A Monoclonal Antibody Against a Symbiont-Synthesized Protein in the Cytosol of Symbiont-Dependent Amoebae1

A monoclonal antibody was obtained against a 29-kD polypeptide in the cytosol of a symbiont-bearing strain (xD) of Amoeba proteus and was used to determine the distribution of the antigen in amoebae. The 29-kD polypeptides (xD protein) are produced by bacterial endosymbionts that are necessary for the survival of host xD amoebae. Results of indirect immunofluorescent and electron-microscopic immunogold-labeling studies showed that the xD protein was present diffusely in the amoeba cytoplasm as well as in the symbiotic bacteria. The native protein containing 29-kD polypeptides was purified using an immunoaffinity column prepared with the monoclonal antibody and its molecular weight was determined to be 87,000.     

Respiratory Studies on the Giant Amoeba Pelomyxa palustris1

ABSTRACT. The large fresh-water microaerobic amoeba Pelomyxa palustris does not contain mitochondria, but three types of bacterial endosymbionts are always present. Thus, it is of interest in the discussion of the possible origin of mitochondria from primitive prokaryotes. Gas exchanges (O2 , CO2 ) and concentration of endosymbionts were determined in individual amoebae, in which the life cycle stage was noted. Grey type (stationary phase) amoebae had a lower O2 uptake and lower endosymbiont concentration than light type (growth phase) amoebae, and highest O2 uptake was found in centrifugal pieces of light type Pelomyxa, centrifuged in vivo, which contained nearly all of the endosymbionts. In light type amoebae, the respiratory activity was independent of O2 concentration between 1 and 21%, and, when compared on the basis of dry weight and protein, of the same order as that of other free-living protozoa. The R.Q. was slightly higher than 1, indicating that glycolysis does not play a significant role in energy metabolism. It is concluded that P. palustris is fully aerobic, and suggestions are presented as to the role of the endosymbionts in its respiratory metabolism.     

Nuclear Lethal Effect and Nucleocytoplasmic Incompatibility Induced by Endosymbionts in Amoeba proteus1

ABSTRACT. The role of bacterial endosymbionts in the acquisition of new phenotypic characters was studied by transplanting nuclei from an uninfected strain of Amoeba proteus into the enucleated cytoplasm of a symbiont-carrying strain. After 1–10 cell cycles, the nuclei were tested for two characters: compatibility with uninfected and infected cytoplasm, and their lethal effect against amoebae of the uninfected parent strain. A significant number of transplanted nuclei displayed both of the new phenotypic traits after a few divisions in the infected cytoplasm. Thus the influence of these endosymbionts on the nucleus of A. proteus was virtually instantaneous.     

Endosymbiosis in amoebae: Recently established endosymbionts have become required cytoplasmic components

A strain of large, free-living amoeba that became dependent on bacterial endosymbionts which had infected the amoebae initially as intracellular parasites, was studied by micrurgy and electron microscopy. The results show that the infected host cells require the presence of live endosymbionts for their survival. Thus, the nucleus of an infected amoeba can form a viable cell with the cytoplasm of a noninfected amoeba only when live endosymbionts are present. The endosymbiotic bacteria are not digested by the host amoebae and are not themselves used as nutritional supplement. While the host amoebae are dependent specifically on the endosymbionts, the latter can live inside amoebae of different strains, indicating that their dependence on the host cells is not yet strain specific.     

Structural and biochemical characteristics of the plasmalemma and vacuole membranes in amoebae

The plasmalemma, phagolysosomes and symbiont-containing vesicles of amoebae were isolated and their membrane components were compared by SDS-polyacrylamide gel electrophoresis and radioautography. Both morphological and compositional changes occurred in the course of plasmalemma-to-phagolysosome membrane transition during phagocytosis; the number of PAS-staining bands and the staining intensity decreased, whereas Coomassie blue-stainable and iodinatable polypeptides increased in the number of bands and staining intensity. The membranes of symbiont-containing vesicles which did not fuse with lysosomes contained one large-molecular-weight component which was not found either in the plasmalemma or phagolysosomal membranes. The significance of these findings is discussed in relation to the observed selectivity of membrane fusion.     

