel secreted protein of Acanthamoeba, an M28 aminopeptidase (M28AP), which targets of the human innate immune defense. We investigated the molecular functions and characteristics of the M28AP protein by anti-M28 antibodies and a M28AP mutant strain generated by the CRISPR/Cas9 system. Human complement proteins such as C3b and iC3b were degraded by Acanthamoeba M28AP. We believe that M28AP is an important factor in human innate immunity. This study provides new insight for the development of more efficient medicines to treat Acanthamoeba infection.

Mimivirus' genome includes parts of 5S, 16S and 23S ribosomal RNAs encoded by Acanthamoeba's mitogenome, the giant virus' host. Two non-exclusive hypotheses for rRNA remnants in giant viruses are examined: 1. mitogenomes invade viral genomes as they do for nuclear chromosomes (producing numts); 2. megaviral genomes evolved from an ancestral mitogenome. Alignment analyses confirm mitochondrial, rather than alphaproteobacterial origins of megaviral rRNAs. Other mitogenes have likely megaviral homologues. These megaviral homologues coevolve to much larger extents than candidate rRNA homologues, suggesting rRNA decay in viruses. Megaviral mitogene homologues overall follow mitochondrial gene order, suggesting mitogenome ancestry. Ancestral synteny decreases with megaviral genome size, suggesting that subsequent mitogene insertions blur ancestral gene order. Putative defenses against DNA invasion conserve mitogene order in short megaviral genomes. Synteny between mitogenome and megaviral genomes confirms the RNA/DNA polymerase-homologies-based hypothesis that giant viruses have mitochondrial-like ancestors, viral rRNA remnants are corollary of mitogenomic origins of megaviral genomes. Note that giant viruses, mitochondria and bacterial spores all have double membranes, spores and viruses have protein coats. Mitochondria might occasionally form spore-like structures that drifted into megaviruses. These missing links could confirm mitogenome ancestry of giant viruses rather than giant virus ancestry of mitochondria., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This paper presents unequivocal results about the presence of trypanothione and its precursor glutathionespermidine from the opportunistic human pathogen Acanthamoeba polyphaga. They were isolated by RP-HPLC as thiolbimane derivatives and characterized using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF). Additionally RP-HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were also present in A. polyphaga. Besides trypanothione, we want to report four new peptides in trophozoites, a tetrapeptide, a hexapeptide, a heptapeptide and a nonapeptide. Trypanothione and two of the thiol peptides, the hexapeptide and heptapeptide, are oxidized since the reduced forms increase in amount when the normal extract is treated by DTT or by electrolytic reduction that convert the oxidized forms to reduced ones. On the other hand, they disappear when the amoeba extract is treated with NEM or when the amoeba culture is treated with various inhibitors of NADPH-dependent disulfidereducing enzymes. Comparison of the thiol peptides, including trypanothione from A. polyphaga with extracts from human lymphocytes showed that they are not present in the latter. Therefore, some of the peptides here reported could be used as antigens for rapid detection of these parasites. In regard to the presence of the enzymes that synthesize and reduce trypanothione in A. polyphaga we suggest that they can be used as drug targets.

Parachlamydia acanthamoebae is an obligate intracellular bacterium that infects free-living amoebae (Acanthamoeba), and is a potential human pathogen associated with hospital-acquired pneumonia. The attachment mechanism of this bacteria to host cells is crucial in bacterial pathogenesis, yet remains undetermined. Hence, we obtained monoclonal antibodies (mAbs) specific to either P. acanthamoebae or amoebae in an attempt to elucidate the attachment mechanism involved. Hybridomas of 954 clones were assessed, and we found that four mAbs (mAb38, mAb300, mAb311, mAb562) that were reactive to the amoebae significantly inhibited bacterial attachment. All mAbs recognized amoebal released molecules, and mAb311 also recognized the amoebal surface. mAbs reacted with the bacteria not only within amoebae, but also when they were released from amoebae (except mAb311). Furthermore, a serine protease inhibitor had an inhibitory effect on the bacterial attachment to amoebae, although none of the mAbs had any synergistic effect on the inhibition of attachment by the protease inhibitor. Taken together, we conclude that concurrent P. acanthoamebae attachment to amoebae is required for several amoebal released molecules and serine protease activity, implying the existence of a complicated host-parasite relationship.

