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The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-activated kinases (PAKs) is relatively low, including only the catalytic domain and a short PAK N-terminal motif (PAN), and even these regions are not highly homologous. In this paper, we report the expression in insect cells of full-length, fully regulated Acanthamoeba MIHCK and further characterize the regulation of this PAK by Rac, calmodulin, and autoinhibition. We map the autoinhibitory region of MIHCK to its PAN region and show that the PAN region inhibits autophosphorylation and kinase activity of unphosphorylated full-length MIHCK and its expressed catalytic domain but has very little effect on either when they are phosphorylated. These properties are similar to those reported for mammalian PAK1. Unlike PAK1, MIHCK is activated by Rac only in the presence of phospholipid. However, peptides containing the PAN region of MIHCK bind Rac in the absence of lipid, and Rac binding reverses the inhibition of the MIHCK catalytic domain by PAN peptides. Our data suggest that a region N-terminal to PAN is required for optimal binding of Rac. Also unlike mammalian PAK, phospholipid stimulation of Acanthamoeba MIHCK and Dictyostelium MIHCK) (which is also a PAK) is inhibited by Ca(2+)-calmodulin. In contrast to Dictyostelium MIHCK, however, Ca(2+)-calmodulin also inhibits Rac-induced activity of Acanthamoeba MIHCK. The basic region N-terminal to PAN is essential for calmodulin binding.

The role played by soluble molecules that may participate in acanthamoebal cytopathogenicity has yet to be fully characterized. We demonstrate here that Acanthamoeba castellanii trophozoites constitutively release ADP in the medium. Cell-free supernatants prepared from A. castellanii, by interaction with specific P(2y2) purinoceptors expressed on the Wish cell membrane, caused a biphasic rise in [Ca(2+)](i), extensive cell membrane blebbing, cytoskeletal disorganization, and the breakdown of nuclei. Cell damage induced by amoebic supernatants was blocked by the P(2y2) inhibitor Suramin. The same results were found in Wish cells exposed to purified ADP. These findings suggest that pathogenic free-living A. castellanii may have a cytopathic effect on human epithelial cells through ADP release, by a process that begins with a rise of cytosolic free-calcium concentration, and culminates in apoptosis.

Extensive colonization of the rhizosphere by beneficial bacteria is desirable but it is not yet clear whether the feeding patterns of protozoa exert any ecological impact on the competence of bacteria in the rhizosphere. To determine whether the successful colonization of the rhizosphere by bacteria can be explained by inherent abilities of the bacteria to resist predation, six bacteria categorized as poor, mediocre or good rhizosphere colonists were tested. The populations of protozoa in the rhizosphere consistently increased to a maximum density in two days but then declined to a stable level. Grazing by protozoa increased the doubling time of all of the test bacteria but did not significantly affect the final densities of bacteria in the rhizosphere. The differences in the colonization ability of the six bacteria tested were not attributable to inherent differences in their susceptibility to predation by protozoa.

Aims: To develop rapid means of distinguishing between cysts and trophozoites of the opportunistic pathogen, Acanthamoeba castellanii, the causative agent of keratitis., Methods and Results: Fluorescence of Congo Red, Calcoflor White was specific for the endocyst wall; trophozoites did not become fluorescent. The anionic oxonol dye, DiBAC4(3), did not penetrate the cytoplasmic membrane after short-term (<5 min) exposure, whereas cysts are permeable and become fluorescent. Confocal scanning laser microscopy confirmed these properties and large populations of organisms were analysed by flow cytometry., Conclusion: These data provide a rapid alternative to traditional haemocytometer or plate counts for discrimination of trophozoites from cysts., Significance and Impact of the Study: Rapid and precise determination of the growth cycle of a dangerous ocular pathogen.

Treatment with the drugs berenil, dibromopropamidine, pentamidine and CGP-4O-215 were found to alter levels of intracellular polyamines of Acanthamoeba polyphagia trophozoites in organisms. While the polyamine, spermidine, consistently remained higher in the control organisms incubated at 8, 16 and 32 h respectively, the level fluctuated significantly from below 5% to 40% in drug-treated organisms. A novel polyamine (polyamine F), yet to be identified, representing 80% of the total polyamine extracts and seen in control organisms after 48 h of incubation (stationary phase), was seen much earlier in the drug treated organisms. Pentamidine, dibromopropamidine and CGP-4O-215 induced the appearance of the novel polyamine in the trophozoites after only 8 h of incubation with the drugs while induction by berenil occurred after 32 h. It is suggested that diamidine drugs either induce encystment or alter the pathway of polyamine metabolism in Acanthamoeba. If inducing encystment is the main mode of reaction, then drugs that induce early encystment will be less effective in treatment than those that delay it.

Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. Since 1995, it has been shown that the uncoupling protein is present in many higher plants and some microorganisms like non-photosynthetic amoeboid protozoon, Acanthamoeba castellanii and non-fermentative yeast Candida parapsilosis. In mitochondria of these organisms, uncoupling protein activity is revealed not only by stimulation of state 4 respiration by free fatty acids accompanied by decrease in membrane potential (these effects being partially released by ATP and GTP) but mainly by lowering ADP/O ratio during state 3 respiration. Plant and microorganism uncoupling proteins are able to divert very efficiently energy from oxidative phosphorylation, competing for deltamicroH+ with ATP synthase. Functional connection and physiological role of uncoupling protein and alternative oxidase, two main energy-dissipating systems in plant-type mitochondria, are discussed.

Mitochondria of amoeba Acanthamoeba castellanii in addition to the conventional cytochrome pathway possess, like plant mitochondria, a cyanide-resistant alternative quinol oxidase. In mitochondria isolated from amoeba batch culture grown temporarily at low temperature (6 degrees C), higher respiration was accompanied by lower coupling parameters as compared to control culture (grown at 28 degrees C). In the presence of benzohydroxamate, respiratory rates and coupling parameters were similar in both types of mitochondria indicating that growth in cold conditions did not disturb the cytochrome pathway. Increased contribution of alternative oxidase in total mitochondrial respiration in low-temperature-grown amoeba cells was confirmed by calculation of its contribution using ADP/O measurements. Furthermore, in mitochondria from low-temperature- grown cells the content of the alternative oxidase was increased and correlated with the increase in the unstimulated and GMP-stimulated cyanide-resistant respiratory activity. A possible physiological role of higher activity of alternative oxidase as response to growth at a low temperature in unicellular organisms, such as amoeba, is discussed.

Phosphorylation of the regulatory light chain of Dictyostelium myosin II increases V(max) of its actin-dependent MgATPase activity about 5-fold under normal assay conditions. Under these assay conditions, unphosphorylated chimeric myosins in which the tail domain of the Dictyostelium myosin II heavy chain is replaced by either the tail domain of chicken gizzard smooth muscle or Acanthamoeba myosin II are 20 times more active because of a 10- to 15-fold increase in V(max) and a 2- to 7-fold decrease in apparent K(ATPase) and are only slightly activated by regulatory light chain phosphorylation. Actin-dependent MgATPase activity of the Dictyostelium/Acanthamoeba chimera is not affected by phosphorylation of serine residues in the tail whose phosphorylation completely inactivates wild-type Acanthamoeba myosin II. These results indicate that the actin-dependent MgATPase activity of these myosins involves specific, tightly coupled, interactions between head and tail domains.

Acanthamoeba myosin IC has a single 129-kDa heavy chain and a single 17-kDa light chain. The heavy chain comprises a 75-kDa catalytic head domain with an ATP-sensitive F-actin-binding site, a 3-kDa neck domain, which binds a single 17-kDa light chain, and a 50-kDa tail domain, which binds F-actin in the presence or absence of ATP. The actin-activated MgATPase activity of myosin IC exhibits triphasic actin dependence, apparently as a consequence of the two actin-binding sites, and is regulated by phosphorylation of Ser-329 in the head. The 50-kDa tail consists of a basic domain, a glycine/proline/alanine-rich (GPA) domain, and a Src homology 3 (SH3) domain, often referred to as tail homology (TH)-1, -2, and -3 domains, respectively. The SH3 domain divides the TH-3 domain into GPA-1 and GPA-2. To define the functions of the tail domains more precisely, we determined the properties of expressed wild type and six mutant myosins, an SH3 deletion mutant and five mutants truncated at the C terminus of the SH3, GPA-2, TH-1, neck and head domains, respectively. We found that both the TH-1 and GPA-2 domains bind F-actin in the presence of ATP. Only the mutants that retained an actin-binding site in the tail exhibited triphasic actin-dependent MgATPase activity, in agreement with the F-actin-cross-linking model, but truncation reduced the MgATPase activity at both low and high actin concentrations. Deletion of the SH3 domain had no effect. Also, none of the tail domains, including the SH3 domain, affected either the K(m) or V(max) for the phosphorylation of Ser-329 by myosin I heavy chain kinase.

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.)

LIM-kinase activated by GST-Pak1 phosphorylates Acanthamoeba actophorin stoichiometrically and specifically on serine 1. The atomic structure of phosphorylated actophorin determined by X-ray crystallography is essentially identical with the structure of unphosphorylated actophorin. We compared biochemical properties of phosphorylated actophorin, unphosphorylated actophorin and mutants of actophorin with serine 1 replaced by aspartic acid or alanine. Phosphorylation strongly inhibits interaction of actophorin with Mg-ADP- or Mg-ATP-actin monomers and Mg-ADP-actin filaments, so Ser1 phosphorylation directly blocks interaction of actin-depolymerizing factor (ADF)/cofilin proteins with actin. About 30 % of actophorin is phosphorylated in live amoebas grown in suspension culture. Phosphorylation of ADF/cofilin proteins by LIM-kinase or other enzymes will tend to stabilize actin filaments by inhibiting the ability of these proteins to sever and depolymerize older actin filaments that have hydrolyzed their bound ATP and dissociated the phosphate., (Copyright 2000 Academic Press.)

