Detection of Acanthamoeba from Acanthamoeba Keratitis Mouse Model Using Acanthamoeba-Specific Antibodies.

Although the prevalence of Acanthamoeba keratitis (AK) is rare, its incidence in contact lens wearers has increased. Acanthamoeba infections can lead to the loss of vision if the diagnosis and treatment are delayed. In this study, we investigated the diagnostic potential of two antibodies raised against the adenylyl cyclase-associated protein (ACAP) and periplasmic binding protein (PBP) of A. castellanii in the AK mouse model. The specificity of ACAP and PBP antibodies to Acanthamoeba was confirmed by immunocytochemistry. AK mouse models were produced by corneal infections with A. castellanii trophozoites for 7 days and 21 days. Enzyme-linked immunosorbent assay results revealed that both ACAP and PBP antibodies successfully detected Acanthamoeba antigens in the tears and eyeball lysates of the AK mouse model. The detection levels of Acanthamoeba antigens were similar at both infection time points. Anti-Acanthamoeba IgG, IgA, and IgM antibodies were evaluated from the sera of the AK mouse model. Notably, IgM and IgA antibody responses were highest and lowest at both time points, respectively. Our findings revealed that both ACAP and PBP antibodies could detect Acanthamoeba antigens in the tears and eyeball lysates of the AK mouse model. These results provide important information for understanding Acanthamoeba infections and developing a new diagnostic tool for AK.

A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts.

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.

Establishment of an Acanthamoeba keratitis mouse model confirmed by amoebic DNA amplification.

Acanthamoeba castellanii, the causative agent of Acanthamoeba keratitis (AK), occurs mainly in contact lens users with poor eye hygiene. The findings of many in vitro studies of AK, as well as the testing of therapeutic drugs, need validation in in vivo experiments. BALB/c mice were used in this study to establish in vivo AK model. A. castellanii cell suspensions (equal mixtures of trophozoites and cysts) were loaded onto 2-mm contact lens pieces and inserted into mouse eyes that were scratched using an ophthalmic surgical blade under anesthesia and the eyelids of the mice were sutured. The AK signs were grossly observed and PCR was performed using P-FLA primers to amplify the Acanthamoeba 18S-rRNA gene from mouse ocular tissue. The experimental AK mouse model was characterized by typical hazy blurring and melting of the mouse cornea established on day 1 post-inoculation. AK was induced with at least 0.3 × 105 A. castellanii cells (optimal number, 5 × 104), and the infection persisted for two months. The PCR products amplified from the extracted mouse eye DNA confirmed the development of Acanthamoeba-induced keratitis during the infection periods. In conclusion, the present AK mouse model may serve as an important in vivo model for the development of various therapeutic drugs against AK.

Molecular detection of free-living amoebae from Namhangang (southern Han River) in Korea.

The free-living amoebae Naegleria spp. and Acanthamoeba spp. exist in the natural environment and are sometimes causal agents of lethal primary amoebic meningoencephalitis (PAM), amoebic keratitis (AK) and granulomatous amebic encephalitis (GAE) in humans, respectively. To ascertain the existence of free-living amoebae in Korea, water samples were collected from the Korean hydrosphere, Namhangang (southern Han River), an active location for water skiing and recreation. Samples underwent two-step filtration and were cultured on non-nutrient agar medium with inactivated E. coli. The remaining samples were subjected to PCR for primarily the 18S small ribosomal RNA gene and gene sequencing. Similarities in 18S rDNA sequences, in comparison with various reference amoebae in GenBank, showed 86~99% homology with N. gruberi, N. philippinensis, N. clarki, A. polyphaga, A. castellannii, and Hartmannella (Vermamoeba) vermiformis. Therefore, this study will be useful for seasonal detection of free-living amoebae from various Korean hydrospheres in future studies.

Cytopathic Change and Inflammatory Response of Human Corneal Epithelial Cells Induced by Acanthamoeba castellanii Trophozoites and Cysts.

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.

Cellular characterization of actin gene concerned with contact-dependent mechanisms in Naegleria fowleri.

Free-living amoeba, Naegleria fowleri, destroys target cells through contact-dependent mechanisms, such as phagocytosis and/or trogocytosis. A previous experiment showed that the nf-actin gene consisted of 1.2 kbp, produced a 50.1 kDa recombinant protein (Nf-actin), and was localized on the cytoskeleton, pseudopodia and amoebastome. In this study, cellular characterization of the nf-actin gene concerned with contact-dependent mechanisms in N fowleri was performed. The nf-actin gene was amplified from a gene-cloned vector, pEXQP5-T7/NT TOPO. The nf-actin gene was introduced into the Ubi-pEGFP-C2 vector, and Ubi-pEGFP-C2/nf-actin was transfected into N fowleri trophozoites. Strong GFP fluorescence was detected in N fowleri trophozoites transfected with Ubi-pEGFP-C2/nf-actin. Expression of EGFP-Nf-actin protein was detected by Western blot analysis. The nf-actin-overexpressing N fowleri showed significantly increased adhesion activity against extracellular matrix components, fibronectin, collagen I and fibrinogen, compared with wild-type N fowleri. Moreover, nf-actin-overexpressing N fowleri showed increased phagocytic activity and cytotoxicity in comparison with wild-type N fowleri. In summary, the overexpressed nf-actin gene has an important function in ability to increase cell adhesion, cytotoxicity and phagocytosis by N fowleri.