Differentiation in Acanthamoeba castellanii is induced by specific monoclonal antibodies

Monoclonal antibodies that bind a large molecular weight plasma membrane protein of Acanthamoeba castellanii cause the cells to differentiate. A different monoclonal antibody that binds specifically to the major plasma membrane protein has no effect upon cell division or differentiation. The induction of differentiation by the monoclonal antibodies requires a bivalent attachment, more than a single binding cycle of the antibody to the plasma membrane protein, does not require cell-cell contact, and appears to be mediated by an inhibition of pinocytosis. These results suggest one of two alternatives: either this free living amoeba possesses a cell surface receptor that serves to initiate the differentiation process when stimulated, or the specific plasma membrane antigen for the differentiation-inducing monoclonal antibodies is an essential component of the pinocytotic mechanism. While it seems more likely on the basis of available evidence that we are observing the biological effects of a cell surface receptor, either of the two alternative circumstances open up investigative areas of large significance.     

Gene discovery in the Acanthamoeba castellanii genome

Acanthamoeba castellanii is a free-living amoeba found in soil, freshwater, and marine environments and an important predator of bacteria. Acanthamoeba castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct diseases in humans. In order to provide a genomic platform for the study of this ubiquitous and important protist, we generated a sequence survey of approximately 0.5 x coverage of the genome. The data predict that A. castellanii exhibits a greater biosynthetic capacity than the free-living Dictyostelium discoideum and the parasite Entamoeba histolytica, providing an explanation for the ability of A. castellanii to inhabit a diversity of environments. Alginate lyase may provide access to bacteria within biofilms by breaking down the biofilm matrix, and polyhydroxybutyrate depolymerase may facilitate utilization of the bacterial storage compound polyhydroxybutyrate as a food source. Enzymes for the synthesis and breakdown of cellulose were identified, and they likely participate in encystation and excystation as in D. discoideum. Trehalose-6-phosphate synthase is present, suggesting that trehalose plays a role in stress adaptation. Detection and response to a number of stress conditions is likely accomplished with a large set of signal transduction histidine kinases and a set of putative receptor serine/threonine kinases similar to those found in E. histolytica. Serine, cysteine and metalloproteases were identified, some of which are likely involved in pathogenicity.     

The role of proteases in the differentiation of Acanthamoeba castellanii

Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii . Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.     

Anaerobiosis-induced differentiation of Acanthamoeba castellanii

Acanthamoeba castellanii is a free living amoeba ubiquitous in soil and also commonly found in aquatic environments. In waterlogged soils, anoxia is quickly established as the dissolved oxygen is consumed by the organisms present. We were interested in the effects of anoxic conditions upon this organism. Batch cultures degassed with N₂ during mid-exponential growth, induced encystation within 12 h of anoxia, and mature cysts were formed within 2–3 days. Excystation (99%) was achieved by subsequent aeration of these cultures after 3–6 days. Anoxia-induced cysts, maintained in anoxic conditions for up to four months, remained viable. Difference spectra, during anaerobiosis, revealed that cytochromes were not lost, suggesting that the organism retains its respiratory components. The growth rate of trophozoites, grown in a chemostat, was dependent on the concentration of O₂ in the head space and glucose uptake increased at lower dissolved O₂ tensions. The results obtained suggest that A. castellanii has a complex adaptive strategy enabling it to cope with microaerobic and anoxic conditions which may be experienced in the environment.     

Cell surface control of differentiation in acanthamoeba

Acanthamoeba castellanii (Neff) is a free-living soil amoeba with close relatives that are opportunistic pathogens. Trophozoites differentiatite into cysts when deprived of nutrients; cysts convert into trophozoites, leaving the well 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 effect on pinocytosis.     

Acid Hydrolase Activity During Growth and Encystment in Acanthamoeba castellanii*

SYNOPSIS. The activity and sedimentation of acid phosphatase (APase), acid deoxyribonuclease (DNase), and acid ribonuclease (RNase) were investigated throughout growth and encystment in Acanthamoeba castellanii. The activities/mg protein of all 3 hydrolases are high in young cultures and decrease to constant levels in postlog cells. The RNase activity/ ameba decreases 50% during growth, whereas the activity/cell of both APase and DNase remains constant. The percent sedimentation at 20,000 g of all 3 enzymes gradually increases from about 40% in midlog to a plateau of 80% in postlog cells. During encystment, the sedimentation behavior of RNase differs from that of APase and DNase. Encystment is characterized by a differential decrease in the activity/cell of the 3 hydrolases, with RNase decreasing most rapidly and APase least rapidly. APase is unique in that a transient increase of its specific activity is noted during encystment, even though its activity/cell is decreasing.     

Preparation of an immunotoxin for Acanthamoeba castellanii

Acanthamoeba castellanii (Neff) is a small free-living amoeba which is closely related to pathogenic organisms that produce almost invariably fatal human infections (1). The f(ab)' fragment of a monoclonal antibody (designated A9) specifically reactive to the A. castellanii cell surface, was covalently linked to the A chain of diphtheria toxin. This immunotoxin inhibited cell division completely, whereas nonspecific mouse IgG or nonspecific f(ab)' derivatized with diphtheria toxin A chain (DTA), A chain alone, or unlinked A chain and A9-f(ab)' in combination, had no effect on cell division.     