We used biochemical fractionation, immunoassays and microscopy of live and fixed Acanthamoeba to determine how much profilin is bound to its known ligands: actin, membrane PIP(2), Arp2/3 complex and polyproline sequences. Virtually all profilin is soluble after gentle homogenization of cells. During gel filtration of extracts on Sephadex G75, approximately 60% of profilin chromatographs with monomeric actin, 40% is free and none voids with Arp2/3 complex or other large particles. Selective monoclonal antibodies confirm that most of the profilin is bound to actin: 65% in extract immunoadsorption assays and 74-89% by fluorescent antibody staining. Other than monomeric actin, no major profilin ligands are detected in crude extracts. Profilin-II labeled with rhodamine on cysteine at position 58 retains its affinity for actin, PIP(2) and poly-L-proline. When syringe-loaded into live cells, it distributes throughout the cytoplasm, is excluded from membrane-bounded organelles, and concentrates in lamellapodia and sites of endocytosis but not directly on the plasma membrane. Some profilin fluorescence appears punctate, but since no particulate profilin is detected biochemically, these spots may be soluble profilin between organelles that exclude profilin. The distribution of profilin in fixed human A431 cells is similar to that in amoebas. Our results show that the major pool of polymerizable actin monomers is complexed with profilin and spread throughout the cytoplasm.

An uncoupling protein (UCP) has been identified in mitochondria from Acanthamoeba castellanii, a nonphotosynthetic soil amoeboid protozoon that, in molecular phylogenesis, appears on a branch basal to the divergence points of plants, animals, and fungi. The existence of UCP in A. castellanii (AcUCP) has been revealed using antibodies raised against plant UCP. Its molecular mass (32,000 Da) was similar to those of plant and mammalian UCPs. The activity of AcUCP has been investigated in mitochondria depleted of free fatty acids. Additions of linoleic acid stimulated state 4 respiration and decreased transmembrane electrical potential (DeltaPsi) in a manner expected from fatty acid cycling-linked H(+) reuptake. The half-maximal stimulation by linoleic acid was reached at 8.1 +/- 0.4 microM. Bovine serum albumin (fatty acid-free), which adsorbs linoleic acid, reversed the respiratory stimulation and correspondingly restored DeltaPsi. AcUCP was only weakly inhibited by purine nucleotides like UCP in plants. A single force-flow relationship has been observed for state 4 respiration with increasing concentration of linoleic acid or of an uncoupler and for state 3 respiration with increasing concentration of oligomycin, indicating that linoleic acid has a pure protonophoric effect. The activity of AcUCP in state 3 has been evidenced by ADP/oxygen atom determination. The discovery of AcUCP indicates that UCPs emerged, as specialized proteins for H(+) cycling, early during phylogenesis before the major radiation of phenotypic diversity in eukaryotes and could occur in the whole eukaryotic world.

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.

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.

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.

The three single-headed monomeric myosin I isozymes of Acanthamoeba castellanii (AMIs)-AMIA, AMIB, and AMIC-are among the best-studied of all myosins. We have used AMIC to study structural correlates of myosin's actin-activated ATPase. This activity is normally controlled by phosphorylation of Ser-329, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively. To determine whether activation status is reflected in structural differences in the mode of attachment of myosin to actin, these mutant myosins were bound to actin filaments in the absence of nucleotide (rigor state) and visualized at 24-A resolution by using cryoelectron microscopy and image reconstruction. No such difference was observed. Consequently, we suggest that regulation may be affected not by altering the static (time-averaged) structure of AMIC but by modulating its dynamic properties, i.e., molecular breathing. The tail domain of vertebrate intestinal brush-border myosin I has been observed to swing through 31 degrees on binding of ADP. However, it was predicted on grounds of differing kinetics that any such effects with AMIC should be small [Jontes, J. D., Ostap, E. M., Pollard, T. D. & Milligan, R. A. (1998) J. Cell Biol. 141, 155-162]. We have confirmed this hypothesis by observing actin-associated AMIC in its ADP-bound state. Finally, we compared AMIC to brush-border myosin I and AMIB, which were previously studied under similar conditions. In each case, the shape and angle of attachment to F-actin of the catalytic domain is largely conserved, but the domain structure and disposition of the tail is distinctively different for each myosin.

The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (ii) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (iii) its Km for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same Km for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.

