Pathogenicity of Korean isolates of Acanthamoeba by observing the experimental infection and zymodemes of five isoenzymes

Amoebae

The amoebae were axenically cultured and maintained at 37℃ in CGV medium (Willaert, 1976) in plastic culture flasks. Five Korean isolates of Acanthamoeba spp. and three reference Acanthamoeba strains used in the present study are listed in Table 1.

Table 1.

Acanthamoeba spp. used in this study

Preparation of amoebae lysate

Live trophozoites were washed twice with 0.85% saline solution, centrifuged and suspended in the equal volume of enzyme stabilizer (1 mM dithiothreithol, 1 mM ε-aminocaproic acid, 1 mM ethylene diaminotetraacetic acid). Then, the amoebae were subjected to 3 to 4 cycles of freezing and thawing by the method of Moura et al. (1992). The lysate was prepared by centrifugation at 24,000 g at 4℃ for 30 min, and stored at -70℃ until use. The protein concentration of the lysate was determined by the method of Bradford (1976).

Observation of experimental GAE development

Mice used for the experimental infection were outbred ICR mice each weighing 15 to 18 g. Trophozoites of Acanthamoeba spp. were harvested, washed twice with saline and suspended in saline (1 × 10⁵ trophozoites per 5 µl saline). Suspended trophozoites were inoculated intranasally into mice under anesthesia that was induced by intraperitoneal injection of secobarbital, 0.06 mg per gram of mouse body weight. The death of mice was observed after day 30 post-inoculation (PI). Autopsy was performed immediately after the death, and pieces of the forebrain tissue were placed into the culture medium for amoebae. In order to compare the virulence between long-term cultured (subcultured since the primary isolation) and brain-passaged amoebae (subcultured from the brain tissue of infected mice), the isolating amoebae were also cultured for subsequent infection.

In vitro cytotoxicity

The chinese hamster ovary (CHO) cells were used as target cells. The CHO cells were cultured and maintained in a Eagle's minimal essential medium (Gibco BRL, NY, USA) in a 5% CO₂ incubator at 37℃ and harvested by treatment of trypsin-EDTA solution (Gibco BRL). After the cells were suspended in 0.4% trypan blue, the number of viable cell was counted using the hemocytometer.

The non-radioactive cytotoxicity assay (Promega, WI, USA) was used to determine the cytopathic effect of live trophozoites of Acanthamoeba and their lysates. The target cells (1 × 10⁴) and various concentrations of lysates were added to each well of 96-well round-bottomed plates. The plates were spun at 250 g for 4 min and incubated at 37℃ in a 5% CO₂ incubator for 4 hr. After centrifugation, 50 µl of supernatant fluid was harvested from each well, transferred to the fresh 96-well microtiter plates containing equal volume of substrate mixture and incubated at the room temperatute for 30 min. The absorbance of red colored formazan, which was developed by lactic dehydrogenase released from the dead target cells, was read at 490 nm by ELISA reader (Dynatech MR 5000, USA). The percent of cytotoxicity was calculated by using the following equation;

Observation of isoenzyme band patterns

The lysates were subjected to thin-layer starch-gel electrophoresis as previously described by Soliman et al. (1982) with some modifications. A constant 75 mA of power was supplied to each gel for 4 hr. Chemicals used for the assay include glutamate oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD), and hexokinase (HK). Preparation of buffers, coenzymes, substrates, and other staining procedures are listed in Table 2.

Table 2.

Staining reagent buffers used for visualizing isoenzymes

Pathogenicity in mice

Mice inoculated intranasally with amoeba began to die after day 4 PI, but no death was observed from mice inoculated with Acanthamoeba sp. YM-3. The mortality rate varied among different strains or isolates of Acanthamoeba, but the majority of mice died from day 6 to day 15 PI (Table 3). The mortality rate was the highest in mice infected with Acanthamoeba sp. YM-4 (Fig. 1). Although some of the infected mice survived, the amoebae were detected from the brains of mice after sacrifice. The virulence between the long-term cultured and second brain-passaged amoeba (especially Acanthamoeba sp. YM-4, YM-5, and A. hatchetti) revealed a significant difference in intensification. Mice inoculated with brain-passaged amoebae died earlier than mice with long-term cultured amoebae. Especially, Acanthamoeba sp. YM-4 produced a low mortality rate (2.8 %) in mice inoculated with the long-term cultured amoeba, but they induced a higher mortality rate (65.5 %) when they went through the brain passages twice. Acanthamoeba culbertsoni, A. hatchetti, Acanthamoeba sp. YM-2, and YM-5 also exhibited the enhanced virulence (P<0.001). According to the above results, the Korean isolates and reference Acanthamoeba spp. can be divided into three groups based on the mortality rate of infected mice: highly virulent (Acanthamoeba sp. YM-4, A. culbertsoni and A. hatchetti), low virulent (Acanthamoeba sp. YM-2, YM-5, YM-7 and A. royreba), and the non-pathogenic groups (Acanthamoeba sp. YM-3).

Table 3.

Mortality of the mice infected intranasally with Acanthamoeba spp.

Fig. 1

The mortality of mice infected intranasally with Korean isolates of Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, and YM-7.

Cytotoxicity against CHO cells

Cytotoxicity of the Korean isolates of Acanthamoeba spp. did not agree with the level of pathogenicity demonstrated by the mortality rate of the infected mice (Table 4.). Nevertheless, the cytotoxicity of the nonpathogenic YM-3 was relatively low when compared to that of the pathogenic Korean isolates, Acanthamoeba sp. YM-4 or YM-7; in addition, the cytotoxicity of the brain-passaged amoeba was relatively higher than that of the long-term cultivated ones.

Table 4.

Cytotoxicity (%) of Acanthamoeba spp. against the chinese hamster ovary cells

Isoenzyme patterns

Three migrating bands (slow, intermediate, and fast) of different electrophoretic mobility in each isoenzyme were observed (Table 5). All strains or isolates of Acanthamoeba spp. exhibited multiple forms of each isoenzyme patterns. But the isoenzyme band patterns were correlated with the pathogenicity or virulence, which was demonstrated by the mortality of the infected mice in this experiment. In case of G6PD, all pathogenic isolates or strains showed a slow band with the same mobility. And, all pathogenic or virulent isolates or strains revealed an intermediate band in migrating pattern of MDH, but did not show a slow band in HK. The isoenzyme band patterns of brain-passaged amoeba was distinctly different from those of long-term cultivated ones (Fig 2) On the other hand, the isoenzyme band patterns of brain-passaged Acanthamoeba spp. changed to those of long-term cultured ones several months later; five and ten months later in case of Acanthamoeba sp. YM-2 and A. culbertsoni, respectively (Table 6). This period was not related to the virulence degree of amoeba.

Table 5.

Rate of flow values of fraction of Acanthamoeba spp. separated electrophoretically

Fig. 2

Photographic and diagrammatic representation of isozyme zymodemes of Korean isolates and reference strains of Acanthamoeba spp. YM-2, YM-3, YM-4, YM-5, and YM-7 represent Korean isolates of Acanthamaeba spp. A. cul, Acanthamoeba culbertsoni; A. hat, A. hatchetti; A. roy, A. royreba; Glucose-6-PD, glucose-6-phosphate dehydrogenase; MDH, malate dehydrogenase; HK, Hexokinase; GOT, glutamate oxaloacetate transaminase; ME, malic enzyme; N, non-passaged amoeba; P, passaged amoeba.

Table 6.

Changes of isoenzyme band patterns of Acanthamoeba spp. subcultured several months after brain passage