Naturally acquired antibody responses to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in Korea.

We expressed a protein in Saccharomyces cerevisiae in order to evaluate the humoral immune responses to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax. This protein (Pv200(18)) had a molecular mass of 18 kDa and was reactive with the sera of individuals with patent vivax malaria on immunoblotting analysis. The levels of immunoglobulin M (IgM) and IgG antibodies against Pv200(18) were measured in 421 patients with vivax malaria (patient group), 528 healthy individuals from areas of nonendemicity (control group 1), and 470 healthy individuals from areas of endemicity (control group 2), using the indirect enzyme-linked immunosorbent assay (ELISA) method. To study the longevity of the antibodies, 20 subjects from the patient group were also tested for the antibody levels once a month for 1 year. When the cutoff values for seropositivity were determined as the mean + 3 x standard deviation of the antibody levels in control group 1, both IgG and IgM antibody levels were negative in 98.5% (465 of 472) of control group 2. The IgG and IgM antibodies were positive in 88.1% (371 of 421) and 94.5% (398 of 421) of the patient group, respectively. The IgM antibody became negative 2 to 4 months after the onset of symptoms, whereas the IgG antibody usually remained positive for more than 5 months. In conclusion, indirect ELISA using Pv200(18) expressed in S. cerevisiae may be a useful diagnostic method for vivax malaria.

Role of cAMP-dependent pathway in eosinophil apoptosis and survival.

The survival and apoptosis of eosinophils is of pivotal importance for controlling allergic diseases such as asthma and rhinitis. In this study we have investigated the role for cAMP in regulating eosinophil survival and apoptosis in the absence of eosinophil-active cytokines. The treatment with dibutyryl cyclic AMP (dbcAMP) increased eosinophil survival with a concomitant decrease of apoptosis in a dose-dependent manner. The pretreatment with a protein kinase A (PKA) inhibitor blocked the effects of dbcAMP on survival and apoptosis of eosinophils. The catalytic subunit of PKA was translocated to nucleus in parallel with a robust increase of intracellular cAMP levels upon exposure to dbcAMP but not IL-5, suggesting the separation of PKA activation from the IL-5-induced suppression of eosinophil apoptosis. When eosinophils were treated with pharmacological inhibitors of protein kinases prior to exposure to dbcAMP or IL-5, only the mitogen-activating protein kinase (MAPK) inhibitor, PD098059, was partly able to block dbcAMP-induced augmentation of eosinophil viability, whereas both Janus kinase 2 and MAPK inhibitors effectively interrupted the IL-5-induced prolongation of eosinophil survival. The effects of dbcAMP and these protein kinase inhibitors on eosinophil apoptosis were confirmed by morphologic analysis. We propose that a cAMP-dependent pathway may constitute an important component for regulating eosinophil survival/apoptosisand that cAMP may inhibit eosinophil apoptosis through the activation of PKA and of subsequent MAPK in part.

Characterization of myosin heavy chain and its gene in Amoeba proteus.

Monoclonal antibodies against the myosin heavy chain of Amoeba proteus were obtained and used to localize myosin inside amoebae and to clone cDNAs encoding myosin. Myosin was found throughout the amoeba cytoplasm but was more concentrated in the ectoplasmic regions as determined by indirect immunofluorescence microscopy. In symbiont-bearing xD amoebae, myosin was also found on the symbiosome membranes, as checked by indirect immunofluorescence microscopy and by immunoelectron microscopy. The open reading frame of a cloned myosin cDNA contained 6,414 nucleotides, coding for a polypeptide of 2,138 amino acids. While the amino-acid sequence of the globular head region of amoeba's myosin had a high degree of similarity with that of myosins from various organisms, the tail region building a coiled-coil structure did not show a significant sequence similarity. There appeared to be at least three different isoforms of myosins in amoebae, with closely related amino acids in the globular head region.

Evidence for symbiont-induced alteration of a host's gene expression: irreversible loss of SAM synthetase from Amoeba proteus.