Transport of molecules across mitochondrial outer membrane is pivotal for a proper function of mitochondria. The transport pathways across the membrane are formed by ion channels that participate in metabolite exchange between mitochondria and cytoplasm (voltage-dependent anion-selective channel, VDAC) as well as in import of proteins encoded by nuclear genes (Tom40 and Sam50/Tob55). VDAC, Tom40, and Sam50/Tob55 are present in all eukaryotic organisms, encoded in the nuclear genome, and have β-barrel topology. We have compiled data sets of these protein sequences and studied their phylogenetic relationships with a special focus on the position of Amoebozoa. Additionally, we identified these protein-coding genes in Acanthamoeba castellanii and Dictyostelium discoideum to complement our data set and verify the phylogenetic position of these model organisms. Our analysis show that mitochondrial β-barrel channels from Archaeplastida (plants) and Opisthokonta (animals and fungi) experienced many duplication events that resulted in multiple paralogous isoforms and form well-defined monophyletic clades that match the current model of eukaryotic evolution. However, in representatives of Amoebozoa, Chromalveolata, and Excavata (former Protista), they do not form clearly distinguishable clades, although they locate basally to the plant and algae branches. In most cases, they do not posses paralogs and their sequences appear to have evolved quickly or degenerated. Consequently, the obtained phylogenies of mitochondrial outer membrane β-channels do not entirely reflect the recent eukaryotic classification system involving the six supergroups: Chromalveolata, Excavata, Archaeplastida, Rhizaria, Amoebozoa, and Opisthokonta.

Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro.

Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of approximately 90% at approximately 250 nm mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from approximately 30 min without mCAH3 to approximately 10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments.

The involvement of the mannose-induced Acanthamoeba cytopathic protein (MIP-133) in tissue injury and activation of metalloproteinase of corneal and stromal cells was examined in vitro. Activation of MMP-1, MMP-2, MMP-3, and MMP-9 induced by MIP-133 on human corneal epithelial and stromal cell cultures was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA. MMP-1, MMP-2, MMP-3, and MMP-9 mRNA were expressed in both cultured human corneal epithelial and stromal cells. When the epithelial cells were exposed to MIP-133 protein, the mRNA expression for MMP-1 and MMP-9 was unchanged. However, the transcript for MMP-2 and MMP-3 was decreased by 2-fold. By contrast, the expression of MMP-2 and MMP-3 was significantly upregulated (2- to 4-fold) in the corneal stromal cells 1, 4, and 8h after MIP-133 stimulation. At the protein level, there was no significant difference in the level of MMPs between the corneal epithelial cells before and after stimulation with MIP-133. By contrast, the levels of MMP-2 and MMP-3 were significantly higher in the corneal stromal cells stimulated with MIP-133. The supernatants from corneal stromal cells stimulated with MIP-133 were incubated with PMSF and MIP-133 antibody and the level of MMP-2 was measured by ELISA. Activation of MMP-2 by MIP-133 was inhibited in the supernatants pretreated with the serine protease inhibitor, PMSF, and anti-MIP-133. Supernatants pretreated with the cysteine protease inhibitor E6 or control antibody produced the same amount of MMP-2 as the untreated supernatants. To verify possible homology between MMPs and Acanthamoeba castellanii proteases, the mRNA from A. castellanii was prepared and analyzed for the expression of MMP genes by PT-PCR. The results showed that A. castellanii did not express mRNA for MMP-1, MMP-2, MMP-3, or MMP-9. Thus, A. castellanii mRNA does not cross-react with human MMPs. Furthermore, ELISA was used to determine the cross-reactivity of MMP antibodies with the MIP-133 protein. Monoclonal antibodies against MMPs did not cross-react with either the MIP-133 protein or BSA (negative control antigen). The results indicate that the MIP-133 protein modulates MMP-2 and -3 expression differently in human corneal epithelial and stromal cells.

Key to the pathogenesis of intracellular pathogens is their ability to manipulate host cell processes, permitting the establishment of an intracellular replicative niche. In turn, the host cell deploys defence mechanisms that limit intracellular infection. The bacterial pathogen Legionella pneumophila, the aetiological agent of Legionnaire's Disease, has evolved virulence mechanisms that allow it to replicate within protozoa, its natural host. Many of these tactics also enable L. pneumophila's survival and replication inside macrophages within a membrane-bound compartment known as the Legionella-containing vacuole. One of the virulence factors indispensable for L. pneumophila's intracellular survival is a type IV secretion system, which translocates a large repertoire of bacterial effectors into the host cell. These effectors modulate multiple host cell processes and in particular, redirect trafficking of the L. pneumophila phagosome and mediate its conversion into an ER-derived organelle competent for intracellular bacterial replication. In this review, we discuss how L. pneumophila manipulates host cells, as well as host cell processes that either facilitate or impede its intracellular survival.