Enhancement of in vitro cytopathogenicity by Acanthamoeba spp. following acquisition of bacterial endosymbionts

Approximately one in five isolates of Acanthamoeba spp. recovered from clinical and environmental sources are found to harbor obligate, uncultured bacterial endosymbionts of unknown clinical significance. To investigate their possible role in amoebic pathogenesis, four uninfected amoebic strains were exposed to four different endosymbionts, from which 12 stably-infected host-symbiont pairs resulted. Standardized inocula of amoebae with and without endosymbionts were placed on fibroblast monolayers to examine for cytopathic effects (CPEs). Eight to 10 days were required for monolayer effacement by endosymbiont-free amoebae; 5–8 days for amoebae containing Gram-negative rod endosymbionts; and 3 days for two amoebic isolates infected with a Chlamydia -like endosymbiont. All endosymbiont-infected amoebae produced a statistically significant enhancement in CPEs in comparison to uninfected amoebae; endosymbionts alone on monolayers produced no CPEs. This report provides evidence that obligate bacterial endosymbionts are able to enhance amoebic pathogenic potential in vitro by some as-yet unknown mechanism.     

Genetic and physiological interactions in the amoeba-bacteria symbiosis

Amoebae of the xD strain of Amoeba proteus that arose from the D strain by spontaneous infection of Legionella-like X-bacteria are now dependent on their symbionts for survival. Each xD amoeba contains about 42,000 symbionts within symbiosomes, and established xD amoebae die if their symbionts are removed. Thus, harmful infective bacteria changed into necessary cell components. As a result of harboring X-bacteria. xD amoebae exhibit various physiological and genetic characteristics that are different from those of symbiont-free D amoebae. One of the recent findings is that bacterial symbionts control the expression of a host's house-keeping gene. Thus, the expression of the normal amoeba sams gene (sams1) encoding one form of S-adenosylmethionine synthetase is switched to that of sams2 by endosymbiotic X-bacteria. Possible mechanisms for the switching of sams genes brought about by endosymbionts and its significance are discussed.     

Bakterien,die in Acanthamöben leben: Dasein im Verborgenen

One fourth of Acanthamoebaisolates studied contain obligate bacterial endosymbionts. These intracellular bacteria have recently been assigned to four different, previously unknown phylogenetic lineages within the Proteobacteriaand the Chlamydiales. The symbiotic association of these amoebae and their bacterial endosymbionts might be a valuable model system for the analysis of bacterial adaptations and mechanisms for intracellular survival. In addition, Chlamydia‐related amoebal endosymbionts have been implicated as causative agents for respiratory disease suggesting that these protozoa may be sources of new emerging pathogens.     

Ultrastructure of the Giant Amoeba Pelomyxa palustris*

SYNOPSIS. The ultrastructure of the herbivorous amoeba Pelomyxapalustris was studied. Nuclear division is not understood in this amoeba, and evidence for the method of nuclear division was sought. This species typically has many spheroidal nuclei which are similar within a given cell. However, some amoebae from our collections differed from this common type in both the number and structure of their nuclei. This suggested stages associated with nuclear division. One current hypothesis of nuclear division in this organism is that of nuclear budding. Our evidence is more in accord with this method than with mitosis. The cytoplasm contained no mitochondria, Golgi bodies, contractile vacuoles or crystals. Most amoebae had 2 types of bacteria (bacteroids or endosymbionts) in their cytoplasm; a separate vesicle enclosed each of these. Characteristically, only 1 type of bacterium (B_n) surrounded the nucleus. Another type (B) was found elsewhere in the cytoplasm. Also in the cytoplasm were the following: food vacuoles enclosing various algae, relatively clear vacuoles and vesicles, glycogen, various electron-opaque particles, and occasional microtubules. The plasmalemma was smooth, lacking the external fringe which characterizes other large fresh-water amoebae.     