Symbiont-bearing xD amoebae no longer produce a 45-kDa cytoplasmic protein that functions as S-adenosylmethionine synthetase in symbiont-free D amoebae. The absence of the protein in xD amoebae is attributable to xD amoeba's failure to transcribe the corresponding gene as a result of harboring bacterial symbionts. However, xD amoebae have about half the level of enzyme activity found in D amoebae, indicating that they use an alternative source for the enzyme. xD amoebae originated from D amoebae by bacterial infection and now depend on their symbionts for survival. xD amoebae exhibit irreversible nucleolar abnormalities when their symbionts are removed, suggesting that X-bacteria supply the needed enzyme. A monoclonal antibody against the 45-kDa protein was produced and used as a probe in cloning its corresponding cDNA. The product of the cDNA was found to have S-adenosylmethionine synthetase activity. These results show how symbiotic X-bacteria may become essential cellular components of amoeba by supplementing a genetic defect for an amoeba's house-keeping gene that is brought about by an action of X-bacteria themselves. This is the first reported example in which symbionts alter the host's gene expression to block the production of an essential protein.

Restriction endonuclease analysis of mitochondrial DNA of Acanthamoeba sp. YM-4 (Korean isolate).

Acanthamoeba sp. YM-4 is similar to A. culbertsoni based upon morphological characteristics of trophozoites and cysts. However, based on other characteristics, pathogenicity to mice, in vitro cytotoxicity and isoenzyme patterns. Acanthamoeba sp. YM-4 was quite different from A. culbertsoni. Restriction fragment length polymorphism (RFLP) analysis of mtDNA is useful in the classification of members belonging to the genus Acanthamoeba. Therefore, in this study, RFLP analysis of Acanthamoeba mtDNAs was accomplished using five restriction enzymes: HaeIII, HindIII, ClaI, PvuII and SalI. Each restriction enzyme produced approximately 3-15 fragments (range: from 0.6 kbp to 34.4 kbp). The mtDNA genome size, calculated by the summation of restriction fragments, averaged 46.4 kbp in Acanthamoeba sp. YM-4, 48.3 kbp in A. culbertsoni and 48.8 kbp in A. polyphaga, respectively. Digested mtDNA fragments of Acanthamoeba sp. YM-4 contained nine and seven same size fragments, respectively, from a total of 67 and 69 fragments observed in A. culbertsoni and A. polyphaga. An estimate of the genetic divergence was 10.1% between Acanthamoeba sp. YM-4 and A. culbertsoni, and 9.9% between Acanthamoeba sp. YM-4 and A. polyphaga.

A symbiont-produced protein and bacterial symbiosis in Amoeba proteus.

Gram symbiotic X-bacteria present in the xD strain of Amoeba proteus as required cell components, synthesize and export a large amount of a 29-kDa protein (S29x) into the host's cytoplasm across bacterial and symbiosome membranes. The S29x protein produced by E. coli transformed with the s29x gene is also rapidly secreted into the culture medium. Inside amoebae, S29x enters the host's nucleus as detected by confocal and immunoelectron microscopy, although it is not clear if S29x is selectively accumulated inside the nucleus. The deduced amino-acid sequence of S29x has a stretch of basic amino acids that could act as a nuclear localization signal, but there is no signal peptide at the N-terminus and the transport of S29x is energy independent. The functions of S29x are not known, but in view of its prominent presence inside the amoeba's nucleus, S29x is suspected to be involved in affecting the expression of amoeba's nuclear gene(s).

The s29x gene of symbiotic bacteria in Amoeba proteus with a novel promoter.

Gram-symbiotic bacteria (called X-bacteria), present in the xD strain of Amoeba proteus as required cell components, synthesize and export a large amount of a 29-kDa protein, S29x. S29x is exported into the host's cytoplasm across the bacterial membranes and the symbiosome membrane. The complete nucleotide (nt) sequence of the s29x gene of X-bacteria has been determined, and the promoter sequence and tsp have also been identified. The gene has a nonconventional promoter with putative nt sequences different from the known consensus sequences. When Escherichia coli cells are transformed with s29x, the gene is expressed and the product is secreted into the culture medium. Functions of S29x are not fully known, but it is suspected that S29x plays an important role in the symbiotic relationship between amoebae and X-bacteria.

Bacterial endosymbiosis in amoebae.

The large, free-living amoebae are inherently phagocytic. They capture, ingest and digest microbes within their phagolysosomes, including those that survive in other cells. One exception is an unidentified strain of Gram-negative, rod-shaped bacteria that spontaneously infected the D strain of Amoeba proteus and came to survive inside them. These bacteria established a stable symbiotic relationship with amoebae that has resulted in phenotypic modulation of the host and mutual dependence for survival.