Purpose: To determine whether the expression of Acanthamoeba mannose-binding protein (MBP) is associated with the pathogenicity of the parasite in vitro., Methods: Both active trophozoites and dormant cysts of a pathogenic strain of A. castellanii were analyzed for their ability to bind to corneal epithelium, express MBP, and produce a cytopathic effect (CPE) on host cells. In addition, host cell binding, CPE-inducing ability, and MBP expression pattern of trophozoites of four different isolates of Acanthamoeba with various degrees of in vitro pathogenicity were analyzed. Binding assays were performed with radiolabeled parasites; CPE assays were performed with rabbit corneal epithelial cells as host cells; and the expression of MBP was detected by affinity chromatography of parasite extracts on mannose affinity columns and by immunohistochemical and Western blot analyses., Results: Trophozoites of A. castellanii bound avidly to corneal epithelial cells in a mannose-inhibitable manner, whereas cysts exhibited little binding. The lack of binding of the cysts to host cells was associated with the downregulation of MBP, along with the concomitant loss of CPE. Analysis of trophozoites of five different species of Acanthamoeba exhibiting various degrees of pathogenic potential revealed that the ability of parasites to bind to host cells and produce CPE is directly correlated with the expression of the MBP. Acanthamoeba strains that bound avidly to host cells and produced potent CPE, robustly expressed MBP. In contrast, parasite strains that produced only weak CPE, expressed markedly reduced levels of MBP., Conclusions: The data demonstrating that the pathogenic potential of Acanthamoeba directly correlates with the expression level of the MBP in conjunction with our published studies showing that Acanthamoeba MBP is a major virulence protein suggest that the amoeba lectin has the potential to serve as a marker of pathogenicity.

Some carbohydrates are known to decrease the attachment of Acanthamoeba sp. to biological surfaces. By a method based on the reduction of a tetrazolium salt (XTT) by the mitochondrial dehydrogenases of the parasites, d-mannose and alpha-d-mannopyranoside have been shown to reduce Acanthamoeba attachment to inert surfaces, indicating that the mannose binding protein of Acanthamoeba trophozoites is involved in adherence to inert surfaces. The reduction in attachment is dose dependant and is not linked with a potential toxicity of the carbohydrates. All the species of Acanthamoeba tested were concerned by this mannose binding protein, but the adhesion of A. culbertsoni was also reduced by the presence of glucose.

Acanthamoebae produce a painful, blinding infection of the cornea. The mannose-binding protein (MBP) of Acanthamoeba is thought to play a key role in the pathogenesis of the infection by mediating the adhesion of parasites to the host cells. We describe here the isolation and molecular cloning of Acanthamoeba MBP. The MBP was isolated by chromatography on the mannose affinity gel. Gel filtration experiments revealed that the Acanthamoeba lectin is a approximately 400-kDa protein that is constituted of multiple 130-kDa subunits. Cloning and sequencing experiments indicated that the Acanthamoeba MBP gene is composed of 6 exons and 5 introns that span 3.6 kb of the amoeba genome and that MBP cDNA codes for a precursor protein of 833 amino acids. That the cloned cDNA encodes authentic MBP was demonstrated by showing that: (i). recombinant MBP possesses mannose binding activity, and (ii). polyclonal antibodies prepared against Acanthamoeba MBP bound to the recombinant protein. Sequence analysis revealed that the MBP contains a large N-terminal extracellular domain, a transmembrane domain, and a short C-terminal cytoplasmic domain. Despite extensive BLAST searches using the MBP sequence, no significant matches were retrieved. The most striking feature of the Acanthamoeba MBP sequence is the presence of a cysteine-rich region containing 14 CXCXC motifs within the extracellular domain. In summary, we have isolated, cloned, and characterized a novel MBP from Acanthamoeba. Because the presence of antibodies to MBP in tears provides protection against infection, the availability of the MBP cDNA sequence and rMBP should help develop: (i). a tear-based test to identify individuals who are at risk of developing the keratitis and (ii). strategies to immunize high-risk individuals.

