Intracellular localization and trafficking of serine proteinase AhSub and cysteine proteinase AhCP of Acanthamoeba healyi.

Proteinases have been proposed to play important roles in pathogenesis and various biologic actions in Acanthamoeba. Although genetic characteristics of several proteases of Acanthamoeba have been reported, the intracellular localization and trafficking of these enzymes has yet to be studied. In the present study, we analyzed the intracellular localization and trafficking of two proteinases, AhSub and AhCP, of Acanthamoeba healyi by transient transfection. Full-length AhSub-enhanced green fluorescent protein (EGFP) fusion protein was found in intracellular vesicle-like structures of transfected amoebae. Time-lapse photographs confirmed the secretion of the fluorescent material of the vesicle toward the extracellular space. The mutated AhSub, of which the pre or prepro region was deleted, was found to localize diffusely throughout the cytoplasm of the amoeba rather than concentrated in the secretory vesicle. Transfection of the construct containing the pre region only showed the same localization and trafficking of the full-length AhSub. A cysteine proteinase AhCP-EGFP fusion protein showed similar localization in the vesicle-like structure in the amoeba. However, using Lyso Tracker analysis, these vesicular structures of AhCP were confirmed to be lysosomes rather than secretory vesicles. The AhCP construct with a deletion of the prepro region showed a dispersed distribution of fluorescence in the cytoplasm of the cells. These results indicated that AhSub and AhCP would play different roles in Acanthameoba biology and that the pre region of AhSub and pro region of AhCP are important for proper intracellular localization and trafficking of each proteinase.

Genetic analyses of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea.

We conducted both the small subunit ribosomal DNA (SSU rDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and mitochondrial (mt) DNA RFLP analyses for a genetic characterization of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea. Twenty-three strains of Acanthamoeba from the American Type Culture Collection and twelve clinical isolates from Korean patients were used as reference strains. Thirty-nine isolates from contact lens storage cases were classified into seven types (KA/LS1, KA/LS2, KA/LS4, KA/LS5, KA/LS7, KA/LS18, KA/LS31). Four types (KA/LS1, KA/LS2, KA/LS5, KA/LS18) including 33 isolates were regarded as A. castellanii complex by riboprints. KA/LS1 type was the most predominant (51.3%) in the present survey area, followed by KA/LS2 (20.9%), and KA/LS5 (7.7%) types. Amoebae of KA/LS1 type had the same mtDNA RFLP and riboprint patterns as KA/E2 and KA/E12 strains, clinical isolates from Korean keratitis patients. Amoebae of KA/LS2 type had the identical mtDNA RFLP patterns with A. castellanii Ma strain, a corneal isolate from an American patient as amoebae of KA/LS5 type, with KA/E3 and KA/E8 strains from other Korean keratitis patients. Amoebae of KA/LS18 type had identical patterns with JAC/E1, an ocular isolate from a Japanese patient. Three types, which remain unidentified at species level, were not corresponded with any clinical isolate in their mtDNA RFLP and riboprint patterns. Out of 39 isolates analyzed in this study, mtDNA RFLP and riboprint patterns of 33 isolates (84.6%) were identical to already known clinical isolates, and therefore, they may be regarded as potentially keratopathogenic. These results suggest that contact lens wearers in Seoul should pay more attention to hygienic maintenance of contact lens storage cases for the prevention of Acanthamoeba keratitis.

Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi.

Randomly selected 435 clones from Acanthamoeba healyi cDNA library were sequenced and a total of 387 expressed sequence tags (ESTs) had been generated. Based on the results of BLAST search, 130 clones (34.4%) were identified as the genes encoding surface proteins, enzymes for DNA, energy production or other metabolism, kinases and phosphatases, protease, proteins for signal transduction, structural and cytoskeletal proteins, cell cycle related proteins, transcription factors, transcription and translational machineries, and transporter proteins. Most of the genes (88.5%) are newly identified in the genus Acanthamoeba. Although 15 clones matched the genes of Acanthamoeba located in the public databases, twelve clones were actin gene which was the most frequently expressed gene in this study. These ESTs of Acanthamoeba would give valuable information to study the organism as a model system for biological investigations such as cytoskeleton or cell movement, signal transduction, transcriptional and translational regulations. These results would also provide clues to elucidate factors for pathogenesis in human granulomatous amoebic encephalitis or keratitis by Acanthamoeba.

The first human case of Trichinella spiralis infection in Korea.

