Phagocytosis-associated genes in Acanthamoeba castellanii feeding on Escherichia coli.

Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.

Identifying the function of genes involved in excreted vesicle formation in Acanthamoeba castellanii containing Legionella pneumophila.

BACKGROUND: Legionella spp. can survive and replicate inside host cells such as protozoa and macrophages. After enough growth, Legionella is released from the host cells as free legionellae or Legionella-filled vesicles. The vesicles support Legionella to survive for a long time in the environment and transmit to a new host. In this study, we identified the differentially expressed genes of Acanthamoeba infected by Legionella (ACA1_114460, ACA1_091500, and ACA1_362260) and examined their roles in the formation of the excreted vesicles and escape of Legionella from the Acanthamoeba. METHODS: After ingestion of Escherichia coli and Legionella pneumophila, expression levels of target genes in Acanthamoeba were measured by real-time polymerase chain reaction (PCR) analysis. The roles of target genes were investigated by transfection of small interfering RNA (siRNA). The formation of Legionella-containing excreted vesicles and the vesicular co-localization with the lysosomes were examined by Giemsa stain and LysoTracker stain. RESULTS: ACA1_114460, ACA1_091500, and ACA1_362260 were upregulated after ingestion of Legionella in Acanthamoeba. ACA1_114460- and ACA1_091500-silenced Acanthamoeba failed to form the Legionella-containing excreted vesicles. Legionella was released as free legionellae from the Acanthamoeba. When the ACA1_362260 of Acanthamoeba was silenced, Legionella-containing excreted vesicles were fused with the lysosome. CONCLUSIONS: These results indicated that ACA1_114460, ACA1_091500, and ACA1_362260 of Acanthamoeba played important roles in the formation of Legionella-containing excreted vesicles and inhibition of the lysosomal co-localization with the phagosome.

Evaluating the Diagnostic Potential of Chorismate Mutase Poly-Clonal Peptide Antibody for the Acanthamoeba Keratitis in an Animal Model.

Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis (AK), a vision-threatening parasitic disease whose primary risk factor has been attributed to poor contact lens hygiene. Unfortunately, differential diagnosis of AK is challenging as the clinical manifestations for AK are similar to those of bacterial, fungal, or even viral keratitis. Since delayed AK diagnosis can incur permanent vision impairment, a rapid and sensitive diagnostic method is urgently needed. Here, the diagnostic potential of polyclonal antibodies targeting the chorismate mutase (CM) of Acanthamoeba spp. was evaluated in AK animal models. CM antibody specificity against Acanthamoeba trophozoites and cysts was confirmed by immunocytochemistry after co-culturing Acanthamoeba with Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus, and human corneal epithelial (HCE) cells. Enzyme-linked immunosorbent assay (ELISA) was performed using CM-specific immune sera raised in rabbits, which demonstrated that the antibodies specifically interacted with the Acanthamoeba trophozoites and cysts in a dose-dependent manner. To evaluate the diagnostic potential of the CM antibody, AK animal models were established by incubating contact lenses with an inoculum containing A. castellanii trophozoites and subsequently overlaying these lenses onto the corneas of BALB/c mice for 7 and 21 days. The CM antibody specifically detected Acanthamoeba antigens in the murine lacrimal and eyeball tissue lysates at both time points. Our findings underscore the importance of antibody-based AK diagnosis, which could enable early and differential AK diagnosis in clinical settings.

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.

Trichinella Infection Ameliorated Vincristine-Induced Neuroinflammation in Mice.

Vincristine (VCR) is a chemotherapeutic agent widely used in treatment of malignancies. However, VCR has a limitation in use since it commonly causes a painful neuropathy (VCR-induced peripheral neuropathy, VIPN). Inflammatory cytokines secreted by immune cells such as macrophages can exacerbate allodynia and hyperalgesia, because inhibiting the inflammatory response is a treatment target for VIPN. In this study, we investigated whether Trichinella spiralis, a widely studied helminth for its immunomodulatory abilities, can alleviate VCR-induced allodynia. Von Frey test showed that T. spiralis infection improved mechanical allodynia at 10 days after VCR injection. We further observed whether the difference was due to mitigated axon degeneration, but no significant difference between the groups in axonal degeneration in sciatic nerves and intra-epidermal nerve fibers was found. Conversely, we observed that number of infiltrated macrophages was decreased in the sciatic nerves of the T. spiralis infected mice. Moreover, treatment of T. spiralis excretory-secretory products caused peritoneal macrophages to secrete decreased level of IL-1ß. This study suggests that T. spiralis can relieve VCR-induced mechanical allodynia by suppressing neuroinflammation and that application of controllable degree of helminth may prove beneficial for VIPN treatment.