Acanthamoeba actophorin is a member of ADF/cofilin family that binds both actin monomers and filaments. We used fluorescence anisotropy to study the interaction of actin monomers with recombinant actophorin labeled with rhodamine on a cysteine substituted for Serine-88. Labeled actophorin retains its affinity for actin and ability to reduce the low shear viscosity of actin filaments. At physiological ionic strength, actophorin binds Mg-ADP-actin monomers (Kd = 0.1 microM) 40 times stronger than Mg-ATP-actin monomers. When bound to actin monomers, actophorin has no effect on elongation at either end of actin filaments by Mg-ATP-actin and slightly increases the rate of elongation at both ends by Mg-ADP-actin. Thus actophorin does not sequester actin monomers. Sedimentation equilibrium ultracentrifugation shows that actophorin and profilin compete for binding actin monomers. Actophorin and profilin have opposite effects on the rate of exchange of nucleotide bound to actin monomers. Despite the high affinity of actophorin for ADP-actin, physiological concentrations of profilin overcome the inhibition of ADP exchange by actophorin. Profilin rapidly recycles ADP-actin back to the profilin-ATP-actin pool ready for elongation of actin filaments.

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.

Amoeba mitochondria possess a respiratory chain with two quinol-oxidizing pathways: the cytochrome pathway and the cyanide-resistant alternative oxidase pathway. The ADP/O method, based on the non-phosphorylating property of alternative oxidase, was used to determine contributions of both pathways in overall state 3 respiration in the presence of GMP (an activator of the alternative oxidase in amoeba) and succinate as oxidizable substrate. This method involves pair measurements of ADP/O ratios plus and minus benzohydroxamate (an inhibitor of the alternative oxidase). The requirements of the method are listed and verified. When overall state 3 respiration was decreased by increasing concentrations of n-butyl malonate (a non-penetrating inhibitor of succinate uptake), the quinone reduction level declined. At the same time, the alternative pathway contribution decreased sharply and became negligible when quinone redox state was lower than 50%, whereas the cytochrome pathway contribution first increased and then passed through a maximum at a quinone redox state of 58% and sharply decreased at a lower level of quinone reduction. This study is the first attempt to examine the steady-state kinetics of the two quinol-oxidizing pathways when both are active and to describe electron partitioning between them when the steady-state rate of the quinone-reducing pathway is varied.

Two Acanthamoeba species, fed at three temperatures, expelled vesicles containing living Legionella pneumophila cells. Vesicles ranged from 2.1 to 6.4 microns in diameter and theoretically could contain several hundred bacteria. Viable L. pneumophila cells were observed within vesicles which had been exposed to two cooling tower biocides for 24 h. Clusters of bacteria in vesicles were not dispersed by freeze-thawing and sonication. Such vesicles may be agents for the transmission of legionellosis associated with cooling towers, and the risk may be underestimated by plate count methods.

Acanthamoeba keratitis is a chronic inflammatory disease of the cornea which is highly resistant to many antimicrobial agents. The pathogenic mechanisms of this disease are poorly understood. However, it is believed that the initial phases in the pathogenesis of Acanthamoeba keratitis involve parasite binding and lysis of the corneal epithelium. These processes were examined in vitro, using Acanthamoeba castellanii trophozoites. Parasites readily adhered to Chinese hamster corneal epithelial cells in vitro; however, parasite binding was strongly inhibited by mannose but not by lactose. Although mannose prevented trophozoite binding, it did not affect cytolysis of corneal epithelial cells. Moreover, mannose treatment induced trophozoites to release cytolytic factors that lysed corneal epithelial cells in vitro. These factors were uniquely induced by mannose because supernatants collected from either untreated trophozoites or trophozoites treated with other sugars failed to lyse corneal cells. The soluble factors were size fractionated in centrifugal concentrators and found to be > or = 100 kDa. Treatment of the supernatants with the serine protease inhibitor phenylmethylsulfonyl fluoride inhibited most, but not all, of the cytopathic activity. These data suggest that the binding of Acanthamoeba to mannosylated proteins on the corneal epithelium may exacerbate the pathogenic cascade by initiating the release of cytolytic factors.