Three cases of human infection by Trichinella spiralis were first confirmed by detecting encysted larvae in the biopsied muscle in December 1997, in Korea. The patients were one 35- and two 39-year-old males residing in Kochang-gun, Kyongsangnam-do. They had a common past history of eating raw liver, spleen, blood and muscle of a badger, Meles meles melanogenys, and complained of high fever, facial and periorbital edema, and myalgia. Hematologic and biochemical examinations revealed leukocytosis and eosinophilia, and highly elevated levels of GOT, GPT, LDH and CPK. In the gastrocnemius muscle of a patient, roundly coiled nematode larvae were detected. The larvae measured 0.775-1.050 (av. 0.908) mm in length, and 0.026-0.042 (av. 0.035) mm in maximum width. The specific IgG antibody levels in three patients' sera were significantly higher when compared with those of normal controls. The patients were treated with flubendazole and albendazole for 15-30 days, and discharged at 13-34 days post-admission. From the above findings, it was confirmed that T. spiralis is present in Korea, and the badger plays a role of as the natural host.

Purification and characterization of a secretory serine proteinase of Acanthamoeba healyi isolated from GAE.

We purified and characterized a serine proteinase secreted by Acanthamoeba healyi to evaluate it as a possible virulence factor in the pathogenesis of granulomatous amoebic encephalitis (GAE). Ammonium sulfate precipitated culture supernatant of A. healyi OC-3A strain was purified by chromatography on CM-Sepharose, Sephacryl-S200, and Q-2 anion-exchange columns. The purified 33-kDa enzyme had a pH optimum of 8.0 and a temperature optimum of 40 C. Phenylmethylsulfonylfluoride and diisopropyl fluorophosphate, serine proteinase inhibitors, diminished activity of the enzyme to near zero. In addition to types I and IV collagen and fibronectin, the main components of the extracellular matrix, other proteins such as fibrinogen, IgG, IgA, albumin, and hemoglobin were also degraded by the enzyme. The broad substrate specificity of this secreted serine proteinase suggests that it may play an important role in pathogenesis of GAE by A. healyi.

Phylogenetic relationships among Acanthamoeba spp. based on PCR-RFLP analyses of mitochondrial small subunit rRNA gene.

We investigated the value of mitochondrial small subunit rRNA gene (mt SSU rDNA) PCR-RFLP as a taxonomic tool for Acanthamoeba isolates with close inter-relationships. Twenty-five isolates representing 20 species were included in the analysis. As in nuclear 18S rDNA analysis, two type strains (A. astronyxis and A. tubiashi) of morphological group 1 diverged earliest from the other strains, but the divergence between them was less than in 18S riboprinting. Acanthamoeba griffini of morphological group 2 branched between pathogenic (A. culbertsoni A-1 and A. healyi OC-3A) and nonpathogenic (A. palestinensis Reich, A. pustulosa GE-3a, A. royreba Oak Ridge, and A lenticulata PD2S) strains of morphological group 3. Among the remaining isolates of morphological group 2, the Chang strain had the identical mitochondrial riboprints as the type strain of A. hatchetti. AA2 and AA1, the type strains of A. divionensis and A. paradivionensis, respectively, had the identical riboprints as A. quina Vil3 and A. castellanii Ma. Although the branching orders of A. castellanii Neff, A. polyphaga P23, A. triangularis SH621, and A. lugdunensis L3a were different from those in 18S riboprinting analysis, the results obtained from this study generally coincided well with those from 18S riboprinting. Mitochondrial riboprinting may have an advantage over nuclear 18S rDNA riboprinting because the mt SSU rDNAs do not seem to have introns that are found in the 18S genes of Acanthamoeba and that distort phylogenetic analyses.

Subgenus classification of Acanthamoeba by riboprinting.

Subgenus classification of Acanthamoeba remains uncertain. Twenty-three reference strains of Acanthamoeba including 18 (neo)type-strains were subjected for classification at the subgenus level by riboprinting. PCR/RFLP analysis of 18S rRNA gene (rDNA). On the dendrogram reconstructed on the basis of riboprint analyses, two type-strains (A. astronyxis and A. tubiashi) of morphological group 1 diverged early from the other strains and were quite distinct from each other. Four type-strains of morphological group 3, A. culbertsoni, A. palestinensis, A. healyi were considered taxonomically valid, but A. pustulosa was regarded as an invalid synonym of A. palestinensis. Strains of morphological group 2 were classified into 6 subgroups. Among them, A. griffini which has an intron in its 18S rDNA was the most divergent from the remaining strains. Acanthamoeba castellanii Castellani, A. quina Vil3, A. lugdunensis L3a, A. polyphaga Jones, A. triangularis SH621, and A. castellanii Ma strains belonged to a subgroup, A. castellanii complex. However, A. quina and A. lugdunensis were regarded as synonyms of A. castellanii. The Chang strain could be regarded as A. hatchetti. Acanthamoeba mauritaniensis, A. divionensis, A. paradivionensis could be considered as synonyms of A. rhysodes. Neff strain was regarded as A. polyphaga rather than as A. castellanii. It is likely that riboprinting can be applied for rapid identification of Acanthamoeba isolated from the clinical specimens and environments.