Adherence of Acanthamoeba to host tissue is believed to be crucial in the establishment of amoebic keratitis or GAE. We have isolated a cDNA from a GAE-causing gymnoamoeba, Acanthamoeba healyi, encoding a protein that binds laminin by screening with a peptide G-specific DNA probe. The cDNA clone (AhLBP) was identified on the basis of sequence homology to the nonintegrin mammalian metastasis-associated 67-kDa laminin receptor (67-LR). The predicted amino acid sequence is 256 residues long with a calculated molecular mass of 28.2kDa and a theoretical pI of 5.48. Southern and Northern blot analyses suggested the gene as a single copy in A. healyi genome and expressed as a single transcript of approximately 1.0kb. Virulent strains of Acanthamoeba revealed higher level of the AhLBP mRNA expression than soil isolates. Specific binding of the purified recombinant protein to laminin was confirmed by sandwich Western blot. The polypeptide encoded by AhLBP shared substantial identity with the acidic class ribosomal proteins involved in protein synthesis. Therefore, the AhLBP may be multifunctional in A. healyi, acting as a laminin-binding molecule but also playing a role in cell division and growth. AhLBP-EGFP fusion protein expressed in A. healyi was localized mainly at the cell membrane and nucleus and at cytoplasm with lesser degree. N-terminal 64 amino acids were important for the localization at the cell membrane. This is the first description of a cDNA encoding a laminin-binding protein from protozoan parasites.

CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the alpha- and beta-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is approximately 0.4 microm or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, approximately 200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer <--> dimer equilibrium with an association constant of 1.0 x 10(6) m(-1). Together, these results indicate that CARMIL self-associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins ( approximately 1 and 2 microm for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.

The pathogenesis of Acanthamoeba keratitis begins when Acanthamoeba trophozoites bind specifically to mannosylated glycoproteins upregulated on the surfaces of traumatized corneal epithelial cells. When Acanthamoeba castellanii trophozoites are grown in methyl-alpha-D-mannopyranoside, they are induced to secrete a novel 133-kDa protein that is cytolytic to corneal epithelial cells. Clinical isolates of Acanthamoeba spp., and not the soil isolates, were proficient at producing a mannose-induced protein (MIP-133) and generating disease in Chinese hamsters. The purified protein was efficient at killing corneal epithelial cells, the first mechanistic barrier, by inducing apoptosis in a caspase 3-dependent pathway. Subsequent steps in pathogenesis require the amoebae to penetrate and degrade collagen. Only the clinical isolates tested were efficient at migrating through a collagenous matrix in vitro, presumably by MIP-133 degradation of both human type I and human type IV collagen. A chicken anti-MIP-133 antiserum effectively bound to the protein and blocked collagenolytic activity, migration, and cytopathic effects (CPE) against corneal cells in vitro. Chinese hamsters orally immunized with MIP-133 displayed a >30% reduction in disease. Immunoglobulin A isolated from immunized animals bound MIP-133 and blocked CPE on corneal cells in vitro. Animals induced to generate severe chronic infections displayed significant reductions in disease symptoms upon oral immunization postinfection. These data suggest that MIP-133 production might be necessary to initiate corneal disease and that it may play an important role in the subsequent steps of the pathogenic cascade of Acanthamoeba keratitis. Furthermore, as antibodies produced both prior to and after infection reduced clinical symptoms of disease, the protein may represent an important immunotherapeutic target for Acanthamoeba keratitis.

The domain organization of Acanthamoeba myosin-I, an oligomodular motor protein, includes a potentially important protein interaction module that is mostly uncharacterized. The Src homology 3, SH3, domain of myosin-I binds Acan125, a protein containing at least two consensus ligand binding domains: C-terminal SH3 binding motifs (PXXP) and N-terminal leucine-rich repeats. We report the first affinities determined for an SH3 domain of any myosin, namely, K(d) = 7 microM for a 21-residue synthetic peptide based on the PXXP domain sequence and K(d) = 0.15 microM for the PXXP domain included in the C-terminus of Acan125. These values are consistent with affinities reported for peptides and proteins that associate with SH3. By deletional analysis we show that only the PXXP domain is required for Acan125 to interact with the SH3 domain of Acanthamoeba myosin-IC (AmyoC(SH3)). The synthetic peptide described above at a concentration near the K(d) for SH3 binding blocked the interaction between native AmyoC and Acan125, mapping the interaction to the PXXP domain of Acan125 and the SH3 domain of myosin-I. These results are consistent with prototypical SH3 binding and suggest that a PXXP module is both necessary and sufficient to interact with an SH3 module of myosin-I., (Copyright 2000 Academic Press.)