From the Macro to the Micro: Gel Mapping to Differentiate between Sporozoites of Two Immunologically Distinct Strains of Eimeria maxima (Strains M6 and Guelph)

Two immunologically distinct strains of E. maxima were examined in this study: the M6 strain and the Guelph strain. The differential expression between the sporozoites of the two strains of E. maxima was determined by image analysis of 100 μg of protein from each strain separated by standard one- and conventional two-dimensional polyacrylamide gel electrophoresis. In addition to differences in both molecular weight and the electrophoretic mobility, differences in the intensity of polypeptide bands for example, GS 136.4 and M6 169 were explored. Pooled gels were prepared from each strain. A representative 2D-PAGE gel spanning a non-linear pH range of 3–10 of E. maxima strain M6 consisted of approximately 694 polypeptide spots with about 67 (9.6%) of the polypeptide spots being unique relative to the other strain. E. maxima strain GS had about 696 discernable polypeptide spots with 69 spots (9.9%) that differed from those of the M6 strain. In-depth characterization of the variable polypeptide spots; unique polypeptide spots (absence or presence) and shared polypeptide spots with modifications may lead to novel vaccine target in the form of multi-component, multi-stage, multi-immunovariant strains, multi-species subunit vaccine, and diagnostic probe for E. maxima.     

The importance of morphological versus chemical defences for the bloom-forming cyanobacterium Microcystis against amoebae grazing

Amoebae grazing can be an important loss factor for blooms of the common cyanobacterium Microcystis. Some Microcystis strains seem to be protected against amoebae grazing, but it is unclear whether this is achieved by their colony morphology or biochemically. These factors were investigated in grazing experiments using two Microcystis-grazing amoebae (Korotnevella sp. and Vannella sp.) and two Microcystis strains with differing colony morphology (aeruginosa and viridis morphotype) and different sensitivity to amoebae grazing. Amoebae did not increase in density and failed to reduce the growth rate of cultures of the amoebae insensitive viridis strain, irrespective of whether the Microcystis strain was colonial or unicellular. This suggests that the extended mucilage matrix surrounding viridis colonies is not the main defence mechanism against amoebae grazing. At the same time, the growth rate of both unicellular and colonial cultures of the amoebae-sensitive aeruginosa strain was heavily reduced by the growing amoebae. The addition of filtered viridis-conditioned medium to aeruginosa cultures significantly decreased both amoebae growth and its effect on aeruginosa growth rates, which indicates that extracellular compounds constitutively produced by viridis are at least partially responsible for their insensitivity to amoebae grazing. These results demonstrate the potential importance of chemical interactions between lower trophic levels (protists) for Microcystis bloom dynamics.     

Novel oligonucleotide probes for in situ detection of pederin-producing endosymbionts of Paederus riparius rove beetles (Coleoptera: Staphylinidae)

Bacterial endosymbionts from female Paederus rove beetles are hitherto uncultured, phylogenetically related to Pseudomonas sp., and produce the polyketide pederin, which exhibits strong cytotoxic effects and antitumoral activities. The location of such endosymbionts inside beetles and on beetles' eggs is hypothesized based on indirect evidence rather than elucidated. Thus, an endosymbiont-specific and a competitor oligonucleotide probe (Cy3-labelled PAE444 and unlabelled cPAE444, respectively) were designed and utilized for FISH with semi-thin sections of Paederus riparius eggs. Cy3-PAE444-positive cells were densely packed and covered the whole eggshell. Hundred percent of EUB338-Mix-positive total bacterial cells were PAE444 positive, indicating a biofilm dominated by Paederus endosymbionts. Analysis of different egg deposition stadiums by electron microscopy and pks (polyketide synthase gene, a structural gene associated with pederin biosynthesis)-PCR supported results obtained by FISH and revealed that the endosymbiont-containing layer is applied to the eggshell inside the efferent duct. These findings suggest that P. riparius endosymbionts are located inside unknown structures of the female genitalia, which allow for a well-regulated release of endosymbionts during oviposition. The novel oligonucleotide probes developed in this study will facilitate (1) the identification of symbiont-containing structures within genitalia of their beetle hosts and (2) directed cultivation approaches in the future.     

Serological Comparison of Selected Parasitic and Free-Living Amoebae in vitro,Using Diffusion-Precipitation and Fluorescent-Antibody Technics*