Acanthamoeba castellanii: characterization of an adhesin molecule.

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.     

A comparison of the growth and starvation responses of Acanthamoeba castellanii and Hartmannella vermiformis in the presence of suspended and attached Escherichia coli K12

The growth and starvation responses of Acanthamoeba castellanii and Hartmannella vermiformis were investigated in the presence and absence of Escherichia coli on an agar surface or within shaken suspensions. The amoebae perceived all the suspended systems to be unfavourable for growth, despite being challenged with high levels of prey, and as a consequence they exhibited a starvation response. However, the response differed between species, with A. castellanii producing characteristic cysts and H. vermiformis producing round bodies. These amoebic forms were reactivated into feeding trophozoites in the presence of bacterial aggregates, which formed in the suspended systems after 68 h of incubation. In contrast, both species of amoebae grew well in the presence of attached E. coli at a concentration of 1 x 10(6) cells cm(-2) of agar and yielded specific growth rates of c. 0.04 h(-1). Starvation responses were induced at the end of the growth phase, and these were equivalent to those recorded in the suspended systems. We conclude that, when suspended, amoebae in the 'floating form' cannot feed effectively on suspended prey, and hence the starvation response is initiated. Thus the majority of amoebic feeding is via trophozoite grazing of attached bacterial prey.     

Spin label studies of microsomal membranes from Acanthamoeba castellanii in different states of differentiation

Two fatty acid spin labels—[I(1,14)], stearic acid bearing a paramagnetic nitroxide group on carbon 16, and [I(12,3)], stearic acid bearing a paramagnetic nitroxide group on carbon 5—have been used to compare the physical properties of lipid in rough and smooth microsomal membranes from trophozoites and cysts of Acanthamoeba castellanii. Arrhenius plots of rotational correlation times (τ_c) calculated from the spectra for I(1,14) showed an abrupt discontinuity in slope for membranes from both trophozoites and cysts. This occurred at temperatures ranging from ?3 to 1 °C for smooth microsomes and from 8 to 11 °C for rough microsomes for both cysts and amoebae. The value of τ_c at 29 °C, the culturing temperature, in effect scores fluidity of the membrane matrix, and did not show any significant difference for either rough or smooth microsomes during the transition from exponential to stationary phase growth. However, smooth microsomes from cysts showed a 14% increase in fluidity relative to trophozoites, and the fluidity of rough microsomes from cysts tended to be lower. An order parameter (S) calculated from spectra for I(12,3) did not change as a function of encystment for the smooth membranes and increased only slightly for rough microsomes. The activation energy (E_a) for Arrhenius plots of τ_c above the inflection temperature increased as a result of encystment, indicating a greater degree of molecular interaction within the cyst membranes. Moreover, the τ_c plots for both rough and smooth microsomal membranes from trophozoites tended to converge at 29 °C, the growth temperature, whereas plots for cyst membranes were virtually parallel, bracketing those for the trophozoite membranes. This suggests that the trophozoite is able to regulate its membrane fluidity and that cysts, which are resting cells, have lost this regulatory capacity.     

Purification and characterization of an extracellular serine proteinase from Acanthamoeba castellanii

An extracellular proteinase of Acanthamoeba castellanii was purified and its biochemical and pathological properties were characterized. The molecular mass of the purified enzyme was approximately 42 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 HR gel-filtration chromatography. Therefore, its structure seemed to be monomeric with a single polypeptide. Its activity was inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride. Its activity was optimum at 30 to 50 degrees C with a maximum at 50 degrees C; optimal pH was 8.0. As much as 70% of the enzyme activity was maintained at 50 degrees C for at least 12 h but was rapidly inactivated thereafter. The purified enzyme degraded collagen and rabbit corneal extract. Furthermore, it exhibited strong cytopathic effects on human corneal epithelial cells and fibroblast cells. These suggest the possible role of this enzyme in the pathogenesis of Acanthamoeba.     

