Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display-polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis, 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC #289 and the oligo dT₁₁-C primer, revealed the highest homology (49.8%) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.

To determine the pathogenicity of Acanthamoeba spp. isolated in Korea and to develop a isoenzymatic maker, the mortality rate of infected mice, in vitro cytotoxicity against target cells and isoenzyme band patterns were observed. Five isolates of Acanthamoeba spp. (YM-2, YM-3, YM-4, YM-5, and YM-7) were used in this study as well as three reference Acanthamoeba spp. (A. culbertsoni, A. hatchetti, and A. royreba). According to the mortality rate of infected mice, Korean isolates could be categorized into three groups: high virulent (YM-4), low virulent (YM-2, YM-5, YM-7) and the nonpathogenic group (YM-3). In addition, the virulence of Acanthamoeba spp. was enhanced by brain passage in mice. In the cytotoxicity assay against chinese hamster ovary cells, especially, the cytotoxicity of brain-passaged amoebae was relatively higher than the long-term cultivated ones. The zymodeme patterns of glucose-6-phosphate dehydrogenase (G6PD), malate dehydrogenase (MDH), hexokinase (HK), glutamate oxaloacetate transaminase (GOT) and malic enzyme (ME) of Acanthamoeba spp. were different among each isolate, and also between long-term cultured amoebae and brain passaged ones. In spites of the polymorphic zymodemes, a slow band of G6PD and HK, and an intermediate band of MDH were only observed in pathogenic Acanthamoeba spp., which should be used as isoenzymatic makers.