Purpose: Selection of an appropriate animal model is crucial for the investigation of the pathogenesis of Acanthamoeba keratitis. To this end, it has been reported that fluorescein isothiocyanate (FITC)-labeled Acanthamoeba castellanii bind to human, pig, and hamster corneas, but not to rabbit corneas in organ culture. However, 35S-labeled A. polyphaga and A. culbertsoni have been found to bind to rabbit corneal epithelium grown in tissue culture. The purpose of the current study was to establish whether A. castellanii bind to rabbit corneal epithelium in organ culture., Methods: Two different adhesion assays were used to determine whether 35S-labeled and FITC-labeled A. castellanii bind to epithelium of corneal buttons in vitro and, if so, whether the binding is temperature-dependent, enhanced by injury, and inhibited by specific saccharides. Ameba binding to rabbit corneal epithelium was also evaluated by scanning electron microscopy. The binding of A. castellanii to corneal epithelium of three different species (human, pig, and rabbit) was compared., Results: Both 35S-labeled as well as FITC-labeled parasites were found to bind to epithelium of rabbit corneal buttons in vitro. Although the parasites bound avidly to the corneas at 25 degrees C and 35 degrees C, little binding was observed at 4 degrees C. Injury enhanced the binding. Methyl alpha-D-mannopyranoside, but not other saccharides (alpha-L-fucose and beta-galactose), inhibited binding of the parasites to the epithelium of rabbit corneas. By scanning EM, A. castellanii were found to adhere, invade, and penetrate the epithelium of rabbit corneas. Compared with rabbit corneas, ameba binding to pig, and human corneas was only 1.2 and 1.4 times higher, respectively., Conclusions: Rabbit animal model may prove useful for investigation of the molecular mechanisms that mediate adhesion of Acanthamoeba to corneal epithelium.

Previous studies have shown that trophozoites of the pathogenic free-living amoeba Acanthamoeba castellanii rapidly lyse a variety of cells in vitro. However, the role played by cytolitic molecules that may participate in Acanthamoebal cytopathogenicity has yet to be completely elucidated. The aim of this work was to study whether soluble molecules released by A. castellanii trophozoites could induce cytopathic effect in human epithelial cells in vitro. The results obtained indicate that A. castellanii trophozoites constitutively elaborate and release soluble factors that immediately elicit a cytosolic free-calcium increase in target cells. This phenomenon is induced by low molecular weight amoebic metabolites and depends on a transmembrane influx of extracellular calcium. Morphological changes, cytoskeletal damage, cell death and cytolysis followed the elevation of cytosolic free-calcium levels. Calcium ions are very important for cell homeostasis, in fact, they control the functions of a variety of cellular responses, including secretion, cell proliferation and apoptosis. Our results suggest that the substained elevation of the cytosolic free-calcium in response to A. castellanii metabolites might play a fundamental role in target cell damage during Acanthamoeba infections.

The class I myosins are single-headed, actin-binding, mechanochemical "motor" proteins with heavy chains in the molecular mass range of 110-130 kDa; they do not form filaments. Each myosin I heavy chain is associated with one to six light chains that bind to specific motifs known as IQ domains. In vertebrate myosin I isoforms, the light chain is calmodulin, which is thought to regulate motor activity. Proteins similar to calmodulin are associated with myosin I isoforms from lower eukaryotes. Some myosin I isoforms from lower eukaryotes are regulated by phosphorylation; however, the phosphorylation site is not present in vertebrate myosin I isoforms. Based on sequence analyses of the amino terminal "head" domains, myosin I can be subdivided into several subclasses. Analyses of the biochemical properties of the isolated molecules and localization studies support the proposal of roles for these molecules in intracellular trafficking and changes in membrane structure. Our present understanding of the properties of these molecules and their proposed roles is reviewed here.

Purpose: This study was undertaken to determine the extent of adhesion of cysts and trophozoites of Acanthamoeba to worn disposable hydrogel lenses and to study the effect of time, lens material, and washing on the adhesion., Methods: Etafilcon A (58% water content) and Polymacon (38% water content) lenses worn on a 6 night extended wear basis and collected aseptically in PBS pH 7.2, were used for the study. Unworn lenses of the same materials were used as controls. Segments of lenses were incubated in Acanthamoeba suspension (cysts and trophozoites) and examined at 0, 5, and 24 hours, either washed or unwashed, for adherent Acanthamoeba. The amoebae adherent per square mm surface of the lens were enumerated. Levels of adhesion in various groups were compared using Wilcoxon sign rank test., Results: At 0 hours, unwashed worn Polymacon lenses showed significantly greater adherence than Etafilcon A lenses compared to the controls (P = 0.0469). However, there was no difference at 5 and 24 hours exposure time between the lenses. At 24 hours the adhesion of Acanthamoeba was greater for washed worn lenses of both types compared to washed lenses of controls (P = 0.002, 0.012)., Conclusions: Amoebae adhered with equal affinity to both types of lenses but adhered with greater affinity to worn lenses than unworn lenses. Adhesion of Acanthamoeba to worn disposable lenses may be a factor in the pathogenesis of Acanthamoeba keratitis in disposable lens wearers.