Elucidation of iron homeostasis in Acanthamoeba castellanii

Acanthamoeba castellanii is a ubiquitously distributed amoeba that can be found in soil, dust, natural and tap water, air conditioners, hospitals, contact lenses and other environments. It is an amphizoic organism that can cause granulomatous amoebic encephalitis, an infrequent fatal disease of the central nervous system, and amoebic keratitis, a severe corneal infection that can lead to blindness. These diseases are extremely hard to treat; therefore, a more comprehensive understanding of this pathogen’s metabolism is essential for revealing potential therapeutic targets. To propagate successfully in human tissues, the parasites must resist the iron depletion caused by nutritional immunity. The aim of our study is to elucidate the mechanisms underlying iron homeostasis in A. castellanii. Using a comparative whole-cell proteomic analysis of cells grown under different degrees of iron availability, we identified the primary proteins involved in Acanthamoeba iron acquisition. Our results suggest a two-step reductive mechanism of iron acquisition by a ferric reductase from the STEAP family and a divalent metal transporter from the NRAMP family. Both proteins are localized to the membranes of acidified digestive vacuoles where endocytosed medium and bacteria are trafficked. The expression levels of these proteins are significantly higher under iron-limited conditions, which allows Acanthamoeba to increase the efficiency of iron uptake despite the observed reduced pinocytosis rate. We propose that excessive iron gained while grown under iron-rich conditions is removed from the cytosol into the vacuoles by an iron transporter homologous to VIT/Ccc1 proteins. Additionally, we identified a novel protein that may participate in iron uptake regulation, the overexpression of which leads to increased iron acquisition.     

Differential stimulation of microglial pro-inflammatory cytokines by Acanthamoeba culbertsoni versus Acanthamoeba castellanii

Acanthamoeba spp. are opportunistic pathogens that cause granulomatous amebic encephalitis. We compared the highly pathogenic species A. culbertsoni to the relatively less pathogenic species A. castellanii for its capacity to elicit from neonatal rat microglia the gene expression of pro-inflammatory cytokines. Acanthamoeba culbertsoni elicited a robust cytokine gene response by neonatal rat microglia in vitro as compared to A. castellanii. The preponderant cytokine elicited at the mRNA and protein levels was interleukin-1beta. In addition, transmission electron microscopy revealed that microglial cells were capable of phagocytozing A. castellanii. In contrast, A. culbertsoni destroyed microglia. Collectively, these results suggest that a combined action of pro-inflammatory cytokines and destruction of host cells by amebae contribute to the pathology caused by the more pathogenic species.     

Purification and characterization of an ATP-sensitive actin gelation protein from Acanthamoeba castellanii

A protein which cross-links actin filaments in a nucleotide-sensitive manner has been purified to homogeneity from Acanthamoeba castellanii. This protein, GF-210, is a slightly asymmetric molecule composed of six subunits, each with an apparent mass of 35,000 Da. As determined by the method of falling ball vicometry, GF-210 was shown to cross-link actin filaments at hexamer:actin molar ratios of 1:500, with gelation occurring at molar ratios of 1:300 and higher. Actin gels did not form in the presence of 10 microM ATP, and filament cross-linking was completely inhibited by 100 microM ATP. Although ATP was the most effective inhibitor of actin filament cross-linking, other phospho-compounds including ADP, GTP, sodium phosphate, and sodium pyrophosphate prevented gelation at concentrations lower than 1.5 mM. In contrast, 50 mM KCl was required to inhibit the formation of actin networks. Direct binding studies showed that GF-210 binds to F-actin with a KD of 1.2 microM in the absence of ATP but with a KD of 72.8 microM in the presence of 2 mM ATP. This weakening of the interaction between F-actin and GF-210 may explain the inhibition of GF-210-induced actin cross-linking by nucleotides and other phospho-compounds.     

Effects of Mannose on Pathogenesis of Acanthamoeba castellanii

Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.     

Differentially expressed genes of Acanthamoeba castellanii during encystation

To examine the expressed gene profile during encystation of Acanthamoeba castellanii Castellani, we used differentially expressed gene (DGE) screening by RT-PCR with 20 sets of random primers. From this analysis, we found that approximately 16 genes showed upregulation during encystation. We chose 6 genes, which had relatively higher expression levels, for further investigation. Based on homology search in database, DEG2 showed 55% of similarity with xylose isomerase, DEG9 showed 37% of similarity with Na P-type ATPase, and DEG14 showed 77% of similarity with subtilisin-like serine proteinase. DEG3 and DEG26 were identified as hypothetical proteins and DEG25 exhibited no significant similarity to any known protein. Encystation of Acanthamoeba has been suggested to be a process to resist adverse environmental or nutritional conditions. Further characterization studies of these genes may provide us with more information on the encystation mechanism of Acanthamoeba.     

Callus formation versus differentiation of cultured barley embryos: Hormonal and ostomic interactions

Summary The effect of various plant growth substances as single agents was evaluated in a complex tissue culture system: whole embryo culture of early differentiating barley embryos. Callus formation in unsupplemented medium derives from the mesocotyl and is uniquely characteristic of cultures initiated at this stage of embryonic development. This phenomenon could be prevented or reversed by incorporation of gibberellic acid in the medium resulting in plantlet formation. Indoleacetic acid enhanced callus growth, whereas kinetin did not promote either callus or meristematic development. Callus tissue markedly accumulated starch, effectively lowering the cellular osmolarity, while inducing a corresponding rise in the osmolarity of the culture medium. This osmotic pattern was reversed by gibberellic acid induction of shoot formation. Thise osmotic-hormonal interactions are interpreted relative to in vivo, in situ normal embryogeny or developmental lesions such as tumours. Research supported by Inst. Grant No. 994.     

Naegleria fowleri: enolase is expressed during cyst differentiation

Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri.