The role of Acanthamoeba castellanii (T4 genotype) antioxidant enzymes in parasite survival under H2O2-induced oxidative stress.

Acanthamoeba castellanii (A. castellanii) is an important opportunistic parasite. Induction of oxidative stress by the host immune system is one of the most important defense strategies against parasites. Hence, parasites partly deal with oxidative stress by different mechanisms. Identifying resistance mechanisms of A. castellanii parasites against oxidative stress is important to achieve a new therapeutic approach. Thus, this study aimed to understand the resistance mechanisms of A. castellanii, against oxidative stress. Trophozoites of A. castellanii were treated with different concentrations of H2O2. The half maximal inhibitory concentration (IC50) of H2O2 was determined using the MTT assay. The induction of oxidative stress was confirmed by flow cytometer. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were determined. The gene expression levels of CAT and SOD were measured by qRT-PCR. Furthermore, 3-amino-1:2:4-triazole (3-AT) and potassium cyanide (KCN) were used as specific inhibitors of CAT and SOD, respectively. Cell cycle assay and the apoptosis were evaluated by flow cytometer. The activities of SOD, CAT, GR, and GPx, showed an increase in oxidative stress. The cell cycle analysis revealed that most of the cellular population was in G0 and G1 phases. The apoptosis increased in oxidative stress conditions. Moreover, the apoptosis significantly increased after the specific inhibition of CAT and SOD under oxidative stress. The gene expression levels of CAT and SOD significantly increased under oxidative stress. A. castellanii can resist the host immune system through various mechanisms, including evoking its antioxidant enzymes. Therefore, by reducing or inhibiting the activity of the parasite's antioxidant enzymes such as SOD and CAT, it is possible to cope with A. castellanii.

Anti-Acanthamoeba castellanii activity of alkaloid-enriched extracts and lycorine from the Amaryllidaceae species

Abstract Free-living amoebae of the genus Acanthamoeba are the causative agents of granulomatous encephalitis and keratitis, severe human infections. Bioactive compounds from plants are recognized as an alternative source for the development of new drugs. The Amaryllidaceae is a botanical family able to synthesize a very specific and consistent group of biologically active isoquinoline-like alkaloids. The alkaloidal fractions from the Brazilian species Hippeastrum canastrense, H. diniz-cruziae, H. puniceum, and Crinum x amabile, along with the alkaloid lycorine, were investigated against Acanthamoeba castellanii. The in vitro assays were performed with distinct concentrations of lycorine and alkaloidal fractions, while the cell viability was evaluated by the MTT method upon MDCK cells. Chlorhexidine 0.02% was used as the positive control. The effect of alkaloid fractions was concentration dependent, and 2000 µg mL-1 of H. canastrense and H. diniz-cruziae provided a 100% inhibition. At concentrations of 250, 500, and 1000 µg mL-1, the H. diniz-cruziae alkaloidal fraction showed the lowest cytotoxic effect (5%-7%) and remarkable anti-amoebic activity, demonstrating values of IC50 285.61 µg mL-1, low cytotoxicity (5%-7%), and selectivity index (7.0). Taken together, the results are indicative of the great potential that the alkaloids from H. diniz-cruziae have as new candidates for anti-amoebicidal compounds

DNA content in Acanthamoeba during two stress defense reactions: Encystation, pseudocyst formation and cell cycle.

During environmental stress, the vegetative cells of the facultative pathogenic amoeba Acanthamoeba castellanii reversibly differentiate into resistant dormant stages, namely, cysts or pseudocysts. The type of resistant stage depends on the nature and duration of the stressor. Cell differentiation is accompanied by changes in morphology and cellular metabolism. Moreover, cell differentiation is also expected to be closely linked to the regulation of the cell cycle and, thus, to cellular DNA content. While the existence of the resistant stages in A. castellanii is well known, there is no consensus regarding the relationship between differentiation and cell cycle progression. In the present work, we used flow cytometry analysis to explore the changes in the DNA content during Acanthamoeba encystation and pseudocyst formation. Our results strongly indicate that A. castellanii enters encystation from the G2 phase of the cell cycle. In contrast, differentiation into pseudocysts can begin in the G1 and G2 phases. In addition, we present a phylogenetic analysis and classification of the main cell cycle regulators, namely, cyclin-dependent kinases and cyclins that are found in the genome of A. castellanii.

Isolates from ancient permafrost help to elucidate species boundaries in Acanthamoeba castellanii complex (Amoebozoa: Discosea).

Acanthamoeba castellanii species complex (genotype T4) comprises of more than ten species with unclear synonymy. Its molecular phylogeny has several conflicts with published morphological data. In this paper, we analyze morphometric traits and temperature preferences in six new strains belonging to A. castellanii complex isolated from Arctic permafrost in the framework of molecular phylogeny. This integrative approach allows us to cross-link genotypic and phenotypic variability and identify species-level boundaries inside the complex. We also analyze previously known and newly found discrepancies between the nuclear and mitochondrial gene-based phylogenies. We hypothesize that one reason for these discrepancies may be the intragenomic polymorphism of ribosomal RNA genes.

Clinical presentations, genotypic diversity and phylogenetic analysis of Acanthamoeba species causing keratitis.

Introduction. Acanthamoeba keratitis is a sight-threatening corneal infection that is commonly reported among contact lens users and those suffering from corneal trauma. The prevalence of Acanthamoeba species or genotypes in causing keratitis infection is not well known.Aim. This study was conducted to identify and genotype Acanthamoeba isolates from keratitis patients, targeting the ribosomal nuclear subunit (Rns) region, and describe the associated clinical presentation and treatment outcome.Methodology. Thirty culture-confirmed patients with Acanthamoeba keratitis, identified in a tertiary eye care centre in South India during the period from December 2016 to December 2018, were included in this study. The data collected from patient records include demographic details, history of illness, mode of trauma, treatment history and follow-up status. The genotype and species were identified based on the Rns sequence and phylogenetic tree analysis.Results. Acanthamoeba culbertsoni was the most predominant keratitis-causing species, followed by Acanthamoeba quina, Acanthamoeba castellanii, Acanthamoeba healyi, Acanthamoeba hatchetti, Acanthamoeba polyphaga and Acanthamoeba stevensoni. Three major genotypes were identified (T4, T11 and T12), with the T4 genotype being the most predominant, with four subclusters, i.e. T4A, T4B, T4D and T4E. This is the first report on corneal infection by the A. stevensoni T11 genotype and the A. healyi T12 genotype. No significant correlation was observed between the clinical outcomes of corneal disease and the genotypes or species.Conclusion. Rns genotyping is very effective in identifying the Acanthamoeba species and genotype in keratitis. Genotyping of Acanthamoeba spp. will help to advance our understanding of genotype-specific pathogenesis and geographical distribution.

Acanthamoeba castellanii: A new high-throughput method for drug screening in vitro.

Despite significant public health impact, there is no specific antiprotozoal therapy for prevention and treatment of Acanthamoeba castellanii infection. There is a need for new and efficient anti-Acanthamoeba drugs that are less toxic and can reduce treatment duration and frequency of administration. In this context a new, rapid and sensitive assay is required for high-throughput activity testing and screening of new therapeutic compounds. A colorimetric assay based on sulforhodamine B (SRB) staining has been developed for anti-Acanthamoeba drug susceptibility testing and adapted to a 96-well microtiter plate format. Under these conditions chlorhexidine was tested to validate the assay using two clinical strains of A. castellanii (Neff strain, T4 genotype [IC50 4.68±0.6µM] and T3 genotype [IC50 5.69±0.9µM]). These results were in good agreement with those obtained by the conventional Alamar Blue assay, OCR cytotoxicity assay and manual cell counting method. Our new assay offers an inexpensive and reliable method, which complements current assays by enhancing high-throughput anti-Acanthamoeba drug screening capabilities.

Effect of non-steroidal anti-inflammatory drugs on biological properties of Acanthamoeba castellanii belonging to the T4 genotype.

Non-steroidal anti-inflammatory drug, Diclofenac, targeting COX have shown promise in the treatment of Acanthamoeba keratitis, but the underlying mechanisms remain unknown. Using various NSAIDs, Diclofenac sodium, Indomethacin, and Acetaminophen, here we determined the effects of NSAIDs on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype. Using amoebicidal assays, the results revealed that Diclofenac sodium, and Indomethacin affected growth of A. castellanii. In contrast, none of the compounds tested had any effect on the viability of A. castellanii. Importantly, all NSAIDs tested abolished A. castellanii encystation. This is a significant finding as the ability of amoebae to transform into the dormant cyst form presents a significant challenge in the successful treatment of infection. The NSAIDs inhibit production of cyclo-oxegenase, which regulates the synthesis of prostaglandins suggesting that cyclooxygenases (COX-1 and COX-2) and prostaglandins play significant role(s) in Acanthamoeba biology. As NSAIDs are routinely used in the clinical practice, these findings may help design improved preventative strategies and/or of therapeutic value to improve prognosis, when used in combination with other anti-amoebic drugs.

The effect of peptidic and non-peptidic proteasome inhibitors on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype.

The treatment of Acanthamoeba infections remains problematic, suggesting that new targets and/or chemotherapeutic agents are needed. Bioassay-guided screening of drugs that are clinically-approved for non-communicable diseases against opportunistic eukaryotic pathogens is a viable strategy. With known targets and mode of action, such drugs can advance to clinical trials at a faster pace. Recently Bortezomib (proteasome inhibitor) has been approved by FDA in the treatment of multiple myeloma. As proteasomal pathways are well known regulators of a variety of eukaryotic cellular functions, the overall aim of the present study was to study the effects of peptidic and non-peptidic proteasome inhibitors on the biology and pathogenesis of Acanthamoeba castellanii of the T4 genotype, in vitro. Zymographic assays revealed that inhibition of proteasome had detrimental effects on the extracellular proteolytic activities of A. castellanii. Proteasome inhibition affected A. castellanii growth (using amoebistatic assays), but not viability of A. castellanii. Importantly, proteasome inhibitors affected encystation as determined by trophozoite transformation into the cyst form, as well as excystation, as determined by cyst transformation into the trophozoite form. The ability of proteasome inhibitor to block Acanthamoeba differentiation is significant, as it presents a major challenge in the successful treatment of Acanthamoeba infection. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic infections.

Comparative proteomic analysis of extracellular secreted proteins expressed by two pathogenic Acanthamoeba castellanii clinical isolates and a non-pathogenic ATCC strain.

Acanthamoeba keratitis (AK) is a serious ocular disease caused by pathogenic Acanthamoeba gaining entry through wounds in the corneal injury; generally, patients at risk for contracting AK wear contact lenses, usually over a long period of time. Moreover, pathogenic Acanthamoeba causes serious consequences: it makes the cornea turbid and difficult to operate on, including procedures such as enucleation of the eyeball. At present, diagnosis of this disease is not straightforward, and treatment is very demanding. We have established the comparative transcriptome and extracellular secreted proteomic database according to the non-pathogenic strain ATCC 30010 and the pathogenic strains NCKU_B and NCKU_D. We identified 44 secreted proteins successfully, 10 consensus secreted proteins and 34 strain-specific secreted proteins. These proteins may provide targets for therapy and immuno-diagnosis of Acanthamoeba infections. This study shows a suitable approach to identify secreted proteins in Acanthamoeba and provides new perspectives for the study of molecules potentially involved in the AK.

Anaerobic respiration: In vitro efficacy of Nitazoxanide against mitochondriate Acanthamoeba castellanii of the T4 genotype.

Acanthamoeba is an opportunistic protist pathogen that is responsible for serious human and animal infection. Being one of the most frequently isolated protists from the environment, it is likely that it readily encounters microaerophilic environments. For respiration under anaerobic or low oxygen conditions in several amitochondriate protists, decarboxylation of pyruvate is catalyzed by pyruvate ferredoxin oxidoreductase instead of pyruvate dehydrogenase. In support, Nitazoxanide, an inhibitor of pyruvate ferredoxin oxidoreductase, is effective and non-mutagenic clinically against a range of amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. The overall aim of the present study was to determine in vitro efficacy of Nitazoxanide against Acanthamoeba castellanii. At micromolar concentrations, the findings revealed that Nitazoxanide neither affected A. castellanii growth or viability nor amoeba-mediated host cell monolayer damage in vitro or extracellular proteolytic activities. Similarly, microaerophilic conditions alone had no significant effects. In contrast, microaerophilic conditions together with Nitazoxanide showed amoebicidal effects and inhibited A. castellanii-mediated host cell monolayer damage as well as extracellular proteases. Using encystation assays, it was observed that Nitazoxanide inhibited trophozoite transformation into cysts both under aerophilic and microaerophilic conditions. Furthermore, pre-treatment of cysts with Nitazoxanide inhibited A. castellanii excystation. These findings are important in the identification of potential targets that could be useful against parasite-specific respiration as well as to understand the basic biology of the life cycle of Acanthamoeba.

Essential Role for an M17 Leucine Aminopeptidase in Encystation of Acanthamoeba castellanii.

Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba.

The role of G protein coupled receptor-mediated signaling in the biological properties of Acanthamoeba castellanii of the T4 genotype.

Despite advances in antimicrobial chemotherapy and supportive care, the prognosis of Acanthamoeba infections remains poor, suggesting that new targets are needed that can affect parasite survival and host-pathogen interactions. G proteins and their coupled receptors are well known regulators of a variety of cellular functions. The overall aim of the present study was to study the role of G-protein coupled receptor, ß adrenergic receptor on the biology and pathogenesis of keratitis isolate of Acanthamoeba castellanii of the T4 genotype. Inhibition of ß adrenergic receptor using antagonist, propranolol had detrimental effects on the extracellular proteolytic activities A. castellanii as determined using zymographic assays. Conversely, ß adrenergic receptor agonist, isoprenaline showed increased proteases. Interestingly, ß adrenergic receptor inhibition affected A. castellanii growth (using amoebistatic assays), viability (using amoebicidal assays by measuring uptake of Trypan blue) and encystation as determined by trophozoite transformation into the cyst form. Pre-treatment of parasites with propranolol hampered A. castellanii-mediated human brain microvascular endothelial cell cytotoxicity, as measured by the lacatate dehydrogenase release. The aforementioned findings suggest that G-protein coupled receptor, ß adrenergic receptor-mediated signaling in A. castellanii biology and pathogenesis may offer new pharmacological targets.

Acanthamoeba castellanii STAT protein.

STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

The effect of environmental and physiological conditions on excystation of Acanthamoeba castellanii belonging to the T4 genotype.

Excystation in Acanthamoeba is an important property for the onset of infection as well as infection recurrence, post-treatment. The overall aim of this study was to determine the effects of several environmental and physiological parameters on excystation in Acanthamoeba castellanii belonging to the T4 genotype. Cysts were prepared by inoculating A. castellanii trophozoites on non-nutrient agar plates for up to 2 weeks. To determine the effects of various conditions on excystation, A. castellanii cysts were inoculated in growth medium i.e. PYG and incubated at varying temperatures (4-40 °C), various pHs (4-9), artificial light/dark cycles and 5% of CO2. Optimum excystation was observed when cysts were incubated at 30 °C in growth medium at neutral pH. Extremes of temperature and pH reduced excystation, while light/dark cycles had no effect on excystation of A. castellanii. On the other hand, 5% of CO2 enhanced excystation and growth of excysting amoebae. To determine the effect of serum on A. castellanii excystation, assays were performed in the presence of varying concentrations of heat-inactivated foetal bovine serum (FBS) (5-100%). The results revealed that FBS promoted excystation. The involvement of G proteins in excystation was also determined. Using propranolol hydrochloride, a G protein inhibitor, the results revealed that G proteins play a role in A. castellanii differentiation. Furthermore, organic solvents (methanol/ethanol) completely blocked excystation. None of the aforementioned conditions had any effect on the viability of A. castellanii. A complete understanding of excystation in A. castellanii will be of value to counter infection recurrence.