Specific Detection of Acanthamoeba species using Polyclonal Peptide Antibody Targeting the Periplasmic Binding Protein of A. castellanii.

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.

Sirtinol Supresses Trophozoites Proliferation and Encystation of Acanthamoeba via Inhibition of Sirtuin Family Protein.

The encystation of Acanthamoeba leads to the development of metabolically inactive and dormant cysts from vegetative trophozoites under unfavorable conditions. These cysts are highly resistant to anti-Acanthamoeba drugs and biocides. Therefore, the inhibition of encystation would be more effective in treating Acanthamoeba infection. In our previous study, a sirtuin family protein-Acanthamoeba silent-information regulator 2-like protein (AcSir2)-was identified, and its expression was discovered to be critical for Acanthamoeba castellanii proliferation and encystation. In this study, to develop Acanthamoeba sirtuin inhibitors, we examine the effects of sirtinol, a sirtuin inhibitor, on trophozoite growth and encystation. Sirtinol inhibited A. castellanii trophozoites proliferation (IC50=61.24 µM). The encystation rate of cells treated with sirtinol significantly decreased to 39.8% (200 µM sirtinol) after 24 hr of incubation compared to controls. In AcSir2-overexpressing cells, the transcriptional level of cyst-specific cysteine protease (CSCP), an Acanthamoeba cysteine protease involved in the encysting process, was 11.6- and 88.6-fold higher at 48 and 72 hr after induction of encystation compared to control. However, sirtinol suppresses CSCP transcription, resulting that the undegraded organelles and large molecules remained in sirtinol-treated cells during encystation. These results indicated that sirtinol sufficiently inhibited trophozoite proliferation and encystation, and can be used to treat Acanthamoeba infections.

Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii.

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

Detection of Acanthamoeba spp. using carboxylesterase antibody and its usage for diagnosing Acanthamoeba-keratitis.

Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.

Comparative analysis of differentially expressed genes in Acanthamoeba after ingestion of Legionella pneumophila and Escherichia coli.

Acanthamoeba spp. feeds on bacteria, fungi, and algae to obtain nutrients from the environment. However, several pathogens can survive and multiply in Acanthamoeba. Mechanisms necessary for the survival and proliferation of microorganisms in Acanthamoeba remain unclear. The object of this study was to identify effective factors for the survival of microorganisms in Acanthamoeba. Differentially expressed genes (DEGs) in A. castellanii infected by Legionella pneumophila or Escherichia coli were identified based on mRNA sequencing. A total of 2342 and 1878 DEGs were identified in Acanthamoeba with L. pneumophila and E. coli, respectively. Among these DEGs, 502 were up-regulated and 116 were down-regulated in Acanthamoeba infected by L. pneumophila compared to those in Acanthamoeba feed on E. coli. Gene ontology analysis showed that the genes encoded small GTPase-mediated signal transduction proteins in the biological process domain, intracellular proteins in the cellular component domain, and ATP binding proteins in the molecular function domain were up-regulated while integral components of membrane proteins in the cellular component domain were down-regulated in Acanthamoeba infected by Legionella compared to those in Acanthamoeba feed on E. coli. During endosymbiosis with Legionella, Acanthamoeba showed various changes in the expression of genes supposed to be involved in phagosomal maturation. Acanthamoeba infected by Legionella also showed high expression levels of aminotransferase, methyltransferase, and cysteine proteinase but low expression levels of RNA pseudouridine synthase superfamily protein and 2OG-Fe(II) oxygenase superfamily. These results provide directions for further research to understand the survival strategy of L. pneumophila in A. castellanii.