Acanthamoeba castellanii is a free-living protozoan that causes keratitis in humans and has been associated with pneumonia and granulomatous amebic encephalitis in dogs, sheep, and other species. Adherence of the Acanthamoeba to epithelial cells is critical to the pathogenesis of this disease. In this study, several mouse monoclonal antibodies (MAb) generated to whole Acanthamoeba trophozoites identified surface membrane epitopes by ELISA and IFA. Nine antibodies inhibited adherence of [(35)S]-methionine-labeled Acanthamoeba trophozoites to hamster corneal epithelial cells by 27-90%. Sodium periodate treatment, but not proteinase K digestion, of whole Acanthamoeba destroyed epitopes recognized by adherence-inhibiting antibodies such as MAb 7H6, suggesting that the adherence epitopes are carbohydrates. Other antibodies, MAb 2A8 for example, recognized surface membrane peptide epitopes that were proteinase K sensitive and sodium periodate resistant. Purified MAb 2A8 was used in an antigen-capture ELISA with peroxidase-labeled MAb 7H6 and demonstrated that the carbohydrate adhesion molecule was linked to the peptide recognized by MAb 2A8. Both MAbs 7H6 and 2A8 recognized a >207-kDa band on a Western blot of eluant from a MAb 2A8 immunoaffinity column, confirming that MAb 7H6 and MAb 2A8 recognize different epitopes on the same adherence molecule. MAbs 7H6 and 2A8 also identified the adhesion molecule in soluble Acanthamoeba membrane preparations and MAb 2A8 immunoaffinity column eluant by ELISA and Western blot. Neither of these antibodies were inhibited from binding to whole trophozoites nor membrane extracts by mannose or mannan in competitive binding assays. When our Acanthamoeba membrane preparations were electrophoresed and immunoblotted with alpha-d-mannosylated-biotin albumin, no bands were recognized in the >207 kDa range by our adherence-associated antibodies. These results suggest that the Acanthamoeba adhesin is not identical to the mannose binding protein of Acanthamoeba but rather is a distinct surface membrane glycoprotein., (Copyright 1999 Academic Press.)

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.

Three methods, fluorescence anisotropy of rhodamine-labeled profilin, intrinsic fluorescence and nucleotide exchange, give the same affinity, Kd = 0.1 microM, for Acanthamoeba profilins binding amoeba actin monomers with bound Mg-ATP. Replacement of serine 38 with cysteine created a unique site where labeling with rhodamine did not alter the affinity of profilin for actin. The affinity for rabbit skeletal muscle actin is about 4-fold lower. The affinity for both actins is 5-8-fold lower with ADP bound to actin rather than ATP. Pyrenyliodoacetamide labeling of cysteine 374 of muscle actin reduces the affinity for profilin 10-fold. The affinity of profilin for nucleotide-free actin is approximately 3-fold higher than for Mg-ATP-actin and approximately 24-fold higher than for Mg-ADP-actin. As a result, profilin binding reduces the affinity of actin 3-fold for Mg-ATP and 24-fold for Mg-ADP. Mg-ATP dissociates 8 times faster from actin-profilin than from actin and binds actin-profilin 3 times faster than actin. Mg-ADP dissociates 14 times faster from actin-profilin than from actin and binds actin-profilin half as fast as actin. Thus, profilin promotes the exchange of ADP for ATP. These properties allow profilin to bind a high proportion of unpolymerized ATP-actin in the cell, suppressing spontaneous nucleation but allowing free barbed ends to elongate at more than 500 subunits/second.

The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 microM. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 +/- 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actin-related proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of approximately 13 x 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 microM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actin-rich regions of Acanthamoeba.

A cytosolic polyamine N-acetyltransferase that preferentially catalyzes the acetylation of spermidine in the N8-position was identified in the free-living pathogenic amoeba Acanthamoeba culbertsoni. In addition to spermidine, the enzyme also catalyzed the acetylation of spermine and putrescine with Michaelis constants (Km values) of 97, 12, and 10 microM, respectively. The Km value for acetylcoenzyme A (acetyl-CoA) was estimated to be 11 microM, whereas CoA had an inhibitory constant of 6 microM. The N-acetylase has a molecular mass of approximately 45 kDa. That the enzyme preferentially catalyzed the acetylation of spermidine at the N8-position, resulting in N8-acetylspermidine, the preferred substrate of the polyamine oxidase found in A. culbertsoni, indicates a role for the enzyme in the production of 1,3-diaminopropane, the major polyamine found in the Acanthamoeba.