Axenically and monoxenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga and different isolates of Hartmannella vermiformis strains were examined by polyacrylamide isoelectric focusing in the pH range 3-10. Isoenzyme patterns of acid phosphatase (AP), propionyl esterase (PE), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) were compared. Zymograms were used to reveal differences in typical isoenzyme patterns between axenically and monoxenically grown amoebae and to compare axenically grown A. castellanii, A. polyphaga and H. vermiformis. Comparison of zymograms for AP, PE and MDH between axenically grown Acanthamoeba and Hartmannella strains revealed different isoenzyme patterns. Acanthamoeba showed strong bands for ADH and extremely weak bands for GPI and PGM, while Hartmannella lacked ADH but possessed bands for GPI and PGM. Comparison of zymograms from axenically and monoxenically grown amoebae revealed a lower intensity and even lack of typical isoenzyme bands in lysates from monoxenic cultures. The observed changes in typical isoenzyme patterns induced by the bacterial substrate can influence the correct isoenzymatic typing of different strains in clinical and phylogenetic studies.

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 non-radioactive method was used to investigate the uptake by Acanthamoeba castellanii of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMD). Based on the Giles et al. (1974) hypothesis, the uptake of CHA by trophozoites appeared to be of the L3 pattern whereas that of cysts was C2. Unlike CHA, trophozoites took up PHMB with an L2 pattern at low concentrations followed by a C-type pattern at higher concentrations, the uptake by cysts was found to be of the C2 pattern with a plateau effect at high concentrations. A diphasic leakage effect was found in trophozoites whereas a relatively low peak of maximal leakage occurred from cysts treated with high biocide concentrations. The amount of pentose release depended on the formulation ingredients. No correlation between pentose leakage and trophozoicidal or cysticidal activity was found.

During capping and phagocytosis the interaction between cluster cell surface receptors and the submembraneous actin-based skeleton may be mediated by spectrin-like proteins. To test this possibility we examined the localization of an alpha-spectrin immunoanalogue, that had been previously identified in whole extracts of Acanthamoeba, during capping of Con A receptors and during phagocytosis of Con A-coated yeast. During capping alpha-spectrin and filamentous actin co-migrated with the Con A receptors and accumulated in the region of cap formation, as demonstrated by double immunofluorescence studies. Immunoelectron microscopy revealed submembraneous location of alpha-spectrin in cells exposed to Con A, both at the time of initial cross-linking and during accumulation of alpha-spectrin in the region of the cap. Phagocytosis studies showed that alpha-spectrin and actin filaments were concentrated around phagocytic cups that enclosed ConA-coated yeast upon internalization. The proteins also surrounded nascent phagosomes present in the vicinity of the plasma membrane but were absent at the later time point of phagosome maturation. These data demonstrate a correlation between clustering of cell surface receptors and submembraneous localization of alpha-spectrin, suggesting an involvement of spectrin-like proteins in mediating the interaction of receptor clusters with the actin cytoskeleton.

The aim of the present study was to examine the biosynthesis of prostaglandins and to investigate factors conditioning their biosynthesis in pathogenic and nonpathogenic strains of Acanthamoeba spp. We established that the activity of the synthase of prostaglandins was almost identical in pathogenic and non-pathogenic strains and that the synthesis of endoperoxide prostaglandins was similar to that of other organisms up to the point at which prostaglandin H2 was produced. The course of biosynthesis in vitro can be activated by various compounds such as glutathione, albumin, and p-chloromercuribenzoic acid (p-CMB), which are either activators or inhibitors of the enzymes. We suggest that the course of biosynthesis of prostaglandins in vivo is most probably activated by tissues or constitutional liquids surrounding the parasites.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin II minifilaments is dependent both on Mg2+ concentration and on the state of phosphorylation of three serine sites at the C-terminal end of the heavy chains. Previous electric birefringence experiments on minifilaments showed a large dependence of signal amplitude on the phosphorylation state and Mg2+ concentration, consistent with large changes in filament flexibility. These observations suggested that minifilament stiffness was important for function. We now report that the binding of nucleotides to dephosphorylated minifilaments at Mg2+ concentrations needed for optimal activity increases the flexibility by about 10-fold, as inferred from the birefringence signal amplitude increase. An increase in flexibility with nucleotide binding is not observed for dephosphorylated minifilaments at lower Mg2+ concentrations or for phosphorylated minifilaments at any Mg2+ concentrations examined. The relaxation times for minifilament rotations that are sensitive to the conformation myosin heads are also observed to depend on phosphorylation, Mg2+ concentration, and nucleotide binding. These latter experiments indicate that the actin-activated Mg2+ concentration, and nucleotide binding. These latter experiments indicate that the actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin II correlates with both changes in myosin head conformation and the ability of minifilaments to cycle between stiff and flexible conformations coupled to nucleotide binding and release.