Chorismate mutase peptide antibody enables specific detection of Acanthamoeba.

Accurate and rapid diagnosis of Acanthamoeba keratitis (AK) is difficult. Although the diagnostic procedure for AK has improved, further development and effective diagnostic tool utilization for AK need to continue. Chorismate mutase is a key regulatory enzyme involved in the shikimate pathway, a metabolic pathway absent in mammals but central for amino acid biosynthesis in bacteria, fungi, algae, and plants. In this study, we describe the identification and production of a polyclonal peptide antibody targeting chorismate mutase secreted by A. castellanii, which could be used for AK diagnosis. Western blot was performed using the protein lysates and conditioned media of the human corneal epithelial (HCE) cells, non-pathogenic Acanthamoeba, pathogenic Acanthamoeba, clinical isolate of Acanthamoeba spp., and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Polyclonal antibodies raised against A. castellanii chorismate mutase specifically interacted with lysates of Acanthamoeba origin and their culture media, while such interactions were not observed from other samples. Acanthamoeba-specificity of chorismate mutase was also confirmed using immunocytochemistry after co-culturing Acanthamoeba with HCE cells. Specific binding of the chorismate mutase antibody to Acanthamoeba was observed, which were absent in the case of HCE cells. These results indicate that the chorismate mutase antibody of Acanthamoeba may serve as a method for rapid and differential Acanthamoeba identification.

Exploring medical educators' readiness and the priority of their educational needs for online teaching.

PURPOSE: This study investigated medical educators' readiness for online teaching by exploring their perceived ability and importance of online teaching competencies and identified the highest priority of their educational needs. METHODS: In this study, 144 medical education faculty members from a university were invited to participate. The faculty online teaching readiness scale was virtually distributed at the end of the spring semester of 2020 and 38 faculty members responded for 2 weeks. The collected data were analyzed with descriptive statistics, paired t-tests, Borich Needs Assessment, and the Locus for Focus model. RESULTS: The overall average perceived ability was 2.76, while the overall average perceived importance was 3.36. The course design and the technical competency categories showed the highest and lowest educational needs, respectively. Five competencies were given the highest priority of educational needs. CONCLUSION: The results revealed that the medical educators are not ready for online teaching; thus, urgent educational needs for online teaching competencies exist.

Differentially Expressed Gene Profile of Acanthamoeba castellanii Induced by an Endosymbiont Legionella pneumophila.

Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term 'integral component of membrane' were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.

Clinical performance of medical students in Korea in a whole-task emergency station in the objective structured clinical examination with a standardized patient complaining of palpitations

This study assessed the clinical performance of 150 third-year medicalstudents in Busan, Korea in a whole-task emergency objective structured clinical examination station that simulated a patient with palpitations visiting the emergency department. The examination was conducted from November 25 to 27, 2019. Clinical performance was assessed as the number and percentage of students who performed history-taking (HT), a physical examination (PE), an electrocardiography (ECG) study, patient education (Ed), and clinical reasoning (CR), which were items on the checklist. It was found that 18.0% of students checked the patient's pulse, 51.3% completed an ECG study, and 57.9% explained the results to the patient. A sizable proportion (38.0%) of students did not even attempt an ECG study. In a whole-task emergency station, students showed good performance on HT and CR, but unsatisfactory results for PE, ECG study, and Ed. Clinical skills educational programs for subjected student should focus more on PE, timely diagnostic tests, and sufficient Ed.

Identification of differentially expressed Legionella genes during its intracellular growth in Acanthamoeba.