Radix auricularia coreana: natural snail host of Clinostomum complanatum in Korea.

An epidemiological survey was carried out to determine the first intermediate host of Clinostomum complanatum among freshwater snails in Korea. Two species of snails belonging to the family Lymnaeidae were collected in Kaum-ji (pond), Uisong-gun, Kyongsangbuk-do. Twelve (0.9%) out of 1,273 Radix auricularia coreana examined were found to liberate cercariae of C. complanatum, which were identified by morphological characteristics and experimental infections in freshwater fish. Pseudorasbora parva. The cercariae were brevifurcate and clinostomatoid. They had a transparent dorsal fin, a well developed penetrating organ and a pair of eye spots. The body measured 119-147 x 33-36 microns, tail stem, 275-370 x 19-26 microns, and furcae, 72-104 microns. Rediae were demonstrated in the infected snail after crushing. Redia, 527-1,630 microns long and 121-368 microns wide, contained 10-45 germ balls and cercariae in various developmental stages. The metacercariae recovered from fish experimentally infected with C. complanatum cercariae were morphologically identical to those from naturally infected fish.

Ultrastructural changes of Acanthamoeba cyst of clinical isolates after treatment with minimal cysticidal concentration of polyhexamethylene biguanide.

In order to understand the action mechanism of polyhexamethylene biguanide (PHMB) to the cyst of Acanthamoeba on the morphological basis, the cysts of four corneal isolates of Acanthamoeba were treated with minimal cysticidal concentration (MCC) of PHMB and their ultrastructural changes were examined by transmission electron microscopy. The most striking change of cysts treated with PHMB compared with normal cysts was the shrinkage of intracystic amoebae, which resulted in the separation of the plasma membrane of intracystic amoeba from endocystic wall. Subplasmalemmal lipid droplets became irregularly shaped. In severely damaged cysts, cytoplasm was aggregated and organelles were severely deformed. Cytoplasmic materials were leaked out through the damaged plasma membrane. Most cysts showed aggregation of nuclear chromatin material. Number of mitochondrial cristae was also reduced. Ecto- and endo-cystic walls were relatively well tolerated. Findings in the present study revealed that PHMB affected mainly on plasma membrane, but lesser on organellar membrane of intracystic amoeba. It seemed likely that PHMB might kill cystic forms of Acanthamoeba by similar mechanism in which this environmental biocide can damage the cell wall of Escherichia coli by binding with acidic phospholipids.

[Comparison of virulence by Acanthamoeba strains in a murine model of acquired immunodeficiency syndrome].

The pathogenic potential of Acanthamoeba strains was evaluated by experimental infection of murine AIDS (MAIDS) model. C57BL/6 mice were induced to immunocompromized state by intraperitoneal injection of LP-BM5 MuLV and revealed the typical splenomegaly and lymphatic enlargement of axillar and inguinal regions on necropsy 4 weeks after viral infection. Although there was no significant difference in the mortality rate of MAIDS mouse according to the culture temperature, it was very different in the mortality rate from strain to strain of Acanthamoeba. A. healyi OC-3A strain isolated from the brain of a GAE patient showed the highest mortality rate and A. culbertsoni A-1 strain from tissue culture was the second. KA/S3 and KA/S2 strains isolated from soil revealed very low virulence. The mice infected by intranasal inoculation of Acanthamoeba showed relatively chronic course than intravenous inoculation. The gross findings of lungs and brains from infected mice were variable among mice. On the microscopic observations, the lungs showed much more severe inflammation and necrosis than the brains microscopically. This MAIDS model would be useful to study the opportunistic protozoan infections of AIDS patients. In the light of these results, the pathogenic potential and the virulence of Acanthamoeba may be determined genetically.

[Bacterial endosymbiosis within the cytoplasm of Acanthamoeba lugdunensis isolated from a contact lens storage case].