Objectives: To determine the effect of Pseudomonas aeruginosa and Staphylococcus epidermidis on the binding of Acanthamoeba species to hydrogel lenses., Methods: Cells of amebae and bacteria were incubated with different types of hydrogel lenses. Densities of amebae that were bound to the lenses after rinsing were determined from direct counts with a cell detachment procedure and from scintillation counts of cells, which were radiolabeled with tritiated leucine., Results: With both methods, amebae showed significantly increased binding to hydrogel lenses with attached P aeruginosa. The numbers of amebae that were retained on lenses with attached S epidermidis were not significantly different from those that were retained on lenses without bacteria. The binding of amebae to unworn hydrogel lenses, in contrast to the irreversible adherence of P aeruginosa, was tenuous., Conclusions: The binding of Acanthamoeba species to unworn hydrogel lenses was tenuous and appeared to be related to water content, surface tensions, and ionic charge. The presence of adhered P aeruginosa on the hydrogel lenses facilitated the binding of Acanthamoeba species. The cocontamination of lens systems with bacteria (eg, P aeruginosa) may be a prime factor in the development of amebic keratitis.

Background: Approximately 85% of Acanthamoeba-contaminated contact lens systems in asymptomatic patients have concurrent bacterial contamination. Pseudomonas aeruginosa and Xanthomonas maltophilia are common contact lens contaminants; we investigated the effect of coincubation of Acanthamoeba adherence to contact lenses., Methods: A. castellanii, 1 x 10(5) organisms/ml, was coincubated with P. aeruginosa or X. maltophilia, 1 x 10(8) CFU/ml in phosphate-buffered saline. Sterile, unworn polymacon, etafilcon A or lidofilcon contact lens were investigated. The experimental groups were: (I) lenses exposed to bacteria for 1 h, then Acanthamoeba for 2 h; (II) lenses exposed concurrently to bacteria and Acanthamoeba for 2 h; (III) Acanthamoeba coincubated with bacteria for 24 h, then lenses exposed for 2 h; (IV) lenses exposed to Acanthamoeba for 2 h (control)., Results: For all experimental groups, Acanthamoeba adherence was greater to lidofilcon than to polymacon and etafilcon. For both P. aeruginosa and X. maltophilia, neither group I nor group II displayed greater Acanthamoeba adherence than group IV. Group III exhibited significantly less adherence than group IV for lidofilcon and polymacon. The decrease in group III adherence reflected an overall decrease in Acanthamoeba trophozoite concentration., Conclusion: Short bacteria/Acanthamoeba coincubation times did not result in increased Acanthamoeba adherence. Twenty-four-hour coincubation resulted in decreased adherence for Pseudomonas and unchanged adherence rates for Xanthomonas. This model suggests that Pseudomonas or Xanthomonas co-contamination does not necessarily facilitate quantitative Acanthamoeba contact lens adherence.

Axenic mass cultivation of Acanthamoeba castellanii in laboratory fermentors (141) yielded after 20 days approximately 3 g cells (wet weight). After a short lag phase amoebal cell numbers increased exponentially to a maximum of 3.5 x 10(5) cells per ml until cell death occurred after 20 days. Optical density and protein concentrations revealed identical patterns. During amoebal growth only 12-19% of the initially added glucose (100 mM) as sole carbon source was used. Large amounts of ammonia (1 g in 10.51 culture volume) were excreted into the medium which subsequently raised the pH from 6.6 to 7.7, and from 6.6 to 6.8 in 2 and 20 mM buffered media, respectively. Growth inhibition and cell death could not be explained by a depletion of glucose or oxygen limitations during growth. The production of ammonia had a growth inhibitory effect, however, the sudden termination of the exponential growth phase and cell death could not be explained by the toxic influence of ammonia only.

Eukaryotic promoter elements specify the direction and efficiency of transcription, as well as the type of RNA polymerase to be used. One such element, the TATA box, is thought to participate in determining the direction of transcription and can function within promoters for RNA polymerase II or III, depending on the sequence context. In this report the ability of four different TATA boxes to support transcription in vitro was determined. It was found that TATA elements are not directional. However, they support transcription by RNA polymerases II and III. An upstream activating sequence was found to stimulate downstream transcription by RNA polymerase II and to inhibit upstream transcription by RNA polymerases II and III. Thus a promoter necessarily consists of a TATA element and upstream sequences in order to specify the direction of transcription and the type of polymerase to be used.

We characterized several deletion and substitution mutations of Acanthamoeba profilin and nine monoclonal antibodies to Acanthamoeba profilin. The results provide two independent lines of evidence about the binding sites for actin and poly-L-proline on the profilin molecule. This new evidence is consistent with the main conclusions about these binding sites from previous structural and mutagenic studies. Mutagenesis also revealed that the native structure of profilin is very sensitive to substitutions and deletions at the C terminus. For example, profilin with a deletion of the eight C-terminal residues has many of the physical properties of a molten globule, yet remarkably still binds to actin. This instability may account for the lack of function of similar mutants in yeast.

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.

We cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin-binding proteins.