SYNOPSIS. The present study has been directed to a serological comparison of three closely similar species of Entamoeba: E. histolytica, E. invadens and E. moshkovskii, and two free-living soil amoebae: Hartmannella rhysodes and Mayorella palestinensis. Except for E. histolytica and E. moshkovskii, the other amoebae used here were grown axenically; this is the first report of the use of antigenic extracts from axenic cultures of parasitic amoebae. The method described here provides a potent antigen that elicits a good antibody titer and is generally applicable to both parasitic and free-living amoebae. Amoebae pooled from well-grown cultures were thoroughly washed, sonicated and mixed with Freund's Adjuvant; this antigenic preparation was injected into rabbits. Two subcutaneous injections were given at three-week intervals, and 2-3 weeks thereafter blood was withdrawn to obtain antiserum. Agar-gel diffusion, cellulose acetate paper and fluorescent antibody technics were used to test the antigen-antibody (Ag-Ab) reactions. Results of the Ag-Ab reactions can be summarized as follows: 1) The homologous Ag-Ab reaction was obtained in all cases tested. 2) There was no serological reaction between the parasitic and free-living amoebae tested. 3) There was a definite serological reaction between H. rhysodes and M. palestinensis. 4) Multiple antigens were found in E. invadens (PZ strain) and E. histolytica (DKB strain) when they were tested against anti-PZ serum and anti-DKB serum, respectively, and no reaction was found when the other test antigens were exposed to these two antisera in gel-diffusion tests. 5) With the fluorescent antibody technic, E. histolytica (Laredo strain), E. moshkovskii (DSR strain) and E. histolytica (DKB strain) showed some degree of serological reaction in descending order when they were stained with conjugated anti-E. invadens serum.     

Cytopathogenicity of Naegleria fowled for Cultured Rat Neuroblastoma Cells1

The cytopathogenicity of Naegleria fowleri strain LEE (ATCC-30894) for cultured rat neuroblastoma cells (B-103) has been investigated. Both live N. fowleri amoebae and Naegleria lysates added to ⁵¹Cr-labeled B-103 cells caused release of radiolabel, which was dependent upon the ratio of amoebae to target cells or to the lysate concentration. Lysates of N. fowleri strains LEE, NF-66, NF-69, and HB-4 were equally injurious to B-103 target cells whereas lysates of strains 6088 and KUL were less cytotoxic. Highly pathogenic mouse-passaged strain LEE were less cytotoxic than axenically grown amoebae. Maximum cytotoxicity was observed in lysates from amoebae in late exponential or early stationary phase of growth. Cytopathogenicity of lysates was reduced after heating at 44°C for 60 min or at 60°C for 30 min. Cytotoxicity was stable during storage at 4°C or at ?20°C for 26 h. Neither live amoebae nor lysates injured B-103 target cells at 4°C. Live amoebae and lysates injured B-103 by a time, temperature, and concentration dependent process.     

Polypeptide compositions of amoebae of the cellular slime mouldDictyostelium discoideum separated by partitioning during development

Amoebae of the cellular slime mouldDictyostelium discoideum at 8 h or l0 h development were separated into two populations by countercurrent distribution in a dextran-poly(ethylene glycol), two-phase system. Two-dimensional, polyacrylamide-gel electrophoresis was then used to separate the polypeptides from the populations of amoebae. The two populations of amoebae at 8 h development differed sn polypeptide composition as did the populations separated at 10 h development. This confirms that cell differentiation is initated inD. discoideum prior to g h development.     

An Acanthamoeba sp. containing two phylogenetically different bacterial endosymbionts

Acanthamoebae are ubiquitous free-living amoebae and important predators of microbial communities. They frequently contain obligate intracellular bacterial symbionts, which show a worldwide distribution. All Acanthamoeba spp. described so far harboured no or only a single specific endosymbiont phylotype, and in some cases evidence for coevolution between the symbiotic bacteria and the amoeba host has been reported. In this study we have isolated and characterized an Acanthamoeba sp. (strain OEW1) showing a stable symbiotic relationship with two morphologically different endosymbionts. 16S rRNA sequence analysis assigned these symbionts to the candidate genus Procabacter (Betaproteobacteria) and the genus Parachlamydia (Chlamydiae) respectively. Fluorescence in situ hybridization and transmission electron microscopy confirmed the affiliation of the endosymbionts and showed their co-occurrence in the amoeba host cells and their intracellular location within separate compartments enclosed by host-derived membranes. Further analysis of this stable relationship should provide novel insights into the complex interactions of intracellular multiple-partner associations.     

Genetic variants of the Bombyx mori silkworm encoding sericin proteins of different lengths

A variant sericin polypeptide originally found by acid gel electrophoresis in the Nd-s mutant strain of the silkworm, Bombyx mori, has been analyzed genetically. The vriant polypeptide (called S-2^v) is encoded by a gene which behaves as a codominant allele of the gene encoding the standard S-2 sericin polypeptide. Linkage analysis locates these alleles at 0.0 map unit on chromosome 11. SDS-polyacrylamide gel electrophoresis shows that the molecular weight of the S-2^v variant polypeptide is lower by approximately 62,500 than that of the S-2 polypeptide. Amino acid analysis indicates that the two sericin polypeptides have similar compositions. These results are consistent with the idea that the variant allele arose by deletion within the S-2 coding sequence in the Src-2 gene locus as the result of unequal recombination.     

A HYPOTHESIS FOR IMPORT OF THE NUCLEAR‐ENCODED PsaE PROTEIN OF PAULINELLA CHROMATOPHORA (CERCOZOA,RHIZARIA) INTO ITS CYANOBACTERIAL ENDOSYMBIONTS/PLASTIDS VIA THE ENDOMEMBRANE SYSTEM1

The cyanobacterial endosymbionts of Paulinella chromatophora can shed new light on the process of plastid acquisition. Their genome is devoid of many essential genes, suggesting gene transfer to the host nucleus and protein import back into the endosymbionts/plastids. Strong evidence for such gene transfer is provided by the psaE gene, which encodes a PSI component that was efficiently transferred to the Paulinella nucleus. It remains unclear, however, how this protein is imported into the endosymbionts/plastids. We reanalyzed the sequence of Paulinella psaE and identified four potential non‐AUG translation initiation codons upstream of the previously proposed start codon. Interestingly, the longest polypeptide, starting from the first UUG, contains a clearly identifiable signal peptide with very high (90%) predictability. We also found several downstream hairpin structures that could enhance translation initiation from the alternative codon. These results strongly suggest that the PsaE protein is targeted to the outer membrane of Paulinella endosymbionts/plastids via the endomembrane system. On the basis of presence of respective bacterial homologs in the Paulinella endosymbiont/plastid genome, we discuss further trafficking of PsaE through the peptidoglycan wall and the inner envelope membrane. It is possible that other nuclear‐encoded proteins of P. chromatophora also carry signal peptides, but, alternatively, some may be equipped with transit peptides. If this is true, Paulinella endosymbionts/plastids would possess two distinct targeting systems, one cotranslational and the second posttranslational, as has been found in higher plant plastids. Considering the endomembrane system‐mediated import pathway, we also discuss homology of the membranes surrounding Paulinella endosymbionts/plastids.     

Isolation and Characterization of the Putative Photoreceptor for Phototaxis in Amoebae of the Cellular Slime Mold,Dictyostelium discoideum

Amoebae of the cellular slime mold Dictyostelium discoideum (strain AX2) produce a pigment with an absorption spectrum that closely resembles the action spectrum for phototaxis. The protein-pigment complex was isolated and purified by sucrose gradient centrifugation, fast protein liquid chromatography (FPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). It is tightly membrane-bound and the bulk of it is located in the mitochondrial membrane fraction, while a small part is located in the cytoplasmic membrane fraction, as indicated by marker enzyme tests (succinate dehydrogenase for mitochondria and alkaline phosphatase for the cytoplasmic membrane). It is speculated that the pigment bound to the cytoplasmic membrane acts as photoreceptor and that bound to the mitochondria operates as a shading pigment in the light direction perception mechanism of Dictyostelium amoebae.     

Living together: the marine amoeba Thecamoeba hilla Schaeffer, 1926 and its endosymbiont Labyrinthula sp

Two protists isolated simultaneously from the same sample of gill tissue of Psetta maxima (L.) were identified as Thecamoeba hilla Schaeffer, 1926 and Labyrinthula sp. A Labyrinthula strain (LTH) derived from a mixed culture of both organisms was well established in a short time, while subcultures of T. hilla continued to be associated with Labyrinthula cells despite all efforts to eliminate them. Ultrastructural examination, repeated several times in the course of long-lasting subculturing of amoebae, revealed that trophozoites of T. hilla host in their cytoplasm multiplying labyrinthulid cells. Comparison of SSU rDNA sequences of the Labyrinthula strain LTH and those from labyrinthulid endosymbionts from T. hilla verified the assumption that the extra- and intra-cellularly multiplying Labyrinthula cells are identical organisms. The association of the marine amoeba T. hilla and Labyrinthula sp. displayed signs of mutualistic symbiosis.