Transmission electron microscopy of an Acanthamoeba isolate (KA/L5) from a contact lens case revealed bacterial endosymbionts within cytoplasm of the amoebae. The Acanthamoeba isolate belonged to the morphological group II. Based on the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of 18S ribosomal RNA coding DNA (rDNA), the isolate was identified as A. lugdunensis. Strain typing by isoenzyme analysis using isoelectric focusing (IEF) and mitochondrial (Mt) DNA RFLP revealed that the isolate was closely related with KA/L1, the most predominant type of isolates from contact lens storage cases, KA/E2, a clinical isolate, KA/W4, previously reported to host endosymbionts, and L3a strains of A. lugdunensis. The endosymbionts were similar to those of KA/W4 in aspects that they were randomly distributed in both trophozoites and cysts, and were rod-shaped bacteria measuring approximately 1.38 x 0.50 microns. But the number of endosymbionts per amoeba was significantly lower than that of KA/W4. They were neither limited by phagosomal membranes nor included in lacunaelike structure.

Species identification and molecular characterization of Acanthamoeba isolated from contact lens paraphernalia.

We applied ribosomal RNA gene (rDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to identify Acanthamoeba isolates from contact lens paraphernalia, and characterized these on the basis of mitochondrial DNA (mtDNA) RFLP and isoenzyme analysis. The 22 Acanthamoeba strains used as reference strains were obtained from the American Type Culture Collection. Twenty-eight isolates were classified into six ribogroups, as follows: Acanthamoeba ribogroup (AcRG) 1 consisted of 18 isolates; AcRG 2, of three, AcRG 3, of three; AcRG 4, of two; AcRG 5, of one, and AcRG 6, of one. AcRG 1, which was the most frequently isolated type, was identified as A. lugdunensis, and AcRG 2 as A. hatchetti. AcRG 4 was identified as A. triangularis, while AcRG 3 and AcRG 5 were closely related to A. triangularis. AcRG 6 was identified as A. castellanii. The mtDNA RFLP patterns and zymograms for five isoenzymes of the isolates belonging to a ribogroup were identical to one another. The mtDNA digestion phenotype and zymogram for acid phosphatase (AcP) of AcRG 1 were identical to those of A. lugdunensis L3a and KA/E2, the type strain and corneal isolates from a Korean keratitis patient, respectively. The mtDNA digestion phenotype and zymogram for AcP of AcRG 6 were identical to those of A. castellanii Castellani and KA/E3, the type strain and another corneal isolate found in Korea, respectively. The mtDNA RFLP and zymogram for AcP of AcRG 2 were very similar to those of A. hatchetti BH-2 and Chang, respectively the type strain and a pathogen. The mtDNA RFLP and zymogram for AcP of AcRG 4 were similar to those of A. triangularis SH621, the type strain. The mtDNA RFLP patterns of AcRG 3 and 5 were unique. These results showed that the riboprints, mtDNA RFLP and zymograms of 22 of 28 Acanthamoeba isolates were the same as or very similar to those of the clinical isolates, which can probably be regarded as keratopathogens. More attention should be paid to the prevention of contamination by Acanthamoeba and to the disinfection of contact lens paraphernalia.

Live female Enterobius vermicularis in the posterior fornix of the vagina of a Korean woman.

A 33-year-old Korean woman, para 2. visited an obstetrics and gynecology clinic, Kumi-shi, Kyongsangbuk-do, due to postcoital spotting and flank pain. She had a tubal ligation 7 years before and demonstrated back pain during menstruation. She revealed a foul smelling discharge without complaint of itching. Enterobius vermicularis eggs were demonstrated during microscopic examination of a smear taken from the posterior fornix of the vagina. On endoscopic examination of her vagina, a live worm was found in the posterior fornix. The worm was removed and identified as a female E. vermicularis based on morphology. This is the first case report of vaginal enterobiasis in Korea.

Close relatedness of Acanthamoeba pustulosa with Acanthamoeba palestinensis based on isoenzyme profiles and rDNA PCR-RFLP patterns.

The taxonomic validity of morphological group III Acanthamoeba spp. is uncertain. In the present study, six type strains of group III Acanthamoeba spp., A. culbertsoni, A. healyi, A. pustulosa, A. palestinensis, A. royreba and A. lenticulata were subjected for the evaluation of their taxonomic validity by comparison of the isoenzyme patterns by isoelectic focusing on polyacrylamide gels, mitochondrial DNA (Mt DNA) restriction fragment length polymorphism (RFLP), and small subunit ribosomal DNA (ssu rDNA) PCR-RFLP patterns. The Mt DNA RFLP patterns were heterogeneous between the species. The type strains of A. palestinensis and A. pustulosa showed almost identical patterns of isoenzymes and rDNA PCR RFLP with an estimated sequence divergence of 2.6%. The other species showed heterogeneous patterns of isoenzymes and rDNA PCR-RFLP. It is likely that A. pustulosa is closely related with A. palestinensis and that the former may be regarded as a junior synonym of the latter.