Inefficacy of marketed contact lens disinfection solutions against keratitis-causing Acanthamoeba castellanii belonging to the T4 genotype.

The aim of this study was to assess the anti-amoebic effects of marketed contact lens disinfecting solutions. Using amoebistatic, amoebicidal, and cysticidal assays, nine different contact lens solutions were tested including: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Replenish, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra+, Kontex Soak. In vitro growth inhibition (amoebistatic) assays were performed by incubating Acanthamoeba castellanii with aforementioned contact lens disinfection solutions as per manufacturer's instructions in the growth medium for up to 48h at 30°C. To determine amoebicidal and cysticidal effects, amoebae were incubated with contact lens solutions in phosphate buffered saline for 24h and viability was determined by haemocytometer counting as well as re-inoculating them in the growth medium. For controls, solutions were tested against bacterial corneal pathogen, Pseudomonas aeruginosa, as well as amoebae were incubated with the solvent alone. Of the nine contact lens solutions tested, none of them showed potent amoebicidal effects. Only DuraPlus and OptiFree Replenish exhibited trophozoite lysis of 85.3% and 73.7% respectively. In contrast, all contact lens disinfection solutions except Kontex Clean, Kontex Normal, Kontex Multisol extra+, tested showed amoebistatic effects. Importantly, none of the contact lens disinfection solutions exhibited cysticidal effects using qualitative assays, i.e., cysts treated with aforementioned solutions re-emerged as viable amoebae upon inoculation in the growth medium. However, more than 3-log reduction was observed when ReNu MultiPlus, DuraPlus and OptiFree Express were tested against P. aeruginosa which is in accordance with the ISO Stand-Alone Primary acceptance criteria. These findings are of great concern for contact lens users.

Identification and quantification of the Acanthamoeba species and genotypes from reservoirs in Taiwan by molecular techniques.

The occurrence of Acanthamoeba was investigated from 21 main reservoirs of Taiwan with 12 (57.1%) testing positive. Analysis of the 18S rRNA gene PCR product was performed in order to identify the Acanthamoeba isolates. Acanthamoeba spp. concentrations were determined according to TaqMan real-time qPCR. Acanthamoeba genotypes of all isolates were identified T4. The species were categorized to Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba hatchetti. The concentration of Acanthamoeba spp. in detected positive reservoir water samples was in the range of 3.0-1.8 × 10(3) cells/L. These results highlight the importance of Acanthamoeba in reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila.

BACKGROUND: Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts. METHODS: Here, we used a keratitis isolate of A. castellanii of the T4 genotype and studied its interactions with two bacterial genera which have not been tested before, Enterobacter aerogenes, and Aeromonas hydrophila, as well as E. coli. Assays were performed to determine bacterial association with and invasion of A. castellanii. Additionally, bacterial survival intracellular of A. castellanii trophozoites as well as cysts was determined. RESULTS: All three bacterial isolates tested, associated, invaded, and survived inside A. castellanii trophozoites as well as A. castellanii cysts. However, E. aerogenes and E. coli exhibited significantly reduced association with and invasion of A. castellanii as compared with A. hydrophila (P < 0.01 using paired T-test, one tail distribution). In the long term survival assays, all three bacterial isolates tested remained viable inside A. castellanii trophozoites, while amoeba remained intact; however A. hydrophila exhibited higher survival inside amoebae (14.54 ± 3.3 bacteria:amoeba ratio) compared with E. aerogenes (3.96 ± 0.7 bacteria:amoeba ratio) and E. coli (5.85 ± 1.1 bacteria:amoeba ratio). A. hydrophila, E. coli, and E. aerogenes remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (14.13 ± 0.89 A. hydrophila:amoeba ratio, 10.13 ± 1.17 E. aerogenes:amoeba ratio, and 11.95 ± 0.7 E. coli:amoeba ratio). CONCLUSIONS: A. hydrophila and E. aerogenes also joined the ranks of other bacteria that could benefit from A. castellanii. Because cysts can be airborne, these findings suggest that Acanthamoeba is a potential vector in the transmission of A. hydrophila and E. aerogenes to susceptible hosts.