Legionella grows intracellularly in free-living amoeba as well as in mammalian macrophages. Until now, the overall gene expression pattern of intracellular Legionella in Acanthamoeba was not fully explained. Intracellular bacteria are capable of not only altering the gene expression of its host, but it can also regulate the expression of its own genes for survival. In this study, differentially expressed Legionella genes within Acanthamoeba during the 24 h intracellular growth period were investigated for comparative analysis. RNA sequencing analysis revealed 3,003 genes from the intracellular Legionella. Among them, 115 genes were upregulated and 1,676 genes were downregulated more than 2 fold compared to the free Legionella. Gene ontology (GO) analysis revealed the suppression of multiple genes within the intracellular Legionella, which were categorized under 'ATP binding' and 'DNA binding' in the molecular function domain. Gene expression of alkylhydroperoxidase, an enzyme involved in virulence and anti-oxidative stress response, was strongly enhanced 24 h post-intracellular growth. Amino acid ABC transporter substrate-binding protein that utilizes energy generation was also highly expressed. Genes associated with alkylhydroperoxidase, glucose pathway, and Dot/Icm type IV secretion system were shown to be differentially expressed. These results contribute to a better understanding of the survival strategies of intracellular Legionella within Acanthamoeba.

The role of the Acanthamoeba castellanii Sir2-like protein in the growth and encystation of Acanthamoeba.

BACKGROUND: The encystation of Acanthamoeba leads to the development of resilient cysts from vegetative trophozoites. This process is essential for the survival of parasites under unfavorable conditions. Previous studies have reported that, during the encystation of A. castellanii, the expression levels of encystation-related factors are upregulated. However, the regulatory mechanisms for their expression during the encystation process remains unknown. Proteins in the sirtuin family, which consists of nicotinamide adenine dinucleotide-dependent deacetylases, are known to play an important role in various cellular functions. In the present study, we identified the Acanthamoeba silent-information regulator 2-like protein (AcSir2) and examined its role in the growth and encystation of Acanthamoeba. METHODS: We obtained the full-length sequence for AcSir2 using reverse-transcription polymerase chain reaction. In Acanthamoeba transfectants that constitutively overexpress AcSir2 protein, SIRT deacetylase activity was measured, and the intracellular localization of AcSir2 and the effects on the growth and encystation of trophozoites were examined. In addition, the sirtuin inhibitor salermide was used to determine whether these effects were caused by AcSir2 overexpression RESULTS: AcSir2 was classified as a class-IV sirtuin. AcSir2 exhibited functional SIRT deacetylase activity, localized mainly in the nucleus, and its transcription was upregulated during encystation. In trophozoites, AcSir2 overexpression led to greater cell growth, and this growth was inhibited by treatment with salermide, a sirtuin inhibitor. When AcSir2 was overexpressed in the cysts, the encystation rate was significantly higher; this was also reversed with salermide treatment. In AcSir2-overexpressing encysting cells, the transcription of cellulose synthase was highly upregulated compared with that of control cells, and this upregulation was abolished with salermide treatment. Transmission electron microscope-based ultrastructural analysis of salermide-treated encysting cells showed that the structure of the exocyst wall and intercyst space was impaired and that the endocyst wall had not formed. CONCLUSIONS: These results indicate that AcSir2 is a SIRT deacetylase that plays an essential role as a regulator of a variety of cellular processes and that the regulation of AcSir2 expression is important for the growth and encystation of A. castellanii.

Histone Deacetylase Inhibitors Enhance the Amoebicidal Effect of Low Concentration of Polyhexamethylene Biguanide by Inducing Apoptosis.

PURPOSE: The aim of this study was to reduce the cytotoxicity and improve the amoebicidal effect of polyhexamethylene biguanide (PHMB) at low concentrations by combining it with histone deacetylase (HDAC) inhibitors. METHODS: To reduce the cytotoxic effect on human corneal epithelial (HCE) cells, the concentration of PHMB was reduced to 0.0002%. To enhance the amoebicidal effect of PHMB, HDAC inhibitors such as suberoylanilide hydroxamic acid, MS275, or MC1568 were combined with it. Acanthamoeba and HCE cells were treated with 3 combinations to evaluate the amoebicidal and cytotoxic effects. Microscopy and fluorescence-activated cell sorting analysis were performed to investigate the apoptotic cell death of Acanthamoeba by these combinatorial treatments. RESULTS: The low concentration of PHMB (0.0002%) alone demonstrated no cytopathic effects (CPEs) on HCE cells. Three combinatorial treatments using 0.0002% PHMB with 10 µM suberoylanilide hydroxamic acid, 10 µM MS275, or 10 µM MC1568 showed higher amoebicidal effects on A. castellanii trophozoites than PHMB alone. Fluorescence-activated cell sorting analysis confirmed that HDAC inhibitors increased the apoptotic cell death of Acanthamoeba. Mild CPEs were observed from HCE cells cotreated with PHMB and the HDAC inhibitors after 24 hours of exposure. CONCLUSIONS: Combinatorial treatments showed high amoebicidal effects on Acanthamoeba and low CPEs on HCE cells, which suggests their potential application for Acanthamoeba keratitis treatment.

Effect of 2, 6-Dichlorobenzonitrile on Amoebicidal Activity of Multipurpose Contact Lens Disinfecting Solutions.

Multipurpose contact lens disinfecting solutions (MPDS) are widely used to cleanse and disinfect microorganisms. However, disinfection efficacy of these MPDS against Acanthamoeba cyst remain insufficient. 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, is capable of increasing the amoebical effect against Acanthamoeba by inhibiting its encystation. In this study, we investigated the possibility of DCB as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba cyst. Eight commercial MPDS (from a to h) were assessed, all of which displayed insufficient amoebicidal activity against the mature cysts. Solution e, f, and h showed strong amoebicidal effect on the immature cysts. Amoebicidal efficacy against mature cysts remained inadequate even when the 8 MPDS were combined with 100 µM DCB. However, 4 kinds of MPDS (solution d, e, f, and h) including 100 µM DCB demonstrated strong amoebicidal activity against the immature cysts. The amoebicidal activity of solution d was increased by addition of DCB. Cytotoxicity was absent in human corneal epithelial cells treated with either DCB or mixture of DCB with MPDS. These results suggested that DCB can enhance the amoebicical activity of MPDS against Acanthamoeba immature cyst in vitro.

Polyhexamethylene biguanide and chloroquine induce programmed cell death in Acanthamoeba castellanii.

Several chemotherapeutic drugs have been described as amoebicidal agents acting against Acanthamoeba trophozoites and cysts. However, the underlying mechanism of action is poorly characterized. Here, we describe programmed cell death (PCD) in A. castellanii induced by polyhexamethylene biguanide (PHMB) and chloroquine. We used four types of amoebicidal agents including 0.02% PHMB, 0.02% chlorhexidine digluconate, 100 µM chloroquine, and 100 µM 2,6-dichlorobenzonitrile to kill Acanthamoeba trophozoites and cysts. Exposure to PHMB and chloroquine induced cell shrinkage and membrane blebbing in Acanthamoeba, observed microscopically. Externalization of phosphatidyl serine on the membranes of Acanthamoeba was detected by annexin V staining. Apoptotic cell death of Acanthamoeba by PHMB and chloroquine was confirmed by FACS analysis. Nuclear fragmentation of Acanthamoeba was demonstrated by DAPI staining. PHMB induced PCD in trophozoites and cysts, and chloroquine induced PCD in cysts. These findings are discussed to establish the most effective treatment for Acanthamoeba-induced keratitis.

Chloroquine as a possible disinfection adjunct of disinfection solutions against Acanthamoeba.

Acanthamoeba keratitis is commonly encountered by contact lens wearers. Contact lens solution plays an important role in the safe use of contact lenses. The most popular products for disinfecting lenses are multipurpose disinfecting solutions (MPDS). However, almost all MPDS retailed in Korea are ineffective in killing Acanthamoeba. The objective of this study was to determine the possibility of using autophagy inhibitor chloroquine as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba, especially the cyst. Amoebicidal effects of eight different MPDSs combined with chloroquine (CQ), an autophagy inhibitor, and their cytotoxicities to human corneal epithelium cells were determined. Almost all MPDS showed strong amoebicidal effect on trophozoites after 8 h of exposure. However, they showed inadequate amoebicidal effect on cysts even after 24 h of exposure. MPDSs combined with 100 µM CQ increased their amoebicidal effects on immature cyst by inhibiting formation of mature cysts. Incubation with 100 µM CQ for 30 min did not have cytotoxicity to human corneal epithelial cells.