Acanthamoeba castellanii (Neff) is a free-living soil amoeba with close relatives that are opportunistic pathogens. Trophozoites differentiate into cysts when deprived of nutrients; cysts convert into trophozoites, leaving the wall behind, in the presence of nutrients. The data presented here, which includes immunoaffinity purification of the receptor, indicate that cell surface molecular signals also control Acanthamoeba differentiation in both directions. Monoclonal antibodies that bind specifically to a 40 kD trophozoite protein initiate the encystment of trophozoites. When bound to cysts the same monoclonal antibodies prevent excystment. Washing away the antibody allows both trophozoites and cysts to resume normal activity. One of these monoclonal antibodies inhibits pinocytosis, while another has no effect on pinocytosis.

We determined the amino acid sequence of the actin monomer binding/actin filament severing protein actophorin from Acanthamoeba castellanii by automated Edman degradation of peptide fragments and by sequencing of full-length cDNA. Actophorin consists of 138 amino acids (calculated molecular weight of 15,543) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially vertebrate cofilin, vertebrate actin depolymerizing factor/destrin, and echinoderm depactin. Actophorin is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that actophorin is a single-copy gene; however, Northern blots show two distinct mRNA species of 1 and 0.9 kb in size. Homogeneous recombinant actophorin purified from Escherichia coli is indistinguishable from the native protein in its physical properties and in biochemical assays of its interaction with actin, but is less reactive with three monoclonal antibodies raised against the native protein. The NH2 terminus of native actophrin is blocked, while the initiating methionine residue is removed from recombinant actophorin. This difference has no measurable effect on activity. By fluorescent antibody staining of Acanthamoeba, actophorin colocalizes with actin filaments in the cortical cytoplasm, especially at the leading edge of the cell. Additionally, actophorin binds phosphatidylinositol 4',5'-bisphosphate. The recombinant actophorin forms X-ray diffraction quality crystals of superior quality in poly(ethylene glycol)/2-propanol and, like the native crystal form, belongs to space group P2(1)2(1)2(1).

Treatment of E. histolytica HM1-IMSS trophozoite extracts to conditions that produce gels of actin and associated cytoskeletal proteins in other ameboid cells caused formation of macroscopic actin rich complexes (ARCs). The one-dimensional PAGE protein profile of this ARC was similar to those of Dictyostelium and Acanthamoeba actin gels. Formation of the E. histolytica ARCs was enhanced by added lipids. In addition to actin, the ARC was enriched with proteins that showed cross-reactivity to antibodies to alpha-actinin and the 50K actin binding protein (elongation factor 1 alpha) from Dictyostelium. E. histolytica ARCs appear to be comprised of a number of actin cytoskeleton proteins and provide a source for their isolation and characterization.

Actobindin is a protein from Acanthamoeba castellanii with bivalent affinity for monomeric actin. Because it can bind two molecules of actin, actobindin is a substantially more potent inhibitor of the early phase of actin polymerization than of F-actin elongation. The complete amino acid sequence of 88 residues has been deduced from the determined sequences of overlapping peptides obtained by cleavage with trypsin, Staphylococcus V8 protease, endoproteinase Asp-N, and CNBr. Actobindin contains 2 trimethyllysine residues and an acetylated NH2 terminus. About 76% of the actobindin molecule consists of two nearly identical repeated segments of approximately 33 residues each. This could explain actobindin's bivalent affinity for actin. The circular dichroism spectrum of actobindin is consistent with 15% alpha-helix and 22% beta-sheet structure. A hexapeptide with sequence LKHAET, which occurs at the beginning of each of the repeated segments of actobindin, is very similar to sequences found in tropomyosin, muscle myosin heavy chain, paramyosin, and Dictyostelium alpha-actinin. A longer stretch in each repeated segment is similar to sequences in mammalian and amoeba profilins. Interestingly, the sequences around the trimethyllysine residues in each of the repeats are similar to the sequences flanking the trimethyllysine residue of rabbit reticulocyte elongation factor 1 alpha, but not to the sequences around the trimethyllysine residues in Acanthamoeba actin and Acanthamoeba profilins I and II.