The effect of different environmental conditions on the encystation of Acanthamoeba castellanii belonging to the T4 genotype.

In this study, Acanthamoeba castellanii was cultivated under different stress conditions to induce possible encystation. The morphological and histological properties were analysed by light and electron microscopy as well as cyst-specific staining. The findings revealed that cysts prepared through liquid medium using higher osmolarity as a trigger (10% glucose with 50mM magnesium chloride for 72 h) are similar to cysts prepared using non-nutrient agar (nutrient deprivation as a trigger in plating assays for 14 days), as determined by SDS-resistance, cyst-specific Calcofluor white staining and transmission electron microscopy. Using liquid medium assay, A. castellanii encystation was studied by exposing trophozoites to media lacking growth ingredients (phosphate buffered saline or distilled water), inappropriate temperatures (4-45°C), pH (3-9), artificial light-dark cycles, 5% CO2, and microaerophilic conditions. Optimal encystation was observed when cells were incubated in PBS with 50mM MgCl2 and 10% glucose at 24-30°C at pH 7. Increasing temperature over 37°C or pH 9 adversely affected encystation, while light-dark cycles, 5% CO2 and microaerophilic conditions had no effect on encystation of A. castellanii. None of the aforementioned conditions had any effect on the viability of A. castellanii, as determined by Trypan blue exclusion assay. A complete knowledge of encystation in A. castellanii is crucial to our understanding of the biology of these ecologically and medically important organisms.

Acanthamoeba genotype T4 detected in naturally-infected feline corneas found to be in homology with those causing human keratitis.

A total of 10 out of 65 cornea swab samples from cats with eye symptoms showed Acanthamoeba-like morphology after cultivation. By PCR and DNA sequencing of Acanthamoeba diagnostic fragment 3 (DF3), all 10 isolates from the positive samples were categorized into two homologous groups of AfC1 (PM1, PM2, PM3, PF6, KM7, KF8, KMK9) and AfC2 (PM4, PM5, KFK10) due to the presence of bases A(354) and G(354), respectively. Furthermore, DF3 of AfC1 and AfC2 showed 100% similarity with Genbank reference isolates with the accession numbers DQ087314, EU146073 and U07401, GU808323, which were Acanthamoeba castellanii strains genotype T4 originating from human keratitis. This finding suggests that A. castellani strains have the capability to infect cats and human under favorable conditions.

Protein kinase C signaling molecules regulate encystation of Acanthamoeba.

Protein kinase C (PKC) is involved in receptor desensitization, membrane biogenesis, transcription regulation, immune response mediation, and cell growth regulation. Results of ESTs analysis and microarray analysis of Acanthamoeba cysts revealed high expression of PKC during encystation of Acanthamoeba. PKC inhibitor, chelerythrine chloride, inhibited cyst maturation of Acanthamoeba. Through domain search analysis, we found 27 types of PKC genes from Acanthamoeba ESTs (AcPKC1-AcPKC27), all of which were defined as atypical PKC isoforms. Results of fluorescence microscopic analysis showed the localization of AcPKC in cell membranes or nuclear membranes during encystation. siRNA against AcPKC reduced the encystation efficiency of Acanthamoeba. These AcPKC may be involved in several signal transduction pathways, especially during encystation of Acanthamoeba.