Jian-Ming Huang is currently studying in National Cheng Kung University. His major project is to study the protein expression in the parasitic protozoan Acanthamoeba. He also developed antibody to identify a rapid and sensitive antibody-based assay and investigate molecular and characteristics of the M28_ AAP. A total of 2 papers were published by him based on his work.
Background: Acanthamoeba keratitis (AK) is a serious disease caused by pathogenic Acanthamoeba through wounds of cornea; by and large, most high-risk patients catching AK wear contact lens generally over a long period of time. However, microscopic examination and polymerase chain reaction following the culture that they are traditional diagnostic tests for pathogenic Acanthamoeba need long time to confirm AK. Hence, we purpose to develop a rapid and sensitive antibody-based assay to improve the diagnostic procedure. Objective: Comparing with proteomic analysis of extracellular secreted proteins expressed by two pathogenic Acanthamoeba castellanii clinical isolates and a non-pathogenic ATCC strain, we will evaluate the efficiency of Acanthamoeba M28 aminopeptidase (M28_AAP) as a diagnostic target for developing a rapid and sensitive antibodybased assay and investigate molecular and characteristics of the M28_AAP. Methods: The M28_AAP antibody was produced to detect Acanthamoeba isolates through an antibody-based diagnostic test such as a western blot or an enzyme-linked immunosorbent assay (ELISA). Results: We have produced successfully M28_AAP antibody to detect Acanthamoeba isolates through a western blot, an immunofluorescence assay (IFA) and an ELISA. Furthermore, the complement can be degraded by M28_AAP in the human innate immunity. We believe that M28_AAP will be the opportunity to develop a rapid diagnostic marker and newer target to decrease the AK patient cases. To evaluate the sensitivity and specificity of the Acanthamoeba M28_AAP antibody, we will plan to develop rapid diagnostic tests such as test strips on clinical diagnosis.
Pin-Ju, Ko is a second-year master’s student now, who is majored in parasitology. She has focused on drug metabolism, especially cytochrome P450 enzymes. Base on her research which may provide a better understanding of CYPs in Acanthamoeba but also provide insight into the development of treatment for AK.
Acanthamoeba is a free-living parasite present in rivers, lakes, seawater and soils that can cause severe infection, such as Granulomatous Amoebic Encephalitis (GAE) and Amoebic Keratitis (AK). Two main stages can be recognized in the life cycle of Acanthamoeba castellanii: the trophozoite and the cyst. Since the transformation process from a trophozoite into a cyst, also called encystation, plays an important role in resisting extreme physical and chemical conditions for Acanthamoeba cell survival. Therefore, we tried to figure out the difference between these two stages by collecting the trophozoites and encysting samples for transcriptomic high-throughput surveys. According to the transcriptome analysis, we found out several cytochrome P450 and antibiotic biosynthesis related genes expressed higher in the cyst form. Cytochromes P450 (CYPs) are proteins of the superfamily which is necessary for the detoxification of foreign chemicals and the metabolism of drugs which might be an essential process to against potential toxicity from the environment that Acanthamoeba uptake. Since previous studies have shown that CYPs play a role in drug metabolism and therefore we will try to use different expression vectors to express CYPs in both prokaryotes and eukaryotes and followed by drugs treatment to investigate the effects of CYPs. To sum up, these findings may not only provide us with a better understanding of CYPs in Acanthamoeba but also provide insight into the development of treatment for AK.