PCR and RFLP variation of conserved region of small subunit ribosomal DNA among Acanthamoeba isolates assigned to either A. castellanii or A. polyphaga.

Twelve isolates of Acanthamoeba spp. assigned to either A. castellanii or A. polyphaga, and type strains of A. culbertsoni, A. healyi, A. palestinensis, and A. astronyxis were examined by restriction fragment length polymorphism (RFLP) of a conserved region of small subunit ribosomal RNA gene (ssu rDNA) amplified by polymerase chain reaction (PCR). The PCR products of the isolates measured approximately 910-930 bp, except for that of A. astronyxis which was extraordinarily long, approximately 1,170 bp. Average of estimated sequence divergence of the amplified DNA among the isolates assigned to A. castellaii was 9.8% whereas that among the isolates assigned to A. polyphaga 9.6%. The maximum intraspecific sequence divergence among the isolates assigned to A. castellanii was observed between the Chang and Ma strains (17.3%) while that among the isolates assigned to A. polyphaga was observed between KA/S3 and KA/S7 strains (16.1%). The both maximum sequence divergences were much greater than the minimum interspecific sequence divergence between A. castellanii and A. polyphaga (2.6%) which appeared between the Castellani (or CCAP 1501/2 g) and KA/S3 strains. The PCR-RFLP patterns of A. culbertsoni, A. healyi, A. palestinensis, and A. astronyxis were quite diverse from one another and from those of isolates assigned to either A. castellanii or A. polyphaga. It is suggested that taxonomic validity of the isolates assigned to either A. castellanii or A. polyphaga should be reevaluated.

Biochemical and molecular characterization of a strain KA/S2 of Acanthamoeba castellanii isolated from Korean soil.

A strain, KA/S2, isolated from Korean soil and morphologically assigned to Acanthamoeba castellanii, was characterized by isoenzyme analysis, and total proteins profile, and mitochondrial (Mt) DNA restriction fragment length polymorphism (RFLP), and compared with four reference strains assigned to the species (the authenitic Castellani, Neff, Ma, and Chang strains). It was found that four isoenzyme, total proteins, and Mt DNA RFLP patterns by eight restriction endonucleases of the strain KA/S2 were identical with those of the Neff strain, isolated from soil of California, USA. The Chang strain was unique in its morphology and total protein patterns. Interstrain polymorphisms of isoenzyme profiles and Mt DNA RFLP patterns were observed among the Castellani, Neff, Ma, and Chang strains. Mt DNA RFLP was confirmed to be highly appropriate for the strain characterization and identification of Acanthamoeba spp.

Demonstration of the second intermediate hosts of Clinostomum complanatum in Korea.

A species of metacercariae recovered from the fresh-water fish, collected from Kaumji (Pond), Kaechonji (Pond) and Ssanggyechon (River). Uisong-gun, Kyongsangbuk-do, Korea, was identified as Clinostomum complanatum by morphological observation and experimental infection to chicks. The excysted metacercariae, tongue-shaped and progenetic, were 3.28-4.27 mm in length and 0.94-1.46 mm in width. The adult flukes recovered from the chicks four days after infection were 4.20-4.86 mm long and 1.14-1.49 mm wide. Twelve species of the fresh-water fish were found to be infected with the metacercariae. The infection rate ranged from 1.6% (Zacco temminkii) to 88.9% (Acheilognathus rhombea and Microphysogobio yaluensis). The intensity was highest in Carassius auratus (13.0/fish infected) and the abundance (relative density) was highest in A. rhombea (7.8/fish examined). This survey demonstrated for the first time the source of human infection by C. complanatum in Korea.

Interstrain polymorphisms of isoenzyme profiles and mitochondrial DNA fingerprints among seven strains assigned to Acanthamoeba polyphaga.

Interstrain polymorphisms of isoenzyme profiles and mitochondrial (Mt) DNA fingerprints were observed among seven strains of Acanthamoeba isolated from different sources and morphologically assigned to A. polyphaga. Mt DNA fingerprints by eight restriction endonucleases (Bgl II, Sca I, Cla I, EcoR I, Xba I, Kpn I, Sal I, and Sst I) revealed considerable interstrain polymorphisms. Isoenzyme profiles revealed considerable interstrain polymorphisms for acid phosphatase, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase while those for glucose phosphate isomerase, leucine aminopeptidase, and malate dehydrogenase showed similarity. Despite of the interstrain polymorphisms, the isoenzyme profiles and Mt DNA fingerprints of the strain Ap were found to be identical with those of the strain Jones. Mt DNA fingerprinting was found to be highly applicable for the strain identification, characterization, and differentiation.