The large, free-living amoebae: wonderful cells for biological studies.

The large, free-living amoebae have been widely used as model cells for studying a variety of biological phenomena, including cell motility, nucleocytoplasmic interactions, membrane function, and symbiosis. Results of studies by our group on amoebae as moving cells, as material for micrurgical manipulations, and as hosts for intracellular symbionts are summarized here. In particular, our recent studies of the amoeba as a microcosm, in which spontaneously infecting foreign microbes have become integrated as necessary cell components, are described in some detail. These processes have involved an initial microbial infection, mutual adaptation by the host and symbionts, and development of obligatory symbiosis. Evidence is presented to show that symbiont-derived macromolecules are involved in the protection of symbionts from digestion, the symbionts have acquired regulatory elements on their chromosomal genes to enhance production of beneficial gene products, and symbionts apparently utilize host-derived macromolecules to their benefit. These studies involved morphological observations both at light and electron microscopic levels, physiological and genetic studies, production and use of poly- and monoclonal antibodies, and molecular-biological approaches including gene cloning and sequencing. It is shown that amoebae are uniquely suited as model cells with which to study these phenomena.

A novel strong promoter of the groEx operon of symbiotic bacteria in Amoeba proteus.

Gram- symbiotic bacteria (called X-bacteria), present in the xD strain of Amoeba proteus as required cell components, contain a large amount of a 67-kDa protein, a GroEL analog. The complete nucleotide (nt) sequence of the groEx operon of X-bacteria has been determined and it has a high degree of nt identity with those of other bacterial groE operons. The groELx gene is expressed in transformed Escherichia coli and has a novel and potent promoter (P2) in addition to the heat-shock consensus promoter (P1). This is shown by the production of GroELx in Escherichia coli transformed with modified DNA clones lacking P1 and by an enhanced production of a GroELx::beta-galactosidase fusion protein when a portion of groEx containing P2 is linked to the lacZ gene. Primer-extension analyses revealed the presence of possible P2 sequences within the open reading frame of the groESx gene. It is suggested that the presence of a potent P2 in the X-bacterial gene is an adaptation for the endosymbiotic bacteria to survive within a potentially hostile intracellular environment.

Bacterial endosymbiont-derived lipopolysaccharides and a protein on symbiosome membranes in newly infected amoebae and their roles in lysosome-symbiosome fusion.

Experimental results are presented to support the view that symbiont-derived lipopolysaccharides are involved in the prevention of lysosome-symbiosome fusion in xD amoebae harboring bacterial endosymbionts. Monoclonal antibodies against lipopolysaccharides and a 96-kDa protein present on symbiosome membranes of amoebae were used to monitor the appearance of the membrane-specific components in newly infected amoebae with endosymbionts from xD amoebae. The lipopolysaccharides and protein appeared on the newly forming symbiosome membranes within 3 to 7 days, as detected by indirect immunofluorescence staining with monoclonal antibodies. The lysosome-symbiosome fusion was followed by double staining of two antigens with different monoclonal antibodies applied to the same amoeba. Antilipopolysaccharide monoclonal antibodies were detected by staining with a fluorescein isothiocyanate-conjugated secondary antibody, and a biotinylated anti-lysosomal protein monoclonal antibody was detected by staining with Texas Red-conjugated streptavidin. In xD amoebae injected with an antilipopolysaccharide antibody, lysosomes fused with some of the symbiosomes that did not fuse with lysosomes in noninjected cells.

Lysosomal membrane proteins of Amoeba proteus, as studied with monoclonal antibodies.

Monoclonal antibodies were prepared against lysosomal membrane proteins of amoebae and used to follow lysosome-phagosome fusion after induced phagocytosis. The specificity of antibodies was checked by indirect immunofluorescence microscopy, immunoelectron microscopy, and localization of the antigen in subcellular fractions. The antibody-recognized proteins started to appear on the membranes of phagolysosomes about 5 min after phagocytosis as detected by indirect immunofluorescence, and the intensity of fluorescence increased for up to 1 h. Results of injection experiments in which purified antibodies had been injected into living cells and probed by indirect fluorescence indicated that the antigens were located on the cytoplasmic side of the lysosomal membranes. Lysosomes fuse with phagosomes on the one hand but not with non-fusible vesicles such as symbiosomes on the other. The results support the view that a membrane component(s) of non-fusible vesicles somehow prevents lysosomes from fusing with them.