Myosins are molecular motors that upon interaction with actin filaments convert energy from ATP hydrolysis into mechanical force. Evidence has emerged for the existence of a large, widely expressed and evolutionarily ancient superfamily of myosin genes. In addition to the well-catheterized conventional, filament-forming, two-headed myosin-II of muscle and nonmuscle cells, at least ten additional classes of myosins have been identified. In vertebrates, at least seven of the eleven classes are expressed, and many myosins can be expressed in a single cell type. This review summarizes known structural and functional features of these novel unconventional myosins.

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.

A technique based on binding of eosin dye to cell was applied to quantitate Acanthamoeba trophozoites in culture. Using this technique in combination with the uptake of radiolabelled adenosine, we assessed the activities of triazole (saperconazole), imidazole (ketoconazole, miconazole) and diamidine (berenil, pentamidine, dibromopropamidine) compounds against A. polyphaga trophozoites. The quantity of dye bound to the trophozoites correlated (r = 0.99) with the number of organisms per well. When inhibition of the growth of Acanthamoeba was the only parameter used to measure the effectiveness of these drugs, saperconazole was found to be most active with IC50 (concentration of drug that inhibited by 50%, the growth of A. polyphaga trophozoites incubated at 28 degrees C for 48 h) of 0.95 microM. The IC50s of the other drugs ranged from > or = 1500 microM for micronazole, the least active, to 3.0 microM for berenil. However, when efficacy was assessed by combining inhibition of growth with the uptake of [14C-8]adenosine by the drug treated organism, the diamidines, particularly berenil and pentamidine were considered most potent.

Benzimidazoles have been widely used since the 1960s as anthelmintic agents in veterinary and human medicine and as antifungal agents in agriculture. More recently, selected benzimidazole derivatives were shown to be active in vitro against two protozoan parasites, Trichomonas vaginalis and Giardia lamblia, and clinical studies with AIDS patients have suggested that microsporidia are susceptible as well. Here, we first present in vitro susceptibility data for T. vaginalis and G. lamblia using an expanded set of benzimidazole derivatives. Both parasites were highly susceptible to four derivatives, including mebendazole, flubendazole, and fenbendazole (50% inhibitory concentrations of 0.005 to 0.16 microgram/ml). These derivatives also had lethal activity that was time dependent: 90% of T. vaginalis cells failed to recover following a 20-h exposure to mebendazole at 0.17 microgram/ml. G. lamblia, but not T. vaginalis, was highly susceptible to five additional derivatives. Next, we examined in vitro activity of benzimidazoles against additional protozoan parasites: little or no activity was observed against Entamoeba histolytica, Leishmania major, and Acanthamoeba polyphaga. Since the microtubule protein beta-tubulin has been identified as the benzimidazole target in helminths and fungi, potential correlations between benzimidazole activity and beta-tubulin sequence were examined. This analysis included partial sequences (residues 108 to 259) from the organisms mentioned above, as well as the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi and the sporozoan Cryptosporidium parvum. beta-tubulin residues Glu-198 and, in particular, Phe-200 are strong predictors of benzimidazole susceptibility; both are present in Encephalitozoon spp. but absent in C. parvum.

Transcription of the Acanthamoeba tbp gene is stimulated by a cis-acting promoter element that is bound by an activator protein, TATA-binding protein promoter binding factor (TPBF). Here, we report the complete purification of TPBF and describe its transcription activating and DNA-binding properties. TPBF contains two polypeptides with molecular weights of 51,000 and 50,000, whereas the native molecular weight of TPBF suggests it is dimeric or trimeric in solution. Phosphatase treatment of TPBF converts the 51,000 molecular weight species to the 50,000 molecular weight form, demonstrating that TPBF is phosphorylated. Phosphorylation reduces DNA binding by TPBF, as assessed by electrophoretic mobility shift assays after phosphatase treatment. TPBF makes numerous contacts with the bases and phosphate backbone of its DNA recognition element, and the pattern of these contacts suggests that it is a novel type of DNA-binding protein. TPBF can bind to additional, low affinity sites within the TBP gene promoter, suggesting that, in addition to positive activation of tbp gene expression, TPBF could also inhibit transcription by competing for binding sites for other proteins within the TBP promoter.

We have isolated and sequenced a 525 bp ubiquitin cDNA (Acantub1) from the soil amoeba Acanthamoeba castellanii. The deduced amino acid (aa) sequence reveals that the Acantub1 gene product is a fusion protein consisting of 128 aa (76 aa ubiquitin and 52 aa 60S ribosomal protein). The Acantub1 protein sequence has unique substitutions at aa 28 (glutamine for alanine) and 121 (tryptophan for leucine) compared to the 8 highest homologous sequences in the databank. A putative 'zinc finger' nucleic acid-binding domain was located in the 52 aa ribosomal protein. Northern analyses reveal that the Acantub1 gene is regulated during development.