Role of spectrin in Amoeba proteus, as studied by microinjection of anti-spectrin monoclonal antibodies.

Spectrin is a major protein accounting for about 5% of whole-cell proteins in Amoeba proteus, and the precipitation of spectrin by intracellular injection of purified anti-spectrin monoclonal antibodies has a profound effect on cell morphology, motility, and movement-related cell activities in amoebae. Thus, amoebae injected with anti-spectrin antibodies show drastic changes in their shape and movement, suggesting that amoeba spectrin plays an important structural role, unlike nonerythroid spectrins in other cells. However, precipitation of spectrin does not affect the distribution of F-actin in amoebae.

Elevated levels of stress proteins associated with bacterial symbiosis in Amoeba proteus and soybean root nodule cells.

Obligatory bacterial endosymbionts of Amoeba proteus and symbiotic Bradyrhizobium japonicum bacteroids in soybean-root nodules contained large amounts of 67-kDa and 65-kDa proteins, respectively, antigenically related to groEL of E. coli and the 58-kDa heat-shock protein of Tetrahymena. Monoclonal antibodies against the 67-kDa protein recognized groEL analogs from several different organisms. The quantity of the stress protein in symbiotic B. japonicum bacteroids was augmented seven times that in the free-living counterparts. The increase in these proteins in endosymbionts, as determined by immunoblot techniques, indicated that intracellular symbiosis is a stress condition even when the symbiotic relationship is considered to be mutually beneficial. Mitochondria and chloroplasts may also be under a stressed condition like endosymbionts in view of the presence of heat-shock proteins in these cell organelles.

Antigenic reactivity of a monoclonal antibody against Amoeba proteus actin.

The reactivity of a monoclonal antibody against actin of Amoeba proteus with actins from other sources was examined. The monoclonal antibody cross-reacted with actins from vertebrate muscles, human erythrocytes, and Acanthamoeba castellanii, but it did not react with Naegleria gruberi actin. The amoeba actin was resolved into 3 bands with isoelectric points of 5.96, 6.03 and 6.10 in electrofocusing gels and they corresponded to 3 peptide spots reacting with the antibody on 2-dimensional immunoblots.

A spectrin-like protein present on membranes of Amoeba proteus as studied with monoclonal antibodies.

Monoclonal antibodies against a spectrin-like membrane-associated protein of xD amoebae. (Amoeba proteus) were used to determine the distribution of the protein and some of its characteristics. A total of 34 monoclonal antibodies recognizing different epitopes of the protein were obtained, of which seven stained cell membranes by indirect immunofluorescence. The spectrin-like protein had two subtypes of 225 and 220 kDa and several monoclonal antibodies cross-reacted with human erythrocyte spectrin when checked by indirect immunofluorescence staining and immunoblotting. Some of the antibodies also cross-reacted with antigens in HeLa cells and chick embryo fibroblasts. Polyclonal and monoclonal antibodies against Drosophila and human erythrocyte spectrins cross-reacted with the spectrin-like protein from amoebae. On the basis of these results, it was concluded that the protein is a spectrin. The protein was found on most cellular membranes of amoebae, including the plasma, nuclear, and phagosomal membranes, as well as symbiosome membranes.

Recycling of membrane proteins during endo- and exocytosis in amoebae.

The fate of a membrane protein of the amoeba plasmalemma was studied by means of 125I iodination by lactoperoxidase, gel electrophoresis, radioautography and gamma counting. There was only one iodinatable polypeptide group with a molecular weight (MW) of 175 000 on the external surface of the plasmalemma. Two hours or more after induced phagocytosis, isolated phagolysosomal membranes contained two other smaller polypeptides with MWs of 70 000 and 35 000, respectively, suggesting that the 175 000 polypeptide was broken down to these smaller components during endocytosis. After 22 h of induced phagocytosis, isolated plasmalemma contained a 35 000 polypeptide group in addition to the 175 000 polypeptide species. The results suggested that some of the iodinatable membrane proteins were altered and recycled during endo- and exocytosis in amoebae, while others were recycled intact.